Class 2 Transferases VI

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Springer Handbook of Enzymes Volume 33

Dietmar Schomburg and Ida Schomburg (Eds.)

Springer Handbook of Enzymes Volume 33 Class 2 Transferases VI EC 2.4.2.1±2.5.1.30 coedited by Antje Chang

Second Edition

13

Professor Dietmar Schomburg e-mail: [email protected] Dr. Ida Schomburg e-mail: [email protected]

University to Cologne Institute for Biochemistry Zülpicher Strasse 47 50674 Cologne Germany

Dr. Antje Chang e-mail: [email protected]

Library of Congress Control Number: 2006926726 ISBN-10 3-540-332588-3

2nd Edition Springer Berlin Heidelberg New York

ISBN-13 978-3-540-32588-8

2nd Edition Springer Berlin Heidelberg New York

The first edition was published as Volume 12 (ISBN 3-540-60703-X) and Volume 13 (ISBN 3-540-62608-5) of the ªEnzyme Handbookº.

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. Springer is a part of Springer Science+Business Media springer.com # Springer-Verlag Berlin Heidelberg 2007 Printed in Germany The use of general descriptive names, registered names, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and free for general use. The publisher cannot assume any legal responsibility for given data, especially as far as directions for the use and the handling of chemicals and biological material are concerned. This information can be obtained from the instructions on safe laboratory practice and from the manufacturers of chemicals and laboratory equipment. Cover design: Erich Kirchner, Heidelberg Typesetting: medionet AG, Berlin Printed on acid-free paper 2/3141m-5 4 3 2 1 0

Attention all Users of the ªSpringer Handbook of Enzymesº Information on this handbook can be found on the internet at http://www.springer.com choosing ªChemistryº and then ªReference Worksº. A complete list of all enzyme entries either as an alphabetical Name Index or as the EC-Number Index is available at the above mentioned URL. You can download and print them free of charge. A complete list of all synonyms (> 25,000 entries) used for the enzymes is available in print form (ISBN 3-540-41830-X).

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Preface

Today, as the full information about the genome is becoming available for a rapidly increasing number of organisms and transcriptome and proteome analyses are beginning to provide us with a much wider image of protein regulation and function, it is obvious that there are limitations to our ability to access functional data for the gene products ± the proteins and, in particular, for enzymes. Those data are inherently very difficult to collect, interpret and standardize as they are widely distributed among journals from different fields and are often subject to experimental conditions. Nevertheless a systematic collection is essential for our interpretation of genome information and more so for applications of this knowledge in the fields of medicine, agriculture, etc. Progress on enzyme immobilisation, enzyme production, enzyme inhibition, coenzyme regeneration and enzyme engineering has opened up fascinating new fields for the potential application of enzymes in a wide range of different areas. The development of the enzyme data information system BRENDAwas started in 1987 at the German National Research Centre for Biotechnology in Braunschweig (GBF) and is now continuing at the University at Cologne, Institute of Biochemistry. The present book ªSpringer Handbook of Enzymesº represents the printed version of this data bank. The information system has been developed into a full metabolic database. The enzymes in this Handbook are arranged according to the Enzyme Commission list of enzymes. Some 4,000 ªdifferentº enzymes are covered. Frequently enzymes with very different properties are included under the same EC-number. Although we intend to give a representative overview on the characteristics and variability of each enzyme, the Handbook is not a compendium. The reader will have to go to the primary literature for more detailed information. Naturally it is not possible to cover all the numerous literature references for each enzyme (for some enzymes up to 40,000) if the data representation is to be concise as is intended. It should be mentioned here that the data have been extracted from the literature and critically evaluated by qualified scientists. On the other hand, the original authors' nomenclature for enzyme forms and subunits is retained. In order to keep the tables concise, redundant information is avoided as far as possible (e.g. if Km values are measured in the presence of an obvious cosubstrate, only the name of the cosubstrate is given in parentheses as a commentary without reference to its specific role). The authors are grateful to the following biologists and chemists for invaluable help in the compilation of data: Cornelia Munaretto and Dr. Antje Chang. Cologne Autumn 2006

Dietmar Schomburg, Ida Schomburg

VII

List of Abbreviations

A Ac ADP Ala All Alt AMP Ara Arg Asn Asp ATP Bicine C cal CDP CDTA CMP CoA CTP Cys d dDFP DNA DPN DTNB DTT EC E. coli EDTA EGTA ER Et EXAFS FAD FMN Fru Fuc G Gal

adenine acetyl adenosine 5'-diphosphate alanine allose altrose adenosine 5'-monophosphate arabinose arginine asparagine aspartic acid adenosine 5'-triphosphate N,N'-bis(2-hydroxyethyl)glycine cytosine calorie cytidine 5'-diphosphate trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid cytidine 5'-monophosphate coenzyme A cytidine 5'-triphosphate cysteine deoxy(and l-) prefixes indicating configuration diisopropyl fluorophosphate deoxyribonucleic acid diphosphopyridinium nucleotide (now NAD+ ) 5,5'-dithiobis(2-nitrobenzoate) dithiothreitol (i.e. Cleland's reagent) number of enzyme in Enzyme Commission's system Escherichia coli ethylene diaminetetraacetate ethylene glycol bis(-aminoethyl ether) tetraacetate endoplasmic reticulum ethyl extended X-ray absorption fine structure flavin-adenine dinucleotide flavin mononucleotide (riboflavin 5'-monophosphate) fructose fucose guanine galactose

IX


GDP Glc GlcN GlcNAc Gln Glu Gly GMP GSH GSSG GTP Gul h H4 HEPES His HPLC Hyl Hyp IAA IC 50 Ig Ile Ido IDP IMP ITP Km lLeu Lys Lyx M mM mMan MES Met min MOPS Mur MW NAD+ NADH NADP+ NADPH NAD(P)H

X

guanosine 5'-diphosphate glucose glucosamine N-acetylglucosamine glutamine glutamic acid glycine guanosine 5'-monophosphate glutathione oxidized glutathione guanosine 5'-triphosphate gulose hour tetrahydro 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid histidine high performance liquid chromatography hydroxylysine hydroxyproline iodoacetamide 50% inhibitory concentration immunoglobulin isoleucine idose inosine 5'-diphosphate inosine 5'-monophosphate inosine 5'-triphosphate Michaelis constant (and d-) prefixes indicating configuration leucine lysine lyxose mol/l millimol/l metamannose 2-(N-morpholino)ethane sulfonate methionine minute 3-(N-morpholino)propane sulfonate muramic acid molecular weight nicotinamide-adenine dinucleotide reduced NAD NAD phosphate reduced NADP indicates either NADH or NADPH


NBS NDP NEM Neu NMN NMP NTP oOrn pPBS PCMB PEP pH Ph Phe PHMB PIXE PMSF p-NPP Pro Q10 Rha Rib RNA mRNA rRNA tRNA Sar SDS-PAGE Ser T tH Tal TDP TEA Thr TLCK Tm TMP TosTPN Tris Trp TTP Tyr U

N-bromosuccinimide nucleoside 5'-diphosphate N-ethylmaleimide neuraminic acid nicotinamide mononucleotide nucleoside 5'-monophosphate nucleoside 5'-triphosphate orthoornithine paraphosphate-buffered saline p-chloromercuribenzoate phosphoenolpyruvate -log10[H+ ] phenyl phenylalanine p-hydroxymercuribenzoate proton-induced X-ray emission phenylmethane-sulfonylfluoride p-nitrophenyl phosphate proline factor for the change in reaction rate for a 10 C temperature increase rhamnose ribose ribonucleic acid messenger RNA ribosomal RNA transfer RNA N-methylglycine (sarcosine) sodium dodecyl sulfate polyacrylamide gel electrophoresis serine thymine time for half-completion of reaction talose thymidine 5'-diphosphate triethanolamine threonine Na-p-tosyl-l-lysine chloromethyl ketone melting temperature thymidine 5'-monophosphate tosyl-(p-toluenesulfonyl-) triphosphopyridinium nucleotide (now NADP+ ) tris(hydroxymethyl)-aminomethane tryptophan thymidine 5'-triphosphate tyrosine uridine

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U/mg UDP UMP UTP Val Xaa XAS Xyl

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mmol/(mg*min) uridine 5'-diphosphate uridine 5'-monophosphate uridine 5'-triphosphate valine symbol for an amino acid of unknown constitution in peptide formula X-ray absorption spectroscopy xylose

List of Deleted and Transferred Enzymes

Since its foundation in 1956 the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) has continually revised and updated the list of enzymes. Entries for new enzymes have been added, others have been deleted completely, or transferred to another EC number in the original class or to different EC classes, catalyzing other types of chemical reactions. The old numbers have not been allotted to new enzymes; instead the place has been left vacant or cross-references given to the changes in nomenclature. Deleted and Transferred Enzymes For EC class 2.4.2.1±2.5.1.30 these changes are: Recommended name

Old EC number Alteration

recommended name never specified glutathione S-alkyltransferase

2.4.2.13

transferred to EC 2.5.1.6

2.5.1.12

glutathione S-aryltransferase

2.5.1.13

glutathione S-aralkyltransferase

2.5.1.14

deleted, included in EC 2.5.1.18 deleted, included in EC 2.5.1.18 deleted, included in EC 2.5.1.18

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Index of Recommended Enzyme Names

EC-No.

Recommended Name

2.4.99.3 2.5.1.7 2.4.99.6 2.4.99.8 2.4.99.7

a-N-acetylgalactosaminide a-2,6-sialyltransferase . . . . . . . . UDP-N-acetylglucosamine 1-carboxyvinyltransferase . . . . . . . N-acetyllactosaminide a-2,3-sialyltransferase . . . . . . . . . . a-N-acetylneuraminate a-2,8-sialyltransferase . . . . . . . . . . a-N-acetylneuraminyl-2,3-b-galactosyl-1,3-N-acetylgalactosaminide a-2,6-sialyltransferase. . . . . . . . . . . . . . . . . . . . . adenine phosphoribosyltransferase . . . . . . . . . . . . . . . adenosylmethionine cyclotransferase . . . . . . . . . . . . . . adenylate dimethylallyltransferase . . . . . . . . . . . . . . . alkylglycerone-phosphate synthase . . . . . . . . . . . . . . . amidophosphoribosyltransferase . . . . . . . . . . . . . . . . anthranilate phosphoribosyltransferase . . . . . . . . . . . . . ATP phosphoribosyltransferase . . . . . . . . . . . . . . . . cob(I)alamin adenosyltransferase. . . . . . . . . . . . . . . . deoxyuridine phosphorylase . . . . . . . . . . . . . . . . . . dihydropteroate synthase . . . . . . . . . . . . . . . . . . . dTDP-dihydrostreptose-streptidine-6-phosphate dihydrostreptosyltransferase . . . . . . . . . . . . . . . . . . dimethylallylcistransferase. . . . . . . . . . . . . . . . . . . dimethylallyltranstransferase. . . . . . . . . . . . . . . . . . dioxotetrahydropyrimidine phosphoribosyltransferase . . . . . . discadenine synthase . . . . . . . . . . . . . . . . . . . . . dolichyl-phosphate D-xylosyltransferase . . . . . . . . . . . . . dolichyl-xylosyl-phosphate-protein xylosyltransferase . . . . . . . farnesyl-diphosphate farnesyltransferase . . . . . . . . . . . . farnesyltranstransferase . . . . . . . . . . . . . . . . . . . . flavone apiosyltransferase . . . . . . . . . . . . . . . . . . . flavonol-3-O-glycoside xylosyltransferase . . . . . . . . . . . . galactose-6-sulfurylase . . . . . . . . . . . . . . . . . . . . b-galactoside a-2,3-sialyltransferase . . . . . . . . . . . . . . b-galactoside a-2,6-sialyltransferase . . . . . . . . . . . . . . galactosyldiacylglycerol a-2,3-sialyltransferase . . . . . . . . . . geranyltranstransferase . . . . . . . . . . . . . . . . . . . . glutathione S-alkyltransferase (deleted, included in EC 2.5.1.18) . . . glutathione S-aralkyltransferase (deleted, included in EC 2.5.1.18) . . glutathione S-aryltransferase (deleted, included in EC 2.5.1.18) . . . glutathione transferase . . . . . . . . . . . . . . . . . . . . glycoprotein 2-b-D-xylosyltransferase . . . . . . . . . . . . . . guanosine phosphorylase . . . . . . . . . . . . . . . . . . . hypoxanthine phosphoribosyltransferase . . . . . . . . . . . . indolylacetylinositol arabinosyltransferase . . . . . . . . . . . . lactosylceramide a-2,3-sialyltransferase . . . . . . . . . . . . . lactosylceramide a-2,6-N-sialyltransferase . . . . . . . . . . . . methionine adenosyltransferase . . . . . . . . . . . . . . . . S-methyl-5-thioadenosine phosphorylase . . . . . . . . . . . .

2.4.2.7 2.5.1.4 2.5.1.27 2.5.1.26 2.4.2.14 2.4.2.18 2.4.2.17 2.5.1.17 2.4.2.23 2.5.1.15 2.4.2.27 2.5.1.28 2.5.1.1 2.4.2.20 2.5.1.24 2.4.2.32 2.4.2.33 2.5.1.21 2.5.1.29 2.4.2.25 2.4.2.35 2.5.1.5 2.4.99.4 2.4.99.1 2.4.99.5 2.5.1.10 2.5.1.12 2.5.1.14 2.5.1.13 2.5.1.18 2.4.2.38 2.4.2.15 2.4.2.8 2.4.2.34 2.4.99.9 2.4.99.11 2.5.1.6 2.4.2.28

Page . . . .

335 443 361 371

. . . . . . . . . . .

367 79 418 599 592 152 181 173 517 215 494

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

233 602 393 199 587 285 287 568 604 221 291 421 346 314 358 470 491 493 492 524 304 168 95 289 378 391 424 236

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Index of Recommended Enzyme Names

2.4.99.2 2.4.2.31 2.4.2.30 2.4.2.37 2.4.2.36 2.4.99.10 2.4.2.12 2.4.2.11 2.4.2.19 2.4.2.21 2.4.2.6 2.4.2.5 2.4.2.10 2.5.1.19 2.4.2.26 2.4.2.1 2.4.2.2 2.4.2.29 2.4.2.13 2.5.1.9 2.5.1.20 2.5.1.16 2.5.1.22 2.5.1.23 2.5.1.2 2.5.1.3 2.4.2.4 2.5.1.30 2.5.1.11 2.5.1.8 2.5.1.25 2.4.2.9 2.4.2.16 2.4.2.3 2.4.2.22 2.4.2.24 2.4.2.39 2.4.2.40

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monosialoganglioside sialyltransferase . . . . . . . . . . . NAD(P)+ -arginine ADP-ribosyltransferase . . . . . . . . . NAD+ ADP-ribosyltransferase . . . . . . . . . . . . . . NAD+ -dinitrogen-reductase ADP-D-ribosyltransferase . . . . NAD+ -diphthamide ADP-ribosyltransferase . . . . . . . . neolactotetraosylceramide a-2,3-sialyltransferase . . . . . . nicotinamide phosphoribosyltransferase . . . . . . . . . . nicotinate phosphoribosyltransferase . . . . . . . . . . . nicotinate-nucleotide diphosphorylase (carboxylating) . . . . nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase . . . . . . . . . . . . . . . . nucleoside deoxyribosyltransferase . . . . . . . . . . . . nucleoside ribosyltransferase . . . . . . . . . . . . . . . orotate phosphoribosyltransferase. . . . . . . . . . . . . 3-phosphoshikimate 1-carboxyvinyltransferase . . . . . . . protein xylosyltransferase . . . . . . . . . . . . . . . . purine-nucleoside phosphorylase . . . . . . . . . . . . . pyrimidine-nucleoside phosphorylase . . . . . . . . . . . queuine tRNA-ribosyltransferase . . . . . . . . . . . . . Recommended Name never specified (transferred to EC 2.5.1.6) riboflavin synthase . . . . . . . . . . . . . . . . . . . rubber cis-polyprenylcistransferase . . . . . . . . . . . . spermidine synthase . . . . . . . . . . . . . . . . . . spermine synthase . . . . . . . . . . . . . . . . . . . sym-norspermidine synthase . . . . . . . . . . . . . . . thiamine pyridinylase . . . . . . . . . . . . . . . . . . thiamine-phosphate diphosphorylase . . . . . . . . . . . thymidine phosphorylase . . . . . . . . . . . . . . . . trans-hexaprenyltranstransferase . . . . . . . . . . . . . trans-octaprenyltranstransferase . . . . . . . . . . . . . tRNA isopentenyltransferase . . . . . . . . . . . . . . . tRNA-uridine aminocarboxypropyltransferase . . . . . . . uracil phosphoribosyltransferase . . . . . . . . . . . . . urate-ribonucleotide phosphorylase . . . . . . . . . . . . uridine phosphorylase . . . . . . . . . . . . . . . . . . xanthine phosphoribosyltransferase . . . . . . . . . . . . 1,4-b-D-xylan synthase . . . . . . . . . . . . . . . . . xyloglucan 6-xylosyltransferase . . . . . . . . . . . . . . zeatin O-b-D-xylosyltransferase . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

330 272 263 299 296 387 146 137 188

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201 66 64 127 546 224 1 34 253 151 458 562 502 578 585 399 413 52 617 483 454 590 116 170 39 206 217 308 311

Description of Data Fields

All information except the nomenclature of the enzymes (which is based on the recommendations of the Nomenclature Committee of IUBMB (International Union of Biochemistry and Molecular Biology) and IUPAC (International Union of Pure and Applied Chemistry) is extracted from original literature (or reviews for very well characterized enzymes). The quality and reliability of the data depends on the method of determination, and for older literature on the techniques available at that time. This is especially true for the fields Molecular Weight and Subunits. The general structure of the fields is: Information ± Organism ± Commentary ± Literature The information can be found in the form of numerical values (temperature, pH, Km etc.) or as text (cofactors, inhibitors etc.). Sometimes data are classified as Additional Information. Here you may find data that cannot be recalculated to the units required for a field or also general information being valid for all values. For example, for Inhibitors, Additional Information may contain a list of compounds that are not inhibitory. The detailed structure and contents of each field is described below. If one of these fields is missing for a particular enzyme, this means that for this field, no data are available.

1 Nomenclature EC number The number is as given by the IUBMB, classes of enzymes and subclasses defined according to the reaction catalyzed. Systematic name This is the name as given by the IUBMB/IUPAC Nomenclature Committee Recommended name This is the name as given by the IUBMB/IUPAC Nomenclature Committee Synonyms Synonyms which are found in other databases or in the literature, abbreviations, names of commercially available products. If identical names are frequently used for different enzymes, these will be mentioned here, cross references are given. If another EC number has been included in this entry, it is mentioned here.

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CAS registry number The majority of enzymes have a single chemical abstract (CAS) number. Some have no number at all, some have two or more numbers. Sometimes two enzymes share a common number. When this occurs, it is mentioned in the commentary.

2 Source Organism For listing organisms their systematic name is preferred. If these are not mentioned in the literature, the names from the respective literature are used. For example if an enzyme from yeast is described without being specified further, yeast will be the entry. This field defines the code numbers for the organisms in which the enzyme with the respective EC number is found. These code numbers (form ) are displayed together with each entry in all fields of BRENDA where organism-specific information is given.

3 Reaction and Specificity Catalyzed reaction The reaction as defined by the IUBMB. The commentary gives information on the mechanism, the stereochemistry, or on thermodynamic data of the reaction. Reaction type According to the enzyme class a type can be attributed. These can be oxidation, reduction, elimination, addition, or a name (e.g. Knorr reaction) Natural substrates and products These are substrates and products which are metabolized in vivo. A natural substrate is only given if it is mentioned in the literature. The commentary gives information on the pathways for which this enzyme is important. If the enzyme is induced by a specific compound or growth conditions, this will be included in the commentary. In Additional information you will find comments on the metabolic role, sometimes only assumptions can be found in the references or the natural substrates are unknown. In the listings, each natural substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included only if the respective authors were able to demonstrate the formation of the specific product. If only the disappearance of the substrate was observed, the product is included without organisms of references. In cases with unclear product formation only a ? as a dummy is given. Substrates and products All natural or synthetic substrates are listed (not in stoichiometric quantities). The commentary gives information on the reversibility of the reaction,

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on isomers accepted as substrates and it compares the efficiency of substrates. If a specific substrate is accepted by only one of several isozymes, this will be stated here. The field Additional Information summarizes compounds that are not accepted as substrates or general comments which are valid for all substrates. In the listings, each substrate (indicated by a bold S) is followed by its respective product (indicated by a bold P). Products are given with organisms and references included if the respective authors demonstrated the formation of the specific product. If only the disappearance of the substrate was observed, the product will be included without organisms or references. In cases with unclear product formation only a ? as a dummy is given. Inhibitors Compounds found to be inhibitory are listed. The commentary may explain experimental conditions, the concentration yielding a specific degree of inhibition or the inhibition constant. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. Cofactors, prosthetic groups This field contains cofactors which participate in the reaction but are not bound to the enzyme, and prosthetic groups being tightly bound. The commentary explains the function or, if known, the stereochemistry, or whether the cofactor can be replaced by a similar compound with higher or lower efficiency. Activating Compounds This field lists compounds with a positive effect on the activity. The enzyme may be inactive in the absence of certain compounds or may require activating molecules like sulfhydryl compounds, chelating agents, or lipids. If a substance is activating at a specific concentration but inhibiting at a higher or lower value, the commentary will explain this. Metals, ions This field lists all metals or ions that have activating effects. The commentary explains the role each of the cited metal has, being either bound e.g. as Fe-S centers or being required in solution. If an ion plays a dual role, activating at a certain concentration but inhibiting at a higher or lower concentration, this will be given in the commentary. Turnover number (min- 1) The kcat is given in the unit min-1 . The commentary lists the names of the substrates, sometimes with information on the reaction conditions or the type of reaction if the enzyme is capable of catalyzing different reactions with a single substrate. For cases where it is impossible to give the turnover number in the defined unit (e.g., substrates without a defined molecular weight, or an undefined amount of protein) this is summarized in Additional Information.

XIX


Specific activity (U/mg) The unit is micromol/minute/milligram of protein. The commentary may contain information on specific assay conditions or if another than the natural substrate was used in the assay. Entries in Additional Information are included if the units of the activity are missing in the literature or are not calculable to the obligatory unit. Information on literature with a detailed description of the assay method may also be found. Km-Value (mM) The unit is mM. Each value is connected to a substrate name. The commentary gives, if available, information on specific reaction condition, isozymes or presence of activators. The references for values which cannot be expressed in mM (e.g. for macromolecular, not precisely defined substrates) are given in Additional Information. In this field we also cite literature with detailed kinetic analyses. Ki-Value (mM) The unit of the inhibition constant is mM. Each value is connected to an inhibitor name. The commentary gives, if available, the type of inhibition (e.g. competitive, non-competitive) and the reaction conditions (pH-value and the temperature). Values which cannot be expressed in the requested unit and references for detailed inhibition studies are summerized under Additional information. pH-Optimum The value is given to one decimal place. The commentary may contain information on specific assay conditions, such as temperature, presence of activators or if this optimum is valid for only one of several isozymes. If the enzyme has a second optimum, this will be mentioned here. pH-Range Mostly given as a range e.g. 4.0±7.0 with an added commentary explaining the activity in this range. Sometimes, not a range but a single value indicating the upper or lower limit of enzyme activity is given. In this case, the commentary is obligatory. Temperature optimum ( C) Sometimes, if no temperature optimum is found in the literature, the temperature of the assay is given instead. This is always mentioned in the commentary. Temperature range ( C) This is the range over which the enzyme is active. The commentary may give the percentage of activity at the outer limits. Also commentaries on specific assay conditions, additives etc.

XX


4 Enzyme Structure Molecular weight This field gives the molecular weight of the holoenzyme. For monomeric enzymes it is identical to the value given for subunits. As the accuracy depends on the method of determination this is given in the commentary if provided in the literature. Some enzymes are only active as multienzyme complexes for which the names and/or EC numbers of all participating enzymes are given in the commentary. Subunits The tertiary structure of the active species is described. The enzyme can be active as a monomer a dimer, trimer and so on. The stoichiometry of subunit composition is given. Some enzymes can be active in more than one state of complexation with differing effectivities. The analytical method is included. Posttranslational modifications The main entries in this field may be proteolytic modification, or side-chain modification, or no modification. The commentary will give details of the modifications e.g.: ± proteolytic modification (, propeptide Name) [1]; ± side-chain modification (, N-glycosylated, 12% mannose) [2]; ± no modification [3]

5 Isolation / Preparation / Mutation / Application Source / tissue For multicellular organisms, the tissue used for isolation of the enzyme or the tissue in which the enzyme is present is given. Cell-lines may also be a source of enzymes. Localization The subcellular localization is described. Typical entries are: cytoplasm, nucleus, extracellular, membrane. Purification The field consists of an organism and a reference. Only references with a detailed description of the purification procedure are cited. Renaturation Commentary on denaturant or renaturation procedure. Crystallization The literature is cited which describes the procedure of crystallization, or the X-ray structure.

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Cloning Lists of organisms and references, sometimes a commentary about expression or gene structure. Engineering The properties of modified proteins are described. Application Actual or possible applications in the fields of pharmacology, medicine, synthesis, analysis, agriculture, nutrition are described.

6 Stability pH-Stability This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. Temperature stability This field can either give a range in which the enzyme is stable or a single value. In the latter case the commentary is obligatory and explains the conditions and stability at this value. Oxidation stability Stability in the presence of oxidizing agents, e.g. O2, H2 O2, especially important for enzymes which are only active under anaerobic conditions. Organic solvent stability The stability in the presence of organic solvents is described. General stability information This field summarizes general information on stability, e.g., increased stability of immobilized enzymes, stabilization by SH-reagents, detergents, glycerol or albumins etc. Storage stability Storage conditions and reported stability or loss of activity during storage.

References

Authors, Title, Journal, Volume, Pages, Year.

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Purine-nucleoside phosphorylase

2.4.2.1

1 Nomenclature EC number 2.4.2.1 Systematic name purine-nucleoside:phosphate ribosyltransferase Recommended name purine-nucleoside phosphorylase Synonyms PNP PNPase PUNP PUNPI PUNPII Pu-NPase adenosine phosphorylase inosine phosphorylase inosine-guanosine phosphorylase nucleotide phosphatase (2.4.2.1) phosphorylase, purine nucleoside purine deoxynucleoside phosphorylase purine deoxyribonucleoside phosphorylase purine nucleoside phosphorylase purine ribonucleoside phosphorylase CAS registry number 9030-21-1

2 Source Organism

Homo sapiens [1, 4, 6, 8, 14, 26, 31, 34, 39, 42, 55, 57, 59, 61] Bos taurus [1, 4, 20, 28, 32, 33, 35, 39, 44, 45, 47, 51, 57, 59, 60, 61] Hyalomma dromedarii [2] Camelus dromedarius [3] Oryctolagus cuniculus [4, 25, 36, 37, 40] Macaca mulatta [4] Rattus norvegicus (adenine phosphorylase activity and a distinct inosineguanosine phosphorylase activity [41]) [4, 24, 29, 41, 61]

1


2

2.4.2.1

Equus caballus [4] Papio hamadryas [4] Felis catus [4] Canis familiaris [4] Sus scrofa [4] Gallus gallus [4, 7, 13, 16] Columba livia [4] Leucisus rusticus [4] Salmo gardneri [4] Escherichia coli (K-12, inosine-guanosine phosphorylase [43]) [4, 5, 9, 33, 38, 39, 43, 45, 50, 52, 54, 61] Bacillus cereus [4, 61] Bacillus brevis [4] Bacillus subtilis (two purine nucleoside phosphorylases: 1. inosine-guanosine phosphorylase, 2. adenosine-specific phosphorylase [11]) [4, 11, 61] Bacillus licheniformis [4] Bacterium cadaveris [4] Brevibacterium acetylicum (ATCC 954 [23]) [23] Corynebacterium sepedonicum [4] Erwinia carotovora (AJ 2992 [21]) [4, 21, 61] Proteus vulgaris [4, 15, 61] Salmonella enteritidis [4] Pseudomonas putrefaciens [4] Aeromonas hydrophila [4] Sarcina lutea [4] Lactobacillus leichmannii [4] Salmonella typhimurium (LT-2 [10]) [10, 24, 38, 61] Plasmodium lophurae [12, 61] Geobacillus stearothermophilus (JTS 859 [17, 22]; TH 6-2 [48]) [17, 22, 48, 61] Enterobacter aerogenes [4, 18, 61] Enterobacter cloacae (KY3074 [19]) [19] Alcaligenes faecalis (KY3106 [19]) [19] Arthrobacter nucleogenes (KY3168 [19]) [19] Brevibacterium vitarumen (KY3459 [19]) [19] Bacterium cadaveris (KY3402 [19]) [19] Cellulomonas cellasea (KY3491 [19]) [19] Micrococcus luteus (KY 3760 [19]) [19, 61] Proteus mirabilis (KY4057 [19]) [19] Xanthomonas campestris (KY4208 [19]) [19] Cricetulus griseus [27, 30] Plasmodium falciparum [42, 61] Cellulomonas sp. [46, 54, 58, 61] Trichomonas vaginalis [49] Serratia marcescens [53, 61] Mus musculus [56, 61]

2.4.2.1


Sulfolobus solfataricus [61] Klebsiella sp. [61] Acholeplasma laidlawii [61] Saccharomyces cerevisiae [61] Trypanosoma cruzi [61] Trypanosoma brucei [61] Fasciola hepatica [61]

3 Reaction and Specificity Catalyzed reaction purine nucleoside + phosphate = purine + a-d-ribose 1-phosphate (, sequential mechanism [2]; , rapid equilibrium bi bi reaction [7]; , Theorell-Chance mechanism [8,32]; , ordered bi-bi mechanism with nucleoside being the first substrate to add and base product to leave [26]; , rapid equilibrium random bi-bi mechanism with formation of abortive complexes [28]; , sequential bireactant mechanism [37]) Reaction type pentosyl group transfer Natural substrates and products S Additional information (, cellular function seems concerned primarily with nucleoside breakdown [4]; , in fish skin the enzyme plays a key role in the deposition of guanine and hypoxanthine crystals [4]; , key enzyme in purine salvage pathway [44]; , children lacking PNP activity exhibit severe T cell immunodeficiency while maintaining normal or exaggerated B cell function. The PNP deficiency results in very low uric acid concentrations and high concentrations of the nucleoside substrate of PNP in plasma and urine [59]; , enzyme plays a key role in the purine salvage pathway. PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function [61]; , in intact cells, the enzyme functions in the direction of phosphorolysis, leading to degradation of purine nucleosides via coupling with guanase and xanthine oxidase. PNP deficiency is a rare disorder associated with an autosomal recessive form of cellular, but not humoral, immunodeficiency, and comprises about 4% of all cases of severe combined immunodeficiency. Mutations identified in cases of PNP deficiency [61]) [4, 44, 59, 61] P ? Substrates and products S 1,6-dihydropurine riboside + phosphate (Reversibility: ? [39]) [39] P a-d-ribose 1-phosphate + 1,6-dihydropurine S 1-methylguanosine + phosphate (Reversibility: ? [33]) [33] P a-d-ribose 1-phosphate + 1-methylguanine

3


S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P 4

2.4.2.1

1-methylinosine + phosphate (Reversibility: ? [33]) [33] a-d-ribose 1-phosphate + 1-methylhypoxanthine 2',3'-dideoxyinosine + phosphate (Reversibility: ? [43]) [43] hypoxanthine + a-d-2,3-dideoxyribose 1-phosphate 2,3-dideoxyinosine + phosphate (, 12% of the activity with inosine [21]; , 22% of the activity with inosine [23]) (Reversibility: ? [21,23]) [21, 23] a-d-2',3'-dideoxyribose 1-phosphate + hypoxanthine 2-amino-6-mercapto-7-methylpurine ribonucleoside + a-d-ribose 1phosphate (Reversibility: ? [60,61]) [60, 61] ? 3-(b-d-ribofuranosyl)adenine + a-d-ribose 1-phosphate (Reversibility: ? [45]) [45] ? 3-(b-d-ribofuranosyl)adenine + a-d-ribose 1-phosphate (Reversibility: ? [61]) [61] ? 3-(b-d-ribofuranosyl)hyopxanthine + a-d-ribose 1-phosphate (Reversibility: ? [61]) [61] ? 3-(b-d-ribofuranosyl)hypoxanthine + a-d-ribose 1-phosphate (Reversibility: ? [45]) [45] ? 3-deazainosine + phosphate (Reversibility: ? [39]) [39] a-d-ribose 1-phosphate + 3-deazahypoxanthine 6-mercaptoguanosine + phosphate (Reversibility: ? [8]) [8] a-d-ribose 1-phosphate + 6-mercaptoguanine 6-mercaptopurine riboside + phosphate (, 14% of the activity with inosine [29]) (Reversibility: ? [29]) [29] a-d-ribose 1-phosphate + 6-mercaptopurine 6-thioxanthine + a-d-ribose 1-phosphate (Reversibility: ? [57]) [57] 6-thioxanthosine + phosphate 7,8-dimethylguanosine + phosphate (Reversibility: ? [33]) [33] 7,8-dimethylguanine + a-d-ribose 1-phosphate 7-(b-d-ribofuranosyl)guanine + a-d-ribose 1-phosphate (Reversibility: ? [61]) [61] ? 7-(b-d-ribofuranosyl)hypoxanthine + a-d-ribose 1-phosphate (Reversibility: ? [61]) [61] ? 7-butylguanosine + phosphate (Reversibility: ? [33]) [33] 7-butylguanine + a-d-ribose 1-phosphate 7-ethylguanosine + phosphate (Reversibility: ? [33]) [33] 7-ethylguanine + a-d-ribose 1-phosphate 7-isobutylguanosine + phosphate (Reversibility: ? [33]) [33] 7-isobutylguanine + a-d-ribose 1-phosphate

2.4.2.1


S 7-isopropylguanosine + phosphate (Reversibility: ? [33]) [33] P 7-isopropylguanine + a-d-ribose 1-phosphate S 7-methyl-8-aminoguanosine + phosphate (Reversibility: ? [33]) [33] P 7-methyl-8-aminoguanine + a-d-ribose 1-phosphate S 7-methyladenosine + phosphate (Reversibility: ? [9,61]) [9, 61] P 7-methyladenine + a-d-ribose 1-phosphate S 7-methylguanosine + phosphate (Reversibility: ? [9,33,35,61]) [9, 33, 35, 61] P 7-methylguanine + a-d-ribose 1-phosphate S 7-methylinosine + phosphate (Reversibility: ? [33,35,54,61]) [33, 35, 54, 61] P 7-methylhypoxanthine + a-d-ribose 1-phosphate S 7-prolylguanosine + phosphate (Reversibility: ? [33]) [33] P 7-prolylguanine + a-d-ribose 1-phosphate S 8-aminoguanine + a-d-ribose 1-phosphate (Reversibility: ? [59,61]) [59, 61] P 8-aminoguanosine + phosphate [59] S 8-azahypoxanthine + a-d-ribose 1-phosphate (Reversibility: ? [61]) [61] P 8-azainosine + phosphate [61] S 8-methylguanosine + phosphate (Reversibility: ? [33]) [33] P 8-methylguanine + a-d-ribose 1-phosphate S adenine + deoxyribose 1-phosphate (Reversibility: r [59,61]) [59, 61] P deoxyadenosine + phosphate S adenine arabinoside + phosphate (, 49% of the activity with adenosine [18]) [18] P a-d-arabinose 1-phosphate + adenine S adenosine + arsenate (Reversibility: ? [38]) [38] P adenine + a-d-ribose 1-arsenate S adenosine + phosphate (, reaction with adenosine-specific phosphorylase, no activity with inosine-guanosine phosphorylase [11]; , best substrate [18]; , 48% of the activity with inosine [21]; , activity with OUNOII, no activity with PUNPI [22]; , 160% of the activity with inosine [22]; , 61% of the activity with deoxyguanosine [38]; , 78% of the activity with deoxyguanosine [38]; , in the reverse reaction the catalytic activity with adenine is higher than that with either hypoxanthine or guanine [49]; , no activity [45, 43, 48]; , wild-type enzyme and mutant enzyme K244Q show no activity with adenosine, mutant enzyme N243D and N243D/K244Q are active with adenosine [56]; , no activity [58]) (Reversibility: r [49]; ? [10, 11, 18, 20, 21, 22, 38, 41, 45, 53, 55, 61]) [10, 11, 18, 20, 21, 22, 38, 41, 45, 49, 53, 56, 61] P adenine + a-d-ribose 1-phosphate 5


2.4.2.1

S deoxyadenosine + phosphate (, reaction with adenosine-specific phosphorylase, no activity with inosine-guanosine phosphorylase [11]; , 2'-deoxyadenosine, 65% of the activity with adenosine. 3'-deoxyadenosine, 8% of the activity with adenosine [18]; , 112.9% of the activity with inosine [19]; , 2'deoxyadenosine, 57% of the activity with inosine. 3'-deoxyadenosine, 7% of the activity with inosine [21]; , 2'-deoxyadenosine, 160% of the activity with inosine [22]; , 61% of the activity with deoxyguanosine [38]; , 78% of the activity with deoxyguanosine [38]) (Reversibility: ? [10, 11, 18, 19, 20, 21, 22, 38, 53, 61]) [10, 11, 18, 19, 20, 21, 22, 38, 53, 61] P a-d-ribose 1-phosphate + adenine S deoxyguanosine + phosphate (, reaction with ionosine-guanosine phosphorylase, very low activity with the adenosine-specific phosphorylase [11]; , 2'-deoxyguanosine, 41% of the activity with inosine. 3'-deoxyguanosine, 16% of the activity with inosine [21]; , 2'-deoxyguanosine, 469% of the activity with inosine [22]; , 11% of the activity with inosine [23]; , 85% of the activity with inosine [48]) (Reversibility: ? [2, 4, 8, 10, 11, 17, 20, 21, 22, 23, 25, 38, 42, 43, 48, 53, 61]) [2, 4, 8, 10, 11, 17, 20, 21, 22, 23, 25, 38, 42, 43, 48, 53, 61] P guanine + a-d-deoxyribose 1-phosphate S deoxyinosine + phosphate (, reaction with inosine-guanosine phosphorylase, no activity with the adenosine-specific phosphorylase [11]; , 2'-deoxyinosine, 43% of the activity with adenosine [18]; , 136% of the activity with inosine [21]; , 2'-deoxyinosine, 691% of the activity with inosine [22]; , 2'deoxyinosine, 87% of the activity with inosine [23]; , 88% of the activity with deoxyguanosine [38]; , 88% of the activity with deoxyguanosine [38]; , 2'-deoxyinosine and 5'-deoxyinosine [43]; , as active as inosine [48]) (Reversibility: r [1, 4, 10, 11, 17, 18, 20, 21, 22, 23, 26, 38, 43, 48, 53, 61]) [1, 4, 10, 11, 17, 18, 20, 21, 22, 23, 26, 38, 43, 48, 53, 61] P hypoxanthine + a-d-deoxyribose 1-phosphate S guanine + ribose 1-phosphate (Reversibility: r [27]) [27] P guanosine + phosphate [27] S guanosine + arsenate (Reversibility: ? [38]) [38] P guanine + a-d-ribose 1-arsenate S guanosine + phosphate (, reaction with inosine-guanosine phosphorylase, no activity with the adenosine-specific phosphorylase [11]; , 2.3% of the activity with inosine [12]; , 47.4% of the reaction with inosine [19]; , 2'-deoxyinosine, 27% of the activity with inosine. 3'-deoxyinosine, 10% of the activity with inosine [21]; , 53% of the activity with inosine [22]; , 21% of the activity with inosine [23]; , 16% of the activity with inosine [29]; , 48% of the activity with deoxyguanosine [38]; , 81% of the activity with 6

2.4.2.1

P S P S P S P S

P S P S P S P S P S


deoxyguanosine [38]; , 84% of the activity with guanosine [48]; , no activity [61]) (Reversibility: r [8, 28, 30, 32, 37, 43, 49, 57, 58]; ? [2, 4, 10, 11, 12, 17, 19, 20, 22, 25, 26, 29, 40, 42, 48, 53, 61]) [2, 4, 8, 10, 11, 12, 15, 17, 19, 20, 21, 22, 23, 25, 26, 28, 29, 30, 32, 37, 38, 40, 42, 43, 48, 49, 53, 57, 58, 61] guanine + a-d-ribose 1-phosphate [2, 49] hypoxanthine + a-d-deoxyribose 1-phosphate (Reversibility: r [28]) [28] deoxyinosine + phosphate hypoxanthine + a-d-ribose 1-phosphate (Reversibility: r [27]) [27] inosine + phosphate inosine + arsenate (, ribosyl group is nearly dissociated from the base prior to attack of the arsenate [47]) (Reversibility: ? [38, 47]) [38, 47] hypoxanthine + ribose 1-arsenate [47] inosine + phosphate (, reaction with ionosine-guanosine phosphorylase, no activity with the adenosine-specific phosphorylase [11]; , 34% of the activity with adenosine [18]; , equilibrium when 65% of inosine has been phosphorylated [18]; , the equilibrium is reached when approximately 14% of inosine is phosphorylated [19]; , reaction reaches an equilibrium when 21% of the inosine is phosphorolyzed [23]; , 46% of the activity with deoxyguanosine [38]; , 82% of the activity with deoxyguanosine [38]; , no activity [61]) (Reversibility: r [4, 8, 12, 18, 19, 23, 26, 28, 30, 37, 38, 43, 49, 55, 57, 58]; ? [1, 2, 3, 4, 9, 10, 11, 13, 14, 15, 17, 20, 21, 22, 24, 25, 29, 33, 35, 36, 42, 45, 53, 54, 56, 61]) [1, 2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 33, 35, 36, 37, 38, 42, 43, 45, 48, 49, 53, 54, 55, 56, 57, 58, 61] hypoxanthine + a-d-ribose 1-phosphate [2, 8, 12, 38, 49, 57] nicotinamide riboside + nicotinamide + a-d-ribose 1-phosphate (Reversibility: ? [61]) [61] ? purine riboside + phosphate (Reversibility: ? [39]) [39] a-d-ribose 1-phosphate + purine ribavirin + phosphate (, 22% of the activity with inosine. Phosphorolysis reaches an equilibrium when 15% of ribavirin is phosphorolyzed [23]) (Reversibility: r [23]) [23] a-d-ribose 1-phosphate + 1,2,4-triazole-3-carboxamide xanthosine + arsenate (Reversibility: ? [57]) [57] xanthine + a-d-ribose 1-arsenate xanthosine + phosphate (, 43.5% of the activity with inosine [19]; , 26% of the activity with inosine [23]; , 7


2.4.2.1

reaction only at high concentrations [37]) (Reversibility: r [57]; ? [19, 20, 23, 37, 43, 48]) [19, 20, 23, 37, 43, 48, 57] P a-d-ribose 1-phosphate + xanthine S Additional information (, two purine nucleoside phosphorylases: 1. inosine-guanosine phosphorylase, 2. adenosine-specific phosphorylase [11]) [11] P ? Inhibitors 1,6-dihydropurine riboside [39] 1-b-d-ribofuranosyl-1,2,4-triazole-3-carboxamidine [35] 1-methylguanine [33] 2-amino-6-chloro-7-deazapurine 2'-deoxyriboside [61] 2-amino-6-mercapto-7-methylpurine ribonucleoside (, in absence of phosphate, the enzyme catalyzes a slow hydrolysis, which is accompanied by inactivation of the enzyme [60]) [60] 2-amino-6-methylthiopurine [4] 2-chloro-6-(3-phenyl-1-propoxy)purine [61] 2-chloroadenosine (, inhibits ribosylation of adenine and N6 -furfuryladenine [41]) [41] 2-mercaptoethanol [2] 5'-chloro-5'-deoxy-8-aminoguanosine [42] 5'-deoxy-5'-iodo-9-deazainosine [61] 5'-deoxy-5'-iodoinosine [61] 5,5'-dithio(2-nitrobenzoic acid) [26, 36] 6-amino-2-chloro-7-deazapurine 2'-deoxyriboside [61] 6-amino-7-phenylethinyl-7-deazapurine 2'-deoxyriboside [61] 6-benzyloxy-2-chloropurine [61] 6-chloro-7-deazapurine 2'-deoxyriboside [61] 6-hydroxy-9-p-aminobenzylpurine [14] 6-mercaptopurine [4] 6-methylformycin [9, 52, 61] 6-methylthiopurine [4] 6-thioxanthine [4] 7-deazainosine [4, 39] 7-deazathioinosine [4] 8-amino-3-(2-thienylmethyl)guanine [61] 8-amino-5'-deoxy-5'-chloroguanosine [42] 8-amino-5'-deoxy-5'-iodoguanosine [61] 8-amino-9-benzylguanine [42, 61] 8-aminoguanine [59] 8-aminoguanosine [12, 42] 8-aminoquinazolinone [61] 8-aza-2,6-diaminopurine (, noncompetitive [40]) [40] 8-bromo-N9 -acycloguanosine [58] 9-(1,3-dihydroxy-2-propoxymethyl)guanine [35] 9-(2-fluoro-3,4-dihydroxybutyl)-guanine [45]

8

2.4.2.1


9-(2-hydroxyethoxymethyl)guanine [35] 9-(3,4-dihydroxybutyl)guanine (, and analogues [61]) [61] 9-(5,5-difluorophosphonopentyl)guanine [61] 9-(5-phosphonopentyl)guanine [61] 9-benzylguanine [61] Acyclovir [58, 61] AgNO3 [21, 23] CuSO4 (, slight [23]) [21, 23] d,l-6-methyl 5,6,7,8-tetrahydropterin (, competitive [40]) [40] Ganciclovir [61] HgCl2 [17, 21, 23] N1 -methylformycin A [9] N6 -methylformycin A (, competitive with respect to inosine, 7methylguanosine and 7-methyladenosine [9]) [9, 52] N7 -acycloguanosine [61] N7 -methylformycin A [52] N9 -acycloguanosine [58, 61] NEM [23] PCMB (, adenosine-specific phosporylase [11]; , no inhibition of PUNPII [22]; , 0.1 mM, 85% loss of activity within 15 min [40]; , dithiothreitol restores activity [43]) [11, 17, 21, 23, 25, 26, 32, 37, 38, 40, 41, 43] adenine [4] adenosine [15, 43, 58] allopurinol [4] a-d-ribose 1-phosphate (, product inhibition [7,8,25]) [7, 8, 25, 28, 32, 37] aminopterin (, noncompetitive [40]) [40] azaguanine [12] deoxyadenosine [43] deoxyinosine [4] diphosphate [4] erythro-9-(2-hydroxy-3-nonyl)adenine (, competitive [40]) [40] folate (, 0.05-0.1 mM, slight competitive inhibitor [28]; , competitive [40]) [28, 40] formycin [61] formycin A [9, 39, 45, 49, 52, 56, 58] formycin B [4, 9, 12, 39, 42, 52, 58, 61] guanine (, product inhibition [7,8]; , product inhibition [25]; , inhibits phosphorolysis of xanthosine [57]) [1, 2, 4, 7, 8, 25, 32, 37, 57, 58] guanosine (, product inhibition [8]; , strong competitive inhibitor with deoxyinosine as substrate [11]; , inhibits phosphorolysis of xynthosine [57]) [8, 11, 57] hypoxanthine (, product inhibition [25]; , inhibits phosphorolysis of xanthosine [57]) [4, 25, 32, 37, 57] hypoxanthine arabinoside [43] 9


2.4.2.1

inosine (, strong competitive inhibitor with deoxyinosine as substrate [11]; , inhibits ribosylation of hypoxanthine and guanine [41]) [4, 11, 41] oxoallopurinol [4] oxoformycin A (, weak [9]) [9] oxoformycin B (, weak [9]) [9] phosphate (, product inhibition [8]) [8] purine riboside [39] quinazolinone [61] xanthine (, poor inhibitor of phosphorolysis of guanosine [57]) [57] xanthosine (, poor inhibitor of phosphorolysis of guanosine [57]) [57] Additional information (, the enzyme inhibitors 8-amino-5'deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine may have some antimalarial potential by limiting hypoxanthine production in the parasiteinfected erythrocyte [42]; , inhibition by 9-cycloaliphatic methyl and 9saturated heterocyclic methyl-9-deazapurines [59]) [42, 59] Activating compounds inosine (, substrate activation [1]; , no substrate activation [1]; , activates the crystalline enzyme [7]) [1, 7] Metals, ions Co2+ (, 1 mM, 1.65fold stimulation [10]) [10] Turnover number (min±1) 3.6 (inosine, , mutant enzyme N243A [55]) [55] 5.4 (phosphate, , reaction with inosine, mutant enzyme N243A [55]) [55] 16.92 (adenosine, , mutant enzyme N243D, pH 7.0 [56]) [56] 23.16 (inosine, , mutant enzyme N243D, pH 7.0 [56]) [56] 30 (hypoxanthine, , mutant enzyme R84A [55]) [55] 34.74 (adenosine, , mutant enzyme N243D/K244Q, pH 7.0 [56]) [56] 42.6 (adenosine, , mutant enzyme N243D, pH 6.0 [56]) [56] 58.2 (hypoxanthine, , mutant enzyme H86A [55]) [55] 66 (inosine, , mutant enzyme S33A [55]) [55] 66 (phosphate, , reaction with inosine, mutant enzyme K244A [55]) [55] 68.4 (adenosine, , mutant enzyme N243D/K244Q, pH 6.0 [56]) [56] 84 (hypoxanthine, , mutant enzyme E201A [55]) [55] 84.6 (adenosine, , mutant enzyme N243D/K244Q, pH 6.0 [56]) [56] 90 (phosphate, , reaction with inosine, mutant enzyme R84A [55]) [55] 105.6 (inosine, , mutant enzyme N243D, pH 6.0 [56]) [56]

10

2.4.2.1


108 (phosphate, , reaction with inosine, mutant enzyme E201A [55]) [55] 120.6 (phosphate, , reaction with 7-methylinosine [54]) [54] 126 (hypoxanthine, , mutant enzyme S33A [55]) [55] 126 (inosine, , mutant enzyme R84A [55]) [55] 154.2 (7-methylinosine) [54] 240 (xanthosine) [28] 252 (inosine, , mutant enzyme E201A [55]) [55] 372 (phosphate, , reaction with inosine, mutant enzyme F200A [55]) [55] 540 (hypoxanthine, , mutant enzyme Y88F [55]) [55] 546 (inosine, , mutant enzyme N243D/K244Q, pH 7.0 [56]) [56] 564 (inosine, , mutant enzyme F200A [55]) [55] 564 (phosphate, , reaction with inosine, mutant enzyme H86A [55]) [55] 600 (hypoxanthine, , mutant enzyme H257A [55]) [55] 720 (phosphate, , reaction with inosine, mutant enzyme T242A [55]) [55] 780 (hypoxanthine, , mutant enzyme N243A [55]) [55] 780 (phosphate, , reaction with inosine, mutant enzyme H257A [55]) [55] 840 (hypoxanthine, , mutant enzyme F200A [55]) [55] 840 (inosine, , mutant enzymes T242A and H86A [55]) [55] 960 (inosine, , mutant enzyme H257A [55]) [55] 1440 (hypoxanthine, , mutant enzyme F159A [55]) [55] 1650 (7-methylinosine) [54] 1668 (phosphate, , reaction with 7-methylinosine [54]) [54] 1800 (phosphate, , reaction with inosine, mutant enzyme F159A [55]) [55] 1920 (inosine, , mutant enzyme F159A [55]) [55] 2184 (inosine, , mutant enzyme N243D/K244Q, pH 6.0 [56]) [56] 2334 (inosine, , wild-type enzyme, pH 6.0 [56]) [56] 2460 (phosphate, , reaction with inosine, mutant enzyme M219A [55]) [55] 2700 (2'-deoxyguanosine) [28] 2760 (inosine, , mutant enzyme E89A [55]) [55] 2838 (inosine, , wild-type enzyme, pH 7.0 [56]) [56] 2940 (inosine, , mutant enzyme K244A [55]) [55] 3120 (inosine, , mutant enzyme M219A [55]) [55] 3120 (phosphate, , reaction with inosine, mutant enzyme K244A [55]) [55] 3180 (phosphate, , reaction with inosine, mutant enzyme E89A [55]) [55] 3240 (inosine, , mutant enzyme Y88F [55]) [55] 3360 (inosine, , wild-type enzyme [55]) [55] 3360 (phosphate, , reaction with inosine, mutant enzyme Y88F [55]) [55] 11


2.4.2.1

3360 (phosphate, , wild-type enzyme [55]) [55] 3480 (hypoxanthine, , mutant enzyme T242A [55]) [55] 3540 (guanosine) [28] 5100 (2'-deoxyinosine) [28] 5880 (hypoxanthine, , mutant enzyme K244A [55]) [55] 6000 (a-d-ribose 1-phosphate, , reaction with hypoxanthine [28]) [28] 6180 (hypoxanthine, , reaction with ribose 1-phosphate [28]) [28] 6600 (hypoxanthine, , wild-type enzyme [55]) [55] 6900 (a-d-deoxyribose 1-phosphate, , reaction with hypoxanthine [28]) [28] 7140 (inosine) [28] 7200 (a-d-ribose 1-phosphate, , reaction with guanine [28]) [28] 7620 (guanine, , reaction with ribose 1-phosphate [28]) [28] 7620 (hypoxanthine, reaction with deoxyribose 1-phosphate [28]) [28] 7800 (hypoxanthine, , mutant enzyme M219A [55]) [55] 8700 (a-d-deoxyribose 1-phosphate, , reaction with guanine [28]) [28] 9480 (guanine, , reaction with deoxyribose 1-phosphate [28]) [28] Additional information [4, 43] Specific activity (U/mg) 0.0141 [43] 4.55 [20] 7.9 [19] 8.7 [12] 21.55 [36] 36 [54] 47.12 [25, 37] 48.5 [21] 50 [40] 50.6 (, PUNPII [22]) [22] 53.3 [34] 55 (, reaction with guanosine [8]) [8] 60 [27, 30] 73 (, adenosine-specific phosphorylase [11]) [11] 77.5 [28] 78.3 [32] 91.6 [48] 93 [26] 95 [14] 100 (, inosine-guanosine phosphorylase [11]) [11] 132 [29] 143 [24] 160 [10, 24] 195 [38]

12

2.4.2.1


195.1 [23] 301 [38] 405 (, PUNPI [22]) [17, 22] Additional information [15, 18, 19, 53, 55] Km-Value (mM) 0.0033 (hypoxanthine) [43] 0.0041 (guanosine) [43] 0.006 (guanine, , pH 7.0 [57]) [57] 0.009 (guanine, , pH 7.5 [57]) [57] 0.01 (guanosine) [42] 0.01 (hypoxanthine, , mutant enzyme Y88F [55]) [55] 0.01 (inosine) [42] 0.011 (guanine, , pH 6.0 [57]) [57] 0.011 (guanosine, , pH 7.0 [57]) [57] 0.012 (adenosine) [45] 0.012 (guanosine, , pH 7.0 [57]) [57] 0.013 (2-amino-6-mercapto-7-methylpurine) [61] 0.013 (7-ethylguanosine) [33] 0.013 (hypoxanthine, , mutant enzyme S33A [55]) [55] 0.013 (inosine) [32, 35, 45] 0.013 (phosphate, , reaction with inosine [15]) [15] 0.0138 (guanine, , pH 7.0 [57]) [57] 0.014 (guanine, , reaction with ribose 1-phosphate [28]) [28] 0.014 (guanosine, , pH 6.5 [57]) [57] 0.0147 (7-methylguanosine) [33] 0.015 (7-methylguanosine) [35, 61] 0.015 (phosphate, , reaction with guanosine [15]) [15] 0.0159 (guanosine, , pH 6.5 [57]) [57] 0.017 (guanine, , reaction with deoxyribose 1-phosphate [28]) [28] 0.0182 (guanine) [37] 0.019 (hypoxanthine, , mutant enzyme H86A [55]) [4, 26, 55] 0.02 (guanosine) [33] 0.02 (hypoxanthine, , with ribose 1-phosphate [28]; , mutant enzyme R84A and H257A [55]) [27, 28, 30, 55] 0.021 (hypoxanthine, , reaction with deoxyribose 1-phosphate [28]) [28] 0.022 (1-methylguanosine) [33] 0.022 (guanine, , pH 7.5 [57]) [57] 0.022 (guanosine) [20] 0.022 (inosine, , mutant enzymes N243A and T242A [55]) [55] 0.024 (2'-deoxyguanosine) [28] 0.024 (hypoxanthine, , mutant enzyme T242A [55]) [55] 0.025 (7-prolylguanosine) [33] 0.025 (hypoxanthine, , mutant enzyme F159A [55]) [55] 0.027 (7-(b-d-ribofuranosyl)guanine) [61] 0.027 (deoxyguanosine) [20]

13


2.4.2.1

0.027 (deoxyinosine) [20] 0.028 (adenosine, , mutant enzyme N243D/K244Q, pH 6.0 [56]) [56] 0.028 (inosine) [20] 0.028 (phosphate, , reaction with inosine [28]) [28] 0.029 (guanine, , pH 6.0 [57]) [57] 0.029 (guanosine) [15, 28] 0.03 (guanine) [32] 0.03 (hypoxanthine, , wild-type enzyme [55]) [55] 0.0308 (guanine) [8] 0.032 (inosine, , wild-type enzyme, pH 6.0 [56]) [12, 33, 56] 0.032 (phosphate) [26] 0.034 (7-butylguanosine) [33] 0.035 (guanine) [27, 30] 0.035 (guanosine) [42] 0.036 (inosine) [28] 0.037 (inosine, , mutant enzyme K244A [55]) [55] 0.038 (deoxyguanosine) [42] 0.039 (hypoxanthine) [8] 0.039 (inosine) [15] 0.04 (a-d-ribose 1-phosphate, , reaction with adenine [38]) [38] 0.04 (hypoxanthine, , mutant enzyme K244A [55]) [55] 0.04 (inosine, , wild-type enzyme [55]) [55] 0.041 (a-d-ribose 1-phosphate, , reaction with guanine [28]) [28] 0.041 (inosine) [42] 0.042 (adenosine, , mutant enzyme N243D/K244Q, pH 7.0 [56]) [56] 0.042 (a-d-ribose 1-phosphate, , reaction with hypoxanthine [28]) [28] 0.043 (2'-deoxyinosine) [28] 0.043 (inosine) [58] 0.0435 (guanosine) [32] 0.044 (deoxyguanosine) [43] 0.0443 (deoxyguanosine) [8] 0.0443 (guanosine) [8] 0.045 (adenosine, , mutant enzyme N243D, pH 6.0 ot pH 7.0 [56]) [56] 0.046 (guanosine, , pH 5.7 [57]) [57] 0.047 (deoxyinosine) [10, 24] 0.047 (guanosine) [26] 0.047 (inosine) [45] 0.048 (inosine) [26] 0.05 (1-methylinosine) [33] 0.05 (guanosine) [25] 0.05 (inosine) [8, 10, 25, 30, 37] 0.051 (xanthosine) [43] 14

2.4.2.1


0.0556 (inosine) [14] 0.057 (deoxyguanosine) [42] 0.0578 (phosphate) [19] 0.058 (inosine, , mutant enzyme Y88F [55]; , wild-type enzyme, pH 7.0 [56]) [55, 56] 0.059 (a-d-ribose 1-phosphate) [43] 0.06 (guanosine) [40] 0.062 (2'-deoxyinosine) [43] 0.065 (7,8-dimethylguanosine) [33] 0.066 (deoxyinosine) [26] 0.069 (7-isoprolylguanosine) [33] 0.07 (6-mercaptopurine riboside) [4] 0.07 (inosine) [36, 38] 0.075 (adenosine) [22] 0.082 (hypoxanthine) [12] 0.0823 (inosine, , mutant enzyme N243D/K244Q, pH 7.0 [56]) [56] 0.09 (inosine) [19, 38] 0.09 (isobutylguanosine) [33] 0.091 (guanosine, , pH 5.7 [57]) [57] 0.095 (xanthosine) [20] 0.1 (2'-deoxyguanosine) [17] 0.1 (8-azaguanine) [61] 0.1 (8-methylguanosine) [33] 0.1 (a-d-deoxyribose 1-phosphate, , reaction with adenine [38]) [38] 0.1 (deoxyguanosine) [25, 37] 0.1 (deoxyribose 1-phosphate, , reaction with adenine [38]) [38] 0.1 (inosine) [29] 0.1 (phosphate, , reaction with inosine [38]) [38] 0.108 (7-(b-d-ribofuranosyl)guanine) [61] 0.11 (guanosine) [43, 58] 0.11 (inosine, , enzyme from vegetative cells [4]) [4] 0.12 (guanosine) [29] 0.12 (phosphate, , reaction with inosine [38]) [38] 0.129 (a-d-deoxyribose 1-phosphate, , reaction with hypoxanthine [28]) [28] 0.13 (adenosine) [20] 0.13 (inosine, , mutant enzyme R84A [55]) [55] 0.133 (a-d-deoxyribose 1-phosphate, , reaction with guanine [28]) [28] 0.133 (inosine) [25, 37] 0.134 (a-d-ribose 1-phosphate) [37] 0.135 (phosphate, , reaction with 7-methylinosine [54]) [54] 0.14 (guanosine) [17] 0.14 (inosine, , enzyme from spores [4]; , mutant enzyme S33A [55]) [4, 55]

15


2.4.2.1

0.145 (deoxyadenosine) [20] 0.15 (3-(b-d-ribofuranosyl)adenine) [45, 61] 0.167 (6-mercaptoguanosine) [8] 0.167 (phosphate, , reaction with 7-methylinosine [54]) [54] 0.169 (7-methylinosine) [54] 0.17 (deoxyinosine) [38] 0.17 (inosine) [4] 0.18 (deoxyguanosine) [4] 0.18 (deoxyinosine) [4, 38] 0.18 (hypoxanthine, , mutant enzyme M219A [55]) [55] 0.19 (2'-deoxyadenosine) [2, 22] 0.19 (guanosine) [53] 0.2 (a-d-ribose 1-phosphate) [27, 30] 0.2 (deoxyinosine) [17] 0.2 (inosine, , inosine-guanosine phosphorylase [11]) [11] 0.2 (phosphate) [27, 30] 0.21 (inosine, , mutant enzyme H257A [55]) [55] 0.216 (guanosine) [4] 0.22 (3-(b-d-ribofuranosyl)hypoxanthine) [45, 61] 0.22 (inosine, , mutant enzyme E89A [55]) [22, 55] 0.22 (phosphate, , reaction with inosine, mutant enzyme T242A [55]) [55] 0.228 (a-d-ribose 1-phosphate, , reaction with guanine [8]) [8] 0.247 (7-methylinosine) [54] 0.26 (3-(b-d-ribofuranosyl)hypoxanthine) [45, 61] 0.27 (7-methylinosine) [61] 0.27 (hypoxanthine) [4] 0.28 (inosine, , mutant enzyme H86A [55]) [55] 0.28 (xanthine, , pH 6.0 [57]) [57] 0.306 (xanthine, , pH 7.5 [57]) [57] 0.31 (inosine, , mutant enzyme F159A [55]) [2, 55] 0.33 (deoxyguanosine) [2] 0.34 (adenine) [4] 0.34 (guanosine) [22] 0.34 (inosine) [43] 0.35 (deoxyguanosine) [53] 0.37 (7-methylinosine) [61] 0.37 (phosphate, , reaction with inosine [10]) [10, 24] 0.376 (phosphate) [4] 0.38 (xanthine, , pH 6.0 [57]) [57] 0.4 (hypoxanthine) [4] 0.4 (xanthosine, , pH 5.7 [57]) [57] 0.41 (adenine) [4] 0.425 (xanthine, , pH 7.0 [57]) [57] 0.43 (phosphate) [42] 0.45 (xanthosine) [28] 0.484 (inosine, , mutant enzyme N243D, pH 7.0 [56]) [56] 16

2.4.2.1


0.5 (ribose 1-phosphate) [4] 0.52 (phosphate) [29] 0.57 (hypoxanthine, , mutant enzyme F200A [55]) [55] 0.58 (xanthosine, , pH 5.7 [57]) [57] 0.6 (5'-deoxyinosine) [43] 0.6 (7-methylguanosine) [33, 61] 0.6 (7-methylinosine) [35] 0.62 (nicotinamide riboside) [61] 0.63 (arsenate) [38] 0.64 (7-(b-d-ribofuranosyl)hypoxanthine) [61] 0.661 (8-azahypoxanthine) [61] 0.67 (deoxyinosine) [2] 0.68 (phosphate) [42] 0.691 (inosine, , mutant enzyme N243D, pH 6.0 [56]) [56] 0.729 (inosine, , mutant enzyme N243D/K244Q, pH 6.0 [56]) [56] 0.74 (phosphate) [4] 0.76 (phosphate) [43] 0.8 (3-(b-d-ribofuranosyl)adenine) [45, 61] 0.8 (arsenate, , reaction with inosine [38]) [38] 0.8 (guanine) [4] 0.8 (inosine, , 40 C [18]) [18] 0.92 (2'-deoxyinosine) [22] 1 (hypoxanthine, , mutant enzyme E201A [55]) [55] 1.1 (phosphate) [53] 1.1 (xanthosine, , pH 6.5 [57]) [57] 1.18 (inosine) [48] 1.26 (7-(b-d-ribofuranosyl)hypoxanthine) [61] 1.29 (guanosine) [48] 1.48 (nicotinamide riboside) [61] 1.52 (7-(b-d-ribofuranosyl)hypoxanthine) [61] 1.6 (xanthosine, , pH 6.5 [57]) [57] 1.8 (arsenate) [4] 1.85 (guanosine, , at 40 C [21]) [21] 1.9 (2'-deoxyguanosine) [22] 1.92 (inosine, , at 40 C [21]) [21] 2.1 (phosphate, , mutant enzyme Y88F [55]) [55] 2.2 (inosine, , 60 C [18]) [18] 2.3 (hypoxanthine, , mutant enzyme N243A [55]) [55] 2.5 (phosphate, , mutant enzymes H257A and F159A [55]) [55] 2.6 (2',3'-dideoxyinosine) [43] 2.7 (phosphate, , reaction with inosine, mutant enzyme K244A [55]) [55] 3.5 (phosphate, , reaction with inosine, mutant enzyme N243A [55]) [55] 3.6 (phosphate, , reaction with inosine, mutant enzyme S33A [55]) [55]

17


2.4.2.1

3.9 (phosphate, , reaction with inosine, inosine-guanosine phosphorylase [11]) [11] 4 (phosphate, , reaction with inosine, wild-type enzyme [55]) [55] 4.3 (phosphate, , mutant enzyme M219A [55]) [55] 4.5 (phosphate, , reaction with inosine, mutant enzyme F200A [55]) [55] 4.5 (phosphate, , reaction with inosine, mutant enzyme R84A [55]) [55] 5.1 (phosphate, , enzyme from vegetative cells [4]) [4] 6.1 (adenosine) [49] 6.1 (phosphate, , reaction with inosine, mutant enzyme E201A [55]) [55] 7.2 (phosphate, , enzyme from spores [4]) [4] 8 (inosine, , mutant enzyme M219A [55]) [55] 8.4 (inosine, , mutant enzyme E201A [55]) [55] 9.2 (phosphate, , reaction with inosine, mutant enzyme H86A [55]) [55] 12.3 (adenine) [49] 13.1 (phosphate, , reaction with guanosine [8]) [8] 15.4 (phosphate) [25, 37] 19 (inosine, , mutant enzyme F200A [55]) [55] 21.5 (phosphate) [32] 31.5 (inosine) [49] 35.9 (guanine) [49] 45.6 (hypoxanthine) [49] 59.7 (guanosine) [49] 91 (phosphate, , reaction with inosine, mutant enzyme E89A [55]) [55] Additional information [1, 4, 60, 61] Ki-Value (mM) 0.00018 (5'-deoxy-5'-iodo-9-deazainosine) [61] 0.0002 (8-amino-9-benzylguanine) [42, 61] 0.0002 (8-aminoguanine) [59] 0.00027 (N6 -methyl-formycin A, , pH 8.0 [52]) [52] 0.0003 (N6 -methyl-formycin A, , pH 7 [9,52]) [9, 52, 61] 0.00039 (formycin B) [12] 0.0004 (5'-chloro-5'-deoxy-8-aminoguanosine) [42] 0.0006 (6-benzyloxy-2-chloropurine) [61] 0.0011 (formycin B) [42] 0.0014 (2-chloro-6-(3-phenyl-1-propoxy)purine) [61] 0.0021 (1-b-d-ribofuranosyl-1,2,4-triazole-3-carboxamidine, , reaction with 7-methylguanosine [35]) [35] 0.0022 (N7 -methylformycin A, , pH 5.5 [52]) [52] 0.0023 (6-chloro-7-deaza-purine 2'-deoxyriboside) [61] 0.0024 (2-amino-6-chloro-7-deazapurine 2'-deoxyriboside) [61] 0.0033 (8-amino-5'-deoxy-5'-iodoguanosine) [61]

18

2.4.2.1


0.0034 (N(9)-acycloguanosine) [58] 0.0036 (guanine) [58] 0.004 (guanine, , inhibition of phosphorolysis of xanthosine [57]) [57] 0.0041 (8-bromo-N(9)-acycloguanosine) [58] 0.0045 (formycin B) [39] 0.0046 (formycin B, , pH 7 [9,52]) [9, 52, 61] 0.005 (6-amino-7-phenyethinyl-7-deazapurine 2'-deoxyribose) [61] 0.005 (6-methylformycin A, , pH 6.0 [52]) [52] 0.005 (N7 -acycloguanosine) [61] 0.005 (formycin) [61] 0.005 (guanine) [1] 0.0053 (formycin A, , pH 7.0 [52]) [9, 52] 0.0055 (formycin A) [39] 0.00568 (PCMB) [25] 0.0062 (1-b-d-ribofuranosyl-1,2,4-triazole-3-carboxamidine, , reaction with inosine [35]) [35] 0.0065 (guanine, , with guanosine as variable substrate [28]) [28] 0.007 (8-aminoguanosine) [42] 0.007 (hypoxanthine, , inhibition of phosphorolysis of xanthosine [57]) [57] 0.008 (6-amino-2-chloro-7-deazapurine 2'-deoxyriboside) [61] 0.008 (8-amino-9-benzylguanine) [42] 0.0085 (a-d-ribose 1-phosphate, , with guanosine as variable substrate [28]) [28] 0.01 (guanosine, , inhibition of phosphorolysis of xanthosine [57]) [57] 0.01 (guanosine, , with guanine as variable substrate and ribose 1phosphate as fixed substrate [8]) [8] 0.01 (hypoxanthine) [4] 0.012 (5'-chloro-5'-deoxy-8-aminoguanosine) [42] 0.012 (guanine, , with guanosine as variable substrate and 200 mM phosphate [32]) [32] 0.0125 (guanine, , with guanosine as variable substrate and phosphate as fixed substrate [8]) [8, 25] 0.013 (8-aminoguanosine) [42] 0.0138 (formycin A, , pH 5.5 [52]) [52] 0.014 (N(9)-acycloguanosine) [61] 0.0145 (guanine, , with guanosine as variable substrate and 40 mM phosphate [32]) [32] 0.017 (8-aminoguanosine) [59] 0.018 (5'-deoxy-5'-iodoinosine) [61] 0.018 (N(6)-methylformycin B, , pH 7 [9]) [9] 0.019 (formycin B) [58] 0.02 (1-methylguanine) [33]

19


2.4.2.1

0.024 (9-(1,3-dihydroxy-2-propoxymethyl)guanine, , reaction with 7-methylguanosine [35]) [35] 0.025 (1,6-dihydropurine riboside) [39] 0.025 (guanine, , with phosphate as variable substrate [28]) [28] 0.025 (hypoxanthine) [25] 0.0252 (6-methylformycin A, , pH 5.5 [52]) [52] 0.027 (9-(1,3-dihydroxy-2-propoxymethyl)guanine, , reaction with inosine [35]) [35] 0.027 (N(1)-methylformycin A, , pH 7 [9]) [9] 0.0313 (guanosine, , with guanine as variable substrate and 0.4 mM ribose 1-phosphate [32]) [32] 0.0337 (folic acid) [40] 0.035 (a-d-ribose 1-phosphate, , with phosphate as variable substrate [28]) [28] 0.038 (d,l-6-methy 5,6,7,8-tetrahydropterine) [40] 0.049 (adenosine) [15] 0.05 (hypoxanthine, , with guanosine as variable substrate and 200 mM phosphate [32]) [32] 0.058 (9-(2-hydroxyethoxymethyl)guanine, , reaction with 7-methylguanosine [35]) [35] 0.06 (7-deazainosine) [39] 0.062 (formycin A) [58] 0.065 (guanine, , with phosphate as variable substrate and 0.2 mM guanosine [32]) [32] 0.068 (9-(2-hydroxyethoxymethyl)guanine, , reaction with inosine [35]) [35] 0.0685 (formycin A, , mutant enzyme N243D/K244Q, with adenosine as substrate [56]) [56] 0.073 (6-mercaptopurine) [4] 0.085 (7-deazainosine) [39] 0.09 (acyclovir) [61] 0.097 (N(7)-acycloguanosine) [58] 0.1 (formycin B) [39] 0.11 (acyclovir) [58] 0.114 (formycin A, , mutant enzyme N243D/K244Q, with inosine as substrate [56]) [56] 0.13 (6-methylthiopurine) [4] 0.13 (formycin B) [42] 0.143 (formycin A, , mutant enzyme N243D, with adenosine as substrate [56]) [56] 0.15 (aminopterin) [40] 0.16 (adenosine) [58] 0.18 (oxoformycin B, , pH 7 [9]) [9] 0.19 (erythro-9-(2-hydroxy-3-nonyl)adenine) [40] 0.195 (8-aza-2,6-diaminopurine) [40] 0.2 (oxoformycin A, , pH 7 [9]) [9] 0.203 (6-hydroxy-9-p-aminobenzylpurine) [14] 20

2.4.2.1


0.26 (inosine) [4] 0.279 (formycin B, , wild-type enzyme, with inosine as substrate [56]) [56] 0.3 (2-amino-6-methylthiopurine) [4] 0.32 (a-d-ribose 1-phosphate, , with phosphate as variable substrate and 0.2 mM guanosine [32]) [32] 0.323 (quinazolinone) [61] 0.33 (7-deazainosine) [4] 0.33 (diphosphate) [4] 0.339 (formycin B, , mutant enzyme N243D/K244Q, with inosine as substrate [56]) [56] 0.36 (a-d-ribose 1-phosphate, , with guanosine as variable substrate and 20 mM phosphate [32]) [32] 0.361 (a-d-ribose 1-phosphate) [25, 37] 0.49 (purine riboside) [39] 0.5 (formycin B) [4] 0.667 (formycin A, , wild-type enzyme, with inosine as substrate [56]) [56] 0.67 (formycin B, , mutant enzyme N243D, with inosine as substrate [56]) [56] 0.705 (formycin B, , mutant enzyme N243D/K244Q, with inosine as substrate [56]) [56] 0.81 (oxoallopurinol) [4] 0.97 (allopurinol) [4] 1.1 (6-thioxanthine) [4] 1.5 (deoxyinosine) [4] 4.4 (phosphate, , with ribose 1-phosphate as variable substrate and guanine as fixed substrate [4]) [4] 6.6 (adenine) [4] 26 (phosphate, , with guanine as variable substrate and ribose 1phosphate as fixed substrate [4]) [4] Additional information [33, 45, 61] pH-Optimum 5-6 ( reaction with xanthine or xanthosine [57]) [57] 5.5-7.5 [12] 5.5-8 [28] 6 (, sharp optimum, mutant enzyme N243D and N243D/ K244Q [56]) [13, 56] 6-8 [25, 37] 6.5 [2] 6.5-7.5 (, wild-type enzyme [56]) [56] 6.6 (, phosphorolysis [43]) [43] 6.8 (, synthesis of inosine from hypoxanthine [38]; , synthetic direction [43]) [18, 38, 43] 7 [20] 7-7.5 (, TES buffer [15]) [15]

21


2.4.2.1

7-7.7 [53] 7-8 (, reaction with guanine, guanosine, hypoxanthine and inosine [57]) [46, 57] 7-8.5 [35] 7-11 [22] 7.1 (, phosphorolysis of deoxyinosine [38]) [38] 7.5 (, phosphorolysis of inosine [38]) [10, 24, 38] 7.5-8 [29] 7.5-8.5 [19] 7.5-11 [17] 8 [48, 54] 8.5 (, phosphorylation of inosine [23]) [21, 23] 8.8-9.3 [54] pH-Range 5-11.5 (, pH 5.0: about 50% of maximal activity, pH 11.5: about 85% of maximal activity [22]) [22] 6-9 (, pH 6.0: about 65% of maximal activity, pH 9.0: about 35% of maximal activity [46]) [46] 6-9.5 (, pH 6.0: about 60% of maximal activity, pH 9.5: about 80% of maximal activity [23]) [23] 6-10 (, pH 6.0: about 60% of maximal activity, pH 10.0: about 75% of maximal activity [19]) [19] 6-12 (, pH 6.0: about 65% of maximal activity, pH 12.0: about 25% of maximal activity [17]) [17] 6.8-7.8 (, pH 6.8: about 70% of maximal activity, pH 7.8: about 40% of maximal activity [15]) [15] Temperature optimum ( C) 40 [21] 50 [19] 60 [18] 70 (, phosphorylation of inosine [23]) [21, 22, 23, 48] 80 [17] Temperature range ( C) 30-70 (, 30 C: about 75% of maximal activity, 70 C: about 80% of maximal activity [19]) [19] 30-80 (, 30 C: about 35% of maximal activity, 80 C: about 85% of maximal activity [23]) [23]

4 Enzyme Structure Molecular weight 45000 (, gel filtration [37]) [37] 56000-58000 [2] 58000 (, non-denaturing PAGE [21,61]) [21, 61]

22

2.4.2.1


61000 (, gel filtration [25]) [25] 67000 (, equilibrium sedimentation, gel filtration, amino acid analysis [8]) [8] 68000 (, gel filtration [17]; [61]) [17, 61] 70000 (, gel filtration [40]) [40] 75000-83000 (, sucrose density gradient centrifugation [36]) [36] 78000-80000 (, gel filtration [32]) [32] 80000-92000 (, gel filtration, equilibrium sedimentation [26]) [26] 81000 (, gel filtration [1]) [1] 84000 (, gel filtration [29]) [29] 87000 (, gel filtration [19]) [19] 89000 (, gel filtration [16,27]) [16, 27] 90000 (, gel filtration [13,48]; , equilibrium sedimentation [13,16]; , gel filtration [20]) [13, 16, 20, 48] 91000 (, equilibrium sedimentation [31]) [31] 93800 (, disc gel electrophoresis [34]) [34] 94000 (, calculation from X-ray analysis [6]; , gel filtration [14]) [6, 14] 95000 (, inosine-guanosine phosphoylase, gel filtration [11]) [11] 102000-108000 [61] 112000 (, gel filtration [58]) [58] 113000 (, gel filtration [22]) [22] 114000 (, gel filtration [46,61]) [46, 61] 120000 (, non-denaturing PAGE [15,61]) [15, 61] 121800 (, non-denaturing PAGE [12]) [12] 128800 (, gel filtration [12]) [12] 130000 (, gel filtration [10]) [10] 138000 (, gel filtration [38,61]) [38, 61] 141000 (, equilibrium sedimentation [10,24]) [10, 24] 147000 (, sucrose density gradient centrifugation [42,61]) [42, 61] 153000 (, adenosine-specific phosphorylase, gel filtration [11]) [11] 156000 [61] 160000 [61] 168000 (, gel filtration [54]) [54] 170000 (, gel filtration [53,61]) [53, 61] 180000 (, gel filtration [43]) [43] Subunits ? (, x * 24000, SDS-PAGE [13]; , x * 28000, inosineguanosine phosphorylase, SDS-PAGE [11]; , x * 30000, SDS-PAGE [26]; , x * 39000, SDS-PAGE [37]) [11, 13, 26, 37] dimer (, 2 * 30000, enzyme behaves as a mixture of dimers and trimers, SDS-PAGE [30]; , 2 * 30500, SDS-PAGE [25]; , 2 * 345000, SDS-PAGE [40]; , 2 * 38000, SDS-PAGE [32]; , 2 * 58000, SDS-PAGE [21]; , 2 * 34000, SDS-PAGE [17]; , 2 * 44000, SDS-PAGE [3]) [3, 17, 21, 25, 30, 32, 40]

23


2.4.2.1

hexamer (, 6 * 23500, SDS-PAGE [10,24]; , 6 * 23700, SDS-PAGE [38]; , 6 * 24000, calculation from X-ray analysis [5]; , 6 * 25500, adenosine-specific phosphorylase, SDS-PAGE [11]; , 6 * 25817, electrospray ionizing mass spectrometry [53]; , 6 * 27000, SDSPAGE [53]; , 6 * 27000 [61]) [5, 10, 11, 24, 38, 53, 54, 61] monomer (, 1 * 31000, SDS-PAGE in absence and presence of 2-mercaptoethanol [23]; , 2 * 32000, SDS-PAGE [8]; , 1 * 56000-58000 [2]) [2, 8, 23] pentamer (, 5 * 23900, SDS-PAGE [12]) [12, 14] tetramer (, 4 * 29000, MALDI-MS [58]) [28, 58] trimer (, 3 * 29035, mass spectrometry [46]; , 3 * 29700, SDS-PAGE [34]; , 3 * 30000, SDS-PAGE [31]; , 3 * 30000, enzyme behaves as a mixture of dimers and trimers, SDS-PAGE [30]; , 3 * 31000-32000, calculation from X-ray analysis [6]; , 1 * 32000 + 2 * 28000, SDS-PAGE [16]; , 3 * 29000, SDS-PAGE [29]; , 3 * 30000, SDS-PAGE [48]; , 1 * 30000 + 2 * 27000, gel filtration with 6 M guanidine HCl [16]; , 3 * 31600, SDS-PAGE [14]) [6, 14, 16, 29, 30, 31, 33, 34, 46, 47, 48] Additional information (, enzyme behaves as a mixture of dimers of 68000 Da and trimers of 89000 Da [30]) [30]

5 Isolation/Preparation/Mutation/Application Source/tissue Novikoff hepatoma cell [29] S-180 cell [41] V-79 cell [27, 30] ascites [4] blood [55] bone marrow [4, 36] brain [4, 32, 36, 40] cardiac muscle [4] embryo [2] erythrocyte [1, 4, 6, 8, 14, 26, 31, 34, 36, 39, 57, 59] gastrointestinal tract [4] heart [4] intestinal mucosa [4] kidney [4, 30, 36] leukocyte [4] liver [3, 4, 7, 13, 16, 24, 25, 29, 30, 36, 37, 41] lung [4] lymph node [4] ocular lens [28] skeletal muscle [4] skin [4] 24

2.4.2.1


skin fibroblast [4] spleen [1, 4, 35, 36, 39, 44, 45, 47, 51, 57, 59, 60] spore [4] thymus [4] thyroid gland [20] vegetative cell [4] Purification (wild-type and recombinant mutant enzymes [55]) [1, 4, 8, 26, 34, 55] [4, 20, 28, 32] [3] (partial [36]) [25, 36, 37, 40] [24, 29] [7, 13] (partial [43]; recombinant enzyme [54]) [38, 43, 54] [4] (two purine nucleoside phosphorylases: 1. inosine-guanosine phosphorylase, 2. adenosine-specific phosphorylase [11]) [11] [23] [4] [4, 21] [15] [4] [10, 24, 38] [12] (PUNPI and PUNPII [22]) [17, 22, 48] (partial [19]) [19] [27, 30] (recombinant enzyme [49]) [49] [53] Crystallization (crystals for X-ray analysis are obtained by vapor-diffusion equilibration of droplets hanging from siliconized coverslips inverted on Linbro plates [6]) [1, 6, 26] (hanging drop method, purine nucleoside phosphorylase complexed with substrates and substrate analogues [44]; X-ray crystal structure for purine nucleoside phosphorylase with bound 9-deazainosine and inorganic sulfate [47]; , high-resolution structure may serve for design of inhibitors with potential pharmacological application [51]) [44, 47, 50] [7] (crystals for X-ray analysis are obtained by vapor-diffusion equilibration of droplets hanging from siliconized coverslips inverted on Linbro plates [5]; , complex of enzyme with hypoxanthine at 2.15 A resolution, structural data from crystal structure may be useful in designing prodrugs that can be activated by E. coli enzyme but not the human enzyme [50]; hanging drop method, crystal structure of the ternary complex of the hexameric en-

25


2.4.2.1

zyme with formycin A derivatives and phosphate or sulfate ions is determined at 2.0 A resolution [52]) [5, 38, 51, 52] [38] [58] Cloning (expression of site-directed mutants in Escherichia coli [55]) [55] (overexpression in Escherichia coli [54]) [54] (expression in Escherichia coli [49]) [49] Engineering H86A (, 10-25fold reduction in catalytic activity [55]) [55] K244Q (, ration of turnover-numer/Km is 83% of that for wildtype enzyme, no activity with adenosine [56]) [56] N243A (, 100fold decrease in turnover-number compared to wildtype enzyme [55]) [55] N243D (, substitution results in an 8fold increase in Km -value for inosine and a 100fold decrease in ratio of turnover-number/Km . Catalyzes phosphorolysis of adenosine with a Km -value of 0.045 mM and ratio of turnover-number/Km 8fold that with inosine, wild-type enzyme shows no activity with adenosine [56]) [56] N243D/K244Q (, 14fold increase in Km -value for inosine and 7fold decrease in the ratio of turnover-number/Km as compared to wild-type enzyme. Phosphorolysis of adenosine with a Km -value of 0.042 mM and a ratio of turnover-number/Km twice that of the single D243D substitution [56]) [56] N243T (, mutant enzyme shows no activity with adenosine [56]) [56] Q89A (, 10-25fold reduction in catalytic activity [55]) [55] Application medicine (, the purine nucleoside phosphorylases may play a role in chemotherapy in two ways: 1. by catalyzing the breakdown of nucleosides that contain purine analogs and by promoting the incorporation of purine and pyrimidine analogs into the nucleotides and nucleic acids of the cell by increasing the intracellular pool of ribose 1-phosphate and deoxyribose 1-phosphate [4]; , the enzyme inhibitors 8-amino-5'-deoxy-5'chloroguanosine and 8-amino-9-benzylguanine may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte [42]; , the enzyme constitutes a target for antitrichomonial chemotherapy [49]; , structural data from crystal structure may be useful in designing prodrugs that can be activated by E. coli enzyme but not the human enzyme [50]; , high-resolution structure may serve for design of inhibitors with potential pharmacological application [51]; , since PNP inhibitors block the catabolism of purine nucleosides to hypoxanthine and guanine, precursors of uric acid, they should be useful in the treatment of goat and other hyperuricemic conditions. A number of parasites, including the malaria parasite, lacking the ability to synthesize purine nucleotides de novo, must utilize host purines, formed by PBP, for DNA synthesis. Thus in-

26

2.4.2.1


hibition of PNP could prevent the spread of parasitic infection [59]; , overview of clinical aspects [61]) [4, 42, 49, 50, 51, 58, 61]

6 Stability pH-Stability 5 (, 37 C, 10 min, 75% loss of activity [46]) [46] 5-9 (, stable from pH 5.0 to pH 9.0 [48]) [48] 5.5-9 (, 40 C, 10 min, stable [25]) [25] 6 (, 37 C, 10 min, 65% loss of activity [46]) [46] 6.2-10 (, 5 min, stable [1]) [1] 6.5 (, 80 C, 30 min, about 75% loss of activity [19]; , rather unstable below [58]) [19, 58] 7 (, 37 C, 10 min, 50% loss of activity [46]) [46] 7-7.3 (, 80 C, 30 min, stable [19]) [19] 8 (, 80 C, 30 min, about 35% loss of activity [19]; , 37 C, 10 min, about 30% loss of maximal activity [46]) [19, 46] 8.5-10 (, stable in presence of orthophosphate [58]) [58] 9 (, 37 C, 10 min, 20% loss of activity [46]) [46] 10 (, 37 C, 10 min, 10% loss of activity [46]) [46] Temperature stability 30 (, 1 month, less than 20% loss of activity [18]) [18] 37 (, 10 min, pH 5 or 8, wild-type enzyme and mutant enzyme N243S show no detectable loss in activity, mutant enzyme N243D/K244Q shows 5% loss at pH 8 and 11% loss at pH 5 [56]) [56] 42 (, stable up to [53]) [53] 45 (, half-life at pH 6-7: 8-9 min. Half-life at pH 7-8: 5-7 min. At pH 6.5 in presence of 5 mg/ml bovine serum albumin half-life is 17 min [43]) [43] 50 (, 5 min, 75-85% loss of activity, 1 mM hypoxanthine significantly protects against thermal denaturation [43]; , pH 7.4, 10 min, about 10% loss of activity [46]; , 30 min, 25% loss of activity [53]; , stable up to [58]) [43, 46, 53, 58] 55 (, rapid inactivation [32]; , 10 min, 20% loss of activity [54]; , 10 min, stable [54]) [32, 54] 57 (, 15 min, 50% inactivation [1]) [1] 60 (, half-life: around 1 week [18]; , 30 min, stable [19]; , pH 7.4, 20 min, about 20% loss of activity [29]; , 20 min, 10% loss of activity [37]; , pH 7.4, 10 min, about 10% loss of activity [46]; , 30 min, 90% loss of activity, 5 mM phosphate stabilizes [53]; , 10 min, about 35% loss of activity [54]; , 10 min, about 10% loss of activity [54]) [18, 19, 29, 37, 46, 53, 54] 62 (, pH 7.4, 50% inactivation [46,58]) [46, 58] 65 (, pH 7.4, 10 min, about 75% loss of activity [46]) [46]

27


2.4.2.1

67 (, 10 min, about 90% loss of activity [54]; , 10 min, 45% loss of activity [54]) [54] 69 (, 0.2 M Tris-succinate buffer, pH 7.1, half-life: 11 min. In 0.2 M potassium phosphate buffer, pH 7.1, with 2 mM deoxyadenosine, half-life: 59 min [38]) [38] 70 (, 30 h, stable [17]; , 30 min, about 90% loss of activity [19]; , half-life of PUNPII is 15 min [22]; , denaturation above [46]; , stable below [48]) [17, 19, 22, 46, 48] 80 (, half-life is 16 h in 20 mM potassium phosphate and 1 mM inosine, pH 7.0 [17]) [17] Additional information (, adenine selectively protects the adenosine phosphorylase against heat inactivation, no effect on inosine-guanosine phosphorylase. Hypoxanthine selectively protects inosine-guanosine phosphorylase activity, no effect on adenosine phosphorylase [41]) [41] Oxidation stability , freezing of the enzyme in presence of dithiothreitol results in greater loss of activity than in the absence of a sulfhydryl reagent [26] , photooxidation in presence of methylene blue [2] , high sensitivity to photooxidation in presence of methylene blue, almost 95% loss of activity after 40 min irradiation [40] , highly susceptible to photooxidation in presence of methylene blue, maximal photoinactivation near pH 8.5 [25, 37] General stability information , freezing and thawing results in complete loss of activity of the pure enzyme [28] , photooxidation in presence of methylene blue is pH-dependent [28] , strong susceptibility to photooxidation in presence of methylene blue. Maximal photoinactivation at pH 8.0, 85% loss of activity after only 1 min of irradiation [32] , unstable in presence of 2-mercaptoethanol [2] , freezing of the solubilized enzyme, especially in dilute solutions, causes complete loss of activity [37] , stabilized by 2-mercaptoethanol during the purification [25] , not stable when frozen [24] , lyophilization decreases the activity by 27% [53] , moderately stable to X-rays [5, 6] Storage stability , -20 C, 50% loss of activity after 2 weeks [34] , 20 C, room temperature, stable for several days in concentrated solution, 1% in 0.02% sodium azide [26] , 4 C or frozen, stable for long periods of time [26] , 4 C, 10 mM sodium phosphate buffer, pH 7.0, stable for at least 4 months provided a weekly dialysis with 2 mM DTT is performed [28]

28

2.4.2.1


, 4 C, enzyme containing 2-mercaptoethanol prior to precipitation with ammonium sulfate, near pH 7, retains maximal catalytic activity for several months when stored as a precipitate [37] , -20 C, 50 mM Tris-HCl, 10 mM 2-mercaptoethanol, pH 7.0, stable for several months [12] , 30 C, 1 month, less than 20% loss of activity [18] , 4 C, pH 7.2, 10 mM Tris-HCl buffer, stable for up to 14 days [53] , 4 C, stable for 2 years [38]

References [1] Agarwal, R.P.; Parks, R.E.: Purine nucleoside phosphorylase from human erythrocytes. IV. Crystallization and some properties. J. Biol. Chem., 244, 644-647 (1969) [2] Kamel, M.Y.; Fahmy, A.S.; Ghazy, A.H.; Mohamed, M.A.: Purification and characterization of purine nucleoside phosphorylase from developing embryos of Hyalomma dromedarii. Biochem. Cell Biol., 69, 223-231 (1991) [3] Osman, A.M.; Del Corso, A.; Mohamed, A.S.; Ipata, P.L.; Mura, U.: Liver purine nucleoside phosphorylase in Camelus dromedarius: purification and properties. Comp. Biochem. Physiol. B, 97, 177-182 (1990) [4] Parks, R.E.; Agarwal, R.P.: Purine nucleoside phosphorylase. The Enzymes, 3rd Ed. (Boyer, P.D., ed.), 7, 483-514 (1972) [5] Cook, W.J.; Ealick, S.E.; Krenitsky, T.A.; Stoeckler, J.D.; Helliwell, J.R.; Bugg, C.E.: Crystallization and preliminary X-ray investigation of purine-nucleoside phosphorylase from Escherichia coli. J. Biol. Chem., 260, 12968-12969 (1985) [6] Cook, W.J.; Ealick, S.E.; Bugg, C.E.; Stoeckler, J.D.; Parks, R.E.: Crystallization and preliminary X-ray investigation of human erythrocytic purine nucleoside phosphorylase. J. Biol. Chem., 256, 4079-4080 (1981) [7] Murakami, K.; Tsushima, K.: Crystallization and some properties of purine nucleoside phosphorylase from chicken liver. Biochim. Biophys. Acta, 384, 390-398 (1975) [8] Lewis, A.S.; Lowy, B.A.: Human erythrocyte purine nucleoside phosphorylase: molecular weight and physical properties. A Theorell-Chance catalytic mechanism. J. Biol. Chem., 254, 9927-9932 (1979) [9] Bzowska, A.; Kulikowska, E.; Shugar, D.: Formycins A and B and some analogues: selective inhibitors of bacterial (Escherichia coli) purine nucleoside phosphorylase. Biochim. Biophys. Acta, 1120, 239-247 (1992) [10] Robertson, B.C.; Hoffee, P.A.: Purification and properties of purine nucleoside phosphorylase from Salmonella typhimurium. J. Biol. Chem., 248, 2040-2043 (1973) [11] Jensen, K.J.: Two purine nucleoside phosphorylases in Bacillus subtilis. Purification and some properties of the adenosine-specific phosphorylase. Biochim. Biophys. Acta, 525, 346-356 (1978)

29


2.4.2.1

[12] Schimandle, C.M.; Tanigoshi, L.; Mole, L.A.; Sherman, I.W.: Purine nucleoside phosphorylase of the malarial parasite, Plasmodium lophurae. J. Biol. Chem., 260, 4455-4460 (1985) [13] Umemura, S.; Nishino, T.; Murakami, K.; Tsushima, K.: Trimeric purine nucleoside phosphorylase from chicken liver having a proteolytic nick on each subunit and its kinetic properties. J. Biol. Chem., 257, 13374-13378 (1982) [14] Osborne, W.R.A.: Human red cell purine nucleoside phosphorylase. Purification by biospecific affinity chromatography and physical properties. J. Biol. Chem., 255, 7089-7092 (1980) [15] Surette, M.; Gill, T.; MacLean, S.: Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris. Appl. Environ. Microbiol., 56, 1435-1439 (1990) [16] Murakami, K.; Tsushima, K.: Molecular properties and a nonidentical trimeric structure of purine nucleoside phosphorylase from chicken liver. Biochim. Biophys. Acta, 435, 205-210 (1976) [17] Hori, N.; Watanabe, M.; Yamazaki, Y.; Mikami, Y.: Purification and characterization of thermostable purine nucleoside phosphorylase of Bacillus stearothermophilus JTS 859. Agric. Biol. Chem., 53, 2205-2210 (1989) [18] Utagawa, T.; Morisawa, H.; Yamanaka, S.; Yamazaki, A.; Yoshinaga, F.; Hirose, Y.: Properties of nucleoside phosphorylase from Enterobacter aerogenes. Agric. Biol. Chem., 49, 3239-3246 (1985) [19] Machida, Y.; Nakanishi, T.: Properties of purine nucleoside phosphorylase from Enterobacter cloacae. Agric. Biol. Chem., 45, 1801-1807 (1981) [20] Moyer, T.P.; Fischer, A.G.: Purification and characterization of a purine-nucleoside phosphorylase from bovine thyroid. Arch. Biochem. Biophys., 174, 622-629 (1976) [21] Shirae, H.; Yokozeki, K.: Purifications and properties of orotidine-phosphorolyzing enzyme and purine nucleoside phosphorylase from Erwinia carotovora AJ 2992. Agric. Biol. Chem., 55, 1849-1857 (1991) [22] Hori, N.; Watanabe, M.; Yamazaki, Y.; Mikami, Y.: Purification and characterization of second thermostable purine nucleoside phosphorylase in Bacillus stearothermophilus JTS 859. Agric. Biol. Chem., 53, 3219-3224 (1989) [23] Shirae, H.; Yokozeki, K.: Purification and properties of purine nucleoside phosphorylase from Brevibacterium acetylicum ATCC 954. Agric. Biol. Chem., 55, 493-499 (1991) [24] Hoffee, P.A.; May, R.; Robertson, B.C.: Purine nucleoside phosphorylase from Salmonella typhimurium and rat liver. Methods Enzymol., 51, 517524 (1978) [25] Glantz, M.D.; Lewis, A.S.: Purine nucleoside phosphorylase from rabbit liver. Methods Enzymol., 51, 524-530 (1978) [26] Stoeckler, J.D.; Agarwal, R.P.; Agarwal, K.C.; Parks, R.E.: Purine nucleoside phosphorylase from human erythrocytes. Methods Enzymol., 51, 530-538 (1978) [27] Milman, G.: Chinese hamster purine nucleoside phosphorylase. Methods Enzymol., 51, 538-543 (1978)

30

2.4.2.1


[28] Barsacchi, D.; Cappiello, M.; Tozzi, M.G.; Del Corso, A.; Peccatori, M.; Camici, M.; Ipata, P.L.; Mura, U.: Purine nucleoside phosphorylase from bovine lens: purification and properties. Biochim. Biophys. Acta, 1160, 163170 (1992) [29] May, R.A.; Hoffee, P.: Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells. Arch. Biochem. Biophys., 193, 398-406 (1979) [30] Milman, G.; Anton, D.L.; Weber, J.L.: Chinese hamster purine-nucleoside phosphorylase: purification, structural, and catalytic properties. Biochemistry, 15, 4967-4973 (1976) [31] Stoeckler, J.D.; Agarwal, R.P.; Agarwal, K.C.; Schmid, K.; Parks, R.E.: Purine nucleoside phosphorylase from human erythrocytes: physiocochemical properties of the crystalline enzyme. Biochemistry, 17, 278-283 (1978) [32] Lewis, A.S.; Glantz, M.D.: Bovine brain purine-nucleoside phosphorylase purification, characterization, and catalytic mechanism. Biochemistry, 15, 4451-4457 (1976) [33] Bzowska, A.; Kulikowska, E.; Darzynkiewicz, E.; Shugar, D.: Purine nucleoside phosphorylase. Structure-activity relationships for substrate and inhibitor properties of N-1-, N-7-, and C-8-substituted analogues; differentiation of mammalian and bacterial enzymes with N-1-methylinosine and guanosine. J. Biol. Chem., 263, 9212-9217 (1988) [34] Zannis, V.; Doyle, D.; Martin, D.W.: Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits. J. Biol. Chem., 253, 504-510 (1978) [35] Kulikowska, E.; Bzowska, A.; Wierzchowski, J.; Shugar, D.: Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7methylguanosine and 7-methylinosine. Biochim. Biophys. Acta, 874, 355363 (1986) [36] Savage, B.; Spencer, N.: Partial purification and properties of purine nucleoside phosphorylase from rabbit erythrocytes. Biochem. J., 167, 703710 (1977) [37] Lewis, A.S.; Glantz, M.D.: Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization. J. Biol. Chem., 251, 407-413 (1976) [38] Jensen, K.F.; Nygaard, P.: Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties. Eur. J. Biochem., 51, 253-265 (1975) [39] Bzowska, A.; Kulikowska, E.; Shugar, D.: Properties of purine nucleoside phosphorylase (PNP) of mammalian and bacterial origin. Z. Naturforsch. C, 45c, 59-70 (1990) [40] Lewis, A.S.: Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds. Arch. Biochem. Biophys., 190, 662-670 (1978) [41] Divekar, A.Y.: Adenosine phosphyorylase activity as distinct from inosineguanosine phosphorylase activity in Sarcoma 180 cells and rat liver. Biochim. Biophys. Acta, 422, 15-28 (1976) 31


2.4.2.1

[42] Daddona, P.E.; Wiesmann, W.P.; Milhouse, W.; Chern, J.W.; Townsend, L.B.; Hershfield, M.S.; Webster, H.K.: Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors. J. Biol. Chem., 261, 11667-11673 (1986) [43] Koszalka, G.W.; Vanhooke, J.; Short, S.A.; Hall, W.W.: Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K12. J. Bacteriol., 170, 3493-3498 (1988) [44] Mao, C.; Cook, W.J.; Zhou, M.; Federov, A.A.; Almo, S.C.; Ealick, S.E.: Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues. Biochemistry, 36, 7135-7146 (1998) [45] Bzowska, A.; Kulikowska, E.; Poopeiko, N.E.; Shugar, D.: Kinetics of phosphorolysis of 3-(b-d-ribofuranosyl)adenine and 3-(b-d-ribofuranosyl)hypoxanthine, non-conventional substrates of purine-nucleoside phosphorylase. Eur. J. Biochem., 239, 229-234 (1996) [46] Wielgus-Kutrowska, B.; Bzowska, A.; Tebbe, J.; Koeller, G.; Shugar, D.: Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence, and by protective effects of ligands on thermal inactivation of the enzyme. Biochim. Biophys. Acta, 1597, 320-334 (2002) [47] Kline, P.C.; Schramm, V.L.: Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction. Biochemistry, 32, 13212-13219 (1993) [48] Hamamoto, T.; Noguchi, T.; Midorikawa, Y.: Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus TH 6-2. Biosci. Biotechnol. Biochem., 60, 1179-1180 (1996) [49] Munagala, N.; Wang, C.C.: The purine nucleoside phosphorylase from Trichomonas vaginalis is a homologue of the bacterial enzyme. Biochemistry, 41, 10382-10389 (2002) [50] Mao, C.; Cook, W.J.; Zhou, M.; Koszalka, G.W.; Krenitsky, T.A.; Ealick, S.E.: The crystal structure of Escherichia coli purine nucleoside phosphorylase: a comparison with the human enzyme reveals a conserved topology. Structure, 5, 1373-1383 (1997) [51] Koeller, G.; Luic, M.; Shugar, D.; Saenger, W.; Bzowska, A.: Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 A resolution. J. Mol. Biol., 265, 202-216 (1997) [52] Koellner, G.; Bzowska, A.; Wielgus-Kutrowska, B.; Luic, M.; Steiner, T.; Saenger, W.; Stepinski, J.: Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism. J. Mol. Biol., 315, 351-371 (2002) [53] Choi, H.S.: Purification and partial characterization of purine nucleoside phosphorylase from Serratia marcescens. Biosci. Biotechnol. Biochem., 62, 667-671 (1998) [54] Lee, J.; Filosa, S.; Bonvin, J.; Guyon, S.; Aponte, R.A.; Turnbull, J.L.: Expression, purification, and characterization of recombinant purine nucleoside 32

2.4.2.1

[55] [56]

[57] [58]

[59] [60]

[61]


phosphorylase from Escherichia coli. Protein Expr. Purif., 22, 180-188 (2001) Erion, M.D.; Takabayashi, K.; Smith, H.B.; Kessi, J.; Wagner, S.; Hoenger, S.; Shames, S.L.; Ealick, S.E.: Purine nucleoside phosphorylase. 1. Structurefunction Studies. Biochemistry, 36, 11725-11734 (1997) Maynes, J.T.; Yam, W.S.; Jenuth, J.P.; Yuan, R.G.; Litster, S.A.; Phipps, B.M.; Snyder, F.F.: Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase: enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase. Biochem. J., 344, 585-592 (1999) Stoychev, G.; Kierdaszuk, B.; Shugar, D.: Xanthosine and xanthine. Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems. Eur. J. Biochem., 269, 4048-4057 (2002) Wielgus-Kutrowska, B.; Tebbe, J.; Schroder, W.; Luic, M.; Shugar, D.; Saenger, W.; Koellner, G.; Bzowska, A.: Cellulomonas sp. purine nucleoside phosphorylase (PNP): comparison with human and E. coli enzymes. Adv. Exp. Med. Biol., 431, 259-264 (1998) Montgomery, J.A.: Purine nucleoside phosphorylase: a target for drug design. Med. Res. Rev., 13, 209-228 (1993) Cheng, J.; Farutin, V.; Wu, Z.; Jacob-Mosier, G.; Riley, B.; Hakimi, R.; Cordes, E.H.: Purine nucleoside phosphorylase-catalyzed, phosphate-independent hydrolysis of 2-amino-6-mercapto-7-methylpurine ribonucleoside. Bioorg. Chem., 27, 307-325 (1999) Bzowska, A.; Kulikowska, E.; Shugar, D.: Purine nucleoside phosphorylases: properties, functions, and clinical aspects. Pharmacol. Ther., 88, 349-425 (2000)

33

Pyrimidine-nucleoside phosphorylase

2.4.2.2

1 Nomenclature EC number 2.4.2.2 Systematic name pyrimidine-nucleoside:phosphate a-d-ribosyltransferase Recommended name pyrimidine-nucleoside phosphorylase Synonyms PYNP PYNP Py-NPase phosphorylase, pyrimidine nucleoside pyrimidine nucleoside phosphorylase pyrimidine ribonucleoside phosphorylase Additional information (enzyme may be identical with EC 2.4.2.3 from some organisms catalyzing both uridine and thymidine phosphorolysis) CAS registry number 9055-35-0

2 Source Organism Bacillus stearothermophilus (JTS 859 [5]) [1, 2, 3, 5] Haemophilus influenzae (TH 6-2 [7]) [4, 6, 7]

3 Reaction and Specificity Catalyzed reaction pyrimidine nucleoside + phosphate = pyrimidine + a-d-ribose 1-phosphate Reaction type pentosyl group transfer Natural substrates and products S Additional information (, reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway [2]) [2] P ?

34

2.4.2.2


Substrates and products S 5-bromodeoxyuridine + phosphate (, 95% of the activity with uridine [1]; , 74% of the activity with uridine [4]) (Reversibility: ? [1, 4, 6]) [1, 4, 6] P 5-bromouracil + deoxyribose 1-phosphate S 5-bromouridine + phosphate (, 95% of the activity with uridine [1]; , 40% of the activity with uridine [4]) (Reversibility: ? [1,4]) [1, 4] P 5-bromouracil + a-d-ribose 1-phosphate S 5-methyluridine + phosphate (, 27% of the activity with uridine [4]) (Reversibility: ? [4,5,6]) [4, 5, 6] P 5-methyluracil + a-d-ribose 1-phosphate S cytidine + phosphate (, 8% of the activity with uridine [1]; , no activity [6]) (Reversibility: ? [1]) [1] P cytosine + a-d-ribose 1-phosphate S deoxyuridine + phosphate (, 2'-deoxyuridine [5]; , 179% of the activity with uridine, by measuring the conversion of uracil to uridine or deoxyuridine a slight preference for ribose 1-phosphate over deoxyribose 1-phosphate is observed [1]; , 12% of the activity with uridine [4]; , 147% of the activity with uridine [7]) (Reversibility: r [1]; ? [4, 5, 6, 7]) [1, 4, 5, 6, 7] P uracil + 2-deoxy-d-ribose 1-phosphate S ribofuranosyl thymine + phosphate (Reversibility: ? [7]) [7] P thymine + a-d-ribose 1-phosphate S thymidine + phosphate (, 139% of the activity with uridine [1]; , 21% of the activity with uridine [4]; , 99% of the activity with uridine [7]) (Reversibility: ? [1]; ? [2, 4, 5, 6, 7]) [1, 2, 4, 5, 6, 7] P thymine + 2-deoxy-d-ribose 1-phosphate S uracil arabinoside + phosphate (Reversibility: ? [4]) [4] P uracil + a-d-arabinose 1-phosphate S uridine + phosphate (, by measuring the conversion of uracil to uridine or deoxyuridine a slight preference for ribose 1-phosphate over deoxyribose 1-phosphate is observed [1]) (Reversibility: r [1]; ? [2, 4, 5, 7]) [1, 2, 4, 5, 6, 7] P uracil + a-d-ribose 1-phosphate [1] S Additional information (, no activity with deoxycytidine [1]) [1] P ? Inhibitors uridine (, above 15 mM, competitive inhibition of cleavage of 5-bromodeoxyuridine [6]) [6] Specific activity (U/mg) 25.5 [4] 99.6 [5] 205 [7] Additional information [1, 6] 35


2.4.2.2

Km-Value (mM) 0.03 (5-bromouridine) [4, 6] 0.07 (5-methyluridine) [4, 6] 0.1 (5-bromodeoxyuridine) [4, 6] 0.11 (thymidine) [4, 6] 0.13 (deoxyuridine) [4, 6] 0.19 (uridine) [5] 0.24 (uridine) [4, 6] 0.25 (uridine) [1] 0.32 (5-methyluridine) [5] 0.38 (phosphate, , reaction with uridine [6]) [6] 0.38 (thymidine) [1] 0.46 (thymidine) [5] 0.58 (2'-deoxyuridine) [5] 1.38 (uridine) [7] 1.9 (thymidine) [7] Ki-Value (mM) 0.25 (uridine) [6] pH-Optimum 6.9 (, deoxynucleoside substrates [6]) [4, 6] 7-11.5 [5] 7.2 (, uridine, thymidine [1]) [1] 7.4 (, ribonucleosides and uracil arabinoside [4,6]) [4, 6] pH-Range 6-12 (, pH 6.0: about 75% of maximal activity, pH 12.0: about 80% of maximal activity [5]) [5] 6.9-7.4 (, pH 6.9: optimum, pH 7.4: 75% of maximal activity [4]) [4] Temperature optimum ( C) 70 [5, 7]

4 Enzyme Structure Molecular weight 78000 (, sedimentation velocity analysis [1]) [1] 85000 (, gel filtration [5]) [5] 92000 (, gel filtration [7]) [7] Subunits dimer (, 2 * 46000, SDS-PAGE [7]; , 2 * 54000, SDS-PAGE [5]) [5, 7]

36

2.4.2.2


5 Isolation/Preparation/Mutation/Application Purification [1, 3, 5] [4, 6, 7] Crystallization (crystal structure of the enzyme with the substrate analog, pseudouridine, in its active site is solved to 2.1 A [2]; hanging-drop vapor diffusion method, crystals of the protein-inhibitor complex with the substrate analog pseudouridine [3]) [2, 3] Cloning (expression in Escherichia coli [3]) [3]

6 Stability pH-Stability 5-9 (, stable [7]) [7] Temperature stability 60 (, 60 min, stable [1]; , stable below [7]) [1, 7] 70 (, half-life: 25 min in 20 mM potassium phosphate, 15.1 h in 20 mM potassium phosphate and 1 mM 5-methyluridine [5]) [5] 80 (, 1 h, 20 mM potassium phosphate, complete inactivation [5]) [5] General stability information , repeated cycles of freezing and thawing inactivate the enzyme if phosphate is absent [4] , the enzyme is quite labile during the course of purification, dithiothreitol and glycerol are required for stability [4] Storage stability , -20 C, in presence of phosphate, glycerol and dithiothreitol, stable for 6 months [4, 6]

References [1] Saunders, P.P.; Wilson, B.A.; Saunders, G.F.: Purification and comparative properties of a pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus. J. Biol. Chem., 244, 3691-3697 (1969) [2] Pugmire, M.J.; Ealick, S.E.: The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation. Structure, 6, 1467-1479 (1998) [3] Zhou, M.; Pugmire, M.J.; Vuong, B.Q.; Ealick, S.E.: Cloning, expression and crystallization of pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus. Acta Crystallogr. Sect. D, D55, 287-290 (1999)

37


2.4.2.2

[4] Scocca, J.J.: Pyrimidine nucleoside phosphorylase from Haemophilus influenzae. Methods Enzymol., 51, 432-437 (1978) [5] Hori, N.; Watanabe, M.; Yamazaki, Y.; Mikami, Y.: Purification and characterization of thermostable pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus. Agric. Biol. Chem., 54, 763-768 (1990) [6] Scocca, J.J.: Purification and substrate specificity of pyrimidine nucleoside phosphorylase from Haemophilus influenzae. J. Biol. Chem., 246, 6606-6610 (1971) [7] Hamamoto, T.; Noguchi, T.; Midorikawa, Y.: Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus TH 6-2. Biosci. Biotechnol. Biochem., 60, 1179-1180 (1996)

38

Uridine phosphorylase

2.4.2.3

1 Nomenclature EC number 2.4.2.3 Systematic name uridine:phosphate a-d-ribosyltransferase Recommended name uridine phosphorylase Synonyms UDRPase [Swissprot] UPH UPase UrdPase phosphorylase, uridine pyrimidine phosphorylase uridine:orthophosphate a-d-ribosyltransferase Additional information (enzyme may be identical with EC 2.4.2.2) CAS registry number 9030-22-2

2 Source Organism Dictyostelium discoideum [8] Escherichia coli (K-12 [9,12,27]; B [10,11,15]; B-96, ATCC 13473, two types of enzyme, I and II [13]) [9-13, 15, 18, 22, 27, 30] Mus musculus (NIH 3T3 [34,35]) [1, 5, 6, 29, 31, 34, 35, 36] Rattus norvegicus [2, 4, 14, 19-21] Homo sapiens [3, 5, 6, 29, 31, 34] Giardia lamblia [7] Schistosoma mansoni [24] Haemophilus influenzae [25] Lactobacillus casei [16, 17] Enterobacter aerogenes (AJ 11125 [23]) [23] Acholeplasma laidlawii [26] Hymenolepis diminuta (tapeworm [28]) [28] Saccharomyces cerevisiae (wild type and uridine phosphorylase deficient strain [32]) [32] Salmonella typhimurium [33] 39


2.4.2.3

3 Reaction and Specificity Catalyzed reaction uridine + phosphate = uracil + a-d-ribose 1-phosphate ( ordered bi-bi mechanism [8, 19, 20]; phosphate binds before uridine and ribose 1phosphate is released after uracil [19, 20]; rapid-equilibrium random mechanism [11]; random mechanism [13]; sequential rather than ping-pong mechanism, addition of substrate is random [16]; sequential mechanism [24, 26, 28]; ordered bi-bi mechanism, phosphate binds before uridine and a-d-ribose 1-phosphate is released after uracil [34]) Reaction type pentosyl group transfer Natural substrates and products S uracil + a-d-ribose 1-phosphate ( in conjunction with uridine kinase, the enzyme provides a route for the conversion of uracil to UMP via uridine [4]; role in degradation of pyrimidine nucleosides as well as in the salvage pathway for nucleic acid synthesis [19]; enzyme of pyrimidine salvage pathway [24, 35]) [4, 19, 24, 35] P uracil + a-d-ribose 1-phosphate Substrates and products S 2'-deoxyuridine + phosphate ( catalyzed by a different protein or by a different active center of the same enzyme [1]; unlike cytosolic enzyme, enzyme from plasma membranes shows little or no deoxyuridine-cleaving activity [21]; activities of uridine, deoxyuridine and thymidine phosphorylase from Giardia lamblia remain associated throughout purification suggesting that a single enzyme is responsible for the 3 activities [7]; 6% of the activity with uridine [22]; 18% of the activity with uridine [23]; 12% of the activity with uridine [25]) (Reversibility: ? [7, 21, 22, 23, 25, 31, 33]; r [1, 24]) [1, 7, 21, 22, 23, 24, 25, 31, 33] P uracil + deoxyribose 1-phosphate [1, 22] S 5'-deoxy-5-fluorouridine + phosphate ( 25% of the activity with uridine [29]) (Reversibility: ? [29,36]) [29, 36] P 5-fluorouracil + 5-deoxyribose-1-phosphate S 5-bromo-2'-deoxyuridine + phosphate ( 27% of the activity with uridine [22]; 75% of the activity with uridine [25]) (Reversibility: ? [22,25]) [22, 25] P 5-bromouracil + 2-deoxribose 1-phosphate S 5-bromouracil + deoxyribose 1-phosphate (Reversibility: ? [1]) [1] P 5-bromodeoxyuridine + phosphate S 5-bromouridine + phosphate ( 69% of the activity with uridine [22]; 40% of the activity with uridine [25]; no substrate [16]) (Reversibility: ? [16, 22, 25, 33]) [16, 22, 25, 33] P 5-bromouracil + a-d-ribose 1-phosphate

40

2.4.2.3


S 5-fluoro-2'-deoxyuridine + phosphate ( 14% of the activity with uridine [22]; 15% of the activity with uridine [29]) (Reversibility: ? [22, 29]) [22, 29] P 5-fluorouracil + 2-deoxyribose-1-phosphate S 5-fluorouridine + phosphate ( 15% of the activity with uridine [29]; 85% of the activity with uridine [29]) (Reversibility: ? [29,33]) [29, 33] P 5-fluorouracil + a-d-ribose-1-phosphate [31] S 5-methyluridine + phosphate ( 27% of the activity with uridine [25]) (Reversibility: ? [1, 16, 25]) [1, 16, 25] P 5-methyluracil + a-d-ribose 1-phosphate S arabinofuranosyl-5-ethyluracil + phosphate ( weak substrate [14]) (Reversibility: ? [14]) [14] P 5-ethyluracil + arabinose-1-phosphate S azathymine + deoxyribose 1-phosphate (Reversibility: ir [1]) [1] P azadeoxythymidine + phosphate S azauracil + deoxyribose 1-phosphate (Reversibility: ? [1]) [1] P azadeoxyuridine + phosphate S azauracil + ribose 1-phosphate (Reversibility: ir [1]) [1] P azauridine + phosphate S cytidine + phosphate (Reversibility: ? [32]) [32] P cytosine + a-d-ribose 1-phosphate S thymidine + phosphate ( 2% of the activity with uridine [22]; 22% of the activity with uridine [23]; 21% of the activity with uridine [25]; the activities of uridine, deoxyuridine and thymidine phosphorylases from Giardia lamblia remain associated throughout purification, suggesting that a single enzyme is responsible for the 3 activities [7]; to a lesser extent compared to uridine [34, 36]) (Reversibility: ? [1, 7, 16, 22-25, 28, 31, 33, 34, 36]) [1, 7, 16, 22-25, 28, 31, 33, 34, 36] P thymine + a-d-ribose 1-phosphate [1] S thymine + deoxyribose 1-phosphate (Reversibility: ? [1]) [1] P deoxythymidine + phosphate S thymine + ribose 1-phosphate (Reversibility: ? [1]) [1] P thymidine + phosphate S thymine ribonucleoside + phosphate ( 19% of the activity with uridine [22]) (Reversibility: ? [22]) [22] P thymine + a-d-ribose 1-phosphate S uracil arabinoside + phosphate ( 10% of the activity with uridine [23, 25]) (Reversibility: ? [16, 23, 25]) [16, 23, 25] P uracil + arabinose-1-phosphate S uridine + arsenate (Reversibility: ? [2]) [2] P ? S uridine + phosphate ( equilibrium position favouring nucleoside synthesis [1]; liver enzyme is highly specific to uridine [29]; broader specificity than human enzyme [29]) (Reversibility: ? [4-10, 12-14, 16, 18, 20, 21, 23, 25, 27, 29, 30, 32, 33, 35, 41


2.4.2.3

36]; r [1-3, 11, 15, 17, 19, 22, 24, 26, 28, 31, 34]) [1-4, 6, 8, 11, 15, 16, 19, 22, 23-26, 28-33, 36] P uracil + a-d-ribose 1-phosphate [1, 8, 15, 22, 25, 26, 31, 33, 34] S Additional information ( no substrates: 3'-azido-2',3'dideoxy-5-methyluridine, 3'-azido-2',3'-dideoxy-5-ethyluridine, 2',3'-dideoxy-5-ethyluridine, 3'-chloro-2',3'-dideoxy-5-ethyluridine, 3'-chloro2',3'-dideoxy-5-methyluridine, 3'-bromo-2',3'-dideoxy-5-ethyluridine, 2'deoxylyxofuranosyl-5-ethyluracil, 3'-O-acetyl-2,2'-anhydro-5-ethyluridine, 2,3'-anhydro-2'-deoxy-5-ethyluridine, 2,5'-anhydro-2'-deoxy-5-ethyluridine [14]; no substrate: 5-substituted-2,2'-anhydrouridine [5]; no substrates: 2-deoxyuridine, adenosine [16]; no substrates: cytosine, deoxycytidine, orotidine [24]; no substrate: cytidine [16, 23-25, 31]; no substrates are: thymidine, deoxyuridine, fluorodeoxyuridiune, 5'-deoxyfluorouridine [29]; no substrates: inosine, adenosine, guanosine, thymidine [32]) [5, 14, 16, 23-25, 29, 32] P ? Inhibitors 1-(1',2'-dihydroxypropyl)-5,6-tetramethyleneuracil ( only the R-enantiomer inhibits, but not the S-enantiomer [15]) [15] 1-(1',3'-dihydroxy-2'-propoxy)methyl-5,6-tetramethyleneuracil ( inhibits forward and reverse reaction [15]) [15] 1-(1',3'-dihydroxy-2'-propoxy)methyl-5-benzyluracil ( i.e. DHPBU, competitive [28]) [28] 2',3'-dideoxy-5-ethyluridine [14] 2'-deoxyglycosylthymine [3] 2'-deoxylyxofuranosyl-5-ethyluracil [14] 2,2'-anhydro-5-ethyluridine ( 0.001 mM, competitive [28]; competitive [36]) [5, 14, 24, 28, 36] 2,3'-anhydro-2'-deoxy-5-ethyluridine [14] 2,3'-anhydro-5-ethyluridine ( competitive [28]) [28] 2,5'-anhydro-2'-deoxy-5-ethyluridine [14] 3'-O-acetyl-2,2'-anhydro-5-ethyluridine [14] 3'-azido-2',3'-dideoxy-5-ethyluridine [14] 3'-azido-2',3'-dideoxy-5-methyluridine [14] 3'-bromo-2',3'-dideoxy-5-ethyluridine [14] 3'-chloro-2',3'-dideoxy-5-ethyluridine [14] 3'-chloro-2',3'-dideoxy-5-methyluridine [14] 3-O-methyl-a-d-glucopyranose [16, 17] 5,5'-dithiobis(2-nitrobenzoic acid) [19] 5-(3'-benzyloxybenzyl)-1-[(1'-aminomethyl-2'-hydroxyethoxy)methyl]uracil [6] 5-(benzyloxybenzyloxybenzyl)acyluridine [24] 5-benzylacyclouridine ( 0.01 mM, 90% inhibition of normal tissues, 40-60% inhibition of carcinoma tissue [31,34]) [31, 34] 5-bromoacyclouridine [3]

42

2.4.2.3


5-fluoro-2'-deoxyuridine [3, 17] 5-fluoroacyclouridine [3] 5-iodoacyclouridine [3] 5-substituted 2,2'-anhydrouridine [5] 6-methyluracil [17] N-ethyl-5-phenylisoxazolium-3'-sulfonate ( Woodward's reagent K [30]) [30] N-ethylmaleimide [19] acyclonucleoside analogues ( consisting of 5- and 5,6-substituted uracils and different acyclic chains [15]) [15] acyclothymidine ( competitive [3]) [3] acyclouridine ( competitive [3]) [3] arabinofuranosyl-5-ethyluracil [14] benzylacyclouridines ( 0.1 mM [29]) [6, 24, 29] deoxyglucosylthymine ( phosphorolysis of uridine and deoxyuridine, synthesis of uridine at concentrations of 0.10 mM, 0.018 mM and 0.14 mM, not: phosphorolysis of deoxyuridine or thymidine at 0.19 mM [19]) [19] deoxythymidine [24] guanidine hydrochloride ( 50% loss of activity at 1.04 M, only little residual activity between 2 and 6 M [27]) [27] iodoacetamide [19] iodoacetic acid [19] o-iodosobenzoate [19] p-chloromercuribenzoate [16, 19] p-mercuriphenylsulfonate [19] phosphate ( product inhibition [11]; substrate inhibition above 10 mM [24]) [8, 11, 24] pyrimidine acyclonucleosides ( competitive [3]) [3] ribose 1-phosphate ( product inhibition [11,13]; inhibition above 0.6 mM [20]) [8, 11, 13, 20] tetramethylene acyclouridine [28] thymidine 5-monophosphate [17] uracil ( product inhibition [11,13]) [11, 13, 19] uridine ( product inhibition [11]; substrate inhibition [24]) [11, 24, 28] Activating compounds Triton X-100 ( 0.1%, stimulates enzyme of isolated plasma membrane [21]) [21] Specific activity (U/mg) 2.4 [19] 16.1 [13] 53 [2] 89.6 [31] 97.83 [23] 100-120 [30] 43


129 [26] 133 [22] 182.8 [10] 230 [9] 510 [25] 636 [36] 2810 [16] 4278 [17] Additional information [21, 24] Km-Value (mM) 0.016 (uridine) [4] 0.017 (ribose 1-phosphate) [26] 0.03 (5-bromouridine) [25] 0.033 (uridine) [28] 0.05 (ribose 1-phosphate) [28] 0.06 (uracil) [8] 0.064 (uridine) [36] 0.07 (5-methyluridine) [25] 0.076 (phosphate) [6] 0.076 (ribose 1-phosphate) [31] 0.088 (ribose 1-phosphate) [4] 0.1 (5-bromodeoxyuridine) [25] 0.11 (thymidine) [25] 0.11 (uracil) [26] 0.12 (phosphate) [26] 0.13 (2'-deoxyuridine) [25] 0.13 (phosphate) [3] 0.14 (ribose 1-phosphate) [8] 0.143 (uridine) [6] 0.148 (thymidine) [36] 0.17 (uridine) [26] 0.189 (uridine) [31] 0.202 (phosphate) [31] 0.204 (uracil) [28] 0.209 (uracil) [31] 0.24 (uridine) [8, 25] 0.242 (uridine) [6] 0.279 (phosphate) [6] 0.36 (uracil) [4] 0.42 (phosphate) [8] 0.7 (uridine, at 40 C [23]) [23] 0.71 (deoxyuridine) [1] 0.756 (5'-deoxy-5-fluorouridine) [36] 0.76 (uridine) [1] 0.806 (phosphate) [28] 0.91 (uridine) [13]

44

2.4.2.3

2.4.2.3


1.8 (uridine, at 60 C [23]) [23] 2.9 (phosphate) [13] 3.8 (uridine) [17] 3.9 (phosphate) [1] 4 (5-methyluridine) [16] Additional information [16, 22, 24, 29] Ki-Value (mM) 0.000018 (5-(3'-benzyloxybenzyl)-1-[(1'-aminomethyl-2'-hydroxyethoxy)methyl]uracil) [6] 0.000025 (2,2'-anhydro-5-ethyluridine) [5] 0.00007 (1-(1',3'-dihydroxy-2'-propoxy)methyl-5-benzyluracil) [28] 0.00007 (2,2'-anhydro-5-ethyluridine) [28] 0.0027 (1-(1',3'-dihydroxy-2'-propoxy)methyl-5,6-tetramethyleneuracil) [15] 0.003 (acyclothymidine) [3] 0.005 (2-deoxyglycosylthymine) [3] 0.0073 (5-benzylacyclouridine) [31] 0.0088 (tetramethylene acyclouridine) [28] 0.013 (5-bromoacyclouridine) [3] 0.014 (2,3'-anhydro-5-ethyluridine) [28] 0.014 (5-fluoroacyclouridine) [3] 0.015 (acyclouridine) [3] 0.03 (5-iodoacyclouridine) [3] 0.047 (uridine) [28] 0.056 (3'-azido-2',3'-dideoxy-5-methyluridine) [14] 0.07 (N-ethyl-5-phenylisoxazolium-3'-sulfonate) [30] 0.107 (arabinofuranosyl-5-ethyluracil) [14] 0.109 (2',3'-dideoxy-5-ethyluridine) [14] 0.21 (3'-chloro-2',3'-dideoxy-5-ethyluridine) [14] 0.225 (3'-azido-2',3'-dideoxy-5-ethyluridine) [14] 0.361 (3'-chloro-2',3'-dideoxy-5-methyluridine) [14] 0.448 (3'-bromo-2',3'-dideoxy-5-ethyluridine) [14] 1.003 (2'-deoxylyxofuranosyl-5-ethyluracil) [14] 1.36 (3-O-methyl-a-d-glucopyranoside) [17] 1.45 (uracil) [17] 2.1 (N-ethyl-5-phenylisoxazolium-3'-sulfonate, addition of uridine [30]) [30] 2.5 (N-ethyl-5-phenylisoxazolium-3'-sulfonate, addition of uracil [30]) [30] 3.5 (a-d-ribose 1-phosphate) [17] Additional information [24] pH-Optimum 6.5 ( deoxyuridine phosphorolysis [19]) [19] 6.9 ( deoxynucleoside phosphorolysis [25]) [25] 7 [16, 17] 7.2-7.4 [33] 45


2.4.2.3

7.3 ( uridine phosphorolysis [22]) [22] 7.4 ( assay at [1]; phosphorolysis of ribonucleosides and uracil arabinoside [25]) [1, 25] 7.5 [31] 7.5-8 [28] 8.2 ( reverse assay, uridine phosphorolysis [19]) [1, 19] 8.5 ( uridine synthesis [19]) [19, 23] pH-Range 5.8-9 ( pH 5.8: about 50% of activity maximum, pH 9.0: about 40% of activity maximum [22]) [22] 6.5-10.5 ( pH 6.5: about 50% of activity maximum, pH 10.5: about 65% of activity maximum [23]) [23] 7-7.75 ( pH 6.5: about 70% of activity maximum, pH 8.5: about 25% of activity maximum [31]) [31] Temperature optimum ( C) 37 ( assay at [1,22]; reverse reaction, assay at [19]) [1, 19, 22] 65 [23] Temperature range ( C) 40-80 ( 40 C: about 40% of activity maximum, 80 C: about 10% of activity maximum [23]) [23]

4 Enzyme Structure Molecular weight 32000-35000 ( SDS-PAGE [31]) [31] 43000 ( gel filtration [7]) [7] 45000 ( gel filtration [4]) [4] 56000 ( gel filtration [24]) [24] 65000 ( gel filtration [26]) [26] 80000 ( non-denaturing PAGE [16]) [16] 102500 ( disc gel electrophoresis [20]) [20] 110000 ( gel filtration [19]) [19] 130000 ( gel filtration, native enzyme [10]) [10] 160000 ( gel filtration [22]) [22] 165000 ( gel filtration [33]) [33] Subunits ? ( x * 29000, SDS-PAGE [10]) [10] hexamer ( 6 * 27500 [12,33]) [12, 27, 33] monomer ( 1 * 38000, SDS-PAGE [7]; in the presence of 2 M guanidine hydrochloride [27]) [7, 27] octamer ( 8 * 22200, SDS-PAGE [22]) [22]

46

2.4.2.3


tetramer ( 4 * 26000, SDS-PAGE in presence of 4 M urea and 0.5% 2-mercaptoethanol [19]; 4 * 26000, SDS-PAGE in presence of 6 M urea, without 2-mercaptoethanol [20]; 4 * 20000, SDS-PAGE in presence of 2-mercaptoethanol [16]; 4 * 33000 [34]) [16, 19, 20, 34]

5 Isolation/Preparation/Mutation/Application Source/tissue Ehrlich ascites carcinoma cell [1] Novikoff hepatoma cell [4] colorectal adenocarcinoma cell ( colon 26 tumor cells transplanted in BALB/c mice [31,35]) [31, 35] fibroblast [34, 35] intestinal mucosa [5, 14] leukemia cell [5] liver [2, 6, 20, 21, 29] placenta [29] tumor [3, 5] Additional information ( present in all human tissue and tumors [34]) [34] Localization cytoplasm [28] cytoplasmic membrane [21, 34] cytoskeleton ( associated with intermediate filament vimentin in fibroblasts and colon 26 cells [34,35]) [34, 35] cytosol [19, 20, 35] soluble ( 60-70% of the enzyme exists in the cytosol as a soluble protein [35]) [2, 35] Purification [8] (near homogeneity [9]; phosphorylase type I [13]; homogeneity [22]) [9, 10, 11, 13, 15, 22, 27, 30] [1, 29, 31, 35, 36] (partial [2]) [2, 4, 19, 20] [29, 31] [7] [24] (400fold [25]) [25] [16, 17] (partial [23]) [23] [26] (partial [28]) [28] (homogeneity [33]) [33]

47


2.4.2.3

Renaturation (dilution of the enzyme preincubated with 2 M guanidine hydrochloride results in partial recovery [27]) [27] Crystallization (hanging drop vapor diffusion method [12]) [9, 12] Cloning [9] [34] (fusion protein with maltose-binding protein [31]) [31] [32] [33]

6 Stability Temperature stability 30 ( 1 month, 20% loss of activity [23]) [23] 55 ( 40 min, 95% loss of activity [17]) [17] 60 ( 20 min, complete loss of activity [17]; half-life: around 1 week, [23]) [17, 23] 65 ( aglycone substrates, nucleoside substrates, phosphate or pentose 1-phosphate ester substrates stabilize against heat inactivation [18]) [18] General stability information , aglycone substrates, nucleoside substrates, phosphate or pentose 1phosphate ester substrates stabilize against heat inactivation [18] , much less stable in Tris buffer than in phosphate buffer [20] , use of dithiothreitol and glycerol is necessary for stabilization during purification [25] Storage stability , -73 C, stable for at least 3 months, purified enzyme [13] , -73 C, stable for at least 6 months, crude extract [13] , -20 C, stable for at least 2 weeks [1] , -40 C, 0.05 M potassium phosphate buffer, pH 7.0, 10 mM 2-mercaptoethanol, 1 mM EDTA, 10% loss of activity after 6 weeks [19] , -20 C, stable for 6 months [25] , 5 C, 90% loss of activity after 3-4 days [16]

References [1] Pontis, H.; phorylases 147 (1961) [2] Canellakis, anabolism. 48

Degerstedt, G.; Reichard, P.: Uridine and deoxyuridine phosfrom Ehrlich ascites tumor. Biochim. Biophys. Acta, 51, 138E.S.: Pyrimidine metabolism. II. Enzymatic pathways of uracil J. Biol. Chem., 227, 329-338 (1957)

2.4.2.3


[3] Niedzwicki, J.G.; El Kouni, M.H.; Chu, S.H.; Cha, S.: Pyrimidine acyclonucleosides, inhibitors of uridine phosphorylase. Biochem. Pharmacol., 30, 2097-2101 (1981) [4] McIvor, R.S.; Wohlhueter, R.M.; Plagemann, P.P.G.: Uridine phosphorylase from Novikoff rat hepatoma cells: purification, kinetic properties, and its role in uracil anabolism. J. Cell. Physiol., 122, 397-404 (1985) [5] Veres, Z.; Szabolcs, A.; Szinai, I.; Denes, G.; Kajtar-Peredy, M.; Otvos, L.: 5Substituted-2,2-anhydrouridines, potent inhibitors of uridine phosphorylase. Biochem. Pharmacol., 34, 1737-1740 (1985) [6] Naguib, F.N.M.; El Kouni, M.H.; Chu, S.H.; Cha, S.: New analogues of benzylacyclouridines, specific and potent inhibitors of uridine phosphorylase from human and mouse livers. Biochem. Pharmacol., 36, 2195-2201 (1987) [7] Lee, C.S.; Jimenez, B.M.; O'Sullivan, W.J.: Purification and characterization of uridine (thymidine) phosphorylase from Giardia lamblia. Mol. Biochem. Parasitol., 30, 271-287 (1988) [8] Albe, K.R.; Wright, B.E.: Purification and kinetic characterization of uridine phosphorylase from Dictyostelium discoiderm. Exp. Mycol., 13, 13-19 (1989) [9] Mikhailov, A.M.; Smirnova, E.A.; Tsuprun, V.L.; Tagunova, I.V.; Vainshtein, B.K.; Linkova, E.V.; Komissarov, A.A.; Siprashvili, Z.Z.; Mironov, A.S.: Isolation, crystallization in the macrogravitation field, preliminary X-ray investigation of uridine phosphorylase from Escherichia coli K-12. Biochem. Int., 26, 607-615 (1992) [10] Vita, A.; Magni, G.: A one-step procedure for the purification of uridine phosphorylase from Escherichia coli. Anal. Biochem., 133, 153-156 (1983) [11] Vita, A.; Huang, C.Y.; Magni, G.: Uridine phosphorylase from Escherichia coli B.: kinetic studies on the mechanism of catalysis. Arch. Biochem. Biophys., 226, 687-692 (1983) [12] Cook, W.J.; Koszalka, G.W.; Hall, W.W.; Narayana, S.V.L.; Ealick, S.E.: Crystallization and preliminary X-ray investigation of uridine phosphorylase from Escherichia coli. J. Biol. Chem., 262, 2852-2853 (1987) [13] Krenitsky, T.A.: Uridine phosphorylase from Escherichia coli. Kinetic properties and mechanism. Biochim. Biophys. Acta, 429, 352-358 (1976) [14] Veres, Z.; Neszmelyi, A.; Szabolcs, A.; Denes, G.: Inhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy. Eur. J. Biochem., 178, 173-181 (1988) [15] Drabikowska, A.K.; Lissowska, L.; Draminski, M.; Zgit-Wroblewska, L.; Shugar, D.: Acyclonucleoside analogues consisting of 5- and 5,6-substituted uracils and different acylic chains: inhibitory properties vs purified E. coli uridine phosphorylase. Z. Naturforsch. C, 42c, 288-296 (1987) [16] Avraham, Y.; Grossowicz, N.; Yashphe, J.: Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei. Biochim. Biophys. Acta, 1040, 287-293 (1990) [17] Avraham, Y.; Yashphe, J.; Grossowicz, N.: Thymidine phosphorylase and uridine phosphorylase of Lactobacillus casei. FEMS Microbiol. Lett., 56, 29-34 (1988) 49


2.4.2.3

[18] Krenitsky, T.A.; Tuttle, J.V.: Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases. Biochim. Biophys. Acta, 703, 247-249 (1982) [19] Yamada, E.W.: Uridine phosphorylase from rat liver. Methods Enzymol., 51, 423-431 (1978) [20] Bose, R.; Yamada, E.W.: Uridine phosphorylase, molecular properties and mechanism of catalysis. Biochemistry, 13, 2051-2056 (1974) [21] Bose, R.; Yamada, E.W.: Uridine phosphorylase activity of isolated plasma membranes of rat liver. Can. J. Biochem., 55, 528-533 (1977) [22] Leer, J.C.; Hammer-Jespersen, K.; Schwartz, M.: Uridine phosphorylase from Escherichia coli. Physical and chemical characterization. Eur. J. Biochem., 75, 217-224 (1977) [23] Utagawa, T.; Morisawa, H.; Yamanaka, S.; Yamazaki, A.; Yoshinaga, F.; Hirose, Y.: Properties of nucleoside phosphorylase from Enterobacter aerogenes. Agric. Biol. Chem., 49, 3239-3246 (1985) [24] El Kouni, M.H.; Fardos, N.M.; Naguib, F.N.M.; Niedzwicki, J.G.; Iltzsch, M.H.; Cha, S.: Uridine phosphorylase from Schistosoma mansoni. J. Biol. Chem., 263, 6081-6086 (1988) [25] Scocca, J.J.: Purification and substrate specificity of pyrimidine nucleoside phosphorylase from Haemophilus influenzae. J. Biol. Chem., 246, 6606-6610 (1971) [26] McIvor, R.S.; Wohlhueter, R.M.; Plagemann, P.G.W.: Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties. J. Bacteriol., 156, 198-204 (1983) [27] Burlakova, A.A.; Kurganov, B.I.; Chernyak, V.; Debabov, V.G.: Denaturation of uridine phosphorylase from Escherichia coli K-12 with guanidine hydrochloride: kinetics of inactivation, dissociation, and reactivation of the enzyme. Biochemistry, 62, 95-103 (1997) [28] Drabikowska, A.K.: Uridine phosphorylase from Hymenolepis diminuta (Cestoda): kinetics and inhibition by pyrimidine nucleoside analogs. Acta Biochim. Pol., 43, 733-741 (1996) [29] el Kouni, M.H.; el Kouni, M.M.; Naguib, F.N.: Differences in activities and substrate specificity of human and murine pyrimidine nucleoside phosphorylases: implications for chemotherapy with 5-fluoropyrimidines. Cancer Res., 53, 3687-3693 (1993) [30] Komissarov, A.A.; Romanova, D.V.; Debabov, V.G.: Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent K. J. Biol. Chem., 270, 10050-10055 (1995) [31] Liu, M.; Cao, D.; Russell, R.; Handschumacher, R.E.; Pizzorno, G.: Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors. Cancer Res., 58, 5418-5424 (1998) [32] Mitterbauer, R.; Karl, T.; Adam, G.: Saccharomyces cerevisiae URH1 (encoding uridine-cytidine N-ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase. Appl. Environ. Microbiol., 68, 1336-1343 (2002)

50

2.4.2.3


[33] Molchan, O.K.; Dmitrieva, N.A.; Romanova, D.V.; Lopes, L.E.; Debabov, V.G.; Mironov, A.S.: Isolation and initial characterization of the uridine phosphorylase from Salmonella typhimurium. Biochemistry (Moscow), 63, 195-199 (1998) [34] Pizzorno, G.; Cao, D.; Leffert, J.J.; Russell, R.L.; Zhang, D.; Handschumacher, R.E.: Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update. Biochim. Biophys. Acta, 1587, 133-144 (2002) [35] Russell, R.L.; Cao, D.; Zhang, D.; Handschumacher, R.E.; Pizzorno, G.: Uridine phosphorylase association with vimentin. Intracellular distribution and localization. J. Biol. Chem., 276, 13302-13307 (2001) [36] Watanabe, S.; Hino, A.; Wada, K.; Eliason, J.F.; Uchida, T.: Purification, cloning, and expression of murine uridine phosphorylase. J. Biol. Chem., 270, 12191-12196 (1995)

51

Thymidine phosphorylase

2.4.2.4

1 Nomenclature EC number 2.4.2.4 Systematic name thymidine:phosphate deoxy-a-d-ribosyltransferase Recommended name thymidine phosphorylase Synonyms PD-ECGF PD-ECGF/TP platelet-derived endothelial cell growth factor TDRPASE animal growth regulators, blood platelet-derived endothelial cell growth factors blood platelet-derived endothelial cell growth factor deoxythymidine phosphorylase gliostatin phosphorylase, thymidine pyrimidine deoxynucleoside phosphorylase pyrimidine phosphorylase thymidine-orthophosphate deoxyribosyltransferase thymidine:orthophosphate deoxy-d-ribosyltransferase CAS registry number 9030-23-3

2 Source Organism Homo sapiens (normal and tumor tissue [19]) [1, 4, 7, 11, 13, 18, 19, 21-26, 28, 29, 30, 31] Rattus norvegicus [1, 7, 20] Cavia porcellus [1] Mesocricetus auratus [1] Escherichia coli (strain B [7]; K12 [8,9]; strain W, ATCC 9637 [15]) [2, 6-9, 14, 15, 22, 27, 30] Giardia lamblia [3] Salmonella typhimurium (LT-2 [12]) [5, 12]

52

2.4.2.4


Mus musculus [10, 18] Lactobacillus casei [16, 17]

3 Reaction and Specificity Catalyzed reaction thymidine + phosphate = thymine + 2-deoxy-d-ribose 1-phosphate ( ordered bi bi mechanism with the nucleoside the first substrate to add, and the pyrimidine base the last product to leave [12]; mechanism [14,16,27]; ordered sequential reaction mechanism, phosphate is the first substrate to bind to and deoxyribose the last product to dissociate from the enzyme [6]; rapid equilibrium random bi-bi mechanism with an enzyme-phosphate-thymine dead-end complex [10]) Reaction type pentosyl group transfer Natural substrates and products S thymidine + phosphate ( intact platelets degrade thymidine but are not able to synthesize thymidine from thymine [11]) [11] P thymine + 2-deoxy-d-ribose S thymine + 2-deoxy-d-ribose 1-phosphate ( in Escherichia coli and Salmonella typhimurium thymidine phosphorylase plays an important role in metabolism of thymine auxotrophs and is necessary for the conversion of exogenous thymine to thymidine [5]; enzyme also catalyzes the nucleoside deoxyribosyltransferase reaction with pyrimidine deoxyribonucleosides, ec2.4.2.6 [7]; degradation of uridine in tumor specimen [23]; enzyme is enantioselective, acts only on the naturally occurring d-thymidine and some d-thymidine analogs, but not on their l-counterparts [30]) [5, 7, 21-23, 25, 30] P thymidine + phosphate S Additional information ( identical to platelet-derived endothelial cell growth factor [19, 28]; angiogenic activity [19, 21, 25, 29, 30, 31]; mitogenic effect, promotes endothelial cell proliferation [22]; role in thymidine metabolism, homeostasis, development and regeneration of central nervous system and formation of blood/brain barrier [30]; formation of blood/brain barrier and repair following brain injury [31]) [19, 21, 22, 25, 28, 29, 30, 31] P ? Substrates and products S 1-(tetrahydro-2-furanyl)-5-fluorouracil + phosphate ( i.e. Tegafur [4,30]) (Reversibility: ? [4]) [4, 30] P ? S 5'-deoxy-5-fluorouridine + phosphate ( 5'-DFUR [4]) (Reversibility: ? [4,18]) [4, 18, 30] P 5-fluorouracil + 5-deoxyribose-1-phosphate

53


2.4.2.4

S 5-deoxythymidine + phosphate (Reversibility: ? [16]) [16] P thymine + 2,5-dideoxyribose 1-phosphate S 5-fluoro-2'-deoxyuridine + phosphate (Reversibility: ? [18,31]) [18, 31] P 5-fluorouracil + 2-deoxyribose-1-phosphate [31] S 5-fluorouridine + phosphate (Reversibility: ? [16,18]) [16, 18] P 5-fluorouracil + ribose 1-phosphate S bromodeoxyuridine + phosphate ( at 40% the rate of thymidine [5, 12]) (Reversibility: ? [5, 11, 12, 16]) [5, 11, 12, 16] P bromouracil + 2-deoxy-d-ribose S deoxythymidine + phosphate (Reversibility: ? [13]) [13] P thymidine + 2-deoxy-d-ribose S deoxyuridine + phosphate ( 1.2-1.4 times the rate of thymidine at pH 5.7 [11]) (Reversibility: r [31]; ? [5, 11-13, 16]) [5, 11-13, 16, 31] P uracil + 2-deoxy-d-ribose S fluorodeoxyuridine + phosphate (Reversibility: ? [11]) [11] P fluorouracil + 2-deoxy-d-ribose S iododeoxyuridine + phosphate (Reversibility: ? [5, 11, 12]) [5, 11, 12] P iodouracil + 2-deoxy-d-ribose S thymidine + arsenate (Reversibility: ? [5, 6, 11, 12]) [5, 6, 11, 12] P ? S thymidine + phosphate ( in the reverse reaction only ad-2-deoxyribose 1-phosphate is accepted as substrate [2]; enzyme is enantioselective, acts only on the naturally occurring d-thymidine and some d-thymidine analogs, but not on their l-counterparts [30]) (Reversibility: r [1, 2, 10, 17, 19, 27, 29, 31]; ir [11]; ? [5, 6, 7, 12, 13, 16, 18, 20, 21, 22, 30]) [1, 2, 5, 6, 7, 10, 11-13, 16-22, 27, 29, 30, 31] P thymine + 2-deoxy-d-ribose 1-phosphate [1, 2, 5, 10, 12, 13, 19, 27, 29, 30] S thymine arabinoside + phosphate (Reversibility: ? 9# [16]) [16] P thymine + arabinose 1-phosphate S uridine + phosphate ( the activities of uridine, deoxyuridine and thymidine phosphorylases from Giardia lamblia remain associated throughout purification, suggesting that a single enzyme is responsible for the 3 activities [3]; no substrate [5, 11-13]) (Reversibility: ? [1, 3, 5, 8, 11-13, 23]) [1, 3, 5, 8, 11-13, 23] P uracil + ribose 1-phosphate [1] S Additional information ( the enzyme in some tissues also catalyzes deoxyribonucleosyltransferase reaction of the type catalyzed by EC 2.4.2.6: 2-deoxy-d-ribosyl-base1 + base2 = 2-deoxy-d-ribosyl-base2 + base1 [1, 7, 10]; purine deoxyribonucleosides are no substrates [11]; specificity towards the deoxyribosyl moiety of the sub54

2.4.2.4


strate [16]; nonsubstituted pyrimidine moiety or one which is substituted in position 5 required [16]; purine deoxyribonucleosides and ribonucleosides are not cleaved [6]; specific for deoxyribonucleosides [6, 12]; the phosphorolytic acitvities towards thymidine, 5'deoxy-5-fluorouridine and 1-(tetrahydro-2-furanyl)-5-fluorouracil remain closely parallel during purification [4]; no substrate: deoxycytidine [5, 6, 12, 13]; no substrates: deoxyadenosine, deoxyguanosine [5, 12]) [1, 4-7, 10, 11-13, 16] P ? Inhibitors 1-(8-phosphonooctyl)-6-amino-5-bromouracil ( competitive [30]) [30] 1-(8-phosphonooctyl)-7-deazaxanthine ( competitive [30]) [30] 2',3'-dideoxy-5-ethyluridine ( 9% inhibition at 10 mM [20]) [20] 2-deoxy-d-ribose ( product inhibition [10,12]; competitive [16]) [10, 12, 16] 3'-azido-2',3'-dideoxy-5-ethyluridine ( 16% inhibition at 10 mM [20]) [20] 3'-azido-2',3'-dideoxy-5-methyluridine ( 6% inhibition at 10 mM [20]) [20] 3'-bromo-2',3'-dideoxy-5-ethyluridine ( 9% inhibition at 5 mM [20]) [20] 3'-chloro-2',3'-dideoxy-5-ethyluridine ( 26% inhibition at 10 mM [20]) [20] 3'-chloro-2',3'-dideoxy-5-methyluridine ( 18% inhibition at 10 mM [20]) [20] 4,6-dihydroxy-5-nitropyrimidine ( 0.1 mM, competitive [24]) [24] 5-benzylacyclouridine [23] 5-bromo-2-deoxyuridine ( 100% inhibition at 10 mM [17]) [17] 5-bromo-6-aminouracil [19, 23, 30, 31] 5-bromouracil ( 0.1 mM, competitive [24]) [24] 5-chloro-6-[1-(imminopyrrolidinyl)methyl]uracil hydrochloride ( i.e. TPI, competitive [30]) [30] 5-fluorodeoxyuridine ( 80% inhibition at 10 mM [17]) [17] 5-fluorouracil ( 0.1 mM, competitive [24]) [24] 5-hydroxymethyluracil ( 35% inactivation at 2 mM [12]) [12] 5-nitrouracil ( 0.1 mM, competitive [24]) [24] 6-(2-aminoethyl)amine-5-chlorouracil ( AEAC [29]) [29] 6-(4-phenlybutylamino)uracil ( PBAU, 50% inhibition at 0.03 mM [30]) [30] 6-amino-5-chlorouracil ( inhibits angiogenic activity, competitive [26]) [26] 6-aminothymine [30] 6-aminouracil ( 0.1 mM, competitive [24]) [24] 6-benzyl-2-thiouracil ( 0.1 mM, mixed inhibition [24]) [24]

55


2.4.2.4

6-methyluracil ( competitive, 86% inhibition at 10 mM and 36% inhibition at 1 mM [17]) [17] 7-deazaxanthine ( competitive, 50% inhibition at 0.04 mM [30]) [30] SO24- ( inhibition reversed by phosphate [7]) [7] allyloxymethylthymine ( 0.1 mM, uncompetitive [24]) [24] arabinofuranosyl-5-ethyluracil ( 10% inhibition at 10 mM [20]) [20] bromouracil ( 100% inactivation at 1 mM [12]) [12] deoxyadenosine ( 20% inactivation at 1 mM [12]) [12] p-chloromercuribenzoate ( above 0.01 mM [7]) [7] ribose 1-phosphate ( competitive [6]; 20% inactivation at 1.5 mM [12]) [6, 12] thymine ( substrate inhibition [10]; product inhibition [10,12]) [10, 12, 16] uracil ( 84% inactivation at 2 mM [12]; inhibits activity in turmor tissue, but not in normal tissue [24]) [12, 24] urea ( inhibition at high concentration, 4 M, stimulation at low concentration [7]) [7] uridine ( competitive 27% inactivation at 1 mM) [12] Additional information ( photoinactivation in presence of thymine, thymidine and some halogenated analogs [15]; a specific thymidine phosphorylase inhibitor TPI completely inhibits [21]; iodination reduces activity, 63% inactivation after 5 min and complete inactivation after 20 min [22]; 5- and 6-substituted uracil analogs [29]) [15, 21, 22, 29] Activating compounds urea ( stimulates at low concentration, inhibits at high concentration, 4 M [7]) [7] Turnover number (min±1) 9.4 (thymidine) [22] Specific activity (U/mg) 5.3 [13] 10 [11] 14.6 [15] 18 [7] 111 [17] 122 [6] 155 [7] 320 [13] 465 [5, 12] 604 [11] 1100 [7, 16] Additional information [18] Km-Value (mM) 0.00166 (thymidine) [17] 0.019 (phosphate) [10] 0.1 (phosphate) [16, 17] 56

2.4.2.4


0.11 (thymidine) [22] 0.141 (thymine) [10] 0.168 (thymidine) [4] 0.38 (thymidine) [6] 0.65 (thymidine, normal tissue [24]) [24] 0.89 (phosphate) [6] 0.945 (thymidine) [10] 1.3 (arsenate) [5, 12] 1.72 (5'-deoxy-5-fluorouridine) [4] 2.1 (thymidine) [5, 12] 2.3 (phosphate) [5, 12] 8 (deoxyuridine) [5, 12] 9.3 (thymidine, tumor tissue [24]) [24] 13.3 (5'-deoxy-5-fluorouridine and 1-(tetrahydro-2-furanyl)-5-fluorouracil) [4] 47.6 (uridine) [4] Additional information [16, 18] Ki-Value (mM) 0.00002 (5-chloro-6-[1-(imminopyrrolidinyl)methyl]uracil hydrochloride) [30] 0.000165 (6-(2-aminoethyl)amine-5-chlorouracil) [29] 0.0011 (5-bromouracil, tumor tissue [24]) [24] 0.0034 (6-amino-5-chlorouracil) [26] 0.0051 (5-nitrouracil, tumor tissue [24]) [24] 0.006 (5-bromouracil, normal tissue [24]) [24] 0.021 (4,6-dihydroxy-5-nitropyrimidine, tumor tissue [24]) [24] 0.0253 (5-nitrouracil, normal tissue [24]) [24] 0.043 (5-fluorouracil, tumor tissue [24]) [24] 0.0728 (5-fluorouracil, normal tissue [24]) [24] 0.075 (6-benzyl-2-thiouracil, tumor tissue [24]) [24] 0.088 (6-aminouracil, tumor tissue [24]) [24] 0.127 (allyloxymethylthymine, tumor tissue [24]) [24] 0.1286 (allyloxymethylthymine, normal tissue [24]) [24] 0.13 (6-aminouracil, normal tissue [24]) [24] 0.137 (6-benzyl-2-thiouracil, normal tissue [24]) [24] 0.158 (uracil, tumor tissue [24]) [24] 0.211 (4,6-dihydroxy-5-nitropyrimidine, normal tissue [24]) [24] 0.67 (deoxyribose-1-phosphate) [16] 1.6 (6-methyluracil) [17] Additional information [29] pH-Optimum 5.3 [22] 5.7 [11] 6 ( around [1]) [1, 16, 17] 6.3 [6] 7.1 ( assay at [6]) [6] 57


2.4.2.4

7.4 ( assay at [1,5]) [1, 5] 7.5 [11] 7.5-8 ( rapid decrease of activity above and below this range [5,12]) [5, 12] 7.8 ( assay at [29]) [29] 10 ( assay at, pH 10 is used because 2-deoxy-d-ribose 1-phosphate is heat labile at pH 7.4 [21]) [21] pH-Range 7.5-8 ( rapid decrease of activity above and below this range [5]) [5] Temperature optimum ( C) 37 ( assay at [5,6,11]) [5, 6, 11]

4 Enzyme Structure Molecular weight 45000 ( SDS-PAGE [31]) [31] 52000 ( recombinant enzyme lacking 10 amino acid residues, SDSPAGE [28]) [28] 55000 ( SDS-PAGE [26,28]) [25, 26, 28] 80000 ( gel filtration [16]) [16] 90000 ( gel filtration [6]) [6, 30] 98000 [8] 100000 ( gel filtration [12]) [7, 12] 110000 ( gel filtration [11]) [11] 120000 ( gel filtration [13]) [13] Subunits dimer ( 2 * 47000 [5]; two identical subunits, SDS-PAGE [6]; 2 * 47000, SDS-disc gel electrophoresis [12]; 2 * 46000 [8]; three-dimensional structure, 2 identical subunits, each subunit is composed of a small a-helical domain and a large a/b domain [9]; 2 * 60000, SDSPAGE [11]; 2 * 38000, SDS-PAGE [16]; 2 * 58000, SDS-PAGE, enzyme is capable of being converted to a less active form larger in MW and possibly trimeric or tetrameric in structure [13]; 2 * 45000 [30]) [5, 6, 8, 9, 11-13, 16, 30] Additional information ( dual substrate enzyme with two domains [27]) [27]

5 Isolation/Preparation/Mutation/Application Source/tissue MCF-7 cell [31] Novikoff hepatoma cell [1] amniochorion [13]

58

2.4.2.4


bladder cancer cell [31] blood platelet [11, 21, 22, 30, 31] breast ( tumor specimen [23,30]; ductal carcinoma in situ and invasive elements [31]) [23, 30, 31] colonic cancer cell [30, 31] esophagus ( tumor [31]) [31] gastrointestinal cancer cell ( poorly differentiated adenocarcinoma [4]) [4, 30] intestinal mucosa [20] kidney [1] liver ( normal tissue [1]) [1, 7, 10, 18, 28] lung cancer cell [30, 31] macrophage [30] ovarian cancer cell ( malignant tissue [30,31]) [30, 31] pancreatic cancer cell [30, 31] placenta [18, 25] renal cell carcinoma [30] spleen [1, 7] stomach ( ulcer [30]; tumor tissue [31]) [30, 31] uterus ( normal and tumor tissue [24]; leiomyoma [30]) [24, 30] Additional information ( no activity in normal brain [30]; overview tumor cells [1]) [1, 30] Localization cytoplasm [11] Purification (partial [4,11]; 98% purity [22]; separation of uridine phosphorylase and thymidine phosphorylase activities [23]; partial [24]; homogeneity [30]) [4, 7, 11, 13, 18, 22-25, 28, 30] [7] (133fold [15]) [6, 7, 9, 15, 22] [3] (257fold [12]) [5, 12] [18] (homogeneity [16]) [16, 17] Crystallization [30] (hanging drop vapor diffusion method [8]) [8, 9, 27, 30] Cloning (transfected into colon cancer cell line Colo320 [21]; expressed in Escherichia coli [22]; recombinant enzyme lacks 10 amino acids at aminoterminus [28]) [21, 22, 23, 25, 28, 29, 31] [2]

59


2.4.2.4

Engineering K115E ( no enzyme activity after transfection in COS-7 cells [26]) [26] L148R ( no enzyme activity after transfection in COS-7 cells [26]) [26] R202S ( no enzyme activity after transfection in COS-7 cells [26]) [26]

6 Stability pH-Stability 6 ( maximal stability [7]) [7] Temperature stability 53 ( 77% loss of activity after 10 min, phosphate or 2'-deoxyribose1-phosphate stabilize [14]) [14] 55 ( 40 min, 10% loss of activity [17]; 99% loss of activity after 10 min, phosphate or 2'-deoxyribose-1-phosphate stabilize [14]) [14, 17] 60 ( 20 min, complete loss of activity [17]) [17] 65 ( 30 min, complete inactivation [11]) [11] Additional information ( phosphate or pentose 1-phosphate ester substrates stabilize against heat inactivation [14]) [14] General stability information , stable to repeated freezing and thawing [7] , crystals are stable to X-rays at room temperature for at least 5 days [8] , phosphate or pentose 1-phosphate ester substrates stabilize against heat inactivation [14] , stability depends on protein concentration [6] , 2-mercaptoethanol and sucrose stabilize during preincubation procedure with ammonium sulfate [5, 12] , quite labile during purification [5, 12] Storage stability , -20 C, phosphate buffer containing 2% mannitol, stable for over 1 year, human enzyme [7] , 4 C, storage results in a decrease of the 120 kDa component and a loss of activity [13] , -20 C, 10 mM Tris-HCl, pH 7.3, stable for several months [6] , 4 C, gradual loss of activity [6] , 4 C, 10 mM potassium phosphate, pH 7.5, 10 mM 2-mercaptoethanol, 20% sucrose, stable for 3 months [5, 12] , 5 C, 90% loss of activity after 3-4 days [16]

60

2.4.2.4


References [1] Zimmerman, M.; Seidenberg, J.: Deoxyribosyl transfer. I. Thymidine phosphorylase and nucleoside deoxyribosyltransferase in normal and malignant tissues. J. Biol. Chem., 239, 2618-2621 (1964) [2] Barbas, C.F.III, Wong, C.H.: Overexpression and substrate specificity studies of phosphodeoxyribomutase and thymidine phosphorylase. Bioorg. Chem., 19, 261-269 (1991) [3] Lee, C.S.; Jimenez, B.M.; O'Sullivan, W.J.: Purification and characterization of uridine (thymidine) phosphorylase from Giardia lamblia. Mol. Biochem. Parasitol., 30, 271-287 (1988) [4] Sugata, S.; Kono, A.; Hara, Y.; Karube, Y.; Matsushima, Y.: Partial purification of a thymidine phosphorylase from human gastric cancer. Chem. Pharm. Bull., 34, 1219-1222 (1986) [5] Hoffee, P.A.; Blank, J.: Thymidine phosphorylase from Salmonella typhimurium. Methods Enzymol., 51, 437-442 (1978) [6] Schwartz, M.: Thymidine phosphorylase from Escherichia coli. Methods Enzymol., 51, 442-445 (1978) [7] Zimmerman, M.: Deoxyribosyl transfer. II. Nucleoside:pyrimidine deoxyribosyltransferase activity of three partially purified thymidine phosphorylases. J. Biol. Chem., 239, 2622-2627 (1964) [8] Cook, W.J.; Koszalka, G.W.; Hall, W.W.; Burns, C.L.; Ealick, S.E.: Crystallization and preliminary x-ray investigation of thymidine phosphorylase from Escherichia coli. J. Biol. Chem., 262, 3788-3789 (1987) [9] Walter, M.R.; Cook, W.J.; Cole, L.B.; Short, S.A.; Koszalka, G.W.; Krenitsky, T.A.; Ealick, S.E.: Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution. J. Biol. Chem., 265, 14016-14022 (1990) [10] Iltzsch, M.H.; El Kouni, M.H.; Cha, S.: Kinetic studies of thymidine phosphorylase from mouse liver. Biochemistry, 24, 6799-6807 (1985) [11] Desgranges, C.; Razaka, G.; Rabaud, M.; Bricaud, H.: Catabolism of thymidine in human blood platelets: purification and properties of thymidine phosphorylase. Biochim. Biophys. Acta, 654, 211-218 (1981) [12] Blank, J.G.; Hoffee, P.A.: Purification and properties of thymidine phosphorylase from Salmonella typhimurium. Arch. Biochem. Biophys., 168, 259-265 (1975) [13] Kubilus, J.; Lee, L.D.; Baden, H.P.: Purification of thymidine phosphorylase from human amniochorion. Biochim. Biophys. Acta, 527, 221-228 (1978) [14] Krenitsky, T.A.; Tuttle, J.V.: Correlation of substrate-stabilization patterns with proposed mechanisms for three nucleoside phosphorylases. Biochim. Biophys. Acta, 703, 247-249 (1982) [15] Voytek, P.: Purification of thymidine phosphorylase from Escherichia coli and its photoinactivation in the presence of thymine, thymidine, and some halogenated analogs. J. Biol. Chem., 250, 3660-3665 (1975)

61


2.4.2.4

[16] Avraham, Y.; Grossowicz, N.; Yashphe, J.: Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei. Biochim. Biophys. Acta, 1040, 287-293 (1990) [17] Avraham, Y.; Yashphe, J.; Grossowicz, N.: Thymidine phosphorylase and uridine phosphorylase of Lactobacillus casei. FEMS Microbiol. Lett., 56, 29-34 (1988) [18] el Kouni, M.H.; el Kouni, M.M.; Naguib, F.N.: Differences in activities and substrate specificity of human and murine pyrimidine nucleoside phosphorylases: implications for chemotherapy with 5-fluoropyrimidines. Cancer Res., 53, 3687-3693 (1993) [19] Pizzorno, G.; Cao, D.; Leffert, J.J.; Russell, R.L.; Zhang, D.; Handschumacher, R.E.: Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update. Biochim. Biophys. Acta, 1587, 133-144 (2002) [20] Veres, Z.; Neszmelyi, A.; Szabolcs, A.; Denes, G.: Inhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy. Eur. J. Biochem., 178, 173-181 (1988) [21] de Bruin, M.; Smid, K.; Laan, A.C.; Noordhuis, P.; Fukushima, M.; Hoekman, K.; Pinedo, H.M.; Peters, G.J.: Rapid disappearance of deoxyribose-1phosphate in platelet derived endothelial cell growth factor/thymidine phosphorylase overexpressing cells. Biochem. Biophys. Res. Commun., 301, 675-679 (2003) [22] Finnis, C.; Dodsworth, N.; Pollitt, C.E.; Carr, G.; Sleep, D.: Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity. Eur. J. Biochem., 212, 201-210 (1993) [23] Liu, M.; Cao, D.; Russell, R.; Handschumacher, R.E.; Pizzorno, G.: Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors. Cancer Res., 58, 5418-5424 (1998) [24] Miszczak-Zaborska, E.; Wozniak, K.: The activity of thymidine phosphorylase obtained from human uterine leiomyomas and studied in the presence of pyrimidine derivatives. Z. Naturforsch.c, 52, 670-675 (1997) [25] Miyadera, K.; Dohmae, N.; Takio, K.; Sumizawa, T.; Haraguchi, M.; Furukawa, T.; Yamada, Y.; Akiyama, S.: Structural characterization of thymidine phosphorylase purified from human placenta. Biochem. Biophys. Res. Commun., 212, 1040-1045 (1995) [26] Miyadera, K.; Sumizawa, T.; Haraguchi, M.; Yoshida, H.; Konstanty, W.; Yamada, Y.; Akiyama, S.: Role of thymidine phosphorylase activity in the angiogenic effect of platelet derived endothelial cell growth factor/thymidine phosphorylase. Cancer Res., 55, 1687-1690 (1995) [27] Rick, S.W.; Abashkin, Y.G.; Hilderbrandt, R.L.; Burt, S.K.: Computational studies of the domain movement and the catalytic mechanism of thymidine phosphorylase. Proteins, 37, 242-252 (1999) [28] Sumizawa, T.; Furukawa, T.; Haraguchi, M.; Yoshimura, A.; Takeyasu, A.; Ishizawa, M.; Yamada, Y.; Akiyama, S.: Thymidine phosphorylase activity 62

2.4.2.4


associated with platelet-derived endothelial cell growth factor. J. Biochem., 114, 9-14 (1993) [29] Klein, R.S.; Lenzi, M.; Lim, T.H.; Hotchkiss, K.A.; Wilson, P.; Schwartz, E.L.: Novel 6-substituted uracil analogs as inhibitors of the angiogenic actions of thymidine phosphorylase. Biochem. Pharmacol., 62, 1257-1263 (2001) [30] Focher, F.; Spadari, S.: Thymidine phosphorylase: a two-face janus in anticancer chemotherapy. Curr. Cancer Drug Targets, 1, 141-153 (2001) [31] Yang, Q.; Yoshimura, G.; Mori, I.; Sakurai, T.; Kakudo, K.: Thymidine phosphorylase and breast carcinoma. Anticancer Res., 22, 2355-2360 (2002)

63

Nucleoside ribosyltransferase

1 Nomenclature EC number 2.4.2.5 Systematic name nucleoside:purine(pyrimidine) d-ribosyltransferase Recommended name nucleoside ribosyltransferase Synonyms nucleoside N-ribosyltransferase ribosyltransferase, nucleoside CAS registry number 9030-31-3

2 Source Organism Escherichia coli [1] Pseudomonas trifolii (IAM-1555 [2]) [2]

3 Reaction and Specificity Catalyzed reaction d-ribosyl-base1 + base2 = d-ribosyl-base2 + base1 Reaction type pentosyl group transfer Substrates and products S guanine + inosine (Reversibility: ? [1,2]) [1, 2] P guanosine + hypoxanthine [2] S guanine + uridine (Reversibility: ? [2]) [2] P guanosine + uracil [2] S inosine + 4,5-diaminouracil (Reversibility: ? [1]) [1] P hypoxanthine + 4,5-diaminouridine S inosine + 4,6-aminouracil (Reversibility: ? [1]) [1] P hypoxanthine + 4,6-aminouridine S inosine + 5-bromouracil (Reversibility: ? [1]) [1]

64

2.4.2.5

2.4.2.5

P S P S P S P

Nucleoside ribosyltransferase

hypoxanthine + 5-bromouridine inosine + adenine (Reversibility: ? [1]) [1] hypoxanthine + adenosine inosine + thymine (Reversibility: ? [1]) [1] hypoxanthine + 2-hydroxy-thymidine inosine + xanthine (Reversibility: ? [1]) [1] hypoxanthine + xanthosine

Inhibitors Ag+ [2] Hg+ [2] Hg2+ [2] Pb2+ [2] pH-Optimum 9 (, in absence of phosphate [2]) [2] 10.5 (, in presence of phosphate [2]) [2] Temperature optimum ( C) 50-55 (, formation of guanosine [2]) [2] Temperature range ( C) 40-60 (, 40 C: about 60% of maximal activity, 60 C: about 80% of maximal activity, formation of guanosine [2]) [2]

References [1] Koch, A.L.: Some enzymes of nucleoside metabolism of Escherichia coli. J. Biol. Chem., 223, 535-549 (1956) [2] Kamimura, A.; Mitsugi, K.; Okumura, S.: Bacterial synthesis of nucleosides by the pentosyl transfer reaction from nucleoside to base. Agric. Biol. Chem., 37, 2063-2072 (1973)

65

Nucleoside deoxyribosyltransferase

2.4.2.6

1 Nomenclature EC number 2.4.2.6 Systematic name nucleoside:purine(pyrimidine) deoxy-d-ribosyltransferase Recommended name nucleoside deoxyribosyltransferase Synonyms DRTase I (, strictly specific for transfer between purine bases [12, 13]) [12, 13] DRTase II (, catalyzes the transfer of the deoxyribosyl moiety between purines or pyrimidines as well as from a purine to a pyrimidine [12, 13]) [12, 13] NdRT-II deoxyribose transferase deoxyribosyltransferase, nucleoside nucleoside deoxyribosyltransferase I (, purine nucleoside:purine deoxyribosyltransferase:strictly specific for transfer between purine bases [3]) [3] nucleoside deoxyribosyltransferase II (, purine(pyrimidine) nucleoside:purine(pyrimidine) deoxyribosyltransferase: catalyzes the transfer of the deoxyribosyl moiety between purines or pyrimidines as well as from a purine to a pyrimidine [3]) [3] nucleoside trans-N-deoxyribosylase purine(pyrimidine) nucleoside:purine(pyrimidine) deoxyribosyl transferase trans-N-deoxyribosylase trans-N-glycosidase trans-deoxyribosylase transdeoxyribosylase CAS registry number 9026-86-2

2 Source Organism Lactobacillus helveticus (nucleoside deoxyribosyltransferase-I and nucleoside deoxyribosyltransferase-I [3]) [1, 3, 4, 5, 6, 7, 10, 13, 14, 15] Crithidia luciliae [2] 66

2.4.2.6


Lactobacillus leichmannii [8, 9, 12, 16] Lactobacillus sp. [11]

3 Reaction and Specificity Catalyzed reaction 2-deoxy-d-ribosyl-base1 + base2 = 2-deoxy-d-ribosyl-base2 + base1 (, ping-pong bi bi mechanism [3,5,6]) Reaction type pentosyl group transfer Natural substrates and products S Additional information (, the enzyme participates in the nucleoside salvage pathway [11]; , specific role for DRTase I in the metabolism of deoxyinosine [13]) [11, 13] P ? Substrates and products S 5'-fluoro-5'-thymidine + adenine (, deoxyribosyltransferase II [3]) (Reversibility: ? [3]) [3] P 5'-fluoro-5'-deoxythymine + adenosine S deoxyadenosine + 2,6-dichloropurine (Reversibility: ? [11]) [11] P adenine + 9-(2-deoxy-b-d-ribofuranosyl)-2,6-dichloropurine S deoxyadenosine + 2-amino 6-methylmercaptopurine (Reversibility: ? [11]) [11] P adenine + 9-(2-deoxy-b-d-ribofuranosyl)-2-amino-6-methylmercaptopurine S deoxyadenosine + 2-amino-6-hydroxy 8-mercaptopurine (Reversibility: ? [11]) [11] P adenine + ? S deoxyadenosine + 2-aminobenzimidazole (Reversibility: ? [11]) [11] P adenine + ? S deoxyadenosine + 2-hydroxybenzimidazole (Reversibility: ? [11]) [11] P adenine + ? S deoxyadenosine + 2-thiouracil (Reversibility: ? [11]) [11] P adenine + deoxy-2-thiouridine S deoxyadenosine + 4-amino-5-imidazole carboxamide (Reversibility: ? [11]) [11] P adenine + ? S deoxyadenosine + 5-fluorouracil (Reversibility: ? [11]) [11] P adenine + deoxy-5-fluorouridine S deoxyadenosine + 5-iodouracil (Reversibility: ? [11]) [11] P adenine + deoxy-5-iodouridine S deoxyadenosine + 6-azathymine (Reversibility: ? [11]) [11]

67


P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P 68

2.4.2.6

adenine + deoxy-6-deazathymidine deoxyadenosine + 6-chloroguanine (Reversibility: ? [11]) [11] adenine + 2'-deoxy-6-chloroguanosine deoxyadenosine + 6-chloropurine (Reversibility: ? [11]) [11] adenine + 9-(2-deoxy-b-d-ribofuranosyl)-6-chloropurine deoxyadenosine + 6-dimethylaminopurine (Reversibility: ? [11]) [11] adenine + 9-(2-deoxy-b-d-ribofuranosyl)-6-dimethylaminopurine deoxyadenosine + 6-methylpurine (Reversibility: ? [11]) [11] adenine + 9-(2-deoxy-b-d-ribofuranosyl)-6-methylpurine deoxyadenosine + adenine (Reversibility: ? [6]) [6] adenine + deoxyadenosine deoxyadenosine + cytosine (, DRTase II [12]) (Reversibility: r [1,6]; ? [12]) [1, 6, 12] adenine + deoxycytidine deoxyadenosine + ethyl 4-amino 5-imidazole carboxylate (Reversibility: ? [11]) [11] adenine + ? deoxyadenosine + hypoxanthine (Reversibility: r [1]; ? [2]) [1, 2] adenine + deoxyinosine + 7-(b-d-2'-deoxyribufuranosyl)hypoxanthine [2] deoxyadenosine + thymine (Reversibility: r [1,11]) [1, 11] adenine + thymidine deoxyadenosine + uracil (Reversibility: r [1,11]) [1, 11] adenine + deoxyuridine deoxyadenosine + xanthine (Reversibility: r [1]) [1] adenine + deoxyxanthosine deoxycytidine + 1,7-dimethylguanine (, DRTase II [12]) (Reversibility: ? [12]) [12] cytosine + deoxy-1,7-dimethylguanosine deoxycytidine + 1,N2 -e-guanine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] cytosine + ? deoxycytidine + 1,N6 -e-adenine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] cytosine + ? deoxycytidine + 3,N4 -e-cytosine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] cytosine + ? deoxycytidine + 5,6,7,9-tetrahydro-7-acetoxy-9-oxoimidazolpurine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] cytosine + ? deoxycytidine + 6-chloroguanine (, DRTase II [12]) (Reversibility: ? [12]) [12] hypoxanthine + deoxy-6-chloroguanosine

2.4.2.6


S deoxycytidine + C8 -aminoguanine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] P cytosine + 2'-deoxy-8-aminoguanosine S deoxycytidine + C8 -methylguanine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] P cytosine + 2'-deoxy-8-methylguanosine S deoxycytidine + N2;3 -e-guanine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] P cytosine + ? S deoxycytidine + N2 -(2-oxoethyl)guanine (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] P cytosine + 2'-deoxy-N2 -(2-oxoethyl)guanosine S deoxycytidine + adenine (Reversibility: r [1, 3, 4, 6, 7, 12]) [1, 3, 4, 6, 7, 12] P cytosine + deoxyadenosine [1, 3] S deoxycytidine + cytosine (Reversibility: ? [1]) [1] P cytosine + deoxycytidine S deoxycytidine + guanine (Reversibility: r [1, 7, 12]) [1, 7, 12] P cytosine + deoxyguanosine S deoxycytidine + hypoxanthine (Reversibility: r [1, 7]) [1, 7] P cytosine + deoxyinosine S deoxycytidine + pyrimidopurin-10-one (, deoxyribosylation at the N9 atom [15]) (Reversibility: ? [15]) [15] P cytosine + ? S deoxycytidine + thymine (Reversibility: r [1]) [1] P cytosine + thymidine S deoxycytidine + xanthine (Reversibility: r [1]) [1] P cytosine + xanthosine S deoxyguanosine + adenine (Reversibility: r [1,7]; ? [6]) [1, 6, 7] P guanine + deoxyadenosine S deoxyguanosine + cytosine (Reversibility: r [1,6]; [12]) [1, 6, 12] P guanine + deoxycytidine S deoxyguanosine + hypoxanthine (Reversibility: r [1]) [1] P guanine + deoxyinosine S deoxyguanosine + thymine (Reversibility: r [1]) [1] P guanine + thymidine S deoxyguanosine + uracil (Reversibility: r [1]) [1] P guanine + deoxyuridine S deoxyguanosine + xanthine (Reversibility: r [1]) [1] P guanine + deoxyxanthine S deoxyinosine + 1,7-dimethylguanine (, DRTase I [12]) (Reversibility: ? [12]) [12] P hypoxanthine + deoxy-1,7-dimethylguanosine

69


2.4.2.6

S deoxyinosine + 6-chloroguanine (, DRTase I [12]) (Reversibility: ? [12]) [12] P hypoxanthine + deoxy-6-chloroguanosine S deoxyinosine + 8-azaguanine (Reversibility: r [1]) [1] P hypoxynthine + deoxy-8-azaguanosine S deoxyinosine + 8-azaxanthine (Reversibility: r [1]) [1] P hypoxynthine + deoxy-8-azaxanthosine S deoxyinosine + adenine (, DRTase I [12]) (Reversibility: r [1, 3, 5, 6, 7, 12]) [1, 3, 5, 6, 7, 12] P hypoxynthine + deoxyadenosine [1, 3] S deoxyinosine + cytosine (, DRTase II [12]) (Reversibility: r [1]; ? [6, 12]) [1, 6, 12] P hypoxynthine + deoxycytidine S deoxyinosine + guanine (, DRTase I [12]) (Reversibility: r [1]; ? [2]) [1, 2] P hypoxanthine + deoxyguanosine [1, 2] S deoxyinosine + thymine (, DRTase II [12]) (Reversibility: r [1, 3]) [1, 12] P hypoxynthine + thymidine S deoxyinosine + uracil (Reversibility: r [1]) [1] P hypoxanthine + deoxyuridine S deoxyinosine + uric acid (Reversibility: r [1]) [1] P hypoxanthine + 9-(2-deoxy-b-d-ribofuranosyl)-7,9-dihydro-3H-purine2,6,8-trione S deoxyuridine + adenine (Reversibility: ? [7]) [7] P deoxyadenosine + uracil S deoxyuridine + guanine (Reversibility: ? [7]) [7] P deoxyguanosine + uracil S deoxyuridine + hypoxanthine (Reversibility: ? [7]) [7] P deoxyinosine + uracil S thymidine + 1-deaza-8-aza-adenine (, 21% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 2'-deoxy-1-deaza-8-aza-adenosine S thymidine + 1-deaza-adenine (, 6.2% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 2'-deoxy-1-deaza-adenosine S thymidine + 2,6-diaminopurine (, 53.4% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 9-(2-deoxy-b-d-ribofuranosyl)-2,6-diaminopurine S thymidine + 2,6-dichloropurine (Reversibility: r [4]) [4] P thymine + 9-(2-deoxy-b-d-ribofuranosyl)-2,6-dichloropurine S thymidine + 2-amino-6-methyl-mercaptopurine (Reversibility: r [4]) [4] P thymine + 9-(2-deoxy-b-d-ribofuranosyl)-2-amino-6-methy-mercaptopurine S thymidine + 2-aminopurine (Reversibility: r [4]) [4] P thymine + 9-(2-deoxy-b-d-ribofuranosyl)-2-aminopurine 70

2.4.2.6


S thymidine + 2-hydroxy-6-mercaptopurine (Reversibility: r [4]) [4] P Additional information (, 2 products [4]) [4] S thymidine + 2-mercaptoadenine (Reversibility: r [4]) [4] P Additional information (, 2 products [4]) [4] S thymidine + 2-mercaptopurine (Reversibility: r [4]) [4] P Additional information (, 2 products [4]) [4] S thymidine + 2-pyrimidinol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 2-thio-4-pyrimidinal (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 2-thio-5-methyl-4-pyrimidinol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 3-deaza-8-aza-2-aminopurine (, 39.7% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 9-(2-deoxy-b-d-ribofuranosyl)-3-deaza-8-aza-aminopurine S thymidine + 3-deaza-8-aza-adenine (, 3.5% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 2'-deoxy-3-deaza-8-aza-adenosine S thymidine + 4,6-pyrimidinediol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 4-amino-2-pyrimidinethiol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 4-amino-5-carboxamide-imidazole (, 12.3% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + ? S thymidine + 4-amino-6-pyrimidinol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 4-amino-pyrazolopyrimidine (, 0.37% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + ? S thymidine + 4-methyl-2-pyrimidinol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 4-pyrimidinol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + 5,6-dimethyl-benzimidazole (, 37.7% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + ? S thymidine + 5-bromouracil (Reversibility: ? [10]) [10] P thymine + deoxy-5-bromouridine S thymidine + 5-fluorouracil (Reversibility: ? [10]) [10] P thymine + deoxy-5-fluorouridine S thymidine + 5-hydroxyuracil (Reversibility: ? [10]) [10] P thymine + deoxy-5-hydroxyuridine 71


S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P

72

2.4.2.6

thymidine + 5-methylcytosine (Reversibility: ? [10]) [10] thymine + deoxy-5-methylcytosine thymidine + 5-nitrouracil (Reversibility: ? [10]) [10] thymine + deoxy-5-nitrouridine thymidine + 5-thiouracil (Reversibility: ? [10]) [10] thymine + deoxy-5-thiouridine thymidine + 6-benzylaminopurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-benzylaminopurine thymidine + 6-chloropurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-chloropurine thymidine + 6-dimethylaminopurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-dimethylaminopurine thymidine + 6-hydrazinepurine (Reversibility: r [4]) [4] Additional information (, several products observed [4]) [4] thymidine + 6-iodopurine (, 65.6% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-iodopurine thymidine + 6-mercaptoguanine (, 52.1% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] thymine + 6-mercaptoguanosine thymidine + 6-mercatopurine (, 56.5% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-mercaptopurine thymidine + 6-methoxypurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-methoxypurine thymidine + 6-methyl-cis-triazine-3,5-diol (Reversibility: ? [10]) [10] thymine + ? thymidine + 6-methylaminopurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-methylaminopurine thymidine + 6-methylpurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-methylpurine thymidine + 6-methylthio-8-trifluoromethylpurine (Reversibility: ? [9]) [9] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-methylthio-8-trifluoromethylpurine thymidine + 6-n-hexylaminopurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-n-hexylaminopurine thymidine + 6-n-hexylmercaptopurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-n-hexylmercaptopurine thymidine + 6-phenylaminopurine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-6-phenylaminopurine thymidine + 7-hydroxy-1,2,5-selenadiazolopyridine (, 29.2% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] thymine + ?

2.4.2.6


S thymidine + 8-aza-2,6-diaminopurine (, 59.6% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 9-(2-deoxy-b-d-ribofuranosyl)-8-aza-2,6-diaminopurine S thymidine + 8-azaguanine (, 16.0% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 2'-deoxy-8-azaguanosine S thymidine + 8-azaxanthine (Reversibility: ? [4]) [4] P thymine + 2'-deoxy-8-azaxanthosine S thymidine + 8-bromoadenine (Reversibility: ? [9]) [9] P thymine + 2'-deoxy-8-bromoadenosine (, 3-(2'-deoxyribofuranosyl)nucleoside is initially the major product, but the 9-(2'-deoxyribofuranosyl)nucleoside becomes the major product after long incubation periods [9]) [9] S thymidine + 8-chloroadenine (Reversibility: ? [9]) [9] P thymine + 2'-deoxy-8-chloroadenosine (, 3-deoxyribonucleoside + 9-deoxyribonucleoside [9]) [9] S thymidine + 8-chlorotheophylline (Reversibility: ? [9]) [9] P thymine + 9-(2-deoxy-b-d-ribofuranosyl)-8-chlorotheophylline S thymidine + 8-methyladenine (Reversibility: ? [9]) [9] P thymine + 2'-deoxy-8-methyladenosine S thymidine + 8-trifluoromethyladenine (Reversibility: ? [9]) [9] P thymine + 2'-deoxy-8-trifluoromethyladenosine S thymidine + adenine (Reversibility: r [4]) [4] P thymine + 2'-deoxyadenosine S thymidine + adenine (, DRTase II [12]) (Reversibility: ? [7, 9, 12, 14]) [7, 9, 12, 14] P thymine + adenosine (, 3-deoxyribonucleoside [9]) [9] S thymidine + benzimidazole (, 33.1% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + ? S thymidine + cis-triazine-3,5-diol (Reversibility: ? [10]) [10] P thymine + ? S thymidine + cytosine (Reversibility: ? [10,12]) [10, 12] P thymine + 2'-deoxycytidine S thymidine + cytosine (Reversibility: ? [6,7,14]) [6, 7, 14] P thymine + 2'-deoxycytidine S thymidine + guanine (Reversibility: ? [7]) [7] P deoxyguanine + thymine S thymidine + guanine (, 42.3% of substrate converted into deoxynucleoside after 2 h [4]) (Reversibility: ? [4]) [4] P thymine + 2'-deoxyguanosine S thymidine + guanine (, DRTase II [12]) (Reversibility: ? [12, 14]) [12, 14] P thymine + 2'-deoxyguanosine S thymidine + hypoxanthine (Reversibility: r [4]) [4] P thymine + deoxyinosine S thymidine + hypoxanthine (Reversibility: ? [7]) [7] 73


P S P S P S P S P S P S P S

P

2.4.2.6

deoxyinosine + thymine thymidine + purine (Reversibility: r [4]) [4] thymine + 9-(2-deoxy-b-d-ribofuranosyl)-purine thymidine + theophylline (Reversibility: ? [4]) [4] thymine + 2'-deoxytheophylline riboside thymidine + uracil (Reversibility: ? [10]) [10] thymine + 2'-deoxyuridine thymidine + uracil (Reversibility: ? [7]) [7] deoxyuridine + thymine thymidine + xanthine (Reversibility: ? [3]) [3] 9-deoxyribosylxanthine + 7-deoxyribosylxanthine (, 20% 9deoxyribosylxanthine + 80% 7-deoxyribosylxanthine [3]) [3] thymidine + xanthine (Reversibility: r [4]) [4] thymine + xanthosine Additional information (, nucleoside deoxyribosyltransferase I is strictly specific for transfer between purine bases. Nucleoside deoxyribosyltransferase II catalyzes the transfer of the deoxyribosyl moiety between purines or pyrimidines as well as from a purine to a pyrimidine [3]; , DRTase I is strictly specific for transfer between purine bases. DRTase II catalyzes the transfer of the deoxyribosyl moiety between purines or pyrimidines as well as from a purine to a pyrimidine [12,13]; , DRTase I is specific for purines with preference for deoxyinosine, deoxyadenosine, deoxyguanosine as donor substrates [13]) [3, 12, 13] ?

Inhibitors Tris (, complete inhibition of deoxyribosyltransferase I at 0.1 M, pH 6.9. 62% inhibition of nucleoside deoxyribosyltransferase I at 0.8 M, pH 5.9 [3]) [1, 3] adenine [3] amino-pyrrolo-pyrimidine (, 12.1 mM, complete inhibition of the reaction with adenine and deoxycytidine [4]) [4] cytosine [3] deoxyadenosine [3] deoxyadenosine (, linear competitive inhibition of deoxyinosine and linear non-competitive inhibition of adenine [5]; , linear competitive inhibition of deoxycytidine [6]) [3, 5, 6] deoxycytidine [3] deoxyinosine [3] hydroxypyrrolopyrimidine (, 0.33 mM, 66% inhibition of the reaction with adenine and deoxycytidine [4]) [4] hypoxanthine [3] imidazole (, 12.1 mM, 76% inhibition of the reaction with adenine and deoxycytidine [4]) [4] Turnover number (min±1) Additional information [12]

74

2.4.2.6


Specific activity (U/mg) 0.8 (, reaction with deoxyinosine and adenine [6]) [6] 7.75 (, reaction with thymidine and cytosine [6]) [6] 23.1 [7] 36.8 (, DRTase II, reaction with thymidine + adenine [12]) [12] 42.9 (, DRTase I, reaction with deoxyinosine and adenine [12]) [12] 43 (, reaction with deoxycytidine and adenine [6]) [6] 60 [5] Additional information (, method for determining enzyme activity by coupling the release of adenine from deoxyadenosine to the reaction catalyzed by pyruvate kinase/lactate dehydrogenase via adenine phosphoribosyltransferase and adenylate kinase [11]) [11] Km-Value (mM) 0.0073 (adenine, , reaction with deoxyinosine [6]) [6] 0.008 (adenine, , reaction with deoxyguanosine [6]) [6] 0.019 (adenine, , reaction with deoxycytidine [3]) [3] 0.019 (adenine, , reaction with deoxycytidine [6]) [6] 0.021 (adenine, , reaction with deoxyadenosine [6]) [6] 0.03 (guanine, , reactioin with deoxyinosine [12]) [12] 0.04 (adenosine, , reaction with deoxyinosine [12]) [12] 0.041 (deoxyinosione) [3] 0.05 (cytosine, , reaction with thymidine [12]) [12] 0.05 (thymidine, , reaction with cytosine [12]) [12] 0.073 (cytosine, , reaction with deoxyinosine [6]) [6] 0.077 (cytosine, , reaction with deoxyguanosine [6]) [6] 0.086 (hypoxanthine) [3] 0.09 (deoxycytidine, , reaction with adenine [6]) [3, 6] 0.092 (deoxyadenosine, , reaction with adenine [6]) [6] 0.095 (deoxycytidine, , reaction with cytosine [6]) [6] 0.1 (deoxyadenosine, , reaction with cytosine [12]) [12] 0.12 (cytosine, , reaction with deoxyadenosine [12]) [12] 0.12 (deoxyadenosine, , reaction with cytosine [6]) [3, 6] 0.13 (thymine, , reaction with deoxyinosine [12]) [12] 0.16 (deoxyguanosine, , reaction with cytosine [12]) [12] 0.17 (cytosine, , reaction with deoxycytidine [6]) [6] 0.18 (cytosine, , reaction with deoxyinosine [12]) [12] 0.19 (cytosine, , reaction with deoxyguanosine [12]) [12] 0.22 (cytosine, , reaction with deoxyadenosine [6]) [3, 6] 0.27 (deoxyinosine, , reaction with guanine [12]) [12] 0.3 (adenine, , reaction with deoxycytidine [12]) [12] 0.3 (deoxyinosine, , reaction with thymine [12]) [12] 0.33 (deoxyinosine, , reaction with adenosine [12]) [12] 0.346 (deoxyguanosine, , reaction with adenine [6]) [6] 0.35 (deoxyinosine, , reaction with cytosine [12]) [3, 12] 0.37 (deoxyguanosine, , reaction with cytosine [6]) [6] 0.45 (deoxyadenosine) [3]

75


2.4.2.6

0.48 (adenine, , reaction with thymidine [12]) [12] 0.48 (guanine, , reaction with deoxycytidine [12]) [12] 0.51 (thymidine, , reaction with adenine [12]) [12] 0.52 (deoxycytidine, , reaction with guanine [12]) [12] 0.52 (guanine, , reaction with thymidine [12]) [12] 0.55 (deoxycytidine, , reaction with adenine [12]) [12] 0.63 (thymidine, , reaction with guanine [12]) [12] 3.4 (deoxyinosine, , reaction with adenine [6]) [6] 3.5 (deoxyinosine, , reaction with cytosine [6]) [6] Ki-Value (mM) 0.021 (adenine) [3, 6] 0.039 (adenine) [3] 0.079 (hypoxanthine) [3] 0.092 (deoxyadenosine) [3, 6] 0.095 (deoxycytidine) [3, 6] 0.17 (cytosine) [3, 6] 0.34 (deoxyinosine) [3] 0.43 (deoxyadenosine) [3] pH-Optimum 5.8 [1, 3] 7.7 (, reaction with deoxyinosine and guanine [2]) [2] Temperature optimum ( C) 45 (, DRTase I [12]) [12] 55 (, DRTase II [12]) [12]

4 Enzyme Structure Molecular weight 82000 (, gel filtration [7]) [7] 86000 [3] 110000 (, gel filtration [8]; , DRTase II, non-denaturing PAGE [12]) [8, 12] 120000 (, DRTase I, nondenaturing PAGE [12]) [12] Subunits ? (, x * 17000, SDS-PAGE [2]; , x * 18000, SDS-PAGE [8]; , x * 24000, SDS-PAGE [13]) [2, 8, 13] hexamer (, 6 * 18000, DRTase II, SDS-PAGE [12]; , 6 * 20000, DRTAse I [12]) [12]

76

2.4.2.6


5 Isolation/Preparation/Mutation/Application Purification [1, 7] [2] (DRTase I and DRTAse II [12]) [12] Crystallization (microdiffusion by means of equilibrium dialysis is used to crystallize the enzyme [7]) [7] [8] Cloning (expression in Escherichia coli [13]) [13] (expression of the catalytically inactive mutant E98A in Escherichia coli [16]) [16] Engineering E98A (, catalytically inactive mutant [16]) [16] Application medicine (, the low substrate specificity of the enzyme is used advantageously for synthesis of nucleoside analogs, some of them of medical interest [11]) [11] synthesis (, development of a practical method for enzymatic synthesis of deoxyguanosine by the combination of transglycosylation with NdRT-II from thymidine to a 2-amino-6-substituted purine, and the hydrolysis reaction with bacterial adenosine deaminase [14]) [14]

6 Stability pH-Stability 4 (, 61 C, 30 min, about 30% loss of activity [1]) [1] 6.5 (, 61 C, 30 min, maximal stability [1]; , optimal stability [3]) [1, 3] 8 (, 61 C, 30 min, about 15% loss of activity [1]) [1] 8.5 (, 61 C, 30 min, about 70% of maximal activity [1]) [1] Temperature stability 55 (, pH 6.3, 10 min, stable below [1]) [1] 90 (, pH 6.3, 10 min, 75% loss of activity [1]) [1] General stability information , crystals are stable to X-rays for at least 5 days [8] Storage stability , -50 C, stable for several months either in the lyophilized form or disolved in freshly distilled water [7]

77


2.4.2.6

References [1] Roush, A.H.; Betz, R.F.: Purification and properties of trans-N-deoxyribosylase. J. Biol. Chem., 233, 261-266 (1958) [2] Steenkamp, D.J.: The purine-2-deoxyribonucleosidase from Crithidia luciliae. Purification and trans-N-deoxyribosylase activity. Eur. J. Biochem., 197, 431-439 (1991) [3] Cardinaud, R.: Nucleoside deoxyribosyltransferase from Lactobacillus helveticus. Methods Enzymol., 51, 446-455 (1978) [4] Holguin, J.; Cardinaud, R.: trans-N-Deoxyribosylase: substrate specificity studies. Purine bases as acceptors. Eur. J. Biochem., 54, 515-520 (1975) [5] Danzin, C.; Cardinaud, R.: Deoxyribosyl transfer catalysis with trans-Ndeoxyribosylase. Kinetic studies of purine-to-purine trans-N-deoxyribosylase. Eur. J. Biochem., 48, 255-262 (1974) [6] Danzin, C.; Cardinaud, R.: Deoxyribosyl transfer catalysis with trans-Ndeoxyribosylase. Kinetic study of purine(pyrimidine) to pyrimidine(purine) trans-N-deoxyribosylase. Eur. J. Biochem., 62, 365-372 (1976) [7] Uerkvitz, W.: Trans-N-deoxyribosylase from Lactobacillus helveticus. Crystallization and properties. Eur. J. Biochem., 23, 387-395 (1971) [8] Cook, W.J.; Short, S.A.; Ealick, S.E.: Crystallization and preliminary X-ray investigation of recombinant Lactobacillus leichmannii nucleoside deoxyribosyltransferase. J. Biol. Chem., 265, 2682-2683 (1990) [9] Huang, M.-C.; Montgomery, J.A.; Thorpe, M.C.; Stewart, E.L.; Secrist III, J.A.; Blakley, R.L.: Formation of 3-(2-deoxyribofuranosyl) and 9-(2-deoxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyribosyltransferase. Arch. Biochem. Biophys., 222, 133-144 (1983) [10] Cardinaud, R.; Holguin, J.: Nucleoside deoxyribosyltransferase-II from Lactobacillus helveticus Substrate specificity studied. Pyrimidine bases as acceptors. Biochim. Biophys. Acta, 568, 339-347 (1979) [11] Pistotnik, E.; Sakamoto, H.; Pochet, S.; Namane, A.; Barzu, O.: Assay of nucleoside 2-deoxyribosyltransferase activity with pyruvate kinase/lactate dehydrogenase coupling system. Anal. Biochem., 271, 192-193 (1999) [12] Becker, J.; Brendel, M.: Rapid purification and characterization of two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii. Biol. Chem. Hoppe-Seyler, 377, 357-362 (1996) [13] Kaminski, P.A.: Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus. J. Biol. Chem., 277, 14400-14407 (2002) [14] Okuyama, K.; Shibuya, S.; Hamamoto, T.; Noguchi, T.: Enzymatic synthesis of 2'-deoxyguanosine with nucleoside deoxyribosyltransferase-II. Biosci. Biotechnol. Biochem., 67, 989-995 (2003) [15] Mueller, M.; Hutchinson, L.K.; Guengerich, F.P.: Addition of deoxyribose to guanine and modified DNA bases by Lactobacillus helveticus trans-N-deoxyribosylase. Chem. Res. Toxicol., 9, 1140-1144 (1996) [16] Porter, D.J.T.; Short, S.A.: Nucleoside 2-deoxyribosyltransferase. Pre-steadystate kinetic analysis of native enzyme and mutant enzyme with an alanyl residue replacing Glu-98. J. Biol. Chem., 270, 15557-15562 (1995) 78

Adenine phosphoribosyltransferase

2.4.2.7

1 Nomenclature EC number 2.4.2.7 Systematic name AMP:diphosphate phospho-d-ribosyltransferase Recommended name adenine phosphoribosyltransferase Synonyms AMP pyrophosphorylase AMP-pyrophosphate phosphoribosyltransferase AMP:pyrophosphate phospho-d-ribosyltransferase APRT adenine phosphoribosylpyrophosphate transferase adenosine phosphoribosyltransferase adenylate pyrophosphorylase adenylic pyrophosphorylase phosphoribosyltransferase, adenine transphosphoribosidase Additional information ( structure analysis: different paths for adenine relative to other purine PRTases [24]) [24] CAS registry number 9027-80-9

2 Source Organism

Catharanthus roseus [8] Triticum aestivum [9] Brassica juncea (cv. Green Wave [15]) [15] Bos taurus [1] Arabidopsis thaliana [2] Escherichia coli [3, 11] Homo sapiens [4, 6, 7, 19] Rattus norvegicus [5, 12] Leishmania donovani (APPB2 and APPB2-640A3, totally enzyme-deficient cell line, clone of APPB2 cell line [28]) [10, 20, 28] Mycoplasma mycoides [11]

79


2.4.2.7

Mus musculus [13] Plasmodium falciparum [14] Plasmodium chabaudi (maintained in NMRI mice [16]) [16] Schizosaccharomyces pombe [17] Artemia sp. [18] Hevea brasiliensis [21] Leishmania donovani [22] Homo sapiens [22] Giardia lamblia [23, 24] Saccharomyces cerevisiae (strain ATCC 24903 [25]) [25, 27] Arabidopsis thaliana (isoform APT1 [26]) [26] Arabidopsis thaliana (isoform APT2 [26]) [26] Arabidopsis thaliana (isoform APT3 [26]) [26] Arabidopsis thaliana (isoform APT4 [26]) [26] Arabidopsis thaliana (isoform APT5 [26]) [26]

3 Reaction and Specificity Catalyzed reaction AMP + diphosphate = adenine + 5-phospho-a-d-ribose 1-diphosphate ( forward reaction follows a random bi-bi mechanism [23]; catalytic site: amino acid residues 106-108 [27]; active site structure [22, 24, 27]; mechanism [21, 24]; ordered bi-bi mechanism, kinetics [20]; ping-pong mechanism [3]) Reaction type pentosyl group transfer Natural substrates and products S adenine + 5-phospho-a-d-ribose 1-diphosphate ( enzyme mediates the translocation of adenine into the cell as AMP [3]; adenine salvage enzyme [8]; may play a role in maintaining the supply of adequate levels of active cytokinin [9]; necessary for appropriate regulation of purine de novo biosynthesis [3]) [3, 8, 9, 21] P AMP + diphosphate Substrates and products S 2,6-diaminopurine + 5-phospho-a-d-ribose 1-diphosphate ( no substrate [13]; specific for adenine or 2,6-diaminopurine [3]) (Reversibility: ? [3, 4, 7, 10]) [3, 4, 7, 10] P 2,6-diaminopurine ribotide + diphosphate S 4-amino-5-imidazolecarboxamide + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [4, 7]) [4, 7] P 4-amino-5-imidazolecarboxamide ribotide + diphosphate S 4-aminopyrazolo-(3,4-d)-pyrimidine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [10]) [10] P 4-aminopyrazolo-(3,4-d)-pyrimidine ribotide + diphosphate

80

2.4.2.7


S 4-carbamoylimidazolium 5-olate + 5-phospho-a-d-ribose 1-diphosphate ( low activity [13]) (Reversibility: ? [13]) [13] P 4-carbamoylimidazolium 5-olate 5'-phosphate + diphosphate S 5-amino-4-imidazolecarboxamide + 5-phospho-a-d-ribose 1-diphosphate ( no activity [3]) (Reversibility: ? [1, 2, 10]) [1, 2, 10] P 5-amino-4-imidazolecarboxamide ribotide + diphosphate [1] S 6-amino-2-hydroxypurine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [10]) [10] P 6-amino-2-hydroxypurine ribotide + diphosphate S 6-mercaptopurine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [4]) [4] P 6-mercaptopurine ribotide + diphosphate S 6-methylpurine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [10]) [10] P 6-methylpurine ribotide + diphosphate S 8-azaadenine + 5-phospho-a-d-ribose 1-diphosphate ( no substrate [13]) (Reversibility: ? [10]) [10] P 8-azaadenosine 5'-phosphate + diphosphate S N6 -furfuryladenine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [9]) [9] P N6 -furfuryladenosine 5'-phosphate + diphosphate S adenine + 5-phospho-a-d-ribose 1-diphosphate ( diphosphate does not bind at the active site, but near the N-terminal side at Arg69 [27]; substrate binding structure [22, 24]; best acceptor substrate [7, 26]; highly specific for the donor substrate [4]; equilibrium lies far in the direction of nucleotide synthesis [1]; specific for adenine or 2,6-diamino-purine [3]) (Reversibility: r [20, 23, 24]; ? [1-19, 21, 22, 25-27]) [1-27] P AMP + diphosphate ( ordered substrate binding in the reverse reaction with AMP bound first followed by diphosphate [23]; AMP is bound in the low energy anti conformation with the ribose in the 2' endo conformation [22]; 5'-AMP [3]) [1-5, 11, 2027] S benzyladenine + 5-phospho-a-d-ribose 1-diphosphate ( N6 -benzyladenine [9]) (Reversibility: ? [2, 9, 26]) [2, 9, 26] P benzyladenosine 5'-phosphate + diphosphate S isopentenyladenine + 5-phospho-a-d-ribose 1-diphosphate ( N6 -(d-isopentenyl)adenine [9]) (Reversibility: ? [2, 9, 26]) [2, 9, 26] P isopentenyladenosine 5'-phosphate + diphosphate S xanthine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [21]) [21] P xanthosine 5'-phosphate + diphosphate S zeatin + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [26]) [26] P zeatin ribotide + diphosphate 81


2.4.2.7

S Additional information ( no activity with hypoxanthine, guanine, cytosine [21]; at 0 C in the absence of Mg2+ but in presence of substrates the enzyme catalyzes a rapid and limited synthesis of AMP [7]; no substrate: hypoxanthine,guanine [4,9]; no substrate: adenosine [4]; no donor substrates: d-ribose 5-phosphate, ribose 1phosphate [7]) [4, 7, 9, 21] P ? Inhibitors 2,6-diaminopurine ( competitive [14]) [14] 4-aminopteridine [10] 4-aminopyrido(2,3-d)pyrimidine [10] 6-mercaptopurine ( competitive [14]) [14] ADP [4, 8, 17] AMP ( competitive against adenine [23]; competitive against 5-phospho-a-d-ribose 1-diphosphate [8, 11, 13, 18, 23]) [2-4, 8, 11, 13, 17, 18, 20, 23, 28] ATP [7, 17] Ba2+ ( in presence of MnCl2 [2]) [2, 3, 18] CTP [17] Ca2+ ( strong [18]; no inhibition [21]; in presence of MnCl2 [2]; activation, competitive to Mg2+ [9]) [2, 18] Cd2+ ( in presence of MnCl2 [2]) [2, 14] Co2+ [14] Cu2+ ( strong [21]) [21] EDTA ( reversible by 2-mercaptoethanol and excess Mg2+ [12]) [12] GDP ( slightly at 5 mM [17]) [17] GMP ( slightly at 5 mM [17]) [4, 8, 11, 17] GTP ( slightly at 5 mM [17]) [17] Hg2+ ( complete inhibition at 5 mM [9]; in presence of MnCl2 [2]; reversed by 2-mercaptoethanol [12]) [2, 4, 9, 12, 14] Mg2+ ( inhibitory effects are noncompetitive against 5-phosphoribose 1-diphosphate [17]; inhibition above 2 mM, activation below [8]; inhibition in presence of MnCl2 , activation in absence [2]) [2, 8, 17] N-ethylmaleimide ( strong, DTT or glutathione protect [21]; no inhibition [14]) [15, 21] Na+ ( no inhibition [21]) [4] SO24- ( no inhibition [21]) [4] UDP ( slightly [17]) [17] UMP [8] Zn2+ ( strong [21]; weak [14]) [3, 14, 21] adenine ( at high concentrations, at low 5-phospho-a-d-ribose 1-diphosphate concentration [11]) [2, 11] a-d-5-phosphoribose 1-diphosphate ( substrate inhibition [2]) [2] benzyladenine ( competitive [2]) [2] citrate [4] dAMP [17]

82

2.4.2.7


dATP [17] diphosphate ( at 20 mM [17]; no inhibition [21]; noncompetitive against 5-phospho-a-d-ribose 1-diphosphate [23]; competitive against adenine and 5-phosphoribose 1-diphosphate [20]) [3, 17, 20, 23, 28] formycin AMP ( competitive against adenine and diphosphate [20]) [20] guanine ( noncompetitive against 5-phosphoribose 1-diphosphate [18]; weak [10]) [10, 18] hypoxanthine ( weak [10]) [10] immucillin ( does not bind at the active site [27]) [27] iodoacetate [4] isopentenyladenine [2] nucleotides ( nucleotide mono-, di- and triphosphates of adenine, guanine and hypoxanthine [4]; effect is strongly influenced by pH, inhibition at pH 7.1: ATP, GMP, activation at pH 7.1: GTP, UMP, UTP, CMP, CTP, IMP, no effect at pH 7.1: AMP, inhibition at pH 8.0: AMP, ATP, GMP, GTP, UTP, CTP, IMP, no effect at pH 8.0: UMP, CMP [11]; higher concentrations of all 5'-nucleotides are most inhibitory, 6-OH purine nucleotides are moderately inhibitory, pyrimidine nucleotides are least inhibitory [3]) [3, 4, 11] p-chloromercuribenzoate ( complete inhibition at 1 mM, DTT or glutathione protect [21]; no inhibition [13, 14]; reversed by 2-mercaptoethanol and excess Mg2+ [12]) [12, 21] p-hydroxymercuribenzoate ( not enzyme from monkey liver [4]; DTT [15]; no effect [13]) [4, 15] succinate [4] sulfhydryl reagents [4, 15] trinitrophenyl-AMP [20] xanthine ( weak [10]) [10] Additional information ( mechanism of product inhibition [17]; no inhibition by GMP, XMP, UMP, CMP, and CDP [17]; no effect of a variety of sugars, amino acids, organic acids and nucleotides tested, except for AMP, have any effect on the enzyme activity [21]; not affected by PO34- [21]; no inhibition by iodoacetamide, DTNB [14]; not affected by 2,6-diaminopurine, 4-carbamoylimidazolium 5olate, 8-azaadenine, and 2-fluoro-6-aminopurine [13]; no substrate inhibition by adenine [2]) [2, 13, 14, 17, 21] Activating compounds nucleotides ( effect is strongly influenced by pH, inhibition at pH 7.1: ATP, GMP, activation at pH 7.1: GTP, UMP, UTP, CMP, CTP, IMP, no effect at pH 7.1: AMP, inhibition at pH 8.0: AMP, ATP, GMP, GTP, UTP, CTP, IMP, no effect at pH 8.0: UMP, CMP [11]) [11] Additional information ( no stimulation by ethylene [21]; not affected by 2,6-diaminopurine, 4-carbamoylimidazolium 5-olate, 8-azaadenine, and 2-fluoro-6-aminopurine [13]) [13, 21]

83


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Metals, ions Ca2+ ( activates [9]; inhibition [3, 18]; inhibition in presence of MnCl2 [2]) [4, 9] Co2+ [4] Mg2+ ( Mg2+ is not bound at the catalytic site [27]; 1 Mg2+ is bound at the catalytic site [24]; Mg2+ ligand binding site [22, 27]; not required [12]; absolute requirement [2-4, 13, 17]; required [1, 3, 4, 8, 9, 13, 15, 18, 21-23]; enzyme requires Mn2+ or Mg2+ [2, 3, 21]; Mg2+ most effective [4, 21]; competitive to 5-phospho-a-d-ribose 1diphosphate [3]; half-maximal activation at 0.4 mM [13]; 0.150.18 mM [18]; optimal concentration: 2 mM [8]; optimal concentration: 5 mM [9]; maximal activity at 1-2 mM [17]; 3 mM at 60 C, at 37 C activity varies little in the range 3-50 mM MgCl2 [15]; inhibition above 2 mM [8]; maximal activity if Mg2+ is twice the concentration of 5-phospho-a-d-ribose 1-diphosphate [3, 4]) [1-4, 7-9, 13, 15, 17, 18, 21-27] Mn2+ ( required [21]; best activating divalent cation [2, 21]; activity depends on presence of divalent cations, Mn2+ or Mg2+ activates [2, 9, 18, 21]; Km : 0.04-0.05 [18]) [2, 4, 9, 18, 21] Ni2+ [4] Zn2+ ( activates, Km : 0.045-0.05 mM [18]) [4, 18] Additional information ( no effect of K+ , Na+ , Fe3+ , Ca2+ [21]; activity absolutely depends on presence of divalent cations [2-4,21]; descending order of effectiveness: Mg2+ , Mn2+ , Ca2+ , Co2+ , Ni2+ , Zn2+ [4]) [2-4, 21] Turnover number (min±1) 0.006 (5-phospho-a-d-ribose 1-diphosphate, mutant E106L [27]) [27] 0.18 (5-phospho-a-d-ribose 1-diphosphate, mutant R69A [27]) [27] 0.57 (AMP) [23] 0.57 (diphosphate) [23] 3 (5-phospho-a-d-ribose 1-diphosphate, mutant E106Q [27]) [27] 18 (5-phospho-a-d-ribose 1-diphosphate, mutant Y107D [27]) [27] 42 (5-phospho-a-d-ribose 1-diphosphate, mutant G108H [27]) [27] 48 (5-phospho-a-d-ribose 1-diphosphate, mutant K90A [27]) [27] 168 (5-phospho-a-d-ribose 1-diphosphate) [23] 168 (adenine) [23] 240 (5-phospho-a-d-ribose 1-diphosphate, mutant Y107F [27]) [27] 560 (adenine, cosubstrate 5-phospho-a-d-ribose 1-diphosphate [3]) [3]

84

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600 (5-phospho-a-d-ribose 1-diphosphate, mutant R89A [27]) [27] 675 (diphosphate) [20] 1074 (5-phospho-a-d-ribose 1-diphosphate) [20] 1800 (5-phospho-a-d-ribose 1-diphosphate, wild-type enzyme, mutant G108A [27]) [27] 2400 (5-phospho-a-d-ribose 1-diphosphate, mutant K93A [27]) [27] 60000 (5-phospho-a-d-ribose 1-diphosphate, mutant Y103F [27]) [27] Specific activity (U/mg) 0.00000004 [14] 0.0413 [9] 0.156 ( purified enzyme [8]) [8] 1.1 ( purified enzyme [5,12]) [5, 12] 2.2 ( purified enzyme [21]) [21] 2.8 [10] 9.15 [15] 9.58 ( purified enzyme [4,7]) [4, 7] 14 ( purified enzyme [3]) [3] 14.2 ( purified enzyme [18]) [18] 15.14 ( purified enzyme [25]) [25] 18 [17] 20.6 ( purified enzyme [13]) [13] 21.8 ( purified enzyme) [6] 300 ( purified enzyme, pH 8.8, 37 C [2]) [2] Additional information ( wild-type and mutant [23]) [23] Km-Value (mM) 0.0007 (5-phospho-a-d-ribose 1-diphosphate) [14] 0.0008 (adenine, APT3, pH 7.4 [26]) [14, 26] 0.0008-0.001 (adenine, wild-type [27]) [5, 27] 0.001 (adenine, APT1, pH 7.4 [26]) [26] 0.0012 (5-phospho-a-d-ribose 1-diphosphate) [13] 0.0013 (adenine) [11] 0.002 (adenine) [18] 0.00233 (adenine) [20] 0.0026 (adenine, APT2, pH 7.4 [26]) [26] 0.0038 (adenine) [15, 21] 0.0042 (adenine) [23] 0.0045 (adenine, at 37 C [2]) [2] 0.005 (5-phospho-a-d-ribose 1-diphosphate) [5] 0.005 (adenine) [16] 0.00523 (AMP) [20] 0.006 (5-phospho-a-d-ribose 1-diphosphate) [4, 6] 0.007 (adenine, with 5 mM Mg2+ [10]; pH 7.6 [8]) [8, 10, 13] 85


2.4.2.7

0.0096 (5-phospho-a-d-ribose 1-diphosphate, pH 7.6 [8]) [8] 0.01 (5-phospho-a-d-ribose 1-diphosphate) [11] 0.015 (5-phospho-a-d-ribose 1-diphosphate) [15] 0.015 (benzyladenine) [26] 0.02 (5-phospho-a-d-ribose 1-diphosphate, wild-type [27]) [27] 0.02 (adenine) [3] 0.025 (5-phospho-a-d-ribose 1-diphosphate) [20, 21] 0.03 (5-phospho-a-d-ribose 1-diphosphate, in presence of Mg2+ [25]) [18, 25] 0.032 (5-phospho-a-d-ribose 1-diphosphate) [17] 0.036 (4-aminopyrazolo-(3,4-d)pyrimidine) [10] 0.069 (adenine) [17] 0.074 (adenine) [9] 0.076 (zeatin) [26] 0.087 (AMP) [23] 0.11 (N6 -furfuryladenine) [9] 0.11 (isopentenyladenine) [26] 0.125 (5-phospho-a-d-ribose 1-diphosphate, in presence of a 2fold excess of Mg2+ [3]) [3] 0.13 (N6 -(D2 -isopentenyl)adenine) [9] 0.14 (adenine) [4] 0.143 (5-phospho-a-d-ribose 1-diphosphate) [23] 0.154 (N6 -benzyladenine) [9] 0.2 (Mn2+ ) [21] 0.24 (8-azaadenine) [10] 0.255 (diphosphate) [20] 0.29 (5-phospho-a-d-ribose 1-diphosphate, at 37 C [2]) [2] 0.3 (Mg2+ ) [21] 0.33 (zeatin) [26] 0.44 (benzyladenine) [26] 0.44 (isopentenyladenine) [26] 0.45 (diphosphate) [23] 0.73 (benzyladenine, at 37 C [2]) [2] 0.89 (2,6-diaminopurine) [10] 1 (5-aminoimidazole-4-carboxamide) [10] 1.4 (6-amino-2-hydroxypurine) [10] 1.8 (zeatin) [26] 2.4 (benzyladenine) [26] 2.5 (isopentenyladenine) [26] 3.7 (6-methylpurine) [10] Additional information ( kinetics [3, 5, 10, 20, 23]; Km values for adenine of diverse enzyme mutants [27]; the 2 separated forms of enzyme in gel filtration without Mg2+ give 2 Km values for 5-phosphoribose 1-diphosphate [25]) [3, 5, 10, 12, 20, 23, 25, 27, 28]

86

2.4.2.7


Ki-Value (mM) 0.0002 (immucillin) [27] 0.002 (Cu2+ ) [21] 0.005 (Zn2+ ) [21] 0.017 (4-aminopyrido(2,3-d)pyrimidine) [10] 0.018 (AMP, competitive versus 5-phosphoribose 1-diphosphate [23]) [23] 0.024 (AMP) [13] 0.026 (AMP) [11] 0.039 (AMP) [20] 0.059 (formycin AMP, competitive versus AMP [20]) [20] 0.062 (formycin AMP, competitive versus diphosphate [20]) [20] 0.2 (guanine, above [10]) [10] 0.51 (diphosphate, competitive versus 5-phosphoribose 1-diphosphate [20]) [20] 0.53 (4-aminopteridine) [10] 0.53 (diphosphate, competitive versus adenine [20]) [20] 0.7 (mercaptopurine) [14] 0.8 (hypoxanthine, above [10]) [10] 0.8 (xanthine, above [10]) [10] 0.95 (4-aminopyrolo(2,3-d)pyrimidine) [10] 1 (2,6-diaminopurine) [14] 1.79 (diphosphate, versus 5-phosphoribose 1-diphosphate [23]) [23] 1.96 (5-phosphoribose 1-diphosphate, at 37 C [2]) [2] 23.5 (AMP, competitive versus adenine [23]) [23] pH-Optimum 7 [21] 7.4 ( assay at [6, 10, 27]) [6, 10, 27] 7.4-7.5 ( APT2 and 3, 37 C [26]) [26] 7.5 ( recombinant enzyme, assay at [20]; two zones of pH-optima at pH 7.5 and pH 8.5 [17]) [9, 17, 20] 7.6-8 [8] 7.8 ( at 60 C [2]) [2, 3] 8 ( around, broad, E. coli [11]) [11] 8-9 [18] 8.4-9.1 [10] 8.5 ( two zones of pH-optima at pH 7.5 and 8.5 [17]) [17] 8.8 ( APT1, 37 C [26]; at 37 C [2]) [2, 26] 9 [14] 9.2 [15] 10 [12] Additional information ( pI: 4.95 [16]; pI: 5.65 [12]; pI: 4.85 [4]; pH-optimum depends on 5-phospho-a-d-ribose 1-diphosphate concentration [11]; temperature dependent [2]) [2, 4, 11, 12, 16]

87


2.4.2.7

pH-Range 5.5-10 ( active over a broad range increasing progressively in activity from pH 5.5-10 [5,12]) [5, 12] 7-9 ( active over a broad range increasing progressively in activity from pH 7.0-9.0 [13]) [13] 7.4-9.5 ( broad maximum [4,7]) [4, 7] Temperature optimum ( C) 25 ( assay at [10]) [10] 27 ( recombinant enzyme, assay at [20]) [20] 30 ( assay at [5, 8, 13]) [5, 8, 13] 37 ( assay at [3, 4, 7, 9, 11, 15, 17, 18, 26, 27]) [3, 4, 7, 9, 11, 15, 17, 18, 26, 27] 50-65 [21] 60 [2, 15] 65 [2] Additional information ( at 0 C in the absence of Mg2+ but in presence of substrates enzyme catalyzes a rapid and limited synthesis of AMP [7]) [7]

4 Enzyme Structure Molecular weight 18000 ( gel filtration [14]) [14] 22000 ( sucrose density gradient sedimentation [5,12]) [5, 12] 23000 ( gel filtration [9]) [9] 25000 ( gel filtration [10]) [10] 28000 ( gel filtration [18]) [18] 34000 ( gel filtration, sucrose density gradient ultracentrifugation [4,7]) [4, 7, 16] 38200 ( sedimentation equilibrium centrifugation [6]) [6] 40000 ( gel filtration [3]) [3] 44000 ( gel filtration [13]; gel filtration, 2 forms: 44000 Da and 50000 Da, in presence of 5 mM MgCl2 dissociation into a single form of apparent MW 30000 [17]) [13, 17] 50000 ( gel filtration, in absence of Mg2+ the enzyme elutes as 2 distinct forms [25]; gel filtration, 2 forms: 44000 Da and 50000 Da, in presence of 5 mM MgCl2 dissociation into a single form of apparent MW 30000 [17]) [17, 25] 54000 ( native PAGE [21]; gel filtration [2,15]) [2, 15, 21] Subunits ? ( x * 29000, SDS-PAGE [28]; x * 17500, SDS-PAGE [5,12]; x * 19481, determination of amino acid sequence [19]; x * 20000-20370, recombinant His-tagged enzyme, SDS-PAGE and DNA sequence determination [23]; x * 28000, SDS-PAGE [26]) [5, 12, 19, 23, 26, 28] 88

2.4.2.7


dimer ( 1 * 20000 + 1 * 34000, SDS-PAGE [25]; 2 * 17000-20000, SDS-PAGE, gel filtration in guanidine hydrochloride, peptide mapping data suggest that the subunits are quite similar if not identical [6]; 2 * 15000, SDS-PAGE [18]; 2 * 23000, SDS-PAGE [13]; 2 * 27000, SDS-PAGE [15]; 2 * 28000, SDS-PAGE [21]) [2, 6, 13, 15, 18, 20-22, 24, 25] trimer ( 3 * 11100, SDS-PAGE [7]) [7] Additional information ( three-dimensional structure, subunit structure [24]; three-dimensional structure, apo-enzyme in complexes with AMP, adenine, sulfate, citrate, subunit model [22,27]; three-dimensional structure, dimeric [20]; association of subunits in presence of 5-phospho-a-d-ribose 1-diphosphate [11]) [11, 20, 22, 24, 27]

5 Isolation/Preparation/Mutation/Application Source/tissue F28-7 cell ( cell line derived from FM3A [13]) [13] FM3A cell ( mammary carcinoma cell line [13]) [13] cell culture [8] cyst [18] erythrocyte [4, 6, 7, 19] flower [26] germ [9] latex [21] leaf [2, 15, 26] liver [1, 5, 12] nauplius [18] promastigote [10, 28] trophozoite [14] Additional information ( human enzyme found in all tissues with the highest specific activity in nucleated cells [4]) [4] Localization chloroplast [26] cytosol ( exclusively [4]) [4, 9, 18, 21, 26] membrane [3] nucleus [26] Purification [8] (partial [9]) [9] [15] [1] [2] [3] [4, 6, 7, 19] 89


2.4.2.7

[1, 5, 12] (recombinant from Escherichia coli [20]) [10, 20, 28] [13] [16] [17] [18] [21] (recombinant His-tagged enzyme from Escherichia coli [23]) [23] (recombinant His-tagged wild-type and mutants from Escherichia coli [25]) [25] (3 isoforms recombinant from Escherichia coli as His-tagged proteins [26]) [26] Crystallization (vapour diffusion method, hanging drops from solution: 10 mg/ml purified apo-enzyme in 10 mM MES, pH 6.0, 1 mM dithiothreitol, 5 mM MgCl2 , 4 C, reservoir solution: 7-11% polyethylene glycol 5000 monomethyl ether, 0.2 M ammonium acetate, 0.1 M sodium citrate, pH 4.9, 10 mM MgCl2 , 1.21.6 M ammonium sulfate, for AMP- or adenine-bound crystals addition of 10 mM AMP or 5 mM adenine in the reservoir solution, structure analysis [22]) [22] (enzyme, 10 mg/ml, in complex with 9-deazaadenine and sulfate or Mgphosphoribosyldiphosphate, 50 mM Hepes, pH 6.0, 8 mM MgCl2 , 1 mM DTT, 1:2 molar ratio of 9-deazaadenine and iminoribitol, 1 mM sodium diphosphate, after 45 min incubation preparation of crystallization drops, crystals are obtained from mother liquid 0.1 M sodium acetate, pH 4.6, 24% polyethylene glycol 4000, 0.2 M ammonium sulfate, 0.05 M urea, 18 C, X-ray diffraction structure analysis, hydrogen bond network in the complexes [24]) [24] (mixing of protein solution 13-15 mg/ml with an equal volume of mother liquid 0.1 M Hepes, pH 7.5, 1.5 M lithium sulfate, then equilibration against mother liquid at 18 C, crystals appear after 3 days, X-ray diffraction structure analysis, also crystallization of the enzyme in presence of diphosphate, Mg2+ or inhibitor immucillin, which do not bind at the active site [27]) [27] Cloning (expression in Escherichia coli [20]) [20] (cloning and DNA sequence determination, expression in Escherichia coli BL21 cells as C-terminally His-tagged protein [23]) [23] (expression of wild-type and mutants as His-tagged proteins in Escherichia coli B25 cells [27]) [27] (overexpression of the 3 isoforms in Escherichia coli Bl21(DE3)pLysS as His-tagged proteins, the majority of the recombiannt enzyme is in the soluble fraction [26]) [26]

90

2.4.2.7


Engineering E106L ( site-directed mutagenesis, decreased turnover, increased Km value for adenine and decreased Km value for 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] E106Q ( site-directed mutagenesis, decreased turnover and Km value for 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] F25W ( site-directed mutagenesis, tryptophan at the adenine binding site, kinetic constants similar to the wild-type [23]) [23] G108A ( site-directed mutagenesis, increased Km value for adenine and 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] G108H ( site-directed mutagenesis, decreased turnover, slightly increased Km value for adenine and 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] K90A ( site-directed mutagenesis, decreased turnover compared to the wild-type [27]) [27] K93A ( site-directed mutagenesis, decreased turnover, increased Km value for adenine and 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] R69A ( site-directed mutagenesis, decreased turnover, increased Km value for adenine compared to the wild-type [27]) [27] R89A ( site-directed mutagenesis, decreased turnover, increased Km value for adenine and 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] Y103F ( site-directed mutagenesis, increased turnover, increased Km value for adenine and 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] Y107D ( site-directed mutagenesis, decreased turnover, increased Km value for adenine compared to the wild-type [27]) [27] Y107F ( site-directed mutagenesis, decreased turnover, increased Km value for adenine and 5-phosphoribose 1-phosphate compared to the wild-type [27]) [27] Additional information ( alignment of amino acid sequences, correlation between human clinical missense mutations and structure, structure taken from Leishmania donovani enzyme [22]) [22]

6 Stability Temperature stability 0 ( in absence of Mg2+ and donor substrate, loss of 50% activity after 18 min [10]) [10] 37 ( in absence of Mg2+ and donor substrate, loss of 97% activity after 10 min [10]) [10] 50 ( 40 min, 5 mM MgCl2 , more than 90% loss of activity, + 0.4 mM 5-phospho-a-d-ribose 1-diphosphate, 30% loss of activity [11]) [11]

91


2.4.2.7

58 ( heat denaturation, 35% ammonium sulfate protects [2]) [2] 60 ( 5 min, stable [3]; 10 min, stable in presence of 10 mM AMP [17]) [3, 17] General stability information , unstable in absence of Mg2+ and glycerol, loss 0f 70% activity during gel filtration [2] , instability after calcium phosphate gel treatment [3] , ammonium sulfate, 1 M, partially protects against inactivation during storage [7] , dithiothreitol, 10 mM, or dimethylsulfoxide, 5%, or 5-phospho-a-d-ribose 1-diphosphate, 1 mM, protects against inactivation during storage [7] , Mg2+ stabilizes [12] , dialysis against 20 mM Tris-HCl, pH 7.5, 20 mM (NH4 )2 SO4 + 5 mM 2mercaptoethanol inactivates [12] , inactivation by isoelectric focusing [10] , 5-phospho-a-d-ribose 1-diphosphate stabilizes Mycoplasma mycoides enzyme markedly [11] , bovine serum albumin or adenine stabilizes Mycoplasma mycoides enzyme slightly [11] , freezing and thawing have little effect on E. coli enzyme, but inactivate Mycoplasma mycoides enzyme [11] Storage stability , -80 C, stable for at least 3 weeks [8] , unstable at 4 C or at -20 C [8] , -70 C, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2 , 10 mM KCl, 10% glycerol, stable [15] , -15 C, stable for months [11] , -20 C, purified enzyme, 2-3 weeks, considerable loss of activity [3] , -79 C, purified enzyme, 0.1 mg/ml protein, 0.1 M potassium phosphate buffer, pH 7, stable for several months [3] , -70 C, purified enzyme, 5 mM MgCl2 , 0.1 mM 5-phospho-a-d-ribose 1diphosphate, stable for upt to 2 months [7] , 2-4 C, purified enzyme in the ampholine gradient solution, half-life of 2-4 weeks, dilution inactivates [5, 12] , -70 C, purified enzyme, at least 6 months [10] , -20 C, purified enzyme, 6 mM 5-phospho-a-d-ribose 1-diphosphate, 2 mM MgCl2 , stable for at least a month [17]

References [1] Flaks, J.G.; Erwin, M.J.; Buchanan, J.M.: Biosynthesis of the purines. XVI. The synthesis of adenosine 5'-phosphate and 5-amino-4-imidazolecarboxamide ribotide by a nucleotide pyrophosphorylase. J. Biol. Chem., 228, 201213 (1957)

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[2] Lee, D.; Moffatt, B.A.: Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana. Physiol. Plant., 87, 483492 (1993) [3] Hochstadt, J.: Adenine phosphoribosyltransferase from Escherichia coli. Methods Enzymol., 51, 558-567 (1978) [4] Arnold, W.J.; Kelley, W.N.: Adenine phosphoribosyltransferase. Methods Enzymol., 51, 568-574 (1978) [5] Groth, D.P.; Young, L.G.; Kenimer, J.G.: Adenine phosphoribosyltransferase from rat liver. Methods Enzymol., 51, 574-580 (1978) [6] Holden, J.A.; Meredith, G.S.; Kelley, W.N.: Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition, and peptide mapping. J. Biol. Chem., 254, 6951-6955 (1979) [7] Thomas, C.B.; Arnold, W.J.; Kelley, W.N.: Human adenine phosphoribosyltransferase. Purification, subunit structure, and substrate specificity. J. Biol. Chem., 248, 2529-2535 (1973) [8] Hirose, F.; Ashihara, H.: Adenine phosphoribosyltransferase of Catharanthus roseus cells: purification, properties and regulation. Z. Pflanzenphysiol., 110, 135-145 (1983) [9] Chen, C.-M.; Melitz, D.K.; Clough, F.W.: Metabolism of cytokinin: phosphoribosylation of cytokinin bases by adenine phosphoribosyltransferase from wheat germ. Arch. Biochem. Biophys., 214, 634-641 (1982) [10] Tuttle, J.V.; Krenitsky, T.A.: Purine phosphoribosyltransferases from Leishmania donovani. J. Biol. Chem., 256, 909-916 (1980) [11] Sin, I.L.; Finch, L.R.: Adenine phosphoribosyltransferase in Mycoplasma mycoides and Escherichia coli. J. Bacteriol., 112, 439-444 (1972) [12] Kenimer, J.G.; Young, L.G.; Groth, D.P.: Purification and properties of rat liver adenine phosphoribosyltransferase. Biochim. Biophys. Acta, 384, 87101 (1975) [13] Okada, G.; Kaneko, I.; Koyama, H.: Purification and characterization of adenine phosphoribosyltransferase from mouse mammary carcinoma FM3A cells in culture. Biochim. Biophys. Acta, 884, 304-310 (1986) [14] Queen, S.A.; Vander Jagt, D.L.; Reyes, P.: Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum. Biochim. Biophys. Acta, 996, 160-165 (1989) [15] Moffatt, B.A.; Somerville, C.R.: Purification of adenine phosphoribosyltransferase from Brassica juncea. Arch. Biochem. Biophys., 283, 484-490 (1990) [16] Walter, R.D.; Koenigk, E.: Hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase from Plasmodium chabaudi, purification and properties. Tropenmed. Parasitol., 25, 227-235 (1974) [17] Nagy, M.; Ribet, A.-M.: Purification and comparative study of adenine and guanine phosphoribosyltransferases from Schizosaccharomyces pombe. Eur. J. Biochem., 77, 77-85 (1977) [18] Montero, C.; LLorente, P.: Artemia purine phosphoribosyltransferases. Purification and characterization. Biochem. J., 275, 327-334 (1991)

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[19] Wilson, J.M.; O'Toole, T.E.; Argos, P.; Shewach, D.S.; Daddona, P.E.; Kelley, W.N.: Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme. J. Biol. Chem., 261, 13677-13683 (1986) [20] Bashor, C.; Denu, J.M.; Brennan, R.G.; Ullman, B.: Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani. Biochemistry, 41, 4020-4031 (2002) [21] Gallois, R.; Prevot, J.-C.; Clement, A.; Jacob, J.-L.: Purification and characterization of an adenine phosphoribosyltransferase from rubber tree latex. Physiological implications. Plant Physiol. Biochem., 34, 527-537 (1996) [22] Phillips, C.L.; Ullman, B.; Brennan, R.G.; Hill, C.P.: Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani. EMBO J., 18, 3533-3545 (1999) [23] Sarver, A.E.; Wang, C.C.: The adenine phosphoribosyltransferase from Giardia lamblia has a unique reaction mechanism and unusual substrate binding properties. J. Biol. Chem., 277, 39973-39980 (2002) [24] Shi, W.; Sarver, A.E.; Wang, C.C.; Tanaka, K.S.; Almo, S.C.; Schramm, V.L.: Closed site complexes of adenine phosphoribosyltransferase from Giardia lamblia reveal a mechanism of ribosyl migration. J. Biol. Chem., 277, 39981-39988 (2002) [25] Alfonzo-Garcia, J.; Sahota, A.; Taylor, M.W.: Characterization of the adenine phosphoribosyltransferase from Saccharomyces cerevisiae. Adv. Exp. Med. Biol., 370, 627-630 (1994) [26] Allen, M.; Qin, W.; Moreau, F.; Moffatt, B.: Adenine phosphoribosyltransferase isoforms of Arabidopsis and their potential contributions to adenine and cytokinin metabolism. Physiol. Plant., 115, 56-68 (2002) [27] Shi, W.; Tanaka, K.S.E.; Crother, T.R.; Taylor, M.W.; Almo, S.C.; Schramm, V.L.: Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae. Biochemistry, 40, 10800-10809 (2001) [28] Allen, T.; Henschel, E.V.; Coons, T.; Cross, L.; Conley, J.; Ullman, B.: Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase activities from Leishmania donovani. Mol. Biochem. Parasitol., 33, 273-281 (1989)

94

Hypoxanthine phosphoribosyltransferase

2.4.2.8

1 Nomenclature EC number 2.4.2.8 Systematic name IMP:diphosphate phospho-d-ribosyltransferase Recommended name hypoxanthine phosphoribosyltransferase Synonyms 6-hydroxypurine phosphoribosyltransferase 6-mercaptopurine phosphoribosyltransferase GMP pyrophosphorylase GPRT ( phosphoribosyltransferases for hypoxanthine and guanine are separate enzymes [10]) [10] HGPRT HGPRTase HPRT ( phosphoribosyltransferases for hypoxanthine and guanine are separate enzymes [10]) [10] IMP pyrophosphorylase IMP-GMP pyrophosphorylase guanine phosphoribosyltransferase guanine-hypoxanthine phosphoribosyltransferase guanosine 5'-phosphate pyrophosphorylase guanosine phosphoribosyltransferase guanylate pyrophosphorylase guanylic pyrophosphorylase hypoxanthine-guanine phosphoribosyltransferase inosinate pyrophosphorylase inosine 5'-phosphate pyrophosphorylase inosinic acid pyrophosphorylase inosinic pyrophosphorylase phosphoribosyltransferase, 6-mercaptopurine phosphoribosyltransferase, hypoxanthine purine-6-thiol phosphoribosyltransferase transphosphoribosidase CAS registry number 9016-12-0

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2 Source Organism

Schistosoma mansoni [21, 23] Mus musculus [17] Bos taurus [1, 2] Rattus norvegicus (3 charge variant forms [14]) [14] Plasmodium lophurae [7] Homo sapiens (purified enzyme [37]; construction of 4 chimeric enzyme with segments of human and Plasmodium falciparum enzymes [35]; 3 charge variant forms [8,9]) [8, 9, 14, 15, 18, 24, 28, 33, 35, 37, 38, 40] Schizosaccharomyces pombe [3] Artemia sp. [4] Plasmodium chabaudi [5] Leishmania donovani (parasites isolated from rabbits [32]) [6, 27, 32] Giardia lamblia (strain Portland I, 3 charge variant forms [22]) [22, 34] Streptomyces cyanogenus [12] Saccharomyces cerevisiae [13, 20] Cricetulus griseus (at least 3 charge variant isoforms [9,16]) [9, 16] Escherichia coli (2 charge variant isoforms with different substrate specificities [19]; strain K12 [10]; phosphoribosyltransferases for hypoxanthine and guanine are separate enzymes [10]) [10, 19] Salmonella typhimurium (strain LT-2 [10]; phosphoribosyltransferases for hypoxanthine and guanine are separate enzymes [10]) [10] Gallus gallus [11] Toxoplasma gondii [25, 26, 36] Trichomonas foetus [29, 31, 41, 42] Cryptosporidium parvum (in MDCK cells [30]) [30] Plasmodium falciparum (construction of 4 chimeric enzymes with segments of human and Plasmodium falciparum enzymes [35]) [33, 35] Trypanosoma cruzi [38] Leishmania tarentolae (gene hgprt [39]) [39]

3 Reaction and Specificity Catalyzed reaction IMP + diphosphate = hypoxanthine + 5-phospho-a-d-ribose 1-diphosphate (guanine and 6-mercaptopurine can replace hypoxanthine; during catalysis a long flexible loop closes over the active site, functional role [38]; residues I99-L121 form the catalytic loop [37]; kinetic mechanism [28]; active site structure [25, 26, 31, 36, 38, 41, 42]; catalytic mechanism [25, 26, 28, 31, 36, 37, 41]; ordered bi-bi mechanism [29, 34]; kinetic model [29]; Thr47 binds diphosphate [29]; essential role of Ser95-Tyr96 dyade in catalysis [27]) Reaction type pentosyl group transfer

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Natural substrates and products S guanine + 5-phospho-a-d-ribose 1-diphosphate [2730, 32, 36] P GMP + diphosphate S hypoxanthine + 5-phospho-a-d-ribose 1-diphosphate [27-30, 32, 36, 38] P IMP + diphosphate S Additional information ( salvage incorporation of exogenous purine nucleotides, no de novo synthesis [29, 30, 32, 36, 38, 39]; no incorporation of xanthine in vivo [30]; enzyme is essential for salvaging exogenous purine bases [29, 30, 39]; locus of Lesch-Nyhan syndrome, activator of the prodrugs 6-mercaptopurine and allopurinol [28, 38]) [28-30, 32, 36, 38, 39] P ? Substrates and products S 2-amino-6-mercaptopurine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [19]) [19] P 2-amino-6-mercaptopurine ribotide + diphosphate S 2-hydroxy-6-mercaptopurine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [19]) [19] P 2-hydroxy-6-mercaptopurine ribotide + diphosphate S 6-mercaptopurine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [1,2,7,19]) [1, 2, 7, 19] P 6-mercaptopurine ribotide + diphosphate [2] S 6-thioguanine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [7]) [7] P 6-thio-GMP + diphosphate S 8-azahypoxanthine + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [7]) [7] P 8-aza-IMP + diphosphate S allopurinol + 5-phospho-a-d-ribose 1-diphosphate ( low activity [33]) (Reversibility: ? [33]) [33] P allopurinol ribonucleoside 5'-monophosphate + diphosphate [33] S guanine + 5-phospho-a-d-ribose 1-diphosphate ( absolute specific for guanine [34]; guanine is utilized more rapidly than hypoxanthine [28]; binds hypoxanthine 67-times less effectively than guanine and 4-times less effectively than xanthine [19]; phosphoribosyltransferases for hypoxanthine and guanine are separate enzymes, and catalyse the 2 reactions with different specificities [10]) (Reversibility: r [1, 27-29, 31, 32, 34, 36-38, 41]; ? [2-25, 30, 33, 35, 39, 40]) [1-41] P GMP + diphosphate ( diphosphate binds at Thr70, diphosphate-binding loop sequence: LTGA [34]) [1-41] S hypoxanthine + 5-phospho-a-d-ribose 1-diphosphate ( 2 assay methods [1]; no activity with hypoxanthine [34]; most

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P S

P S

P

2.4.2.8

effective substrate [29]; unlike the hypoxanthine-guanine phosphoribosyltransferase from other sources this enzyme binds hypoxanthine 67times less effectively than guanine and 4-times less effectively than xanthine [19]; equilibrium lies far in direction of IMP formation [1, 28]; phosphoribosyltransferases for hypoxanthine and guanine are separate enzymes, and catalyse the 2 reactions with different specificities [10]) (Reversibility: r [1, 27-29,31, 32, 3638, 41]; ? [2-26, 30, 33, 35, 39, 40]) [1-33, 35-41] IMP + diphosphate ( diphosphate is bound by Thr47 [29]) [1-33, 35-41] xanthine + 5-phospho-a-d-ribose 1-diphosphate ( recombinant chimeric mutant DS1 [35]; poor substrate [12]; no activity [1, 20, 34, 35]) (Reversibility: r [29, 31]; ? [10, 12, 19, 22, 25, 30, 33, 35]) [10, 12, 19, 22, 25, 26, 29-31, 33, 35] XMP + diphosphate [25, 26, 29-31, 33, 35] Additional information ( a mentally retarded child and its asymptomatic uncle have a partial enzyme deficiency, homozygous, while mother and grandmother are heterozygous and not enzyme-deficient [40]; forward reaction: transfer of the phosphoribosyl group to N9 position of 6-oxopurines [38]; dynamic and conformational properties of purified enzyme alone, in complex with GMP and Mg2+ , and in equilibration mixture of enzyme with IMP, Mg2+ /diphosphate and hypoxanthine, Mg2+ /5-phosphoribosyl 1-diphosphate, and in transition-state analogue complex of enzyme, immucillin-GP and Mg2+ / diphosphate [37]; 9-deazaguanine is no substrate [36]; kinetic study [23, 27, 28, 31]; no substrate: adenine [1, 12, 20]; no substrates: 5-formamido-4-imidazolecarboxamide, uric acid, 8-azaguanine, 2,6-diaminopurine, orotic acid, phosphate [1]) [1, 12, 20, 31, 36-38, 40] ?

Inhibitors 2-amino-6-mercaptopurine ( competitive to hypoxanthine [5]) [5] 5-phospho-a-d-ribose 1-diphosphate ( product inhibition [29,34]; competitive against GMP [34]) [29, 34] 6-aminopurine nucleotides [10] 6-chloropurine [7] 6-hydroxypurine nucleotides [10] 6-mercaptopurine ( competitive to hypoxanthine [5]) [5] 6-thioguanine ( competitive to hypoxanthine [5,20]) [5, 20] 6-thioinosine [7] 9-b-arabinofuranosylhypoxanthine [7] AMP ( mutant K134S, competitive [31]; no inhibition [16]) [3, 31]

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ATP [12] Ba2+ ( strong [10]) [4, 10] CDP [3] Ca2+ ( strong [4,10]) [4, 10] EDTA [12] GDP [3, 12] GMP ( product inhibition [16,29,34]; competitive against 5-phospho-a-d-ribose 1-diphosphate [34]; mixed inhibition [4]) [3, 4, 6, 12, 16, 29, 34] GTP [3, 6, 12] Hg2+ ( complete inhibition at 3 mM after 3 min at 0 C [33]) [33] IDP [3, 12] IMP ( product inhibition [16,29]) [3, 12, 16, 29] ITP ( weak [3]) [3] KCl ( inactivation [33]; no inhibition [33]) [33] Mg2+ ( inhibitory effects are noncompetitive against 5-phosphoribose 1-diphosphate [3]) [3] UDP ( weak [3]) [3] XMP ( product inhibition [29]) [3, 29] Zn2+ ( strong [10]) [10] adenine ( no inhibition [30]) [20] azaguanine [7] diethyl dicarbonate ( alkylation of Arg155, complete inactivation at pH 9.0, pH dependent [27]) [27] diphosphate ( product inhibition [29]) [3, 6, 29] guanine ( competitive [30]; competitve against hypoxanthine [13,20]; substrate inhibition [4]) [4, 13, 20, 30] hypoxanthine ( competitive [30]; competitive against guanine [13]; substrate inhibition [4]) [4, 13, 30] immucillin-H ( transition state analogue, binds tightly to the active site, inhibition mechanism and kinetics [41]) [41] immucillin-H 5'-phosphate ( transition state analogue, binds tightly to the active site, inhibition mechanism and kinetics [41]) [41] inosine ( slight inhibition [16]) [16] nucleotides ( all free 5'-nucleotides are inhibitory, 6-OH purine nucleotides are most inhibitory, while 6-NH2 purine only at high concentrations [10]) [10] p-chloromercuribenzoate ( reversed by dithiothreitol or 2-mercaptoethanol [19]) [19] phenylglyoxal ( irreversible, complete inactivation, alkylation of Arg155, GMP protects, no alkylation of mutant R155K [31]) [31] ppGpp ( strong [10]) [10] purine nucleotides ( and analogues, overview [19]) [12, 19] tetranitromethane ( complete inactivation at pH 9.0, pH dependent, modifies Tyr96 in the active site [27]) [27] xanthine ( weak, competitive [30]) [7, 20, 30] 99


2.4.2.8

Additional information ( no effect of iodoacetate, phenylglyoxal, p-chloromercuribenzoate, acetic anhydride, ethyl dimethylaminopropylcarbodiimide/ammonium acetate, and diisopropyl fluorophosphate [27]; mechanism of product inhibition [3]; no inhibition by ADP, ATP, dAMP, UMP, and UTP [3]; overview: inhibition constant of purines and purine analogs [19]) [3, 19, 27] Activating compounds acetate ( slightly activating [10]) [10] Metals, ions Mg2+ ( required [1, 4, 9, 13, 28, 38, 41]; absolute requirement [3]; absolute requirement for Mg2+ or Mn2+ [10, 11]; Km : 0.04-0.05 mM [4]; activating effect of Mn2+ is higher than that of Mg2+ [12]; maximal activity at: 0.5-1 mM MgCl2 [3]; maximal activity at: 1-10 mM [13]; maximal activity at: 1 mM [4]) [1, 3, 4, 9-13, 23, 27, 28, 30, 33-38, 41] Mn2+ ( absolute requirement for Mg2+ or Mn2+ [10, 11]; can replace Mg2+ [4]; Km : 0.015-0.020 mM [4]; activating effect of Mn2+ is higher than that of Mg2+ [12]) [4, 10-12] Zn2+ ( activates, can replace Mg2+ , Km : 0.066-0.075 mM [4]) [4] Turnover number (min±1) 0.06 (inosine monophosphate, loop II-deletion mutant [38]) [38] 0.12 (diphosphate, loop II-deletion mutant [38]) [38] 0.78 (guanine, purified recombinant chimeric enzyme DS1 [35]) [35] 2.34 (guanine, loop II-deletion mutant [38]) [38] 3 (5-phosphoribosyl 1-diphosphate, loop II-deletion mutant [38]) [38] 3.6 (hypoxanthine, loop II-deletion mutant [38]) [38] 3.72 (xanthine, purified recombinant chimeric enzyme DS1 [35]) [35] 4.38 (hypoxanthine, purified recombinant chimeric enzyme DS1 [35]) [35] 13.8-18 (inosine monophosphate, recombinant enzyme, reverse reaction [28]) [28] 28.3 (inosine monophosphate, wild-type enzyme [38]) [38] 28.6 (diphosphate, wild-type enzyme [38]) [38] 144 (hypoxanthine, native enzyme [23]) [23] 156 (hypoxanthine, recombinant enzyme [23]) [23] 258 (guanine, native enzyme [23]) [23] 300 (guanine, recombinant enzyme [23]) [23] 342 (guanine, wild-type enzyme [34]) [34] 342 (hypoxanthine, wild-type enzyme [32,35]) [32, 35] 348 (GMP, wild-type enzyme, reverse reaction [34]) [34] 372 (hypoxanthine, mutant enzyme [32]) [32] 402.6 (hypoxanthine, mutant T47K [29]) [29]

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510-558 (hypoxanthine, recombinant enzyme [28]) [28] 512.4 (hypoxanthine, wild-type enzyme [27]) [27] 628.8 (guanine, wild-type enzyme [35]) [35] 726 (guanine, wild-type enzyme [32]) [32] 948 (guanine, mutant enzyme [32]) [32] 1374 (hypoxanthine, wild-type enzyme [38]) [38] 1392 (5-phosphoribosyl 1-diphosphate, wild-type enzyme [38]) [38] 1974 (guanine, wild-type enzyme [38]) [38] 2178 (5-phospho-a-d-ribose 1-diphosphate, wild-type enzyme [27]) [27] 2476 (guanine, wild-type enzyme [27]) [27] 4488 (5-phospho-a-d-ribose 1-diphosphate, wild-type enzyme, forward reaction [34]) [34] 4602 (guanine, wild-type, forward reaction [34]) [34] 4620 (inosine monophosphate, wild-type enzyme [27]) [27] 5400 (diphosphate, wild-type enzyme [27]) [27] Additional information ( kcat of mutant enzymes [34]; kinetics [28]; wild-type and mutants: kcat for substrates hypoxanthine, guanine, xanthine, IMP, GMP, XMP, diphosphate, 5-phosphoribosyl 1-diphosphate [31]) [28, 31, 34] Specific activity (U/mg) 0.032 ( purified recombinant chimeric enzyme DS1, substrate guanine [35]) [35] 0.108 ( substrate xanthine [30]) [30] 0.147 ( purified recombinant chimeric enzyme DS1, substrate xanthine [35]) [35] 0.226 ( purified recombinant chimeric enzyme DS1, substrate hypoxanthine [35]) [35] 0.27 ( purified recombinant enzyme, substrate allopurinol, pH 8.0 [33]) [33] 0.28 ( substrate hypoxanthine [30]) [30] 0.346 ( substrate guanine [30]) [30] 0.39 ( purified enzyme, reverse reaction [1]) [1] 0.57-0.76 ( recombinant enzyme, reverse reaction [28]) [28] 0.65 ( purified enzyme [4]) [4] 1.4 ( purified recombinant enzyme, substrate hypoxanthine, pH 8.0 [33]) [33] 3 ( purified recombinant enzyme, substrates allopurinol and xanthine, pH 8.0 [33]; about, purified enzyme [17]) [17, 33] 5.4 ( purified recombinant enzyme, substrate guanine, pH 8.0 [33]; purified enzyme, substrate hypoxanthine [23]) [23, 33] 5.75 [3] 5.9 ( recombinant enzyme, substrate hypoxanthine [23]) [23] 7.6 ( purified enzyme, substrate guanine [23]) [23] 8.1 ( recombinant enzyme, substrate guanine [23]) [23]

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9 ( purified enzyme, brain [9,16]) [9, 16] 9.25 ( substrate hypoxanthine [21]) [21] 11.9 ( purified recombinant enzyme, substrate hypoxanthine [35]) [35] 13.25 ( substrate guanine [21]) [21] 17.5 ( purified enzyme, erythrocytes [9,18]) [9, 18] 21-23 ( recombinant enzyme, forward reaction [28]) [28] 25.8 ( purified recombinant enzyme, substrate guanine [35]) [35] 27 ( purified recombinant enzyme, substrate hypoxanthine, pH 8.0 [33]) [33] 46 ( purified recombinant enzyme, substrate guanine, pH 8.0 [33]) [33] 66.5 ( purified enzyme [12]) [12] 698.1 ( purified enzyme, method 1 [11]) [11] 705 ( purified enzyme, immunopurification [11]) [11] Additional information ( kinetics in forward and reverse reaction [29, 38]; kinetics [23, 27, 28, 31]) [2, 10, 13, 20, 22-24, 27-29, 31, 38] Km-Value (mM) 0.00052 (hypoxanthine) [9, 16] 0.0009 (hypoxanthine, recombinant enzyme, with other purine [33]) [33] 0.001 (guanine, below [4]) [4] 0.001 (hypoxanthine, below [4]; with 6-mercaptopurine [1]; wild-type enzyme [35]) [1, 4, 35] 0.0011 (guanine, recombinant chimeric mutant DS1 [35]) [9, 16, 35] 0.0014 (guanine, recombinant enzyme, with other purine [33]) [33] 0.0014 (hypoxanthine, recombinant chimeric mutant DS1 [35]) [35] 0.0016 (hypoxanthine) [12] 0.0018 (guanine) [11] 0.0019 (guanine, recombinant enzyme, with other purine [33]) [33] 0.002 (guanine) [5] 0.0021 (guanine, native enzyme [23]) [23] 0.0024 (guanine) [7] 0.0024 (hypoxanthine, recombinant enzyme [28]) [28] 0.0025 (hypoxanthine) [5] 0.0027 (guanine, native enzyme [23]) [12, 23] 0.0028 (guanine, recombinant enzyme, with 5-phospho-a-d-ribose 1-diphosphate [39]) [39] 0.0031 (hypoxanthine, recombinant enzyme, with other purine [33]) [33] 0.0035 (guanine, recombinant enzyme [28]) [28] 0.0037 (hypoxanthine, native enzyme [23]) [23]

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0.0038 (hypoxanthine) [7] 0.0042 (hypoxanthine, recombinant enzyme [23]) [23] 0.0044 (hypoxanthine, recombinant enzyme, with 5-phospho-ad-ribose 1-diphosphate [39]) [39] 0.0048 (hypoxanthine, mutant enzyme [32]) [32] 0.005 (guanine, wild-type enzyme [27,35]) [27, 35] 0.0052 (hypoxanthine) [11] 0.0053 (5-phospho-a-d-ribose 1-diphosphate) [9, 16] 0.0054 (inosine monophosphate, recombinant enzyme [28]) [28] 0.0061 (hypoxanthine, wild-type enzyme [27]) [27] 0.0062 (6-mercaptopurine) [7] 0.0064 (hypoxanthine, wild-type enzyme [32]) [32] 0.0076 (6-thioguanine) [7] 0.0086 (hypoxanthine, wild-type enzyme [38]) [38] 0.01 (guanine, wild-type enzyme [32]) [32] 0.011 (hypoxanthine) [20] 0.0117 (allopurinol, recombinant enzyme [33]) [33] 0.012 (guanine, wild-type enzyme [38]; mutant enzyme [32]) [32, 38] 0.015 (5-phospho-a-d-ribose 1-diphosphate) [4] 0.016 (guanine, wild-type enzyme [34]) [34] 0.018 (guanine) [13] 0.0225 (GMP, wild-type enzyme, reverse reaction [34]) [34] 0.023 (hypoxanthine) [13] 0.025 (diphosphate, recombinant enzyme, with inosine monophosphate [28]) [28] 0.026 (5-phospho-a-d-ribose 1-diphosphate, wild-type enzyme [34]) [34] 0.027 (inosine monophosphate, wild-type enzyme [38]) [38] 0.028 (guanine) [3] 0.032 (5-phospho-a-d-ribose 1-diphosphate, wild-type enzyme [38]) [38] 0.033 (5-phospho-a-d-ribose 1-diphosphate) [19] 0.0346 (adenine, mutant K134S [31]) [31] 0.035 (5-phosphoribosyl 1-diphosphate, recombinant enzyme, with hypoxanthine [28]) [28] 0.035 (diphosphate, wild-type enzyme [38]) [38] 0.037 (guanine) [19] 0.038 (hypoxanthine, mutant K134S [31]) [31] 0.05 (5-phospho-a-d-ribose 1-diphosphate) [13] 0.06 (hypoxanthine, recombinant enzyme, with 5-phosphoribosyl 1-diphosphate [33]) [33] 0.063 (5-phospho-a-d-ribose 1-diphosphate) [12] 0.065 (5-phosphoribosyl 1-diphosphate, recombinant enzyme, with guanine [28]) [28] 0.09 (inosine monophosphate, wild-type enzyme [27]) [27] 0.1 (5-phospho-a-d-ribose 1-diphosphate) [3] 103


2.4.2.8

0.103 (diphosphate, wild-type enzyme [27]) [27] 0.126 (hypoxanthine, recombinant enzyme, with 5-phosphoribosyl 1-diphosphate, pH 8.5 [33]) [33] 0.127 (5-phosphoribosyl 1-diphosphate, recombinant enzyme, with guanine [39]) [39] 0.134 (5-phospho-a-d-ribose 1-diphosphate, wild-type enzyme [27]) [27] 0.134 (guanine, recombinant enzyme, with 5-phosphoribosyl 1-diphosphate, pH 8.5 [33]) [33] 0.135 (allopurinol, recombinant enzyme [33]) [33] 0.138 (5-phosphoribosyl 1-diphosphate, recombinant enzyme, with hypoxanthine [39]) [39] 0.17 (xanthine, recombinant enzyme, with 5-phosphoribosyl 1diphosphate, pH 8.5 [33]) [33] 0.3 (xanthine, above, purified recombinant chimeric enzyme DS1 [35]) [35] 0.324 (diphosphate, wild-type enzyme, reverse reaction [34]) [34] 0.36 (8-azahypoxanthine) [7] 0.362 (guanine, recombinant enzyme, with 5-phosphoribosyl 1diphosphate [33]) [33] 0.42 (xanthine, recombinant enzyme, pH 8.5 [33]) [33] 0.5 (guanine) [1] Additional information ( Km -values, loop II-deletion mutant [38]; Km -values of mutant enzymes [34]; wild-type and mutants: Km values for substrates hypoxanthine, guanine, xanthine, IMP, GMP, XMP [31]; Km values of mutant enzymes for hypoxanthine, guanine, diphosphate, 5-phosphoribosyl 1-diphosphate, inosine monophosphate [27]; kinetics [16, 27, 28, 29]; wild-type and mutant T47K [29]) [6, 16, 27, 28, 29, 31, 34, 38] Ki-Value (mM) 0.0008 (6-thioguanine) [20] 0.004 (guanine) [20] 0.01 (guanine) [4] 0.018 (guanine) [13] 0.023 (hypoxanthine) [13] 0.0254 (ADP, mutant K134S, competitive versus 5-phospho-a-dribose 1-diphosphate [31]) [31] 0.08 (hypoxanthine) [4] 0.14 (adenine) [20] 0.14 (xanthine) [20] Additional information ( kinetics for product inhibition in forward and reverse reaction, wild-type enzyme [34]; kinetics of product inhibition in forward reaction [29]; overview, inhibition constants for diverse purine nucleotides and analogues [19]) [19, 29, 34]

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pH-Optimum 6-10 ( substrate hypoxanthine, broad maximum [7]) [7] 7 ( 2 buffer-independent pH-optima: pH 7.0 and pH 9.5 [4]) [4] 7-9 ( broad maximum [22]) [22] 7.4 ( assay at [1,6,35]) [1, 6, 35] 7.4-8.2 [19] 7.5 ( assay at [34]) [34] 7.5-9.5 ( substrate guanine, broad maximum [7]) [7] 7.6-8 ( 2 zones of pH-optima: pH 7.6-8.0 and pH 9.2-9.5 [3]) [3] 7.8 ( assay at [9,23,30]) [9, 23, 30] 7.9 ( guanine phosphoribosyltransferase [10]) [10] 8 ( assay at [27]) [27] 8.4 ( hypoxanthine phosphoribosyltransferase [10]) [10] 8.5 ( assay at [33]) [12, 13, 33] 8.6 ( substrate xanthine [10]) [10] 9.2-9.5 ( 2 zones of pH-optima: pH 7.6-8.0 and pH 9.2-9.5 [3]) [3] 9.5 ( 2 buffer-independent pH-optima: pH 7.0 and pH 9.5 [4]) [4] 10 [9, 11, 16] Additional information ( recombinant enzyme, pI: 8.2 [39]; 3 charge variant forms: pIs of 6.75, 5.3, 5.2 [22]; pI of 4.8 for the hypoxanthine specific enzyme, pI of 5.5 for the guanine specific enzyme [19]; 3 charge variant forms with pIs of 5.6, 5.85, 5.9 [14]; pI: 4.4 [12]; 3 charge variant forms with pIs of 5.6, 5.7 and 5.9 [9,18]; 3 charge variant forms with pIs of 6.2, 6.4 and 6.6 [9]; 3 charge variant forms with pIs of 5.7, 5.5 and 5.0 [8]; pI: 7.6 [7]; pI: 5.25 [5]) [5, 7, 8, 12, 14, 18, 19, 22, 39] pH-Range 5-9.5 [13] 5.5-11 [9, 16] 5.8-9.7 [12] 6.3-9.7 ( pH 6.3: about 50% of activity maximum, pH 9.7: about 75% of activity maximum [12]) [12] 7-8.5 ( pH 7.0: about 80% of activity maximum, pH 8.5: about 70% of activity maximum [19]) [19] Temperature optimum ( C) 25 ( assay at [33]) [33] 30 ( assay at [28]) [28] 37 ( assay at [4, 9, 11, 16-18, 21, 23, 24, 27, 30, 34]) [4, 9, 11, 16-18, 21, 23, 24, 27, 30, 34] 38 ( assay at [1]) [1]

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4 Enzyme Structure Molecular weight 42000 ( gel filtration in presence of MgCl2 [3]) [3] 48000 ( gel filtration in absence of MgCl2 [3]) [3] 50000 ( recombinant enzyme, gel filtration [39]) [39] 51000 ( gel filtration [13]) [13] 54500-54800 ( equilibrium sedimentation analysis, gel filtration [20]) [20] 58000-63000 ( gel filtration, isokinetic sucrose gradient centrifugation [22]) [22] 66000 ( gel filtration [4]) [4] 68000 ( gel filtration [15]) [15] 71000 [5] 72000 ( brain enzyme, gel filtration [14]) [14] 78000-85000 ( gel filtration [9,16]) [9, 16] 79000 [7] 80000 ( gel filtration [17]) [17] 80000-85000 ( gel filtration, acrylamide gel electrophoresis [9]) [9] 81000-83000 ( sedimentation equilibrium centrifugation [9,18]) [9, 18] 85000 ( gel filtration [11]; sedimentation equilibrium method [24]) [11, 24] 100000 ( 3 charge variant forms, native gradient gel electrophoresis [8]) [8] 105000 ( pore gradient electrophoresis [21]) [21] 150000 ( gel filtration [12]) [12] Subunits ? ( x * 24352-24353, recombinant mutant enzymes, mass spectroscopy and DNA sequence determination [33]; x * 26000, SDSPAGE [23]; x * 25000-27000, SDS-PAGE [14]; x * 24000, SDSPAGE [6]; x * 41000-45000, sedimentation equilibrium in presence of guanidine-HCl [24]; x * 26000, SDS-PAGE [24]) [6, 14, 23, 24, 33] dimer ( 2 * 23000, recombinant enzyme, SDSPAGE [39]; 2 * 26229-26232, in presence of KCl, SDS-PAGE, mass spectroscopy and DNA sequence determination [33]; 2 * 34000-34700, SDSPAGE [15]; 2 * 29000, SDS-PAGE [22]; 2 * 29500, SDS-PAGE [20]; 2 * 64000, SDS-PAGE [21]) [15, 20-22, 33, 39] monomer ( 1 * 51000, SDS-PAGE [13]) [13] octamer ( 8 * 18000, SDS-PAGE [12]) [12] tetramer ( 4 * 26229-26232, in absence of KCl, SDSPAGE, mass spectroscopy and DNA sequence determination [33]; 4 * 19000, SDS-PAGE [4]; 4 * 24000, SDS-PAGE [8]; 4 * 26000, SDSPAGE [11]) [4, 8, 11, 25, 26, 28, 33]

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2.4.2.8


trimer ( 3 * 25000, SDS-PAGE [9,16]; 3 * 27000, SDSPAGE [17]; 3 * 26000, SDS-PAGE [9,18]) [9, 16-18] Additional information ( stereoview of the three-dimensional structure of subunit, subunit interaction [37]; structure determination and analysis [25,26,36]) [25, 26, 36, 37] Posttranslational modification no glycoprotein ( no carbohydrate [15]; no glucosamine, sialic acid and hexose [24]) [15, 24]

5 Isolation/Preparation/Mutation/Application Source/tissue V-79 cell ( tissue culture cells [16]) [16] brain [8, 9, 11, 14, 16] cyst [4] erythrocyte ( of a mentally retarded child and its family members, partial enzyme deficiency [40]) [9, 14, 15, 18, 24, 28, 40] liver [1, 2, 16, 17] nauplius [4] promastigote [6, 32] sporulated oocyst [30] trophozoite [22] Localization cytosol [4] glycosome ( a fuel-metabolizing microbody unique to this parasite, exclusively [32]) [32] membrane [10] periplasm [10] Additional information ( the mutant enzyme, lacking the targeting signal, is located throughout the parasite, including subcellular organelles such as nucleus and flagellum [32]) [32] Purification [21, 23] [17] (partial [2]) [1, 2] [14] [7] (recombinant from Escherichia coli [33]; recombinant enzyme [28]; 2 methods [24]; from erythrocytes and from brain [9]; 3 isoenzymes [15]) [8, 9, 14, 15, 18, 24, 28, 33] [3] [4] [5]

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(recombinant wild-type and mutant from Escherichia coli [32]; recombinant from Escherichia coli [27]) [6, 27, 32] (recombinant wild-type enzyme and mutants from Escherichia coli [34]) [22, 34] [12] [13, 20] (from brain and liver [16]) [9, 16] [10] [10] (2 methods [11]) [11] (recombinant enzyme [36]) [36] (recombinant from Escherichia coli [41]; recombinant wild-type enzyme and mutant/s from Escherichia coli [29,31]) [29, 31, 41] (recombinant from Escherichia coli , large scale [33]) [33] (recombinant wild-type enzyme and loop II-deletion mutant from Escherichia coli [38]) [38] (recombinant from Escherichia coli [39]) [39] Crystallization (the recombinant enzyme is complexed with Mg2+ , 5-phosphoribosyl 1diphosphate and inactive substrate analogue 9-deazaguanine, hanging drop method, enzyme complex, 20 mg/ml, is precipitated by 0.1 M Tris-HCl, pH 8.0, 30% polyethylene glycol 4000, 0.2 M Li2 SO4, 0.5% b-octylglucoside at 4 C, X-ray diffraction structure analysis [36]; mutant enzyme D150A, crystallization complexed with xanthosine 5'-monophosphate, diphosphate and 2 Mg2+ , post transition state structure analysis, active site structure [25]; crystallization in complex with GMP and IMP, structure analysis [26]) [25, 26, 36] (crystallization and X-ray structure determination and analysis, 1 molecule of GMP bound per dimer [42]; analysis of crystal structure [29]) [29, 42] (recombinant enzyme, 7 mg/ml, hanging-drop vapour-diffusion method, TMD buffer, pH 7.5, + equal volume of reservoir solution: 18 C or 4 C, pH 5.6 , 19% isopropanol, 19% polyethylene glycol 4000, 5% glycerol, or 17% polyethylene glycol 4000, 5% glycerol [39]) [39] Cloning (DNA sequence determination, expression in Escherichia coli [23]; mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone [21]) [21, 23] (expression of wild-type and chimeric enzymes in enzyme-deficient Escherichia coli strain, complementation study [35]; overexpression in Escherichia coli [33,35]) [28, 33, 35] (functional expression of wild-type enzyme and mutants in an enzymedeficient Escherichia coli strain [27,32]) [27, 32] (functional overexpression of wild-type enzyme and expression of mutants in Escherichia coli [34]) [34] [36]

108

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(functional expression in Escherichia coli [41]; functional expression of wild-type enzyme and mutants in an enzyme-deficient Escherichia coli strain [31]; functional expression of wild-type and mutant T47K in an enzyme-deficient Escherichia coli strain [29]) [29, 31, 41] (expression of wild-type and chimeric enzymes in enzyme-deficient Escherichia coli strain, complementation study, overexpression of chimeric mutant DS1 [35]; overexpression in an enzyme-deficient Escherichia coli strain [33]) [33, 35] (functional expression of wild-type and loop II-deletion mutant in Escherichia coli [38]) [38] (DNA and amino acid sequence determination and analysis, functional expression in Escherichia coli BL21(DE3) [39]) [39] Engineering A72G ( site-directed mutagenesis, exchange in diphosphate binding site, decreased Km -value for diphosphate and guanine, increased Km -value for 5-phosphoribosyl 1-diphosphate, reduced activity [34]) [34] C105A ( prepared via splicing by overlap extension, reduced oxidation ofthe enzyme during storage [33]) [33] C205A ( prepared via splicing by overlap extension, reduced oxidation ofthe enzyme during storage [33]) [33] C22A ( prepared via splicing by overlap extension, reduced oxidation ofthe enzyme during storage [33]) [33] D150A ( reduced activity compared to wild-type, kcat for hypoxanthine, guanine, and xanthine are reduced by 11fold, 296fold, and 8.6fold, respectively, Km value for a-d-5-phosphoribosyl 1-diphosphate is reduced by 6.5fold [25]) [25] D163E ( site directed mutagenesis, slightly changed substrate affinities compared to wild-type [31]) [31] D163N ( site-directed mutagensis, exchange of xanthine binding residue, loss of the binding ability and activity against xanthine and XMP [31]) [31] F162L ( site directed mutagenesis, no effect on purine base specificity [31]) [31] G71A ( site-directed mutagenesis, no activity [34]) [34] G71E ( site-directed mutagenesis, no activity [34]) [34] G71R ( site-directed mutagenesis, no activity [34]) [34] I104G ( site directed mutagenesis, increased Km values for hypoxanthine, guanine, and xanthine [31]) [31] K134Q ( site directed mutagenesis, mutant recognizes adenine as substrate in addition, but less efficient than mutant K134S [31]) [31] K134S ( site directed mutagenesis, mutant recognizes adenine as substrate in addition, increased Km values for hypoxanthine, guanine, and xanthine [31]) [31] K68A ( conformational changes, shifted catalytic loop closer to the active site [37]) [37]

109


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L147F ( natural occuring point mutation leading to enzyme deficiency, which is not correlated with a physiological syndrome [40]) [40] R155E ( site directed mutagenesis, reduced affinity to GMP and XMP, catalysation of the forward reaction with guanine and xanthine at accelerated rates, 15fold increased Km for xanthine [31]) [31] R155K ( site directed mutagenesis, reduced affinity to GMP and XMP, catalysation of the forward reaction with guanine and xanthine at accelerated rates, insensitive to phenylglyoxal [31]) [31] S95A ( site-directed mutagenesis, dramatic reduction of catalytic activity, weak complementation of bacterial enzyme deficient strain [27]) [27] S95C ( site-directed mutagenesis, 2-3fold reduction of kcat , weak complementation of bacterial enzyme deficient strain [27]) [27] S95E ( site-directed mutagenesis, dramatic reduction of catalytic activity, no complementation of bacterial enzyme deficient strain [27]) [27] S95T ( site-directed mutagenesis, 2-3fold reduction of kcat , complementation of bacterial enzyme deficient strain [27]) [27] T47K ( site-directed mutagenesis, exchange of diphosphate binding site residue, 4-10fold decreased Km for diphosphate compared to the wildtype [29]) [29] T70K ( site-directed mutagenesis, exchange in diphosphate binding site, 6.7fold lower Km -value for diphosphate, lower Km for guanine, 2fold increase in Km -value for 5-phosphoribosyl 1-diphosphate, reduced activity [34]) [34] T70K/A72G ( site-directed mutagenesis, exchange in diphosphate binding site, decreased Km -value for diphosphate and guanine, increased Km value for 5-phosphoribosyl 1-diphosphate, reduced activity [34]) [34] Y156F ( site directed mutagenesis, weakened binding of GMP and XMP [31]) [31] Y156W ( site directed mutagenesis, slightly changed substrate affinities compared to wild-type [31]) [31] Y96F ( site-directed mutagenesis, dramatic reduction of catalytic activity, no complementation of bacterial enzyme deficient strain, 4-5fold decrease of Km value for 5-phosphoribosyl 1-diphosphate [27]) [27] Y96V ( site-directed mutagenesis, dramatic reduction of catalytic activity, no complementation of bacterial enzyme deficient strain, 4-5fold decrease of Km value for 5-phosphoribosyl 1-diphosphate [27]) [27] Additional information ( construction of deletion mutant lacking 7 amino acid residues, Y82-S88, of the active site loop II, resulting in highly reduced kcat -values and in increased Km -values for the substrates [38]; construction of 4 chimeric enzymes with segments of human and Plasmodium falciparum enzymes, altered substrate specificities [35]; construction of mutant lacking the Ser-Lys-Val C-terminal targeting signal, mutant enzyme is located throughout the parasite, including subcellular organelles such as nucleus and flagellum [32]; wild-type enzyme complements enzyme deficiency of the bacterial enzyme in Escherichia coli [27]) [27, 32, 35, 38]

110

2.4.2.8


Application medicine ( target for antiparasite drug design [42]; target for anti-trichomonial therapy [29, 31]; potential target for antiparasitic chemotherapy [23, 28, 33, 36, 38, 39]) [23, 28, 29, 31, 33, 36, 38, 39, 42]

6 Stability pH-Stability 4.2 ( 10 min, 50% loss of activity [19]) [19] 7-9.3 ( 10 min, stable [19]) [19] 11 ( 10 min, 50% loss of activity [19]) [19] Additional information ( preincubation for 10 min at pH 5.0 or pH 10.5 prior to enzyme assay at pH 8.5 does no affect the enzyme activity [13]) [13] Temperature stability 30 ( pH 7.4, stable [12]) [12] 40 ( pH 7.4, gradual decrease of activity [12]) [12] 60 ( mutant and wild-type enzyme, 8 min, stable [40]; 1.5 mM GMP, 10 min, complete loss of activity [3]) [3, 40] 60-65 ( stable [1]) [1] 85 ( if first incubated in 1 mM 5-phospho-a-d-ribose 1diphosphate, remarkably stable [9,16]; half-life: 3 min [11]) [9, 11, 16] Additional information [19] General stability information , glycerol or sucrose or dimethylsulfoxide stabilizes the purified enzyme at -70 C [15] , 5-phospho-a-d-ribose 1-diphosphate stabilizes against heat inactivation [12] , DTT and hypoxanthine stabilize [33] , 5-phospho-a-d-ribose 1-diphosphate stabilizes [10] Storage stability , frozen, partially purified enzyme, 4 months without loss of activity [2] , -80 C, mutant and wild-type enzyme, stable up to 3 years [40] , 0 C, 10 mM phosphate, pH 7.1, 1 mM DTT, 10 mM Mg2+ , 2 h, no loss of activity [33] , -20 C, purified enzyme, 6 mM 5-phospho-a-d-ribose 1-diphosphate, 2 mM MgCl2 , stable for at least a month [3] , -70 C, wild-type enzyme and mutants, except mutant S95C, stable up to 6 months [27] , -80 C [22] , 4 C, 50 mM Tris-HCl, pH 7.8, 1 M KCl, 10 mM MgCl2 , stable at least 1 month [12] , 4 C, 50 mM Tris-HCl, pH 7.8, complete loss of activity after 4 days [12]

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, -20 C, several months [13] , 4 C, freshly purified enzyme, 10 mM phosphate, pH 6.8, 10 mM DTT, 1 mM 5-phosphoribosyl 1-diphosphate, rapid loss of 90% activity within 48 h [33] , 5 C, purified enzyme, 10 mM phosphate, pH 6.8, 1 mM DTT, 0.2 mM 5phosphoribosyl 1-diphosphate, 0.06 hypoxanthine, loss of 7% activity after 48 weeks [33] , -20 C, dilute aqueous purified enzyme preparation, a few weeks [10]

References [1] Flaks, J.G.: Nucleotide synthesis from 5-phosphoribosylpyrophosphate. Methods Enzymol., 6, 136-158 (1963) [2] Lukens, L.N.; Herrington, K.A.: Enzymic formation of 6-mercaptopurine ribotide. Biochim. Biophys. Acta, 24, 432-433 (1957) [3] Nagy, M.; Ribet, A.-M.: Purification and comparative study of adenine and guanine phosphoribosyltransferases from Schizosaccharomyces pombe. Eur. J. Biochem., 77, 77-85 (1977) [4] Montero, C.; Llorente, P.: Artemia purine phosphoribosyltransferases. Purification and characterization. Biochem. J., 275, 327-334 (1991) [5] Walter, R.D.; Koenigk, E.: Hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase from Plasmodium chabaudi, purification and properties. Tropenmed. Parasitol., 25, 227-235 (1974) [6] Allen, T.; Henschel, E.V.; Coons, T.; Cross, L.; Conley, J.; Ullman, B.: Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase activities from Leishmania donovani. Mol. Biochem. Parasitol., 33, 273-281 (1989) [7] Schimandle, C.M.; Mole, L.A.; Sherman, I.W.: Purification of hypoxanthineguanine phosphoribosyltransferase of Plasmodium lophurae. Mol. Biochem. Parasitol., 23, 39-45 (1987) [8] Smithers, G.W.; O'Sullivan, W.J.: Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization. Biochem. Med., 32, 106-121 (1984) [9] Olsen, A.S.; Milman, G.: Hypoxanthine phosphoribosyltransferase from Chinese hamster brain and human erythrocytes. Methods Enzymol., 51, 543-549 (1978) [10] Hochstadt, J.: Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase from enteric bacteria. Methods Enzymol., 51, 549558 (1978) [11] Veres, G.; Monostori, E.; Rasko, I.: Purification and characterisation of chicken brain hypoxanthine-guanine phosphoribosyltransferase. FEBS Lett., 184, 299-303 (1985) [12] Ohe, T.; Watanabe, Y.: Purification and properties of hypoxanthine phosphoribosyltransferase from Streptomyces cyanogenus. Agric. Biol. Chem., 44, 1999-2006 (1980) 112

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[13] Schmidt, R.; Wiegand, H.; Reichert, U.: Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae. Eur. J. Biochem., 93, 355-361 (1979) [14] Gutensohn, W.; Huber, M.; Jahn, H.: Facilitated purification of hypoxanthine phosphoribosyltransferase. Hoppe-Seyler's Z. Physiol. Chem., 357, 1379-1385 (1976) [15] Arnold, W.J.; Kelley, W.N.: Human hypoxanthine-guanine phosphoribosyltransferase. Purification and subunit structure. J. Biol. Chem., 246, 73987404 (1971) [16] Olsen, A.S.; Milman, G.: Chinese hamster hypoxanthine-guanine phosphoribosyltransferase. Purification, structural, and catalytic properties. J. Biol. Chem., 249, 4030-4037 (1974) [17] Hughes, S.H.; Wahl, G.M.; Capecchi, M.R.: Purification and characterization of mouse hypoxanthine-guanine phosphoribosyltransferase. J. Biol. Chem., 250, 120-126 (1975) [18] Olsen, A.S.; Milman, G.: Human hypoxanthine phosphoribosyltransferase. Purification and properties. Biochemistry, 16, 2501-2505 (1977) [19] Miller, R.L.; Ramsey, G.A.; Krenitsky, T.A.; Elion, G.B.: Guanine phosphoribosyltransferase from Escherichia coli, specificity and properties. Biochemistry, 11, 4723-4731 (1972) [20] Nussbaum, R.L.; Caskey, C.T.: Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae. Biochemistry, 20, 4584-4590 (1981) [21] Dovey, H.F.; McKerrow, J.H.; Aldritt, S.M.; Wang, C.C.: Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schistosoma mansoni. A potential target for chemotherapy. J. Biol. Chem., 261, 944-948 (1986) [22] Aldritt, S.M.; Wang, C.C.: Purification and characterization of guanine phosphoribosyltransferase from Giardia lamblia. J. Biol. Chem., 261, 85288533 (1986) [23] Yuan, L.; Craig, S.P.; McKerrow, J.H.; Wang, C.C.: The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli. J. Biol. Chem., 265, 13528-13532 (1990) [24] Muench, H.; Yoshida, A.: Purification and characterization of human hypoxanthine/guanine phosphoribosyltransferase. Eur. J. Biochem., 76, 107112 (1977) [25] Heroux, A.; White, E.L.; Ross, L.J.; Davis, R.L.; Borhani, D.W.: Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg2+ Ions Bound: insights into the catalytic mechanism. Biochemistry, 38, 14495-14506 (1999) [26] Heroux, A.; White, E.L.; Ross, L.J.; Borhani, D.W.: Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex. Biochemistry, 38, 14485-14494 (1999)

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[27] Jardim, A.; Ullman, B.: The conserved serine-tyrosine dipeptide in Leishmania donovani hypoxanthine-guanine phosphoribosyltransferase is essential for catalytic activity. J. Biol. Chem., 272, 8967-8973 (1997) [28] Xu, Y.; Eads, J.; Sacchettini, J.C.; Grubmeyer, C.: Kinetic mechanism of human hypoxanthine-guanine phosphoribosyltransferase: rapid phosphoribosyl transfer chemistry. Biochemistry, 36, 3700-3712 (1997) [29] Munagala, N.R.; Chin, M.S.; Wang, C.C.: Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47. Biochemistry, 37, 4045-4051 (1998) [30] Doyle, P.S.; Kanaani, J.; Wang, C.C.: Hypoxanthine, guanine, xanthine phosphoribosyltransferase activity in Cryptosporidium parvum. Exp. Parasitol., 89, 9-15 (1998) [31] Munagala, N.R.; Wang, C.C.: Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein. Biochemistry, 37, 16612-16619 (1998) [32] Shih, S.; Hwang, H.Y.; Carter, D.; Stenberg, P.; Ullman, B.: Localization and targeting of the Leishmania donovani hypoxanthine-guanine phosphoribosyltransferase to the glycosome. J. Biol. Chem., 273, 1534-1541 (1998) [33] Keough, D.T.; Ng, A.-L.; Winzor, D.J.; Emmerson, B.T.; de Jersey, J.: Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme. Mol. Biochem. Parasitol., 98, 29-41 (1999) [34] Page, J.P.; Munagala, N.R.; Wang, C.C.: Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis. Eur. J. Biochem., 259, 565-571 (1999) [35] Sujay Subbayya, I.N.; Sukumaran, S.; Shivashankar, K.; Balaram, H.: Unusual substrate specificity of a chimeric hypoxanthine-guanine phosphoribosyltransferase containing segments from the Plasmodium falciparum and human enzymes. Biochem. Biophys. Res. Commun., 272, 596-602 (2000) [36] Heroux, A.; White, E.L.; Ross, L.J.; Kuzin, A.P.; Borhani, D.W.: Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis. Structure Fold Des., 8, 1309-1318 (2000) [37] Wang, F.; Shi, W.; Nieves, E.; Angeletti, R.H.; Schramm, V.L.; Grubmeyer, C.: A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase. Biochemistry, 40, 8043-8054 (2001) [38] Lee, C.C.; Medrano, F.J.; Craig, S.P., 3rd; Eakin, A.E.: Investigation of the functional role of active site loop II in a hypoxanthine phosphoribosyltransferase. Biochim. Biophys. Acta, 1537, 63-70 (2001) [39] Monzani, P.S.; Alfonzo, J.D.; Simpson, L.; Oliva, G.; Thiemann, O.H.: Cloning, characterization and preliminary crystallographic analysis of Leishmania hypoxanthine-guanine phosphoribosyltransferase. Biochim. Biophys. Acta, 1598, 3-9 (2002) [40] Micheli, V.; Gathof, B.S.; Rocchigiani, M.; Jacomelli, G.; Sestini, S.; Peruzzi, L.; Notarantonio, L.; Cerboni, B.; Hayek, G.; Pompucci, G.: Biochemical and molecular study of mentally retarded patient with partial deficiency of hy114

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poxanthine-guanine phosphoribosyltransferase. Biochim. Biophys. Acta, 1587, 45-52 (2002) [41] Sauve, A.A.; Cahill, S.M.; Zech, S.G.; Basso, L.A.; Lewandowicz, A.; Santos, D.S.; Grubmeyer, C.; Evans, G.B.; Furneaux, R.H.; Tyler, P.C.; McDermott, A.; Girvin, M.E.; Schramm, V.L.: Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases. Biochemistry, 42, 5694-5705 (2003) [42] Somoza, J.R.; Chin, M.S., Wang, C.C.; Fletterick, R.J.: Crystal structure of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from the protozoan parasite Tritrichomonas foetus. Biochemistry, 35, 7032-7040 (1996)

115

Uracil phosphoribosyltransferase

2.4.2.9

1 Nomenclature EC number 2.4.2.9 Systematic name UMP:diphosphate phospho-a-d-ribosyltransferase Recommended name uracil phosphoribosyltransferase Synonyms UMP pyrophosphorylase UMP:pyrophosphate phosphoribosyltransferase UPRT UPRTase phosphoribosyltransferase, uracil uridine 5'-phosphate pyrophosphorylase uridine monophosphate pyrophosphorylase uridylate pyrophosphorylase uridylic pyrophosphorylase Additional information ( PyrR protein is a bifunctional protein, that primarily regulates the expression of pyrimidine biosynthetic pyr genes, but also catalyses the uracil phosphoribosyltransferase reaction, little amino acid sequence similarities to other bacterial UPRTases [14]) [14] CAS registry number 9030-24-4

2 Source Organism

Lactobacillus leichmannii (strain ATCC 4797 [7]) [7] Crithidia luciliae [2] Tetrahymena pyriformis (strain GL-7 [3]) [3] Acholeplasma laidlawii [6] Lactobacillus bifidus (strain ATCC 4963 [1]) [1] Escherichia coli (gene upp [4,5,12,15]; enzyme expression is inducible upon uracil starvation [4,5]; strain ATCC 9637 [1]; K12 [4,5]; strain SO 1344 and SO 1346 [5]; not: Escherichia coli B [1]) [1, 4, 5, 12, 15] Lactobacillus bulgaricus (strain 09X [1]) [1] Saccharomyces cerevisiae [8]

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Candida albicans [9] Toxoplasma gondii [10, 17] Gardia intestinalis (strain Portland I, ATCC 30888 [11]) [11] Bacillus caldolyticus (strain DSM 405 [13,18]; gene upp [13,18]) [13, 18] Bacillus subtilis (PyrR protein [14]; strain 168 [13]) [13, 14] Toxoplasma gondii [16]

3 Reaction and Specificity Catalyzed reaction UMP + diphosphate = uracil + 5-phospho-a-d-ribose 1-diphosphate ( active sites pointing away from each other, a long arm from each monomer subunit wraps around the other subunit [18]; modelling of enzyme-5phospho-a-d-ribose 1-diphosphate complex [18]; Pro131 is of little importance for 5-phospho-a-d-ribose 1-diphosphate binding, but critical for binding of uracil to the enzyme-5-phospho-a-d-ribose 1-diphosphate complex [15]; ping pong steady state kinetic pattern, ordered bi-bi mechanism, model, no formation of the phosphoribosyl-enzyme intermediate predicted by classic ping pong kinetics [14]; sequential reaction mechanism [15,17]; rapid-random equilibrium mechanism [11]; pingpong reaction mechanism [6]) Reaction type pentosyl group transfer Natural substrates and products S uracil + 5-phospho-a-d-ribose 1-diphosphate ( incorporation of uracil into nucleotides [6]; pyrimidine salvage enzyme, no de novo synthesis of pyrmidines [4,5,10,17]) [4-6, 10, 17] P UMP + diphosphate Substrates and products S 2-thiouracil + 5-phospho-a-d-ribose 1-diphosphate ( substrate is anti-thyroid drug [7]) (Reversibility: ? [7]) [7] P 2-thio-5'-UMP + diphosphate [7] S 5-fluorouracil + 5-phospho-a-d-ribose 1-diphosphate ( no activity [10]; 1.6fold higher specific activity compared to uracil [9]; 216% of the activity with uracil [5]) (Reversibility: r [1,5]; ? [9,12]) [1, 5, 9, 12] P 5-fluoro-UMP + diphosphate S 6-azauracil + 5-phospho-a-d-ribose 1-diphosphate ( no activity [3]; 97% of the activity with uracil [5]) (Reversibility: ? [5]) [5] P 6-aza-UMP + diphosphate S uracil + 5-phospho-a-d-ribose 1-diphosphate ( 5phospho-a-d-ribose 1-diphosphate binding site [4, 15, 17, 18]; equilibrium lies far in the direction of UMP formation [1, 14]; highly specific for uracil [2, 3, 10]; highly specific for uracil and ana-

117


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logues [5]; transfer to N1 of uracil [17]) (Reversibility: r [1, 4, 5, 14, 15]; ? [2, 3, 6-13, 16-18]) [1-18] P UMP + diphosphate [1-18] S Additional information ( no catalysis of exchange reaction between uracil-UMP and diphosphate-5-phospho-a-d-ribose 1-diphosphate [14]; orotic acid is no substrate [1]; no substrates: cytosine, orotic acid [3,5]; no substrates: thymine, hypoxanthine [5]) [1, 3, 5, 14] P ? Inhibitors 1,3-dimethyl-uracil [11] 5-acetoxyuracil [11] 5-bromouracil [11] 5-fluoro-deoxyuracil [11] 5-fluorouracil [10] 5-n-propyl-2'-deoxyuridine [11] 5-nitrouracil [11] 5-phosphoribose 1-diphosphate ( inhibitory when higher concentrated as Mg2+ [12]) [12] 6-benzyl-2-thiouracil [11] 6-formyluracil [11] DTNB ( 4 mM, 87% inhibition after 30 min [11]) [11] GTP ( slight inhibition [2,10]) [2, 10] K+ ( slight inhibition at 0.1 M [7]) [7] Na+ ( slight inhibition at 0.1 M [7]) [7] TTP ( allosteric inhibition [8]) [8] UDP ( allosteric inhibition [8]) [8] UMP ( product inhibition, forward reaction [14]; competitive against uracil, noncompetitive against [11]; allosteric inhibition [8]) [8, 11, 14] UTP [3] dCMP ( allosteric inhibition [8]) [8] dCTP ( allosteric inhibition [8]) [8] dUMP ( allosteric inhibition [8]) [8] dUTP [4] diphosphate ( product inhibition forward reaction [14]; noncompetitive against uracil, competitive against 5-phosphoribose 1-diphosphate [11]) [11, 14] phosphate ( 80% inhibition at 0.05 M [1]) [1] tetrahydrouridine [11] Additional information ( no inhibition by thymine, cytosine, orotic acid, and hypoxanthine [10]; no inhibition by cytosine, orotic acid, 6-azauracil [3]; no inhibition by diphosphate [5]) [3, 5, 10] Activating compounds GTP ( activation, stabilization of the tetrameric structure at 2 mM, without GTP enzyme forms dimers [17]; activation of wild-type 118

2.4.2.9


and mutant P131D, maximal at 0.08 mM [15]; kinetics [12]; highly activating at low 5-phosphoribose 1-diphosphate concentration, e.g. 0.2 mM, by modification of the structure from dimer/trimer to pentamer/hexamer [12]; about 5-7fold increase of Km for 5-phosphoribose 1-diphosphate, unaltered Vmax [12,17]; highly activating, no effect on Km values [11]; no activation [10]; effect is pH-dependent, pH 7.5: lowers Km value for 5-phospho-a-d-ribose 1-diphosphate and increases Vmax 2-fold, no effect on Km for uracil, pH 8.0: no effect [5]) [5, 11, 12, 15, 17] dGTP ( highly activating, no effect on Km values [11]) [11] glutathione ( omission of glutathione reduces the reaction rate about 25% [1]) [1] guanosine-3',5'-diphosphate ( activates in the same range as GTP [12]) [12] Metals, ions Ca2+ ( no activation [10]; activates [11]) [11] Cd2+ ( activates [11]) [11] Co2+ ( can substitute Mg2+ [10]; poor activator [12]) [10-12] Mg2+ ( required [1, 6, 9, 10, 12]; only 5% of the activity remaining in absence [1]; optimal concentration : 2 mM [9]; optimal concentration: 5 mM [6]) [1, 6-12, 14, 15] Mn2+ ( can substitute for Mg2+ [10]; can partly substitute for Mg2+ at 5 mM, less effective, inhibitory at higher concentration [12]; required, 10 mM [11]; better activator than Mg2+ [11]) [10-12] Ni2+ ( activates [11]) [11] Zn2+ ( can partly substitute for Mg2+ at 5 mM, less effective, inhibitory at higher concentration [12]) [11, 12] divalent cations ( absolute requirement [10]; preference of activation by divalent cations in decreasing order: Mn2+ , Co2+ , Cd2+ , Mg2+ , Ni2+ , Ca2+ , all 1 mM [11]; broad specificity for activating divalent metal ions [2]) [2, 10, 11] Additional information ( no activation by Ba2+ [10]; no inhibition by any metal ion up to 10 mM [11]) [10, 11] Turnover number (min±1) 0.09 (UMP, reverse reaction [14]) [14] 306 (uracil, forward reaction [14]) [14] Additional information ( recombinant wild-type and mutant enzymes [17]) [17] Specific activity (U/mg) 0.0057 ( substrate 2-thiouracil [7]) [7] 0.0067 ( substrate uracil [7]) [7] 0.025 ( purified enzyme [1]) [1] 0.068 ( partially purified enzyme [9]) [9] 0.07 ( 37 C [13]) [13] 0.09 ( 60 C [13]) [13]

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0.192 ( strain SO 1346 [5]) [5] 0.22 ( partially purified enzyme [6]) [6] 3.1 ( purified enzyme [11]) [11] 6.602 ( purified enzyme, strain SO 1346 [5]) [5] 7.5 ( purified recombinant enzyme [12]) [12] 19.2 ( purified recombinant enzyme, 37 C [13]) [13] Additional information ( 1051 DA252/min/mg [10]; activity in different recombinant strain [4]) [4, 10, 15] Km-Value (mM) 0.0004 (uracil) [3] 0.00063 (uracil, recombinant wild-type [15]) [15] 0.002 (uracil) [13] 0.0035 (uracil, recombinant enzyme [10]) [10] 0.0042 (uracil) [6] 0.0069 (5-phospho-a-d-ribose 1-diphosphate) [3] 0.007 (uracil) [5] 0.0077 (uracil) [7] 0.021 (uracil) [8] 0.024 (uracil) [11] 0.026 (5-phospho-a-d-ribose 1-diphosphate) [8] 0.037 (5-phospho-a-d-ribose 1-diphosphate, recombinant wildtype, with GTP [17]) [17] 0.044 (5-phospho-a-d-ribose 1-diphosphate) [1] 0.05 (5-phospho-a-d-ribose 1-diphosphate) [13] 0.06 (uracil, recombinant mutant P131D [15]) [15] 0.066 (5-phospho-a-d-ribose 1-diphosphate) [6] 0.068 (5-phospho-a-d-ribose 1-diphosphate) [14] 0.0705 (uracil) [9] 0.073 (5-phospho-a-d-ribose 1-diphosphate, recombinant wildtype [15]) [15] 0.13 (UMP) [14] 0.159 (uracil) [14] 0.186 (5-phospho-a-d-ribose 1-diphosphate) [9] 0.2 (5-phospho-a-d-ribose 1-diphosphate) [11] 0.216 (5-phospho-a-d-ribose 1-diphosphate, recombinant wildtype, without GTP [17]) [17] 0.243 (5-phospho-a-d-ribose 1-diphosphate, recombinant enzyme [10]) [10] 0.3 (5-phospho-a-d-ribose 1-diphosphate) [5] 0.67 (2-thiouracil) [7] 1 (diphosphate) [14] Additional information ( kinetics [12]; Km -values, recombinant mutant enzymes [17]; Km -value for 5-phosphoribose 1-diphosphate of the mutant P131D is very low and difficult to determine [15]) [12, 15, 17]

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Ki-Value (mM) 0.015 (UMP, competitive versus uracil [11]) [11] 0.017 (tetrahydrouridine) [11] 0.025 (5-fluorouracil) [10] 0.24 (diphosphate, competitive versus 5-phosphoribose 1-diphosphate [11]) [11] Additional information ( product inhibition pattern [14]) [14] pH-Optimum 6 [11] 6-6.5 ( plateau, substrate uracil [7]) [7] 7.4 ( assay at [1]) [1] 7.5 [9] 7.5-8 ( substrate 2-thiouracil [7]) [3, 6, 7, 16] 7.5-8.5 [5] 7.6-7.8 [8] 7.8 ( 2-thiouracil [7]) [7, 14] 8.5 ( assay at [12,15]) [12, 15] 8.7 ( assay at [14]) [14] pH-Range 5.5-8.5 [11] 6.5-10 [8] 7.2-8 ( pH 7.2: about 50% of activity maximum, pH 8.0: about 70% of activity maximum [6]) [6] Temperature optimum ( C) 30 ( assay at [11]) [11] 37 ( assay at [1, 5-7, 10, 12, 14, 15]) [1, 5-7, 10, 12, 14, 15] 40 [3] 60 ( recombinant enzyme [13]) [13] Temperature range ( C) 22-42 [8]

4 Enzyme Structure Molecular weight 24000-27600 ( recombinant enzyme, gel filtration and DNA sequence determination [10]) [10] 36000 [3] 46000 ( gel filtration [13]) [13] 63000 ( sucrose density gradient centrifugation and gel filtration in absence of substrates [12]) [12] 75000 ( gel filtration [5]) [5] 76000 ( gel filtration [11]) [11] 80000 ( gel filtration [6]; gel filtration [8]) [2, 6, 8] 121


2.4.2.9

111000 ( sucrose density gradient centrifugation and gel filtration in presence of substrates [12]) [12] Additional information ( gel filtration and sedimentation analyses: a smaller oligomeric, less active form, dimeric or trimeric, predominates in absence of substrates, a larger aggregated, more active form, pentameric or hexameric, predominates in presence of substrates [12]) [12] Subunits ? ( x * 28000, recombinant protein, SDS-PAGE [16]; x * 47000 + x * 38000, SDS-PAGE [9]; x * 22500, SDS-PAGE [12]; x * 22500, DNA sequence determination [4]) [4, 9, 12, 16] dimer ( 2 * 36000, SDS-PAGE [11]) [2, 11, 18] monomer ( 1 * 27000, recombinant enzyme, SDS-PAGE [10]) [10] trimer ( 3 * 23500, SDS-PAGE [5]) [5] Additional information ( monomer subunit fold with UMP [18]; 2 conserved sequences: Asp131-Ser139 contains the 5-phosphoribose 1-diphosphate binding motif and binds the ribose 5'-phosphate part of UMP, Tyr193-Ala201 is specific for uracil phosphoribosyltransferases and binds the uracil part of UMP [18]; three-dimensional structure [17,18]; a tetramer of 2 dimers, equilibrium between dimeric and tetrameric form [17]) [17, 18]

5 Isolation/Preparation/Mutation/Application Source/tissue trophozoite [11] Purification [2] [3] (partial [6]) [6] [1] (recombinant wild-type and mutant P131D from overexpressing Escherichia coli [15]; recombinant from overexpressing strain [12]; from strain SO 1346 [5]) [5, 12, 15] [8] (only partial, due to the labile nature of the enzyme further purification is unsuccessful [9]) [9] (recombinant from Escherichia coli [10]) [10] [11] (recombinant from enzyme-deficient Escherichia coli [13]) [13] (recombinant from Escherichia coli [16]) [16] Crystallization (hanging drop-vapour diffusion method, enzyme complexed with uracil and 5-phosphoribose 1-diphosphate, 20 mg/ml, mixing of equal volumes, 15 mM sodium citrate/phosphate, 0.2 M NaCl, pH 4.7, 10% polyethylene gly-

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col 3400, X-ray diffraction structure analysis, dynamic light scattering [17]) [17] (vapour diffusion using hanging or sitting drops, room temperature, protein solution 20 mg/ml, pH 7.0, 5 mM phosphate, 0.003 ml + 0.003 ml reservoir solution, pH 7.5, 6-10% polyethylene glycol 4000, 0.1 M HEPES, crystals appeared after 3 weeks, structure determination and analysis [18]) [18] (hanging drop-vapour diffusion technique, 2.0 M ammonium phosphate as precipitant, pH 8.0, room temperature, X-ray diffraction structure analysis, crystals are enzymatically active, wild-type and mutant enzymes [16]) [16] Cloning (overexpression of wild-type and mutant P131D in Escherichia coli [15]; overexpressing from plasmid in Escherichia coli strain NF1815 [12]; DNA sequence determination and analysis, gene upp belongs to purMN operon, complementation of deficient Escherichia coli strain [4]) [4, 12, 15] (wild-type and mutant C128V [17]; functional overexpression in Escherichia coli JM109 [10,17]) [10, 17] (DNA and amino acid sequence determination and analysis, overexpression in enzyme-deficient Escherichia coli [13,18]) [13, 18] (DNA sequence determination, functional overexpression in Escherichia coli BL21(DE3)plyS [16]) [16] Engineering C128V ( site-directed mutagenesis, required for structural sudy [17]) [17] K150A ( site-directed mutagenesis, slightly reduced kcat , increased Km for 5-phosphoribose 1-diphosphate in presence of GTP, structural sudy [17]) [17] K59A ( site-directed mutagenesis, slightly enhanced kcat , increased Km for 5-phosphoribose 1-diphosphate in presence of GTP, structural sudy [17]) [17] P131D ( site-directed mutagenesis, 2-step mutagenic PCR, exchange of proline in 5-phosphoribose 1-diphosphate binding site, 50-60fold reduction of catalytic rate in both reaction directions, about 100fold increase in KM for uracil, strongly reduced Km for 5-phosphoribose 1-diphosphate [15]) [15] R68A ( site-directed mutagenesis, slightly reduced kcat , increased Km for 5-phosphoribose 1-diphosphate in presence of GTP, structural sudy [17]) [17] Application medicine ( enzyme is a possible therapeutic target [10]) [10] pharmacology ( potential target for the development of new antibiotics [18]) [18]

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6 Stability Temperature stability 40 ( irreversible inactivation above [3]) [3] 45 ( 10 min, 90% remaining activity [13]) [13] 50 ( begins to unfold irreversibly [10]) [10] 55 ( 10 min, 20% remaining activity [13]; 4 min, complete loss of activity [9]) [9, 13] 56-57 ( 10 min, 50% loss of activity, recombinant wild-type and mutant, scanning calorimetry [15]) [15] 60 ( 10 min, inactivation [13]; 2 min, complete loss of activity, 1 mM uracil + 0.1 EDTA protect partially [8]) [8, 13] 65 ( complete inactivation after 5 min [10]) [10] 70 ( 10 min, 70% remaining activity [13]) [13] 75 ( 10 min, 5% remaining activity [13]) [13] 80 ( 10 min, inactivation [13]) [13] General stability information , UMP stabilizes the enzyme [6] , dialysis against buffers of low ionic strength invariably results in complete inactivation, whereas after 3 h dialysis against 0.5 M KCl only 40% of the activity is lost [1] , 2-mercaptoethanol, not essential for stability in long-term storage [5] , GTP, 5 mM with 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 2 mM 2-mercaptoethanol, labilizes with only 15% activity remaining after 13 days, labilizing effect abolished in presence of 5-phospho-a-d-ribose 1-diphosphate [5] , ethylene glycol, significantly increases stability with virtually no loss of activity after storage at 4 C for 13 days or after 1 year at-70 C [5] , enzyme is highly unstable, dimethyl sulfoxide stabilizes during purification [8] , enzyme is extremly unstable, UMP stabilizes during storage [9] , rapid precipitation of purified enzyme without thiol reducing agents [10] , 1 mM DTT and 0.1 mM EDTA stabilize the purified enzyme [11] , bovine serum albumin at 2 mg/ml stabilizes [13] Storage stability , 4 C, rapid loss of activity when stored in presence of Mg2+ [6] , -15 C, partially purified enzyme, loss of 10% activity within 2 weeks [1] , 4 C for 13 days or 1 year at -70 C, ethylene glycol, significantly increases stability with virtually no loss of activity [5] , 4 C, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 2 mM 2-mercaptoethanol, 44% loss of activity after 8 d [5] , 4 C, Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM DTT, 1 mM UMP, loss of 40% activity in 1 week [9] , 4 C, Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM DTT, loss of 70% activity in 4 days [9]

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, 4 C, purified enzyme, TMD50 buffer, stable for at least 1 week [10] , -20 C, 20 mM Hepes-KOH, pH 7.4, 1 mM DTT, stable for 3 days [11] , -70 C, 20 mM Hepes-KOH, pH 7.4, 1-2 months without loss of activity [11] , 0 C, enzyme concentration 0.001 mg/ml, 50% loss of activity in 1 h [13] , 4 C, concentrated enzyme is stable for several months, on dilution occurs rapid loss of activity [13]

References [1] Flaks, J.G.: Nucleotide synthesis from 5-phosphoribosylpyrophosphate. Methods Enzymol., 6, 136-158 (1963) [2] Asai, T.; Lee, C.S.; Chandler, A.; O'Sullivan, W.J.: Comp. Biochem. Physiol. B Comp. Biochem., 95, 159-163 (1990) [3] Plunkett, W.; Moner, J.G.: UMP pyrophosphorylase of Tetrahymena pyriformis. Partial purification and properties. Arch. Biochem. Biophys., 187, 264271 (1978) [4] Andersen, P.S.; Smith, J.M.; Mygind, B.: Characterization of the upp gene encoding uracil phosphoribosyltransferase of Escherichia coli K12. Eur. J. Biochem., 204, 51-56 (1992) [5] Rasmussen, U.B.; Mygind, B.; Nygaard, P.: Purification and some properties of uracil phosphoribosyltransferase from Escherichia coli K12. Biochim. Biophys. Acta, 881, 268-275 (1986) [6] McIvor, R.S.; Wohlhueter, R.M.; Plagemann, P.G.W.: Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties. J. Bacteriol., 156, 192-197 (1983) [7] Lindsay, R.H.; Tillery, C.R.; Yu, M.-Y.W.: Conversion of the antithyroid drug 2-thiouracil to 2-thio-5-UMP by UMP pyrophosphorylase. Arch. Biochem. Biophys., 148, 466-474 (1972) [8] Natalini, P.; Ruggieri, S.; Santarelli, I.; Vita, A.; Magni, G.: Bakers yeast UMP:pyrophosphate phosphoribosyltransferase. Purification, enzymatic and kinetic properties. J. Biol. Chem., 254, 1558-1563 (1979) [9] Alloush, H.M.; Kerridge, D.: Characterization of a partially purified uracil phosphoribosyltransferase from the opportunistic pathogen Candida albicans. Mycopathologia, 125, 129-141 (1994) [10] Carter, D.; Donald, R.G.K.; Roos, D.; Ullman, B.: Expression, purification, and characterization of uracil phosphoribosyltransferase from Toxoplasma gondii. Mol. Biochem. Parasitol., 87, 137-144 (1997) [11] Dai, Y.-P.; Lee, C.S.; O'Sullivan, W.J.: Properties of uracil phosphoribosyltransferase from Giardia intestinalis. Int. J. Parasitol., 25, 207-214 (1995) [12] Jensen, K.F.; Mygind, B.: Different oligomeric states are involved in the allosteric behavior of uracil phosphoribosyltransferase from Escherichia coli. Eur. J. Biochem., 240, 637-645 (1996) [13] Jensen, H.K.; Mikkelsen, N.; Neuhard, J.: Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, pur125


[14] [15] [16]

[17]

[18]

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ification, and partial characterization. Protein Expr. Purif., 10, 356-364 (1997) Grabner, G.K.; Switzer, R.L.: Kinetic studies of the uracil phosphoribosyltransferase reaction catalyzed by the Bacillus subtilis pyrimidine attenuation regulatory protein PyrR. J. Biol. Chem., 278, 6921-6927 (2003) Lundegaard, C.; Jensen, K.F.: Kinetic mechanism of uracil phosphoribosyltransferase from Escherichia coli and catalytic importance of the conserved proline in the PRPP binding site. Biochemistry, 38, 3327-3334 (1999) Barchue, J.; Symersky, J.; Narayana, S.V.; Moore, J.K.; DeLucas, L.J.; Chattopadhyay, D.: Expression, purification, crystallization and preliminary X-ray diffraction analysis of uracil phosphoribosyltransferase of Toxoplasma gondii. Acta Crystallogr. Sect. D, 55, 347-349 (1999) Schumacher, M.A.; Bashor, C.J.; Song, M.H.; Otsu, K.; Zhu, S.; Parry, R.J.; Ullman, B.; Brennan, R.G.: The structural mechanism of GTP stabilized oligomerization and catalytic activation of the Toxoplasma gondii uracil phosphoribosyltransferase. Proc. Natl. Acad. Sci. USA, 99, 78-83 (2002) Kadziola, A.; Neuhard, J.; Larsen, S.: Structure of product-bound Bacillus caldolyticus uracil phosphoribosyltransferase confirms ordered sequential substrate binding. Acta Crystallogr. Sect. D, 58, 936-945 (2002)

Orotate phosphoribosyltransferase

2.4.2.10

1 Nomenclature EC number 2.4.2.10 Systematic name orotidine-5'-phosphate:diphosphate phospho-a-d-ribosyltransferase Recommended name orotate phosphoribosyltransferase Synonyms OPRT OPRTase OPRTase orotate PRTase orotate phosphoribosyl pyrophosphate transferase orotate phosphoribosyltransferase orotic acid phosphoribosyltransferase orotidine 5'-monophosphate pyrophosphorylase orotidine 5'-phosphate pyrophosphorylase orotidine monophosphate pyrophosphorylase orotidine phosphoribosyltransferase orotidine-5'-phosphate pyrophosphorylase orotidylate phosphoribosyltransferase orotidylate pyrophosphorylase orotidylic acid phosphorylase orotidylic acid pyrophosphorylase orotidylic phosphorylase orotidylic pyrophosphorylase phosphoribosyltransferase, orotate Additional information ( constitutes together with orotidylate decarboxylase (EC 4.1.1.23) UMP-synthase, former complex U or multienzyme pyr-5,6 [1]; enzyme from mammals is a bifunctional polypeptide, it also catalyzes the reaction listed as EC 4.1.1.23 [2, 10, 11, 14]) [1, 2, 10, 11, 14] CAS registry number 9030-25-5

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2 Source Organism Toxoplasma gondii [16] Escherichia coli (K-12, overproducing strains C-600(pNE24) (wild-type) and its purine-sensitive mutant PS100(pNE31), both harbouring hybrid multi-copy plasmids [8]) [8, 9, 24] Salmonella typhimurium (structural gene pyrE cloned and overexpressed via multi-copy plasmid in Escherichia coli strain MB13 [12]) [3, 12, 13, 15, 17, 25] Plasmodium falciparum (FCB strain [10]) [10] Saccharomyces cerevisiae (Budweiser brand [5,7]) [4-7, 23] Phaseolus mungo (black gram [14]) [14] Thermus thermophilus (recombinant enzyme [19]) [18, 19] Homo sapiens [10, 20, 21] Mus musculus [1, 2, 11, 20] Rhizobium leguminosum (biovar trifolium [22]) [22]

3 Reaction and Specificity Catalyzed reaction orotidine 5'-phosphate + diphosphate = orotate + 5-phospho-a-d-ribose 1diphosphate ( mechanism [5, 12, 17]; mechanism is bi bi ping pong [7]; ping pong mechanism is not operable, NMR exchange experiments [23]) Reaction type pentosyl group transfer Natural substrates and products S orotate + 5-phospho-a-d-ribose 1-diphosphate ( involved in de novo synthesis of pyrimidine nucleotides [8,12]; final steps in biosynthesis of UMP [10]; nucleotide-forming step in pyrimidine biosynthesis [13]) (Reversibility: ? [8, 10, 12, 13]) [8, 10, 12, 13] P orotidine 5'-phosphate + diphosphate Substrates and products S 5-fluoroorotate + phosphoribose diphosphate (Reversibility: ? [7]) [7] P 5-fluoroorotidine 5'-phosphate + diphosphate [7] S orotate + 5-phospho-a-d-ribose 1-diphosphate ( high specificity for orotate [7]; predominant species of phosphoribose diphosphate: metal ion complex [7]; catalyzes stereospecific formation of b-glycosidic bond between orotate and ribose 5'-phosphate portion of phospho-ribose diphosphate [7]; reverse reaction: tri-, tetrapoly- or trimetaphosphate can replace diphosphate with 29%, 70% or 78% efficiency [8]) (Reversibility: r [4, 5, 7, 8, 12]; ? [2, 3, 10, 16, 18, 22]) [1-8, 10, 16, 18, 22]

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P orotidine 5'-phosphate + diphosphate ( i.e. orotidylate or OMP [1]) [1, 4, 5, 7] S orotate methylester + phosphoribose diphosphate (Reversibility: ? [3]) [3] P diphosphate + orotidine 4-methylester S uracil + phosphoribose diphosphate ( not substrate [14]) (Reversibility: ? [3]) [3] P diphosphate + uridine S Additional information ( enzyme from mammals is a bifunctional polypeptide, it also catalyzes the reaction listed as EC 4.1.1.23 [2, 10, 11, 14]) [2, 10, 11, 14] P ? Inhibitors 1-deazaorotic acid [16] 5-azaorotic acid [16] 5-bromouracil [1] 5-chlorouracil [1] 5-fluoroorotate ( at 0.05 M [1]) [1] 5-phospho-a-d-ribose 1-diphosphate ( phosphorolysis, product inhibition, kinetics [7,12]) [7, 12] ADP ( not inhibitory [8]) [1] AMP ( not inhibitory [8]) [1] ATP ( not inhibitory [8]) [1] Co2+ ( inhibits Mg2+ -activation [5]) [5] EDTA ( 5 mM, complete inhibition [4]) [4, 14] GMP [1] HgCl2 ( slight inhibition of mutant, not wild-type [8]) [8] IMP [1] NEM ( slight inhibition of mutant, not wild-type [8]) [8] PCMB ( reversible by DTT [8]; 0.01 mM, complete inhibition [10]) [8] UDP [1, 14] UMP ( 1 mM, not 0.1 mM [1]) [1, 14] UTP ( not inhibitory [8]) [1] Zn2+ [8] adenine ( not inhibitory [8]) [1] adenosine ( not inhibitory [8]) [1] allopurinol [1] anthranilate ( weak [7]) [7] arabinose 5-phosphate ( at higher concentrations [7]) [7] azauracil [1] azauridine [1] barbituric acid [1] cytosine [1] dUMP [1] dihydroorotate ( 10 mM [1]) [1]

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diphosphate ( product inhibition, kinetics [7,12]) [1, 7, 12] erythrose 4-phosphate ( at higher concentrations [7]) [7] fructose 1-phosphate ( at higher concentrations [7]) [7] fructose 6-phosphate ( at higher concentrations [7]) [7] iodoacetamide ( slight inhibition of mutant, not wild-type [8]) [8] nicotinate ( weak [7]) [7] orotate ( phosphorolysis, product inhibition, kinetics [7,12]; above 0.4 mM [22]) [7, 12, 22] orotidine ( 10 mM [1]) [1] orotidylate ( product inhibition, kinetics [7,12]) [4, 7, 12] oxipurinol [1] phosphate ( at higher concentrations [7]) [1, 7] d-ribose 5-phosphate ( at higher concentrations [7]; not inhibitory [1]) [7] uracil [1, 16] uridine [1] xanthosine-5-phosphate [14] Additional information ( structural features of pyrimidine nucleobase inhibitors [16]; no inhibition by SH-group reagents [10]; not inhibitory: CMP, TMP, 6-aza-UMP or 5-bromo-UMP [1]) [1, 10, 16] Activating compounds dithiothreitol ( stimulating [14]) [14] Metals, ions Ba2+ ( slight activation [8]) [8] Ca2+ ( activation, 34% as effective as Mn2+ or Mg2+ [8]; not activating [5]) [8] Co2+ ( slight activation [8]; not activating [5]) [8] Mg2+ ( requirement [1, 3, 5, 7, 8, 14]; best with 2 mM [3]; Km -value: 3 mM [1]; mechanism [3, 5]; no orotate-Mg-complex formation, weak Mg-enzyme-complex [3]) [1, 3, 5, 7, 8, 14] Mn2+ ( activation [3, 5, 8]; can replace Mg2+ [3, 8]; mechanism [3,5]) [3, 5, 8] Specific activity (U/mg) 0.115 [1] 0.41 ( mutant [8]) [8] 0.69 [2] 15.2 ( wild-type [8]) [8] 17 [6] 27 [9] 40 [5] 45 ( in the absence of added OMP-decarboxylase, EC 4.1.1.23 [7]) [7] 61 [22]

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65 ( in the presence of added OMP-decarboxylase, EC 4.1.1.23 [7]) [7] 81.6 [4] 95.7 [12] 300 [19] Additional information [8, 14] Km-Value (mM) 0.0016 (5-phospho-a-d-ribose 1-diphosphate) [14] 0.002 (orotate) [1] 0.0031-0.0036 (orotidine 5'-phosphate, wild-type [8]) [8, 12] 0.0045 (orotate) [14] 0.008-0.0083 (orotidine 5'-phosphate, phosphorolysis [7]) [4, 7] 0.013 (diphosphate, wild-type [8]) [8] 0.016 (5-phospho-a-d-ribose 1-diphosphate) [1] 0.025 (orotate) [18] 0.027-0.0275 (orotate, 5-fluoroorotate [7]) [3, 7] 0.0276 (orotate) [22] 0.03 (orotidine 5'-monophosphate) [8] 0.03-0.035 (orotate, wild-type [8]) [4, 7, 8] 0.034 (5-phospho-a-d-ribose 1-diphosphate) [18] 0.038-0.04 (5-phospho-a-d-ribose 1-diphosphate, wild-type [8]) [7, 8] 0.044 (5-phospho-a-d-ribose 1-diphosphate) [3, 12] 0.062 (5-phospho-a-d-ribose 1-diphosphate) [4] 0.075 (orotate) [19] 0.096 (diphosphate, phosphorolysis [7]) [7] 0.19 (orotate methylester) [3] 0.22-0.25 (diphosphate) [4, 8] 0.36 (5-phospho-a-d-ribose 1-diphosphate, mutant [8]) [8] 0.44 (orotate, mutant [8]) [8] 2.63 (uracil) [3] Additional information ( principles of assay [1,4]; kinetic study [7]; HPLC microassay [20]) [1, 7, 4, 12, 20] Ki-Value (mM) 0.00047 (1-deazaorotic acid) [16] 0.0021 (5-azaorotic acid) [16] 0.0063 (orotydilate, plus 5-phospho-a-d-ribose 1-diphosphate [4]) [4] 0.0083 (orotydilate, plus orotate [4]) [4] 0.01 (orotydilate) [7] 0.042 (5-phospho-a-d-ribose 1-diphosphate) [7] 0.063 (orotate) [7] 0.131 (diphosphate) [7]

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pH-Optimum 7-7.5 ( preferred buffer: Tris-HCl, when substrates are non-saturating, phosphate buffer, when substrate are saturating, not: maleate, imidazole, N-tris[hydroxymethyl] methyl-2-aminoethane-sulfonic acid (i.e. TES) or 3[N-morpholino]-2-hydroxypropanesulfonic acid (i.e. MOPS) buffer [1]) [1] 8 [14] 8.5 ( mutant [8]) [8] 8.5-9 [4] 9 [19] 9.5 ( wild-type [8]) [8] 10 ( recombinant enzyme [22]) [22] Additional information ( 2 isozymes: pI: 5.65 (minor form), pI: 5.85 (major form) [2]) [2] pH-Range 7.2-9.2 ( about half-maximal activity at pH 7.2 and about 70% of maximal activity at pH 9.2 [14]) [14] 7.2-9.5 ( about half-maximal activity at pH 7.2 and 9.5, mutant [8]) [8] 8.5-10.5 ( about half-maximal activity at pH 8.5 and about 70% of maximal activity at pH 10.5, wild-type [8]) [8] Temperature optimum ( C) 20 ( assay at [9]) [9] 25 ( assay at [4,7]) [4, 7] 30 ( assay at [12,14]) [12, 14] 37 ( assay at [1,2,6]) [1, 2, 6] 75-80 [19] 80 [18]

4 Enzyme Structure Molecular weight 39000 ( gel filtration [4]) [4] 40000 ( gel filtration [7]) [7] 47000 ( K-12, gel filtration [8]) [8] 50000 ( sucrose density gradient centrifugation [2]) [2] Subunits ? ( x * 51500, SDS-PAGE, sedimentation studies reveal monomer or dimer, enzyme activity and orotidine 5'-monophosphate decarboxylating activity on a single polypeptide [11]; x * 2000, SDS-PAGE [19]; x * 24700, SDS-PAGE, recombinant enzyme [22]) [11, 19, 22] dimer ( 2 * 20000, SDS-PAGE [7]; 2 * 22000, SDS-PAGE [9]; 2 * 23000, SDS-PAGE [12]; 2 * 23500, SDS-PAGE [8]; homodimer, crystalline structure [24]; homodimer, with catalytically important

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residues from one subunit available for the other, crystalline structure [25]) [7-9, 12, 24, 25] monomer ( 1 * 51500, SDS-PAGE, enzyme activity and orotidine 5'monophosphate decarboxylating activity on a single polypeptide [2]) [2] Additional information ( active site of enzyme requires D125 of one subunit and K103 of second subunit [15]) [15]

5 Isolation/Preparation/Mutation/Application Source/tissue Ehrlich ascites carcinoma cell [1, 2, 11] erythrocyte [10, 20] seedling [14] Localization cytosol [14] soluble [6, 9, 10, 14] Purification (OMP-agarose affinity chromatography [8]) [8, 9] [12] (partial, affinity chromatography on Blue-Dextran- or Cibacron Blue F3GA-Sepharose [6]) [4, 6] (partial, not separable from EC 4.1.1.23 [14]) [14] (recombinant enzyme [19]) [19] (partial, rapid tandem affinity column method [2]) [1, 2] (recombinant enzyme [22]) [22] Crystallization (ligated with sulfate, homodimer [24]) [24] (complexed with orotidine monophosphate, homodimer, with catalytically important residues from one subunit available for the other [25]) [13, 25] Cloning (structural gene pyrE, subcloned from a genomic library in pBR328(pAV002), transduced into pyr-auxotroph Salmonella typhimurium strain SA2434 with phage P22, finally cloned to and expressed in overproducing strain Escherichia coli MB13 [12]) [12] (structural gene pyrE [22]) [22] Engineering D125N ( active site of enzyme requires D125 of one subunit and K103 of second subunit [15]) [15] K103A ( active site of enzyme requires D125 of one subunit and K103 of second subunit [15]) [15] Additional information ( point mutations for negative complementation of enzyme activity [15]; deletion of 1-5 C-terminal amino

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acids, activities reduced to 22%-75% of wild type activity [18]; mutational separation of catalytic activity of enzyme and EC 4.1.1.23 activity results in active but unstable proteins [21]) [15, 18, 21]

6 Stability pH-Stability 4-9 ( stable [1]) [1] 7.5-9.5 ( at least 6 months stable, -20 C [4]) [4] Temperature stability 90 ( 20 min, 70% of activity remaining [18]) [18] General stability information , 0.05 mM UMP and 1% polyethyleneglycol stabilize during final stage of purification [2] , 5 mM Mg2+ , 1 mM phosphoribose diphosphate and 2 mM DTT stabilize dilute enzyme solutions [1] , dialysis against phosphate buffer, pH 7 or 7.5, unstable to, 2 mM DTT protect [1] , dilution inactivates [1] , phosphate, 0.3 M and above, stabilizes [1] , proteins, e.g. albumin, do not stabilize dilute enzyme solutions [1] Storage stability , -25 C, in 50% v/v glycerol, 0.1 mM DTT, about 35% loss of activity within 40 days [9] , 4 C, t1=2 : 1.5 days [10] , -20 C, pH 7.5-9.5, at least 6 months [4] , -76 C, at least 6 months [5] , 4 C, gradual loss of activity at pH 7.5-9.5 [4] , -20 C, concentrated enzyme solution, at least 4 months [2] , -60 C to-20 C, crude enzyme or ammonium sulfate preparation, 2 years [1] , frozen, partially purified enzyme in phosphate buffer, pH 7 or 7.5, 2 months [1]

References [1] Jones, M.E.; Kavipurapu, P.R.; Traut, T.W.: Orotate phosphoribosyltransferase: orotidylate decarboxylase (Ehrlich ascites cell). Methods Enzymol., 51, 155-167 (1978) [2] McClard, R.W.; Black, M.J.; Livingstone, L.R.; Jones, M.E.: Isolation and initial characterization of the single polypeptide that synthesizes uridine 5monophosphate from orotate in Ehrlich ascites carcinoma. Purification by

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[3] [4] [5] [6] [7] [8]

[9] [10] [11] [12] [13] [14] [15]

[16]


tandem affinity chromatography of uridine-5-monophosphate synthase. Biochemistry, 19, 4699-4706 (1980) Bhatia, M.B.; Grubmeyer, C.: The role of divalent magnesium in activating the reaction catalyzed by orotate phosphoribosyltransferase. Arch. Biochem. Biophys., 303, 321-325 (1993) Yoshimoto, A.; Amaya, T.; Kobayashi, K.; Tomita, K.: Orotate phosphoribosyltransferase (yeast). Methods Enzymol., 51, 69-74 (1978) Victor, J.; Leo-Mensah, A.; Sloan, D.L.: Divalent metal ion activation of the yeast orotate phosphoribosyltransferase catalyzed reaction. Biochemistry, 18, 3597-3604 (1979) Reyes, P.; Sandquist, R.B.: Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives. Anal. Biochem., 88, 522-531 (1978) Victor, J.; Greenberg, L.B.; Sloan, D.L.: Studies of the kinetic mechanism of orotate phosphoribosyltransferase from yeast. J. Biol. Chem., 254, 26472655 (1979) Shimosaka, M.; Fukuda, Y.; Murata, K.; Kimura, A.: Purification and properties of orotate phosphoribosyltransferases from Escherichia coli K-12, and its derivative purine-sensitive mutant. J. Biochem., 98, 1689-1697 (1985) Dodin, G.: A rapid purification by affinity chromatography of orotate phosphoribosyltransferase from Escherichia coli K-12. FEBS Lett., 134, 20-24 (1981) Rathod, P.K.; Reyes, P.: Orotidylate-metabolizing enzymes of the human malarial parasite, Plasmodium falciparum, differ from host cell enzymes. J. Biol. Chem., 258, 2852-2855 (1983) Floyd, E.E.; Jones, M.E.: Isolation and characterization of the orotidine 5'monophosphate decarboxylase domain of the multifunctional protein uridine 5'-monophosphate synthase. J. Biol. Chem., 260, 9443-9451 (1985) Bhatia, M.B.; Vinitsky, A.; Grubmeyer, C.: Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium. Biochemistry, 29, 10480-10487 (1990) Scapin, G.; Sacchettini, J.C.; Dessen, A.; Bhatia, M.; Grubmeyer, C.: Primary structure and crystallization of orotate phosphoribosyltransferase from Salmonella typhimurium. J. Mol. Biol., 230, 1304-1308 (1993) Ashihara, H.: Orotate Phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase of black gram (phaseolus mungo) seedlings. Z. Pflanzenphysiol., 87, 225-241 (1978) Ozturk, D.H.; Dorfman, R.H.; Scapin, G.; Sacchettini, J.C.; Grubmeyer, C.: Structure and function of Salmonella typhimurium orotate phosphoribosyltransferase: protein complementation reveals shared active sites. Biochemistry, 34, 10764-10770 (1995) Javaid, Z.Z.; el Kouni, M.H.; Iltzsch, M.H.: Pyrimidine nucleobase ligands of orotate phosphoribosyltransferase from Toxoplasma gondii. Biochem. Pharmacol., 58, 1457-1466 (1999)

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[17] Tao, W.; Grubmeyer, C.; Blanchard, J.S.: Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase. Biochemistry, 35, 14-21 (1996) [18] Hamana, H.; Shinozawa, T.: Effects of C-terminal deletion on the activity and thermostability of orotate phosphoribosyltransferase from Thermus thermophilus. J. Biochem., 125, 109-114 (1999) [19] Bunnak, J.; Hamana, H.; Ogino, Y.; Saheki, T.; Yamagishi, A.; Oshima, T.; Date, T.; Shinozawa, T.: Orotate phosphoribosyltransferase from Thermus thermophilus: Overexpression in Escherichia coli, purification and characterization. J. Biochem., 118, 1261-1267 (1995) [20] Krungkrai, J.; Wutipraditkul, N.; Prapunwattana, P.; Krungkrai, S.R.; Rochanakij, S.: A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase. Anal. Biochem., 299, 162-168 (2001) [21] Yablonski, M.J.; Pasek, D.A.; Han, B.D.; Jones, M.E.; Traut, T.W.: Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine5'-phosphate decarboxylase. J. Biol. Chem., 271, 10704-10708 (1996) [22] Bayles, D.O.; Fennington, G.J., Jr.; Hughes, T.A.: Sequence and phylogenetic analysis of the Rhizobium leguminosarum biovar trifolii pyrE gene, overproduction, purification and characterization of orotate phosphoribosyltransferase. Gene, 195, 329-336 (1997) [23] Witte, J.F.; Tsou, R.; McClard, R.W.: Cloning, overproduction, and purification of native and mutant recombinant yeast orotate phosphoribosyltransferase and the demonstration from magnetization inversion transfer that a proposed oxocarbocation intermediate does not have a kinetic lifetime. Arch. Biochem. Biophys., 361, 106-112 (1999) [24] Henriksen, A.; Aghajari, N.; Jensen, K.F.; Gajhede, M.: A flexible loop at the dimer interface is a part of the active site of the adjacent monomer of Escherichia coli orotate phosphoribosyltransferase. Biochemistry, 35, 38033809 (1996) [25] Scapin, G.; Grubmeyer, C.; Sacchettini, J.C.: Crystal structure of orotate phosphoribosyltransferase. Biochemistry, 33, 1287-1294 (1994)

136

Nicotinate phosphoribosyltransferase

2.4.2.11

1 Nomenclature EC number 2.4.2.11 Systematic name nicotinate-nucleotide:diphosphate phospho-a-d-ribosyltransferase Recommended name nicotinate phosphoribosyltransferase Synonyms NAPRTase [13, 14] NPRTase [7, 8] niacin ribonucleotidase nicotinate phosphoribosyltransferase [5, 9, 14] nicotinate-nucleotide:pyrophosphate phospho-a-d-ribosyltransferase nicotinic acid mononucleotide glycohydrolase nicotinic acid mononucleotide pyrophosphorylase [1, 2, 10] nicotinic acid phosphoribosyltransferase [13, 14] CAS registry number 9030-26-6

2 Source Organism Bacillus subtilis (SB19 [10]) [10] Escherichia coli (K-12 [1]) [1] Saccharomyces cerevisiae (bakersÂyeast [3,6]; Budweiser brand [6]) [3, 6, 9, 12] Bos taurus (beef [2]) [2] Homo sapiens [4, 5, 15] Sus scrofa (hog [7]) [7, 8] Rattus norvegicus (male Wistar strain [7]) [7, 8] Salmonella typhimurium [11-14] Mycobacterium tuberculosis [16] Saccharomyces cerevisiae (SwissProt-ID: P39683) [16] Saccharomyces cerevisiae (SwissProt-ID: S51867) [16] Salmonella typhimurium [16]

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3 Reaction and Specificity Catalyzed reaction nicotinate d-ribonucleotide + diphosphate = nicotinate + 5-phospho-a-d-ribose 1-diphosphate ( ordered ping pong kinetic mechanism [6]; sequential steady state mechanism [9]) Reaction type pentosyl group transfer Natural substrates and products S nicotinate + 5-phospho-a-d-ribose 1-diphosphate ( first step in NAD+ -(salvage) biosynthesis [3,5]; lack of the de novo pathway for NAD+ synthesis from tryptophan, utilizes both nicotinic acid and nicotinamide as NAD+ precursors [15]) [1, 2, 3, 5, 6, 15] P nicotinic acid mononucleotide + diphosphate Substrates and products S nicotinate + 5-phospho-a-d-ribose 1-diphosphate ( favoured reaction [10]; the pyridine N and carboxyl groups are important for interaction with the enzyme [4]; no phosphoribosylation of nicotinamide, quinolic acid, adenine or hypoxanthine [7]; the enzyme is a facultative ATPase that uses ATP hydrolysis to drive the synthesis of nicotinate mononucleotide and diphosphate from nicotinic acid and phosphoribosyl diphosphate in a steady-state reaction, the enzyme undergoes phosphorylation in His-219 by bound ATP, phosphorylated enzyme has higher affinity for substrates than does nonphosphorylated enzyme [1114]; mutants in the His-219 residue catalyze the slow formation of nicotinic acid mononucleotide in the absence of ATP similarly to the wild type enzymes [12]; in the presence of Mg2+ and phosphate [15]) (Reversibility: r [1,2,10,11,14]) [1-16] P nicotinate d-ribonucleotide + diphosphate [1-5, 9, 10] S Additional information ( ATPase activity in the presence of either product and in the absence of phosphoribose diphosphate [6]; one mol ATP is cleaved per mol product formed [4,5,10]; no ATPase activity in the absence of substrates [4,5]; ATP and phosphoribose diphosphate compete for ATP-binding site [6]; negligible ATPase activity in the absence of nicotinic acid [12]) [4-6, 10, 12] P ? Inhibitors 3-acetylpyridine [10] 3-pyridylcarbinol ( weak inhibitor [10]) [10] 3-pyridylcarboxaldehyde [10] 5-fluoronicotinic acid [10] 5-phospho-a-d-ribose 1-diphosphate ( at high concentrations [6]; at high concentrations, when Mg2+ below 0.03 mM [9]) [6, 9]

138

2.4.2.11


ATP ( in the presence of Mn2+ [7]; at high concentrations with Mg2+ below 0.03 mM [9]; 3 mM, competitive inhibition of nicotinic acid mononucleotide formation in mutants enzymes [12]) [7, 9, 12] GDP [7] GMP [7] IDP [7] IMP [7] ITP [7] KCN [10] Mg2+ ( above 10 mM, significant pH alterations [3]; at higher concentrations than ATP, when free Mg2+ ions occur [2]) [2, 3] MgADP- ( non-competitive to Mg-ATP or Mg-diphosphate [9]) [9] N-ethylmaleimide ( 0.1 mM and above [7]) [7] NAD+ ( weak inhibitor [7]; not [10]) [7] NADH ( weak inhibitor [7]) [7] NADP+ ( weak inhibitor [7]; not [10]) [7] NADPH ( weak inhibitor [7]) [7] PCMB ( 0.01 mM and above [7]) [7] UTP ( weak [2]) [2] diphosphate [2] methyl 3-pyridylcarbinol ( weak inhibitor [10]) [10] monoiodoacetic acid ( 1 mM and above [7]) [7] nicotinate analogues ( not [7]) [4] nicotinate mononucleotide ( competitive to both substrates [2]; competitive to MgATP2- [9]; non-competitive to nicotinate [9]; competitive to nicotinate and noncompetitive to phosphoribobose diphosphate [7]; not [10]) [2, 6, 7, 9] nucleoside diphosphates ( e.g. ADP or GDP [10]) [10] phosphate ( 0.1 M and above [3]) [3] pyrazine 2-carboxylic acid [7] pyridazine-4,5-dicarboxylic acid ( weak, 10% less activity [7]) [7] tripolyphosphate [2] urea ( competitive to nicotinate mononucleotide [10]) [10] Additional information ( no inhibition by nicotinate, up to 10 mM [2]; no inhibition by nicotinamide [2, 10]; no inhibition by nicotinamide mononucleotide, deamino-NAD+ [7]; no inhibition by 4-hydroxy-nicotinate [4]; no inhibition by quinolinic acid, AMP, GMP [10]; no inhibition by antibodies against pig kidney quinolinate phosphoribosyltransferase [7]) [2, 4, 7, 10] Cofactors/prosthetic groups ATP ( activation [1, 2, 5, 7, 8]; 60-70% stimulation with different enzyme preparations, the largest effects are observed with aged preparations of the enzyme [2]; requirement in the absence of phosphate [8]; together with Mg2+ [1-4, 7]; together with Co2+ [7]; 1 mol ATP is hydrolyzed to ADP plus phosphate per mol product formed [3, 4, 5, 10, 12, 14, 16]; reaction accelerated 10-30fold [11]; the Vmax

139


2.4.2.11

of nicotinic acid mononucleotide synthesis activity is stimulated 10fold [12]; 2000fold increase in the Kcat /KM [13]; 1640fold increase in the product/substrate ratio [16]) [1-5, 7, 8, 10-14, 16] CTP ( activation, about 70% as effective as ATP [10]; one third the activity of ATP [3]; 20-30% as effective as ATP [2]) [2, 3, 10] GTP ( activation, equally effective as ATP [10]; shows almost as much activity as ATP [3]; 20-30% as effective as ATP [2]; not [7]) [2, 3, 10] ITP ( activation, about 90% as effective as ATP [10]; shows almost as much activity as ATP [3]; 20-30% as effective as ATP [2]; not [7]) [2, 3, 10, 11] TTP ( activation, about 30% as effective as ATP [10]; not [3]) [10] UTP ( activation, about 50% as effective as ATP [10]; one third the activity of ATP [3]) [1, 3, 10] Activating compounds GSH ( activation [1]) [1] tripolyphosphate ( activation, can replace ATP [10]) [10] Additional information ( no activation by ADP [2,3,7]; no activation by AMP, 2'-AMP [7]; no activation by 3'-AMP, 5'-AMP [3, 7]; 3,5-cycloadenosine, borate and sulfate do not affect the reaction rate [2]) [2, 3, 7] Metals, ions Co2+ ( requirement, slight synergism with ATP, more effective than Mg2+ with or without ATP [7]) [7] Fe2+ ( slight stimulation, synergism with ATP [7]) [7] Mg2+ ( requirement [1-4, 7, 9]; 1-10 mM [3]; 0.515 mM [9]; synergism with ATP [1-4, 7]; maximal activity: 20 mM [1]) [1-4, 7, 9] MgATP2- ( maximal activation at equimolar levels of Mg2+ and ATP, presumably enzyme structure stabilizing [2]) [2, 3] Mn2+ ( requirement, 7.5 mM, no synergism with ATP, more effective than Mg2+ with or without ATP [7]) [7] NaF ( activation [1]) [1] Zn2+ ( slight stimulation [7]) [7] phosphate ( activation, 0.01-0.1 M, in the presence of Mg2+ [3]; markedly stimulates even in the absence of ATP [8]; 50 mM, strongly stimulates [9]) [2, 3, 8, 9] Additional information ( no activation by borate, sulfate [2]; absolute requirement of divalent cation, no activation with Ba2+ , Ca2+ , Cu2+ , Cd2+ , Al3+ , Fe3+ [7]) [2, 7] Specific activity (U/mg) 0.0012 [2] 0.052 [5] 0.095 ( crude extract [7]) [7]

140

2.4.2.11


2.3 [3] 3 [14] 160.5 ( DEAE-Sephadex fraction [7]) [7] 421.2 ( Sephacryl fraction [7]) [7] 457.3 ( phenyl-Sepharose fraction [7]) [7] Km-Value (mM) 0.0005 (nicotinate, in the presence of ATP [5]) [5] 0.0008-0.0015 (nicotinate) [10] 0.001 (nicotinate) [2] 0.0015 (nicotinate, pncB gene, in the coupled reaction [14]) [14] 0.002 (nicotinate) [1] 0.013 (5-phospho-a-d-ribose 1-diphosphate, reduced to 0.002 mM in the presence of ATP [8]) [8] 0.02 (Mg-5-phospho-a-d-ribose 1-diphosphate, in the coupled reaction, 200fold lower than in the uncoupled reaction [11]) [11] 0.022 (5-phospho-a-d-ribose 1-diphosphate, pncB gene, in the coupled reaction [14]) [14] 0.023 (nicotinate) [6] 0.024 (5-phospho-a-d-ribose 1-diphosphate) [6] 0.024 (nicotinate, reduced to 0.0005 mM in the presence of ATP [5]) [4, 5] 0.03 (5-phospho-a-d-ribose 1-diphosphate) [1] 0.05 (5-phospho-a-d-ribose 1-diphosphate) [2] 0.06-0.1 (5-phospho-a-d-ribose 1-diphosphate) [5, 10] 0.07 (ATP) [6] 0.09 (ATP) [3] 0.1 (5-phospho-a-d-ribose 1-diphosphate, 0.005 mM in the presence of ATP [5]) [5] 0.22 (nicotinate, reduced to 0.053 mM in the presence of ATP [8]) [8] 0.29 (nicotinate, pncB gene, in the uncoupled reaction [14]) [14] 0.3 (nicotinate) [12] 0.33 (ATP) [11] 0.33-0.4 (nicotinate, mutant enzymes [12]) [12] 0.34 (GTP) [10] 0.35 (ATP) [10] 0.4 (ATP) [1] 1.16 (ITP, good alternative substrate for ATP [11]) [11] 2.3-48 (5-phospho-a-d-ribose 1-diphosphate, mutant enzymes [12]) [12] 4.5 (5-phospho-a-d-ribose 1-diphosphate, pncB gene, in the uncoupled reaction [14]) [12, 14]

141


2.4.2.11

pH-Optimum 6.5-8 ( broad, 0.2 M potassium phosphate buffer or 0.05 M Tris-Cl [5]) [5] 7.2 ( broad, phosphorolysis [2]) [2] 7.3-7.4 [7] 7.5-8.5 ( broad [3]) [3] 8.5 [1, 10] Additional information ( pI: 4.8 [7]; pI: 6.9 [6]) [6, 7] pH-Range 5.5-8 ( about half-maximal activity at pH 5.5 and about 85% of maximal activity at pH 8 [2]) [2] 5.5-10 ( about half-maximal activity at pH 5.5 and 10 [3]) [3] 6.5-9 [10] Additional information ( active over a wide range [1,2]) [1, 2] Temperature optimum ( C) 37 ( assay at [1-5,7-10]) [1-5, 7-10]

4 Enzyme Structure Molecular weight 43000 ( gel filtration [3]) [3] 45000 ( SDS-PAGE in the presence and absence of mercaptoethanol [6]) [6] 45000 ( pncB gene, SDS-PAGE [14]) [14] 45500 ( mutant enzyme, SDS-PAGE [12]) [12] 45530 ( predicted from DNA sequence [16]) [16] 45540 ( SDS-PAGE [12]) [12] 45550 ( mutant enzyme, SDS-PAGE [12]) [12] 86000 ( gel filtration [5]) [5] 120000 ( liver, SDS-PAGE [7]) [7] Subunits dimer ( 2 * 63000, SDS-PAGE [7]) [7] monomer ( 1 * 45529, gel filtration [12]) [12, 14, 16]

5 Isolation/Preparation/Mutation/Application Source/tissue blood clot [7] blood platelet [4] erythrocyte [5, 15] heart [7] kidney [7] liver [2, 7, 8]

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pancreas [7] small intestine [7] spleen [7] Additional information ( distribution in rat tissues: trace activities in brain, lung or stomach, not in thigh muscle, testis or serum [7]) [7] Localization soluble [2, 14] Purification (partial, extraction, ammonium sulfate fractionation and chromatography on DEAE-cellulose, 60fold purified [10]) [10] (bacteria disruption by alumina grinding, protamine sulfate precipitation and chromatography on DEAE-cellulose [1]) [1] (extraction, ammonium sulfate fractionation, chromatography on DEAE-cellulose and hydroxylapatite, 1000fold purified [3]; extraction, fractionation chromatography on phosphocellulose, hydroxyapatite, blue Sephadex and DEAE-cellulose [6]) [3, 6, 9] (extraction, ammonium sulfate fractionation and chromatography on DEAE-cellulose [2]) [2] (ammonium sulfate fractionation, chromatography on DEAE-cellulose, chromatography on Sephadex, affinity column chromatography and hydroxylapatite column chromatography, 30000fold purified [5]; heparinised blood lysates [15]) [5, 15] (ammonium hydroxide precipitation, chromatography on DEAE-Sephadex, Sephacryl and phenyl-Sepharose [7]) [7] (homogenates [7]) [7] (bacterial suspension disruption and centrifugation, chromatography on Q-Sepharose and phenyl-Sepharose [13]; bacterial suspension disruption and centrifugation, chromatography on Sephadex-G, DEAE-Toyopearl, Cibacron Blue and Sephacryl gel filtration [14]) [13, 14] Cloning (expression in E.coli [12]) [12] (expression of pncB gene on a multicopy pUC19 plasmid in Salmonella typhimurium [14]) [14]

6 Stability pH-Stability 5-10 ( stable, 0.2 M potassium phosphate buffer [5]) [5] 6.5 ( inactivation below, phosphorolysis [2]) [2] 8.3 ( native enzyme, unstable [16]) [16] Temperature stability 50 ( 5 min: 95% loss of activity, MgATP2-, Mg-phosphoribose diphosphate, ATP, GTP or nicotinic acid protect, with 27%, 37%, 52%, 54% or 61% loss of activity, respectively. 10 min: 97% loss of activity, MgATP2-, Mg143


2.4.2.11

5-phospho-a-d-ribose 1-diphosphate, ATP, GTP or nicotinic acid protect, with 43%, 52%, 60%, 63% or 69% loss of activity, respectively [3]) [3] Additional information ( thermostability of the enzyme is increased by ATP and 5-phospho-a-d-ribose 1-diphosphate [16]) [16] General stability information , unstable to heat, substrates protect [3] , 50% activity recovered at salt concentrations of 50 mM, inactivation with salt concentrations below 20 mM, 20-35% glycerol partially protects [5] , glycerol, 20-35%, stabilizes during dialysis against low buffer concentrations, more highly purified preparations require higher glycerol concentrations [5] , first order activity loss in the presence of 1/200 trypsin/enzyme [12, 13, 16] , rapidly inactivates the ATPase and nicotinic acid mononucleotide synthesis activities with cleavages at Arg-384 and Lys-374, ATP and 5-phosphoa-d-ribose 1-diphosphate increase the t1=2 for inactivation 8fold and 4fold, respectively, and 32fold, together [13, 16] , sensitive to trypsin, partial proteolysis, 4fold increase in t1=2 for inactivation in the presence of 1 mM MgATP2- [12, 16] , dialysis against 50 mM Tris-HCl buffer at pH 7.3 in the absence of potassium phosphate, significant loss of activity [8] Storage stability , -25 C, at least 1 month [3] , -12 C, partially purified enzyme preparation, t1=2 : at least 3 months [2] , -70 C, t1=2 : 3 months [5] , -70 C, no significantly decreased activity for up to 3 years, no significantly decreased after 8 years [15] , 4 C, as 70% saturated ammonium sulfate suspensions in 200 mM phosphate buffer, pH 8, 10% glycerol and 5 mM DTT [12-14] , t1=2 : at least 2 months [14]

References [1] Imsande, J.: Pathway of diphosphopyridine nucleotide biosynthesis in Escherichia coli. J. Biol. Chem., 236, 1494-1497 (1961) [2] Imsande, J.; Handler, P.: Biosynthesis of diphosphopyridine nucleotide. III. Nicotinic acid mononucleotide pyrophosphorylase. J. Biol. Chem., 236, 525530 (1961) [3] Kosaka, A.; Spivey, H.O.; Gholson, R.K.: Nicotinate phosphoribosyltransferase of yeast. Purification and properties. J. Biol. Chem., 246, 3277-3283 (1971) [4] Gaut, Z.N.; Solomon, H.M.: Inhibition of nicotinate phosphoribosyltransferase in human platelet lysate by nicotinic acid analogs. Biochem. Pharmacol., 20, 2903-2906 (1971) 144

2.4.2.11


[5] Niedel, J.; Dietrich, L.S.: Nicotinate phosphoribosyltransferase of human erythrocytes. Purification and properties. J. Biol. Chem., 248, 3500-3505 (1973) [6] Hanna, L.S.; Hess, S.L.; Sloan, D.L.: Kinetic analysis of nicotinate phosphoribosyltransferase from yeast using high pressure liquid chromatography. J. Biol. Chem., 258, 9745-9754 (1983) [7] Hayakawa, T.; Shibata, K.; Iwai, K.: Purification and some properties of nicotinate phosphoribosyltransferase from hog liver. Agric. Biol. Chem., 48, 445-453 (1984) [8] Hayakawa, T.; Shibata, K.; Iwai, K.: Nicotinate phosphoribosyltransferase from hog liver: regulatory effect of ATP at a physiological concentrations of 5-phosphoribosyl-1-pyrophosphte. Agric. Biol. Chem., 48, 455-460 (1984) [9] Kosaka, A.; Spivey, H.O.; Gholson, R.K.: Yeast nicotinic acid phosphoribosyltransferase. Studies of reaction paths, phosphoenzyme, and Mg2+ effects. Arch. Biochem. Biophys., 179, 334-341 (1977) [10] Imsande, J.: A cross-linked control system. I. Properties of a triphosphatedependent nicotinic acid mononucleotide pyrophosphorylase from Bacillus subtilis. Biochim. Biophys. Acta, 85, 255-264 (1964) [11] Gross, J.W.; Rajavel, M.; Grubmeyer, C.: Kinetic mechanism of nicotinic acid phosphoribosyltransferase. Implications for energy coupling. Biochemistry, 37, 4189-4199 (1998) [12] Rajavel, M.; Lalo, D.; Gross, J.W.; Grubmeyer, C.: Conversion of a cosubstrate to an inhibitor. Phosphorylation mutants of nicotinic acid phosphoribosyltransferase. Biochemistry, 37, 4181-4188 (1998) [13] Rajavel, M.; Gross, J.W.; Segura, E.; Moore, W.T.; Grubmeyer, C.: Limited proteolysis of Salmonella typhimurium nicotinic acid phosphoribosyltransferase reveals ATP-linked conformational change. Biochemistry, 35, 39093916 (1996) [14] Vinitsky, A.; Grubmeyer, C.: A new paradigm for biochemical energy coupling. Salmonella typhimurium nicotinate phosphoribosyltransferase. J. Biol. Chem., 268, 26004-26010 (1993) [15] Pescaglini, M.; Micheli, V.; Simmonds, H.A.; Rocchigiani, M.; Pompucci, G.: Nicotinic acid phosphoribosyltransferase activity in human erythtocytes: studies using a niw HPLC method. Clin. Chim. Acta, 229, 15-25 (1994) [16] Grubmeyer, C.T.; Gross, J.W.; Rajavel, M.: Energy coupling through molecular discrimination: nicotinate phosphoribosyltransferase. Methods Enzymol., 308, 28-48 (1999)

145

Nicotinamide phosphoribosyltransferase

2.4.2.12

1 Nomenclature EC number 2.4.2.12 Systematic name nicotinamide-nucleotide:diphosphate phospho-a-d-ribosyltransferase Recommended name nicotinamide phosphoribosyltransferase Synonyms NAmPRTase NMN pyrophosphorylase NMN synthetase PBEF nicotinamide mononucleotide pyrophosphorylase nicotinamide mononucleotide synthetase phosphoribosyltransferase, nicotinamide pre-B-cell colony-enhancing factor CAS registry number 9030-27-7

2 Source Organism

Homo sapiens [1, 4, 6] Rattus norvegicus (male Sprague-Dawley [5]) [2, 3, 5] Mus musculus (identified as pre-B-cell colony-enhancing factor [7]) [7] Haemophilus ducreyi [8]

3 Reaction and Specificity Catalyzed reaction nicotinamide d-ribonucleotide + diphosphate = nicotinamide + 5-phosphoa-d-ribose 1-diphosphate ( mechanism [3]) Reaction type pentosyl group transfer

146

2.4.2.12


Natural substrates and products S nicotinamide + 5-phospho-a-d-ribose 1-diphosphate ( first step in NAD+ synthesis from nicotinamide [2]; pre-B-cell colony enhancing factor [7]) (Reversibility: ? [1-8]) [1-8] P nicotinamide d-ribonucleotide + diphosphate [1-6] Substrates and products S nicotinamide + 5-phospho-a-d-ribose 1-diphosphate ( b-nicotinamide [3]; specific for nicotinamide, no substrates: thymine, 5bromouracil, nicotinic acid adenine dinucleotide [4]) (Reversibility: ? [1-8]) [1-8] P nicotinamide d-ribonucleotide + diphosphate [1-6] Inhibitors 3-acetylpyridine ( weak, kinetics [3]) [3] 3-acetylpyridine adenine dinucleotide ( kinetics [3]) [3] 5-fluoronicotinamide ( weak, kinetics [3]) [3] 6-aminonicotinamide ( weak, kinetics [3]) [3] NAD+ ( strong (thigh muscle) [5]; 1 mM, 50%-90% inhibition in various tissues, at 0.2 mM not inhibitory in lung and liver [5]; kinetics [3]) [3-5] NADH ( weak [3]) [3] NADP+ [3] NADPH ( weak [3]) [3] a-NAD+ ( weak [3]) [3] hypoxanthine ( phosphorolysis [1]) [1] nicotinamide hypoxanthine dinucleotide [3] nicotinamide mononucleotide ( kinetics [3]) [3] nicotinamide mononucleotide-H2 ( strong, kinetics [3]) [3] nicotinamide riboside ( kinetics [3]) [3] nicotinate adenine dinucleotide ( weak [3]; not inhibitory [4]) [3] thionicotinamide ( weak, kinetics [3]) [3] thionicotinamide adenine dinucleotide [3] Additional information ( no inhibition by NaF [1]; nicotinic acid, thymine, 5-bromouracil [4]) [1, 4] Cofactors/prosthetic groups ATP ( requirement [2-5]; activation, up to 0.4 mM [6]) [2-6] Activating compounds EDTA ( 50% activation, 1 mM [2]) [2] Metals, ions Mg2+ ( requirement [2-5]; activation, up to 10 mM [6]; Km -value: 0.6 mM [1]) [1-6] Specific activity (U/mg) 0.000225 [2] 0.003 [3] Additional information ( activity per g hemoglobin [6]) [6] 147


2.4.2.12

Km-Value (mM) 0.000067 (nicotinamide) [2] 0.001 (nicotinamide, in the presence of ATP [3]) [3] 0.00124 (nicotinamide) [7] 0.00127 (nicotinamide) [6] 0.0016 (nicotinamide) [4] 0.0038 (5-phospho-a-d-ribose 1-diphosphate) [2] 0.00537 (5-phospho-a-d-ribose 1-diphosphate) [6] 100 (nicotinamide, in the absence of ATP [3]) [1, 3] Additional information ( HPLC linked assay method [6]) [6] pH-Optimum 6.8-7.6 [1] 8.5-9 ( assay at pH 8.8. where NAD glycohydrolase is not active [2]) [2] pH-Range 6.1-8.5 ( about half-maximal activity at pH 6.1 and pH 8.5 [1]) [1] 7.8-9.2 ( about half-maximal activity at pH 7.8 and about 80% of maximal activity at pH 9.2 [2]) [2] Temperature optimum ( C) 35 ( assay at [1]) [1] 37 ( assay at [2-6]) [2-6]

4 Enzyme Structure Subunits ? ( x * 55600, deduced from gene sequence [8]) [8]

5 Isolation/Preparation/Mutation/Application Source/tissue brain [5] erythrocyte [1, 2, 6] fibroblast (diploid, GM 1362, from patient with Lesch-Nyhan disease [4]) [4] heart [5] kidney [5] liver [3, 5] lung [5] lymphocyte [7] pancreas [5] small intestine [5] stomach [5] testis [5]

148

2.4.2.12


thigh muscle [5] Additional information ( tissue distribution [5]; ubiquitous in all tissues [7]) [5, 7] Localization cytoplasm [7] soluble [1, 2, 4, 6] Purification (partial [1]) [1] (partial [2]) [2] Cloning (pre-B-cell colony enhancing factor [7]) [7] (significant homology to human pre-B-cell colony enhancing factor [8]) [8]

6 Stability pH-Stability 7-10 ( stable [2]) [2] 8.8 ( rapid inactivation below [2]) [2] Temperature stability 65 ( 10 min, stable, heat treatment during purification [1]) [1] 70 ( 5 min, stable, heat treatment during purification [1]) [1] General stability information , freeze-thawing inactivates [4] , bovine serum albumin, glutathione, cysteine, DTT, EDTA, cystine or H2 O2 does not stabilize during storage [2] Storage stability , -70 C, in crude erythrocyte lysates, at least 30 days [6] , -80 C, crude, 4-6 months [4] , 4 C, crude, inactivation overnight [4] , -20 C to +20 C, about 70% loss of activity overnight [2] , storage at room temperature, 4 C, or frozen, unstable upon [2]

References [1] Preiss, J.; Handler, P.: Enzymatic synthesis of nicotinamide mononucleotide. J. Biol. Chem., 225, 759-770 (1957) [2] Lin, L.F.H.; Lan, S.J.; Richardson, A.H.; Henderson, L.V.M.: Pyridine nucleotide synthesis. Purification of nicotinamide mononucleotide pyrophosphorylase from rat erythrocytes. J. Biol. Chem., 247, 8016-8022 (1972) [3] Dietrich, L.S.; Muniz, O.: Inhibition of nicotinamide phosphoribosyltransferase by pyridine nucleotides. Biochemistry, 11, 1691-1695 (1972) 149


2.4.2.12

[4] Elliott, G.C.; Rechsteiner, M.C.: Evidence for a physiologically active nicotinamide phosphoribosyltransferase in cultured human fibroblasts. Biochem. Biophys. Res. Commun., 104, 996-1002 (1982) [5] Shibata, K.; Taguchi, H.; Nishitani, H.; Okumura, K.; Shimabayashi, Y.; Matsushita, N.; Yamazaki, H.: End product inhibition of the activity of nicotinamide phosphoribosyltransferase from various tissues of rats by NAD. Agric. Biol. Chem., 53, 2283-2284 (1989) [6] Rocchigiani, M.; Micheli, V.; Duley, J.A.; Simmonds, H.A.: Determination of nicotinamide phosphoribosyltransferase activity in human erythrocytes: high-performance liquid chromatography-linked method. Anal. Biochem., 205, 334-336 (1992) [7] Rongvaux, A.; Shea, R.J.; Mulks, M.H.; Gigot, D.; Urbain, J.; Leo, O.; Andris, F.: Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis. Eur. J. Immunol., 32, 3225-3234 (2002) [8] Martin, P.R.; Shea, R.J.; Mulks, M.H.: Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence. J. Bacteriol., 183, 1168-1174 (2001)

150

Recommended Name never specified

2.4.2.13

1 Nomenclature EC number 2.4.2.13 (transferred to EC 2.5.1.6) Recommended name Recommended Name never specified

151

Amidophosphoribosyltransferase

2.4.2.14

1 Nomenclature EC number 2.4.2.14 Systematic name 5-phosphoribosylamine:diphosphate phospho-a-d-ribosyltransferase (glutamate-amidating) Recommended name amidophosphoribosyltransferase Synonyms 5'-phosphoribosylpyrophosphate amidotransferase 5-phosphoribosyl-1-pyrophosphate amidotransferase 5-phosphoribosylpyrophosphate amidotransferase 5-phosphororibosyl-1-pyrophosphate amidotransferase ATASE GPAT GPATase a-5-phosphoribosyl-1-pyrophosphate amidotransferase amidotransferase, phosphoribosyl pyrophosphate glutamine 5-phosphoribosylpyrophosphate amidotransferase glutamine phosphoribosylpyrophosphate amidotransferase glutamine ribosylpyrophosphate 5-phosphate amidotransferase phosphoribose pyrophosphate amidotransferase phosphoribosyl pyrophosphate amidotransferase phosphoribosyldiphosphate 5-amidotransferase phosphoribosylpyrophosphate glutamyl amidotransferase CAS registry number 9031-82-7

2 Source Organism Escherichia coli (strains K-12 or B-96, purine requiring strain [3]) [3, 10, 11, 19, 21, 22, 23, 26] Bacillus subtilis [4, 5, 9-11, 15, 25, 26] Artemia sp. (brine shrimp, activity increases 7fold during early larval development in 2 d old Artemia [6]) [6] Gallus gallus [11, 18]

152

2.4.2.14


Cricetulus griseus [10] Homo sapiens [8, 10, 13, 14, 29] Mus musculus [10, 24] Columba livia (pigeon [1]) [1, 2, 10, 12] Rattus norvegicus [1, 7, 10] Schizosaccharomyces pombe (wild type strain 972h- and mutant strain ade 2h- devoid of adenylosuccinate synthase [17]) [17] Glycine max (soybean, Merr. cv. Williams [16]) [16, 20] Bos taurus [27] Aquifex aeolicus [28]

3 Reaction and Specificity Catalyzed reaction 5-phospho-b-d-ribosylamine + diphosphate + l-glutamate = l-glutamine + 5-phospho-a-d-ribose 1-diphosphate + H2 O ( mechanism of glutamine amide transfer [11]; binding of 5-phospho-a-d-ribose 1-diphosphate activates the enzyme by a structural change that lowers the Km for glutamine 100fold and couples glutamine hydrolysis to synthesis of 5-phospho-b-d-ribosylamine [23]) Reaction type amino group transfer Natural substrates and products S l-glutamine + 5-phospho-a-d-ribose 1-diphosphate + H2 O ( first reaction in de-novo pathway of purine biosynthesis [3, 4, 10]; rate-limiting enzyme of purine biosynthesis [7]; regulating enzyme of purine biosynthesis [13]; enzyme of purine biosynthetic pathway, regulatory enzyme in the flow of recently fixed nitrogen from initial assimilation into amino acids via purine biosynthesis [16]) (Reversibility: ? [3, 7, 10, 13, 16]) [3, 4, 7, 10, 13, 16] P 5-phospho-b-d-ribosylamine + diphosphate [3, 4, 7, 10, 13, 16] Substrates and products S l-glutamine + 5-phospho-a-d-ribose 1-diphosphate + H2 O ( reaction at 33% the rate of amminotransferase activity [14]; reaction at 50% the rate of aminotransferase activity [16]; reaction at 70% the rate of aminotransferase activity [11]; glutamine binding site distinct from NH3 -site [10]; no activity with carbamoyl phosphate [14]) (Reversibility: ? [3, 10, 11, 14, 16, 17, 27]; ir [2, 6]) [1-18, 27] P 5-phospho-b-d-ribosylamine + l-glutamate + diphosphate [1-18, 27]

153


2.4.2.14

S l-glutamine + H2 O ( enzyme exhibits glutaminase activity in the absence of other substrates or effectors [3]) (Reversibility: ? [3]) [3, 22, 23] P l-glutamate + NH3 [3, 22, 23] S NH3 + 5-phospho-a-d-ribose 1-diphosphate + H2 O ( NH3 binding site is distinct from glutamine-site [10]; no activity with NH+4 [11]; 2.8fold higher aminotransferase activity compared to amidotransferase activity [3]; lower aminotransferase activity compared to amidotransferase activity [6]; 3fold higher aminotransferase activity compared to amidotransferase activity [14]; 2fold higher aminotransferase activity compared to amidotransferase activity [16]) (Reversibility: ? [3,6,10,11,14,16]) [3, 6, 10, 11, 14, 16] P 5-phospho-b-d-ribosylamine + diphosphate [3, 6, 10, 11, 14, 16] Inhibitors 1-a-diphosphoryl-2-a,3-a-dihydroxy-4-b-cyclopentane-methanol-5-phosphate ( competitive vs. 5-phospho-a-d-ribose 1-diphosphate in wild-type and P410W mutant, competition for the amidotransferase C site [22]) [22] 2-amino-4-oxo-5-chloropentanoate ( approx. 80% inactivation of NH3 -dependent activity after 30 min [3]) [3] 5',5'''-P1,P4 -diguanosine tetraphosphate ( 0.4 mM, 505 inhibition, 2 mM, approx. 80% inhibition [6]) [6] 5'-p-fluorosulfonylbenzoyladenosine ( inactivation follows pseudofirst order kinetic, AMP, GMP and 5-phospho-a-d-ribose 1-diphosphate protect [19]) [19] 6-diazo-5-oxo-l-norleucine ( approx. 98% inactivation of amidotransferase activity after 30 min [3]; competitive vs. glutamine, inactivation half-life: 11 min [14]) [3, 14] 6-iodopurine ( 5 mM, 39% inhibition [13]) [13] 6-mercaptopurine ribonucleotide ( 5 mM, 64% inhibition [13]) [13] ADP ( 1 mM, approx. 95% inhibition [6]; 5 mM, 40% inhibition [13]; 50% inhibition of wild-type, S283A, K305Q, R307Q and S347A mutant enzyme at 4.7 mM, 24 mM, 31 mM, 28 mM and 8.1 mM respectively [25]; 4.1 mM, 50% inhibition [28]) [1, 3, 6, 13, 25, 28] AMP ( approx. 10 mM, complete inhibition, sigmoidal inhibition curve, competitive vs. 5-phospho-a-d-ribose 1-diphosphate, GMP and AMP together have a synergistic effect on inhibition [3]; 1 mM, approx. 40% inhibition [6]; no inhibition in the presence of 5-phospho-a-d-ribose 1-diphosphate at high concentrations [7]; 1.8 mM, 50% inhibition [8]; 5 mM, 76% inhibition, noncompetive vs. glutamine [13]; 5 mM, 79% and 24% inhibition of aminotransferase and amidotransferase activity at 1 mM 5-phospho-a-d-ribose 1-diphosphate [14]; competitive vs. 5-phospho-a-d-ribose 1-diphosphate [16]; 2.7 mM and 1.2 mM, 50% inhibition of stable and unstable enzyme preparation respectively [17]; 5 mM, 50% inhibition [18]; 4.7 mM, 50% in-

154

2.4.2.14


hibition [19]; competitive vs. 5-phospho-a-d-ribose 1-diphosphate, noncompetitive vs. glutamine [24]; 50% inhibition of wild-type, S283A, K305Q, R307Q and S347A mutant enzyme at 0.9 mM, 6.1 mM, 2.5 mM, 2.6 mM and 1.5 mM respectively [25]; 6.12 mM, 50% inhibition [28]) [1, 3, 6, 7, 8, 13, 14, 16, 17, 18, 19, 24, 25, 28] ATP ( 5 mM, 21% inhibition [13]) [1, 13] CMP ( 5 mM, 22% inhibiiton [13]) [13] GDP ( 1 mM, approx. 75% inhibition [6]; 5 mM, 40% inhibition [13]; 8.5 mM, 50% inhibition [28]) [1, 3, 6, 13, 28] GMP ( approx. 2.5 mM, complete inhibition, highly sigmoidal inhibition curve, competitive vs. 5-phospho-a-d-ribose 1diphosphate, GMP and AMP together have an synergistic effect on inhibition [3]; 1 mM, approx. 60% inhibition [6]; no inhibition in the presence of 5-phospho-a-d-ribose 1-diphosphate at high concentrations [7]; 0.5 mM; 50% inhibition [8]; 5 mM, 70% inhibition [13]; 5 mM, 56% and 12% inhibition of aminotransferase and amidotransferase activity at 1 mM 5-phospho-a-d-ribose 1-diphosphate [14]; 0.44 mM and 0.25 mM, 50% inhibition of stable and unstable enzyme preparation respectively [17]; 2 mM, 50% inhibition [18]; 1.2-1.4 mM, 50% inhibition [19]; 50% inhibition of wild-type, S283A, K305Q, R307Q and S347A mutant enzyme at 9.4 mM, 6.6 mM, 50 mM, 50 mM and 145 mM respectively [25]; 17.9 mM, 505 inhibition [28]) [1, 3, 6, 7, 8, 13, 14, 16, 17, 18, 19, 25, 28] GTP ( 5 mM, 79% inhibition [3]; 5 mM, 13% inhibition [13]) [3, 13] IDP ( 5 mM, 29% inhibition [13]) [13] IMP ( 5 mM, 86% inhibition [3]; 5 mM, 41% inhibition [13]; 50% inhibition of wild-type and S347A mutant enzyme at 26 mM and 41 mM respectively [25]) [1, 3, 13, 16, 25] 8-aza-AMP ( 1.2-1.4 mM, 50% inhibition, AMP and GMP protect [19]) [19] NH3 ( inhibition of amidotransferase activity [14]) [14] NH+4 ( competitive vs. glutamine [16]) [16] OMP ( 5 mM, 25% inhibiiton [13]) [13] TMP ( 5 mM, 42% inhibiiton [13]) [13] TTP ( 5 mM, 12% inhibiiton [13]) [13] UDP ( 5 mM, 19% inhibiiton [13]) [13] UMP ( 5 mM, 25% inhibiiton [13]) [13] UTP ( 5 mM, 16% inhibiiton [13]) [13] XDP ( 5 mM, 27% inhibiiton [13]) [13] XMP ( 5 mM; 51% inhibition [3]; 5 mM, 41% inhibition [13]; competitive vs. 5-phospho-a-d-ribose 1-diphosphate [16]) [3, 13, 16] XTP ( 5 mM, 44% inhibition [13]) [13] allopurinol ribonucleotide ( 5 mM, 68% inhibition [13]) [13] azaserine ( competitive vs. glutamine, inactivation half-life: 12 min [14]) [14] dCMP ( 5 mM, 26% inhibiiton [13]) [13] 155


2.4.2.14

diphosphate ( uncompetitive vs. 5-phospho-a-d-ribose 1-diphosphate [16]) [16] glutamate ( competitive vs. glutamate [16]) [16] iodoacetamide ( approx. 60% inactivation of amidotransferase activity after 30 min, very weak inactivation of NH3 -dependent activity [3]) [3] methyl-dCMP ( 5 mM, 47% inhibiiton [13]) [13] p-mercuribenzoate ( 0.001 mM, complete inhibition [3]) [3] phosphate ( competitive vs. 5-phospho-a-d-ribose 1-diphosphate [13]; 20 mM, 50% inhibition [18]) [13, 18] piritrexim ( noncompetitive vs. 5-phospho-a-d-ribose 1-diphosphate, binds with positive cooperativity at 2 allosteric sites of an inactive dimer [24]) [24] Additional information ( not inhibited by ribose 5-phosphate, purine ribonucleosides or bases, 2'- or 3'-phosphate or deoxyribose phosphate analogues or by pyrimidine ribonucleotides [1]; synergistic inhibition of glutaminase activity by AMP plus GMP and N3 -AMP plus GMP [19]; synergistic inhibition by AMP and GMP, binding of GMP to an allosteric i.e. A site and AMP to a proximal catalytic i. e. C site are necessary for synergistic inhibition [21]; strong synergistic inhibition with ADP and GMP [25]) [1, 19, 21, 25] Activating compounds Mg-phosphoribosyldiphosphate ( requirement, phosphoribosyldiphosphate-Mg3- is the reactive molecular species of phosphoribosyldiphosphate, Km -value: 0.62 mM [16]; no glutamine-binding site in the absence of Mg-phosphoribosyldiphosphate [3]) [3, 8, 16] phosphoribosyl-5-phosphate ( 3-4fold activation of glutaminase activity in the presence of phosphate or diphosphate [3]) [3] Additional information ( complex allosteric enzyme whose activity is regulated by a series of conformational changes induced by a number of ligands [12]) [12, 18] Metals, ions Ca2+ ( activation, approx. 15% as effective as Mg2+ or Mn2+ [13]) [13] Co2+ ( activation, approx. 40% as effective as Mg2+ or Mn2+ [13]) [13] Fe ( required, iron-sulfur protein [4]; enzyme contains a dimagnetic [4Fe-4S] cluster essential for activity [11]; enzyme contains a [4Fe-4S] cluster [4,9,10]; iron is oxidized by O2 to enzyme-bound Fe3+ [9]; low temperature magnetic circular dichroism, electron paramagnetic resonance and resonance Raman spectroscopy of oxidized and reduced [4Fe-4S] cluster, native enzyme contains an [4Fe-4S]2+ cluster [15]; iron-sulfur center can be removed with 1,10-phenanthroline, resulting apoprotein is devoid of amino- and amidotransferase activity [4]; loss of activity after oxidation of the iron-sulfur center [10]) [4, 9-10, 15, 26] Mg2+ ( required [2, 8, 13, 16]; maximal activation at concentrations 2.5-5 times higher than that of the corresponding 5-phos156

2.4.2.14


pho-a-d-ribose 1-diphosphate concentration [8]; Km -value: 0.65 mM, inhibition above 10 mM [13]; 5-phospho-a-d-ribose 1-diphosphate-Mg3is the reactive molecular species [16]) [2, 3, 8, 13, 16] Mn2+ ( required, equally effective as Mg2+ [13]) [13] Additional information ( not activated by Ba2+ , Cd2+ , Cu2+ , Fe2+ , Hg2+ , Ni2+ , Zn2+ [13]; not activated by iron or sulfide [3]; sulfur required, iron-sulfur protein [4]; probably diamagnetic [11]; 4Fe-4S-cluster [4, 9, 10]; S2- is oxidized by O2 to a mixture of sulfur oxides bound as thiocysteine and yet unidentified products [9]; low temperature magnetic circular dichroism, electron paramagnetic resonance and resonance Raman spectroscopy [15]; non-heme iron is not present in significant amounts [3, 11]; prototype for a metal-free amidophosphoribosyltransferase [26]) [3, 4, 9-11, 13, 15, 26] Specific activity (U/mg) 0.001 ( l-glutamine [14]) [14] 0.00125-0.0014 [8] 0.00297 ( NH3 [14]) [14] 0.0055 [13] 0.02 [17] 0.043 [11] 1.82 [7] 15.6 [16] 17.2 [3, 11] Additional information ( assay method to measure both amidotransferase and aminotransferase activity [20]) [20] Km-Value (mM) 0.031 (5-phospho-a-d-ribose 1-diphosphate, P410W mutant enzyme [22]) [22] 0.053 (5-phospho-a-d-ribose 1-diphosphate) [22] 0.067 (5-phospho-a-d-ribose 1-diphosphate) [3] 0.072 (5-phospho-a-d-ribose 1-diphosphate) [11] 0.086 (5-phospho-a-d-ribose 1-diphosphate) [1] 0.1 (l-glutamine) [1] 0.14-0.48 (5-phospho-a-d-ribose 1-diphosphate) [10] 0.24 (5-phospho-a-d-ribose 1-diphosphate) [1] 0.4 (5-phospho-a-d-ribose 1-diphosphate, cosubstrate NH3 [6]) [6, 16] 0.47 (5-phospho-a-d-ribose 1-diphosphate, adenocarcinoma 755 [10]) [10] 0.48 (5-phospho-a-d-ribose 1-diphosphate, in Tris buffer [13]) [13] 0.53 (l-glutamine) [1] 0.57 (5-phospho-a-d-ribose 1-diphosphate) [7, 10] 0.64 (l-glutamine, N101G mutant enzyme, 5-phospho-a-d-ribose 1diphosphate-dependent glutaminase activity [23]) [23] 0.65 (Mg2+ ) [13] 0.66 (5-phospho-a-d-ribose 1-diphosphate) [27] 157


2.4.2.14

0.7 (5-phospho-a-d-ribose 1-diphosphate, cosubstrate glutamine [6]) [6] 0.75 (l-glutamine) [27] 0.87 (5-phospho-a-d-ribose 1-diphosphate, at 90 C [28]) [28] 1-4.5 (l-glutamine) [10, 13] 1.24-1.5 (l-glutamine) [7, 10] 1.42 (l-glutamine, N101D mutant enzyme, 5-phospho-a-d-ribose 1-diphosphate-dependent glutaminase activity [23]) [23] 1.6 (l-glutamine) [8, 13] 1.7 (l-glutamine) [3] 1.72 (l-glutamine, 5-phospho-a-d-ribose 1-diphosphate-dependent glutamine hydrolysis [23]) [23] 1.72 (l-glutamine, wild-type enzyme, 5-phospho-a-d-ribose 1-diphosphate-dependent glutaminase activity [23]) [23] 1.8 (l-glutamine, adenocarcinoma 755 [10]) [10] 2.1 (l-glutamine) [23] 2.43 (l-glutamine, G102A mutant enzyme, 5-phospho-a-d-ribose 1diphosphate-dependent glutaminase activity [23]) [23] 3 (l-glutamine, at 90 C [28]) [28] 4.3 (l-glutamine) [11] 6.03 (l-glutamine, N101G mutant enzyme, aminotransferase activity [23]) [23] 6.08 (l-glutamine, D127A mutant enzyme, aminotransferase activity [23]) [23] 7.31 (l-glutamine, R73L mutant enzyme, aminotransferase activity [23]) [23] 7.34 (l-glutamine, wild-type enzyme, aminotransferase activity [23]) [23] 7.34 (NH3 ) [23] 7.67 (l-glutamine, G102A mutant enzyme, aminotransferase activity [23]) [23] 8.8 (NH3 ) [3] 9.17 (l-glutamine, N101D mutant enzyme, aminotransferase activity [23]) [23] 9.76 (l-glutamine, R73H mutant enzyme, aminotransferase activity [23]) [23] 16 (NH3 ) [16] 16 (NH+4 ) [16] 18 (l-glutamine) [16] 101 (l-glutamine, R73H mutant enzyme, 5-phospho-a-d-ribose 1diphosphate-dependent glutaminase activity [23]) [23] 110 (l-glutamine, R73L mutant enzyme, 5-phospho-a-d-ribose 1diphosphate-dependent glutaminase activity [23]) [23] 193 (glutamine, 5-phospho-a-d-ribose 1-diphosphate-independent glutamine hydrolysis [23]) [23] 236 (l-glutamine, D127A mutant enzyme, 5-phospho-a-d-ribose 1diphosphate-dependent glutaminase activity [23]) [23] 158

2.4.2.14


Additional information ( kinetic study [12,16]; effect of AMP on kinetic parameters [13]; kinetic properties of amido- and aminotransferase activity [14]; kinetic constants of Arg73 and Tyr74 mutants for basal and total glutaminase activity [23]) [12-14, 16, 23] Ki-Value (mM) 0.0034 (6-diazo-5-oxo-l-norleucine) [14] 0.031-1.1 (ATP) [1] 0.038-0.64 (ADP) [1] 0.04 (AMP) [24] 0.053 (1-a-diphosphoryl-2-a,3-a-dihydroxy-4-b-cyclopentane-methanol-5-phosphate, P410W mutant enzyme [22]) [22] 0.058 (1-a-diphosphoryl-2-a,3-a-dihydroxy-4-b-cyclopentane-methanol-5-phosphate) [22] 0.066 (piritrexim) [24] 0.086-0.35 (GMP) [1] 0.092-2.5 (AMP) [1] 0.18-3.5 (IMP) [1] 0.36 (AMP) [3] 0.38-5.4 (GDP) [1] 0.39 (5'-p-fluorosulfonylbenzoyladenosine) [19] 0.65 (diphosphate) [16] 1.2 (XMP) [16] 2 (AMP) [16] 3.3 (NH3 ) [14] 4 (azaserine) [14] 16 (NH+4 ) [16] 30 (glutamate) [16] pH-Optimum 6-8 ( broad, phosphate buffer [13]) [13] 6.5 ( imidazole/HCl buffer [13]) [13] 6.5-8.5 ( broad, Tris or phosphate buffer, constant activity [7]; glutaminase activity [28]) [7, 28] 6.6-7.7 ( broad, amidotransferase activity, phosphate buffer [3]) [3] 6.8-7.4 ( Tris/HCl buffer preferred [8]) [8] 7.5 ( Tris/HCl buffer [13]; unstable enzyme [17]) [13, 17] 7.8-8.6 ( broad, amidotransferase activity, Tris/HCl buffer [3]) [3] 8 ( stable enzyme [17]) [16, 17] 8.5 ( aminotransferase activity, Tris buffer [3]) [3] pH-Range 6-10 ( approx. 70% of maximal activity between pH 6 and pH 10 [16]) [16] 7-9 ( stable enzyme, approx. 65% and unstable enzyme approx. 70% of maximal activity at pH 7, stable enzyme, approx. 75% and unstable enzyme approx. 65% of maximal activity at pH 9 [17]) [17]

159


2.4.2.14

Temperature optimum ( C) 25 ( assay at [16, 18]) [16, 18] 37 ( assay at [3, 6-8, 14]) [3, 6-8, 14] 38 ( assay at [2]) [2] 90 ( both glutaminase and amidotransferase activity [28]) [28]

4 Enzyme Structure Molecular weight 93000 ( sucrose density gradient centrifugation, enzyme exists in equilibrium of tetrameric, dimeric and monomeric forms [4]) [4] 102000 ( sedimentation equilibrium centrifugation, phosphate, phosphoribosyldiphosphate or purine mononucleotides influence the sedimentation profile [12]) [12] 110000 ( ligand-induced alteration of sedimentation coefficient and Stokes radius, gel filtration or sedimentation equilibrium centrifugation [18]) [18] 127000 ( liver small form [10]) [10] 133000 ( placenta small form, large form is converted to small form by incubation with phosphoribosyldiphosphate [10]) [10] 170000 ( placenta large form, in the presence of AMP or GMP [10]) [10] 172000 ( sedimentation equilibrium centrifugation in the presence of phosphoribosyldiphosphate, the sedimentation profile is influenced by phosphate, phosphoribosyldiphosphate or purine mononucleotides [12]) [12] 180000 ( mutant unstable enzyme, gel filtration [17]) [17] 181000 ( sedimentation equilibrium centrifugation in the presence of phosphate, the sedimentation profile is influenced by phosphate, phosphoribosyldiphosphate or purine mononucleotides [12]) [12] 185000 ( highly concentrated enzyme solution, sucrose density gradient centrifugation, enzyme exists in equilibrium of tetramer, dimer and monomeric forms, conversion of dimer to tetramer within 10fold increase in protein concentration [4]) [4] 194000 ( sedimentation equilibrium centrifugation [3,11]) [3, 11] 195000 ( sucrose density gradient centrifugation [7]) [7] 200000 ( highly concentrated enzyme solution, gel filtration, enzyme exists in equilibrium of tetrameric, dimeric and monomeric forms, conversion of dimer to tetramer within 10fold increase in protein concentration, AMP and GMP stabilize the dimeric form, GDP stabilizes the tetrameric form [4]; native PAGE [7]; ligand-induced alteration of sedimentation coefficient and Stokes radius in the presence of phosphoribosyldiphosphate or phosphate, gel filtration and sedimentation equilibrium centrifugation [18]) [4, 7, 18] 215000 ( gel filtration [7,10]; only one enzyme form [10]) [7, 10] 224000 ( gel filtration [3,11]) [3, 11] 160

2.4.2.14


270000 ( placenta, large form, small form is converted to large form by incubation with purine nucleotides, large form presumably catalytically inactive [10]) [10] 292000 ( liver, large form [10]) [10] 360000 ( wild-type stable enzyme, gel filtration [17]) [17] Additional information ( enzymes from human placenta, Chinese hamster fibroblasts and mouse liver exist in two molecular weight forms, the larger one is observed when incubated with purine nucleotides, the smaller one when incubated with phosphoribosyldiphosphate [10]; the smaller molecular weight form of the liver enzyme is observed when incubated with purine nucleotides, the smaller one when incubated with phosphoribosyldiphosphate [10,12]; partially purified enzyme shows 3 molecular forms: an inactive tetramer formed in the presence of AMP, an active dimer formed with 5-phospho-a-d-ribose 1-diphosphate and an inactive dimer formed with piritrexim [24]; 2 molecular forms: homodimeric in the presence of 5-phospho-a-d-ribose 1-diphosphate, homotetrameric in the presence of purine ribonucleotides [29]) [3, 4, 10-12, 24, 29] 8000000 ( probably an aggregation of several purine synthetic activities, gel filtration [16]) [16] Subunits ? ( 2-4 * 50000, SDS-PAGE [4,11]; 3-4 * 56395, calculated from nucleotide sequence [11]; 3-4 * 57000, SDS-PAGE [3,11]; x * 58000, SDS-PAGE [18]) [3, 4, 11, 18]

5 Isolation/Preparation/Mutation/Application Source/tissue adenocarcinoma cell ( adenocarcinoma 755 [10]) [10] erythroleukemia cell [10] fibroblast [10] leukemia cell ( L1210 leukemia cells [24]) [24] leukocyte [10] liver [1, 2, 7, 10-12, 18] lymphoblast ( diploid wild 2 line, tissue culture [8]) [8] nauplius [6] placenta [10, 13, 14] retina [27] root nodule [16] Localization cytosol ( little or no activity in mitochondria or microsomes [13]) [13] soluble [1, 6, 7, 13]

161


2.4.2.14

Purification (ammonium sulfate, heat treatment, DEAE-Sepharose, Blue Dextran-Sepharose, hydroxylapatite [3]; recombinant wild-type and P410W mutant enzyme [22]) [3, 19, 22] (partial, ATP-agarose affinity chromatography [5]; protamine sulfate, DEAE-cellulose [4]) [4, 5] (partial [6]) [6] [18] (partial [13]; DEAE-cellulose, partial purification [14]) [13, 14] (ammonium sulfate, heat treatment, calcium phosphate gel, DEAE-cellulose [1]; partial [1,2,12]) [1, 2, 12] (partial [1]; heat treatment, acid precipitation, ammonium sulfate, ethanol, Sephadex G-150, DE-52 [7]) [1, 7] (streptomycin, ammonium sulfate, Sephadex g-200 [17]) [17] (Sepharose CL-2B, glycerol gradient [16]) [16] [27] (recombinant enzyme [28]) [28] Crystallization (crystal structure of 6-diazo-5-oxonorleucine inactivated enzyme at 2.3 A resolution [23]; crystal structure in the absence of ligands at 2.0 A, and with bound AMP at 2.5 A [26]) [23, 26] (crystals of the ternary enzyme-ADP-GMP complex are grown in glass melting point capillaries by the microbath method, 20 mg/ml enzyme are incubated with 1 mM ADP, 1 mM GMP, and 5 mM MgCl2 and mixed with an equal volume of 24% polyethylene glycol 8000, 200 mM KCl, 50 mM N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid, pH 7.9, brown crystals emerge after 6-12 weeks [25]; crystal structure at 3.0 A [26]) [25, 26] Cloning (expression of wild-type, Y74A, Y258A, Y258F, K326Q, Y329A, G331I, N351A and Y465A mutant enzyme in Escherichia coli [19]; overexpression of wild-type and P410W mutant enzyme in Escherichia coli [22]) [11, 19, 22] (expression in Escherichia coli [11]) [11] (expression of wild-type and K338Q/P422W mutant enzyme in amidophosphoribosyltransferase deficient CHO ade -A cells and in transgenic mice [29]) [29] (expression in Escherichia coli [28]) [28] Engineering G331I ( 50% of wild-type glutaminase activity, reduced inhibition by GMP, enhanced inhibition by AMP [19]) [19] K326Q ( similar glutaminase and amidotransferase activity as wildtype, not inhibited by GMP [19]; insensitive to inhibition by GMP, reduced synergistic inhibition [21]) [19, 21] K326Q/P410W ( binding of GMP and AMP is abolished resulting in loss of inhibition [21]) [21]

162

2.4.2.14


N351A ( approx. 50% of wild-type glutaminase activity, completely insensitive to inhibition by GMP, partially resistent to inhibition by AMP [19]) [19] P410W ( reduced inhibition by AMP, strong synergistic inhibition by AMP and GMP [21]) [21] R26H ( extremely labile enzyme [23]) [23] Y258A ( complete loss of glutaminase and amidotransferase activity [19]) [19] Y258F ( approx. 50% loss of glutaminase activity, very weak amidotransferase activity [19]) [19] Y329A ( normal glutaminase activity, approx. 20% amidotransferase activity, less sensitive to GMP inhibition than wild-type [19]) [19] Y465A ( 100% of wild-type glutaminase activity, 28% of wild-type amidotransferase activity, inhibition by GMP and AMP is similar to wild-type [19]) [19] Y74A ( complete loss of glutaminase activity, little loss of amidotransferase activity [19]) [19]

6 Stability Temperature stability 37 ( glutaminase half-life: 25 h [28]) [28] 48 ( wild-type, stable enzyme from cells of mid-exponential growth phase, t1=2 : 34 min, from cells of stationary growth phase, t1=2 : 2-5 min, mutant enzyme, t1=2 : 2 min, in the presence of ADP or adenosine 3 min, and 4 min in the presence of IMP [17]) [17] 60 ( 6 min, about 25% loss of activity, [1]; at least 15 min stable [8]; after 3-6 min loss of ATP-sensitivity [1]) [1, 8] 80 ( glutaminase half-life: 65 min [28]) [28] Additional information ( AMP and GMP enhance thermal stability [3]) [3] Oxidation stability , low concentrations of thiols e.g. dithiothreitol or 2-mercaptoethanol accelerate aerobic inactivation [3] , oxygen inactivates during purification [3] , O2, rather than peroxide, superoxide, hydroxyl radical or singlet oxygen, inactivates, allosteric inhibitors, such as AMP, ADP, GMP or GDP modulate the rate of inactivation [9] , in vitro O2 oxidizes iron in the Fe-S cluster to the high spin ferric state and sulfur to a mixtur of S in thiocystine residues plus other unidentified products [11] , oxygen-labile in vivo and in vitro, AMP protects [4] , amido- and aminotransferase activity is lost after exposure to oxygen [10] , anaerobic conditions stabilize [3, 4] , oxygen sensitive enzyme [10]

163


2.4.2.14

General stability information , stable during all stages of purification provided thiols and oxygen are avoided [3] , ADP or ADP and GMP, ratio 1/1, stabilize equilibrium between dimeric and tetrameric form [4] , AMP or GMP stabilizes dimeric enzyme form [4, 11] , AMP stabilizes against inactivation by O2 [4, 9] , GDP stabilizes tetrameric enzyme form [4, 11] , phosphoribosyldiphosphate and other nucleotides antagonize stabilizing effect of AMP [9] , Mg2+ and phosphate are essential for stability [13] , 2-mercaptoethanol stabilizes during purification [1] , prolonged dialysis against distilled water leads to precipitation [1] , stability profile depends on growth rate and growth phase of cell culture, PMSF improves stability [17] , freezing inactivates, not stabilized by 30% glycerol [16] , high concentrations of thiol reagents stabilize during purification [16] Storage stability , -20 C or 4 C, cell-free extract, overnight, more than 50% loss of activity [8] , -20 C, crude, more than 50% loss of activity overnight [8] , 4 C, 50 mM phosphate bufer, pH 7.4, 5 mM MgCl2 , 60 mM 2-mercaptoethanol, at least 4 weeks, no loss of activity [13] , 4 C, partially purified preparation in 50 mM phosphate buffer, pH 7.4, 5 mM Mg2+ and 60 mM 2-mercaptoethanol, at least 4 weeks [13] , 4 C, phosphate buffer, 10 d, 40% loss of activity [13] , 2-mercaptoethanol and phosphoribosyldiphosphate stabilize during storage at 4 C [12] , 2-mercaptoethanol stabilizes during storage [1] , 4 C, in phosphate buffer, pH 7.4, 60 mM 2-mercaptoethanol and phosphoribosyldiphosphate, at least 10 days [12] , phosphate stabilizes during storage [12] , storage in 25 mM Tris buffer inactivates with 26% or 4% residual activity after 6 or 10 days, respectively [12] , 3 C, crude mutant enzyme extract, inactivation overnight, partially purified mutant enzyme: inactivation within 72 h, PMSF stabilizes [17] , 3 C, crude or partially purified wild-type enzyme, at least 72 h stable [17] , PMSF stabilizes during storage [17] , 4 C, 25% loss of activity per day [16]

164

2.4.2.14


References [1] Caskey, C.T.; Ashton, D.M.; Wyngaarden, J.B.: The enzymology of feedback inhibition of glutamine phosphoribosylpyrophosphate amidotransferase by purine ribonucleotides. J. Biol. Chem., 239, 2570-2579 (1964) [2] Hartman, S.C.; Buchanan, J.M.: Biosynthesis of purines. XXI. 5-phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem., 233, 451-455 (1958) [3] Messenger, L.J.; Zalkin, H.: Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties. J. Biol. Chem., 254, 3382-3392 (1979) [4] Wong, J.Y.; Bernlohr, D.A.; Turnbough, C.L.; Switzer, R.L.: Purification and properties of glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis. Biochemistry, 20, 5669-5674 (1981) [5] Wong, J.Y.; Switzer, R.L.: Affinity chromatography of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase. Arch. Biochem. Biophys., 196, 134-137 (1979) [6] Liras, A.; Argomaniz, L.; Llorente, P.: Presence, preliminary properties and partial purification of 5-phosphoribosylpyrophosphate amidotransferase from Artemia sp. Biochim. Biophys. Acta, 1033, 114-117 (1990) [7] Tsuda, M.; Katunuma, N.; Weber, G.: Rat liver glutamine 5-phosphoribosyl1-pyrophosphate amidotransferase [EC 2.4.2.14]. Purification and properties. J. Biochem., 85, 1347-1354 (1979) [8] Wood, A.W.; Seegmiller, J.E.: Properties of 5-phosphoribosyl-1-pyrophosphate amidotransferase from human lymphoblasts. J. Biol. Chem., 248, 138-143 (1973) [9] Bernlohr, D.A.; Switzer, R.L.: Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with oxygen: chemistry and regulation by ligands. Biochemistry, 20, 5675-5681 (1981) [10] Holmes, E.W.: Kinetic, physical, and regulatory properties of amidophosphoribosyltransferase. Adv. Enzyme Regul., 19, 215-231 (1981) [11] Zalkin, H.: Structure, function, and regulation of amidophosphoribosyltransferase from prokaryotes. Adv. Enzyme Regul., 21, 225-237 (1983) [12] Itoh, R.; Holmes, E.W.; Wyngaarden, J.B.: Pigeon liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular and kinetic properties. J. Biol. Chem., 251, 2234-2240 (1976) [13] Holmes, E.W.; McDonald, J.A.; McCord, J.M.; Wyngaarden, J.B.; Kelley, W.N.: Human glutamine phosphoribosylpyrophosphate amidotransferase. Kinetic and regulatory properties. J. Biol. Chem., 248, 144-150 (1973) [14] King, G.L.; Boounous, C.G.; Holmes, E.W.: Human placental amidophosphoribosyltransferase. Comparison of the kinetics of glutamine and ammonia utilization. J. Biol. Chem., 253, 3933-3938 (1978) [15] Onate, Y.A.; Vollmer, S.J.; Switzer, R.L.; Johnson, M.K.: Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem., 264, 1838618391 (1989)

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[16] Reynoldds, P.H.S.; Blevins, D.G.; Randall, D.D.: 5-Phosphoribosylpyrophosphate amidotransferase from soybean root nodules: kinetic and regulatory properties. Arch. Biochem. Biophys., 229, 623-631 (1984) [17] Nagy, M.; Reichert, U.; Ribet, A.M.: Regulation of purine metabolism in Schizosaccharomyces pombe. IV. Variations in the stability and kinetic parameters of amidophosphoribosyltransferase depending on growth phase and growth conditions. Biochim. Biophys. Acta, 370, 85-95 (1974) [18] Itoh, R.; Gorai, I.; Usami, C.; Tsushima, K.: Chicken liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular properties. Biochim. Biophys. Acta, 581, 142-152 (1979) [19] Zhou, G.; Charbonneau, H.; Colman, R.F.; Zalkin, H.: Identification of sites for feedback regulation of glutamine 5-phosphoribosylpyrophosphate amidotransferase by nucleotides and relationship to residues important for catalysis. J. Biol. Chem., 268, 10471-10481 (1993) [20] Taha, T.S.; Deits, T.L.: Detection of glycinamide ribonucleotide by HPLC with pulsed amperometry: application to the assay for glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14). Anal. Biochem., 213, 323-328 (1993) [21] Zhou, G.; Smith, J.L.; Zalkin, H.: Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem., 269, 6784-6789 (1994) [22] Kim, J.H.; Wolle, D.; Haridas, K.; Parry, R.J.; Smith, J.L.; Zalkin, H.: A stable carbocyclic analog of 5-phosphoribosyl-1-pyrophosphate to probe the mechanism of catalysis and regulation of glutamine phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem., 270, 17394-17399 (1995) [23] Kim, J.H.; Krahn, J.M.; Tomchick, D.R.; Smith, J.L.; Zalkin, H.: Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site. J. Biol. Chem., 271, 15549-15557 (1996) [24] Schoettle, S.L.; Crisp, L.B.; Szabados, E.; Christopherson, R.I.: Mechanisms of inhibition of amido phosphoribosyltransferase from mouse L1210 leukemia cells. Biochemistry, 36, 6377-6383 (1997) [25] Chen, S.; Tomchick, D.R.; Wolle, D.; Hu, P.; Smith, J.L.; Switzer, R.L.; Zalkin, H.: Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides. Biochemistry, 36, 10718-10726 (1997) [26] Muchmore, C.R.; Krahn, J.M.; Kim, J.H.; Zalkin, H.; Smith, J.L.: Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Protein Sci., 7, 39-51 (1998) [27] Kian, I.A.; Etingof, R.N.: Purine biosynthesis de novo in bovine retina: purification and characterization of amidophosphoribosyl transferase and phosphoribosyl pyrophosphate synthetase. Biochemistry, 64, 648-651 (1999) [28] Bera, A.K.; Chen, S.; Smith, J.L.; Zalkin, H.: Temperature-dependent function of the glutamine phosphoribosylpyrophosphate amidotransferase am-

166

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monia channel and coupling with glycinamide ribonucleotide synthetase in a hyperthermophile. J. Bacteriol., 182, 3734-3739 (2000) [29] Yamaoka, T.; Yano, M.; Kondo, M.; Sasaki, H.; Hino, S.; Katashima, R.; Moritani, M.; Itakura, M.: Feedback inhibition of amidophosphoribosyltransferase regulates the rate of cell growth via purine nucleotide, DNA, and protein syntheses. J. Biol. Chem., 276, 21285-21291 (2001)

167

Guanosine phosphorylase

2.4.2.15

1 Nomenclature EC number 2.4.2.15 Systematic name guanosine:phosphate a-d-ribosyltransferase Recommended name guanosine phosphorylase Synonyms phosphorylase, guanosine CAS registry number 9030-28-8

2 Source Organism Oryctolagus cuniculus [1] Trichomonas vaginalis [2]

3 Reaction and Specificity Catalyzed reaction guanosine + phosphate = guanine + a-d-ribose 1-phosphate Reaction type pentosyl group transfer Natural substrates and products S guanosine + phosphate (, enzyme may play an important role in the utilization by bone marrow of the corresponding preformed base and nucleoside reaching this tissue via blood [1]) (Reversibility: ? [1]) [1] P guanine + d-ribose 1-phosphate [1] Substrates and products S deoxyguanosine + phosphate [1] P guanine + deoxyribose 1-phosphate S guanosine + phosphate (, equilibrium constant is 0.019 [1]) (Reversibility: r [1]; ? [2]) [1, 2] P guanine + d-ribose 1-phosphate [1]

168

2.4.2.15

Guanosine phosphorylase

Inhibitors PCMB (, 0.2 mM, complete inhibition [1]) [1] Tris buffer (, 0.1 M, pH 7.5, 22% inhibition [1]) [1] Activating compounds 2-mercaptoethanol (, required for optimal activity [1]) [1] Specific activity (U/mg) 4.87 [1] Km-Value (mM) 0.21 (deoxyguanosine) [1] 0.216 (guanosine) [1] 0.376 (phosphate, , reaction with guanosine [1]) [1] pH-Optimum 7 (, phosphorolysis of deoxyguanosine [1]) [1] 7-7.4 (, phosphorolysis of guanosine [1]) [1] pH-Range 5-8.5 (, pH 5.0: about 35% of activity maximum, pH 8.5: about 45% of activity maximum [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue bone marrow [1] intestinal mucosa [1] liver [1] Purification [1]

6 Stability Storage stability , -10 C, stable for 1 month [1]

References [1] Yamada, E.W.: The phosphorolysis of nucleosides by rabbit bone marrow. J. Biol. Chem., 236, 3043-3046 (1961) [2] Heyworth, P.G.; Gutteridge, W.E.; Ginger, C.D.: Purine metabolism in Trichomonas vaginalis. FEBS Lett., 141, 106-110 (1982)

169

Urate-ribonucleotide phosphorylase

2.4.2.16

1 Nomenclature EC number 2.4.2.16 Systematic name urate-ribonucleotide:phosphate a-d-ribosyltransferase Recommended name urate-ribonucleotide phosphorylase Synonyms UAR phosphorylase phosphorylase, urate ribonucleotide urate ribonucleotide phosphorylase urate-ribonucleotide:orthophosphate d-ribosyltransferase CAS registry number 9030-29-9

2 Source Organism

Rattus norvegicus [1] Columba livia [1] Cavia porcellus [1] Canis familiaris [1]

3 Reaction and Specificity Catalyzed reaction urate d-ribonucleoside + phosphate = urate + a-d-ribose 1-phosphate Reaction type pentosyl group transfer Natural substrates and products S uric acid ribonucleoside + phosphate (Reversibility: ir [1]; ? [1]) [1] P urate + d-ribose 1-phosphate [1]

170

2.4.2.16


Substrates and products S uric acid ribonucleoside + arsenate ( reverse reaction not demonstrated, arsenate can replace phosphate at a lower reaction rate [1]) (Reversibility: ir [1]; ? [1]) [1] P urate + d-ribose 1-phosphate [1] S uric acid ribonucleoside + phosphate (Reversibility: ir [1]; ? [1]) [1] P urate + d-ribose 1-phosphate [1] Inhibitors 1-deoxyribose 5-methyluracil ( 1 mM, about 95% inhibition [1]) [1] 1-ribosyluracil ( 1 mM, about 85% inhibition [1]) [1] 2-thio-6-oxopurine ( 1 mM, 73% inhibition [1]) [1] 2-thiouracil ( 1 mM, 96% inhibition [1]) [1] 4-amino-5-imidazolecarboxamide ( 1 mM, about 15% inhibition [1]) [1] 5-methyluracil ( 1 mM, about 90% inhibition [1]) [1] colchicine ( 1 mM, 43% inhibition, competitive [1]) [1] guanosine ( 1 mM, about 20% inhibition [1]) [1] inosine ( 1 mM, about 25% inhibition [1]) [1] p-chloromercuribenzoate ( reversed by cysteine [1]) [1] phenylbutazone ( 1 mM, 30% inhibition, competitive [1]) [1] uracil ( 1 mM, about 85% inhibition [1]) [1] xanthine ( 1 mM, 35% inhibition [1]) [1] Specific activity (U/mg) 4.6 [1] Ki-Value (mM) 1.23 (colchicine, , 37 C, pH 8.2 [1]) [1] 1.76 (phenylbutazone, , 37 C, pH 8.2 [1]) [1] pH-Optimum 7.5-8 (, reaction with phosphate or arsenate [1]) [1] pH-Range 6-9.5 (, pH 6.0: about 60% of maximal activity, pH 9.5: 45% of maximal activity [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue heart [1] kidney [1] liver [1] skeletal muscle [1] small intestine mucosa [1] spleen ( low activity [1]) [1] testis [1] 171


2.4.2.16

Purification [1]

6 Stability pH-Stability 4.5 (, 0 C, 10 min, 30% loss of activity, partially purified enzyme [1]) [1] Temperature stability 50 ( pH 7.8, 5 min, 5% loss of activity, partially purified enzyme [1]) [1] 60 ( pH 7.8, 5 min, 40% loss of activity, partially purified enzyme [1]) [1] 70 ( pH 7.8, 5 min, 95% loss of activity, partially purified enzyme [1]) [1] 80 ( pH 7.8, 5 min, 100% loss of activity, partially purified enzyme [1]) [1] Storage stability , -10 C, partially purified enzyme is stable for at least 2 months [1]

References [1] Laster, L.; Blair, A.: An intestinal phosphorylase for uric ribonucleoside. J. Biol. Chem., 238, 3348-3357 (1963)

172

ATP phosphoribosyltransferase

2.4.2.17

1 Nomenclature EC number 2.4.2.17 Systematic name 1-(5-phospho-d-ribosyl)-ATP:diphosphate phospho-a-d-ribosyltransferase Recommended name ATP phosphoribosyltransferase Synonyms 1-(5-phospho-d-ribosyl)-ATP:pyrophosphate phospho-a-d-ribosyltransferase ATP-PRT adenosine triphosphate phosphoribosyltransferase phosphoribosyl ATP synthetase phosphoribosyl ATP:pyrophosphate phosphoribosyltransferase phosphoribosyl-ATP pyrophosphorylase phosphoribosyl-ATP:pyrophosphate-phosphoribosyl phosphotransferase phosphoribosyladenosine triphosphate pyrophosphorylase phosphoribosyladenosine triphosphate synthetase phosphoribosyltransferase, adenosine triphosphate CAS registry number 9031-46-3

2 Source Organism

Salmonella typhimurium (LT2 strain TA2165 [6,10]) [1, 3-6, 9-11] Escherichia coli [2, 7, 8, 14] Corynebacterium glutamicum [12] Arabidopsis thaliana (ecotype Columbia; At-ATP-PRT1 [13]) [13] Arabidopsis thaliana (ecotype Columbia; At-ATP-PRT2 [13]) [13] Mycobacterium tuberculosis [15]

173


2.4.2.17

3 Reaction and Specificity Catalyzed reaction 1-(5-phospho-d-ribosyl)-ATP + diphosphate = ATP + 5-phospho-a-d-ribose 1-diphosphate ( sequential kinetic mechanism in biosynthetic direction, ordered bi-bi mechanism with ATP binding first to free enzyme and phosphoribosyl-ATP dissociating last from enzyme-product complexes [2,3, 10, 11]; double displacement mechanism [2]) Reaction type pentosyl group transfer Natural substrates and products S ATP + 5-phospho-a-d-ribose 1-diphosphate ( first step in histidine biosynthesis [1-15]) [1-15] P diphosphate + N-1-(5Â-phosphoribosyl)-ATP Substrates and products S ATP + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: r [3, 5, 6, 10, 11]; r [7]; ? [12]; ? [14]; r [15]) [1-15] P 1-(5-phospho-d-ribosyl)-ATP + diphosphate [1, 3] S Additional information ( not: ribose 5-phosphate, AMP, ADP, UTP, CTP, GTP [3,10,11]) [3, 10, 11] P ? Inhibitors 1,2,4-triazole-3-alanine [13] 1-(5-phospho-a-d-ribosyl)-ATP ( product inhibition, competitive to both substrates [10]) [10] 2-methyl-l-histidine [1] 2-thiazol-dl-alanine [1] 5-phospho-a-d-ribose 1-diphosphate ( noncompetitive inhibitor with respect to both substrates in the reaction producing ATP and 5-phospho-a-d-ribose 1-diphosphate [11]) [11] ADP ( competitive to ATP, in the presence of histidine inhibition by AMP and ADP becomes positively cooperative and much more potent [1,6]) [1, 6] AMP ( competitive inhibitor to 5-phospho-a-d-ribose 1-diphosphate, in the presence of histidine inhibition by AMP and ADP becomes positively cooperative and much more potent [1,6]; linear competitive inhibitor with respect to ATP, stabilizes the enzyme to thermal inactivation, protect the ordered enzymatic structure against thermodenaturation [2,8]) [1, 2, 6, 8] ATP ( inhibits the reaction at high concentrations [2]) [2] Ag+ [1] Cd2+ [1] Co2+ [1] Cu2+ [1]

174

2.4.2.17


Hg2+ [1] l-histidine ( feed-back inhibition [1-15]; reversed by Hg2+ , p-hydroxymercuribenzoate, methylmercuric bromide, Ni2+ [1]; inhibits reverse reaction cooperatively and completely [11]; the l-configuration is essential, substitution of a-amino group appreciable reduces inhibition, non competitive inhibitor with respect to both substrates [1]; stabilizes the enzyme to thermal inactivation, protects the ordered enzymatic structure against thermodenaturation, no interaction with binding sites [2,8]; allosteric inhibition, synergistically favored by AMP [15]) [1-15] N-ethylmaleimide [1] Ni2+ [1] Zn2+ [1] adenine ( competitive to ATP and 5-phospho-a-d-ribose 1-diphosphate [1]) [1] b,g-methylene-ATP ( inhibitor of the reaction producing ATP and 5phospho-a-d-ribose 1-diphosphate [11]; competitive with respect to N1-(5-phosphoribosyl)-ATP, noncompetitive with respect to diphosphate [11]) [11] dicoumarol ( competitive with respect to ATP, inhibitor in both directions, diminishes yield of phosphoribosyladenosine triphosphate by acting as parasite substrate [7]) [7] dinitrophenol ( diminishes yield of phosphoribosyladenosine triphosphate by acting as parasite substrate [7]) [7] diphosphate ( non competitive to both substrates [10]) [10] guanosine 5'-diphosphate-3'-diphosphate ( in presence of partially inhibiting concentrations of histidine guanosine 5'-diphosphate-3'-diphosphate becomes a potent inhibitor of the residual activity of ATP phosphoribosyltransferase, no inhibition in absence of histidine, inhibition is slowly reversible [5]) [5] methylmercuric bromide [1] p-hydroxymercuribenzoate [1] pentachlorophenol ( competitive to ATP, inhibitor in both directions, diminishes yield of phosphoribosyladenosine triphosphate by acting as parasite substrate [7]) [7] Additional information ( carbonylcyanide m-chlorophenylhydrazone, which is a potent inhibitor of several enzymes with adenine-containing substrates or coenzymes has no effect [7]) [7] Activating compounds potassium chloride ( stimulates ATP-PRT activity in crude extracts, adding KCl to a final concentration of 0.13 M in reaction mixture increases the reaction rate by about 40% [4]) [4] Additional information ( other salts like ammonium sulfate or ammonium chloride show the same effect as KCl [4]) [4]

175


2.4.2.17

Metals, ions Mg2+ ( magnesium could have an effect on the conformation of the enzyme and its activity which would be independent of the state of substrate complexation [10]) [10] Additional information ( maximal activity is achieved when the concentration of free magnesium reaches about 2 mM, concentrations higher than 5 mM lead to inhibition perhaps due to the formation of MgATP2- complex [10]) [10] Specific activity (U/mg) Additional information ( description of assay method [1,9]; data for genetically-modified Corynebacterium glutamicum [12]) [1, 9, 12] Km-Value (mM) 0.067 (5-phospho-a-d-ribose 1-diphosphate) [1] 0.13 (5-phospho-a-d-ribose 1-diphosphate, recombinant At-ATPPRT1 [13]) [13] 0.2 (ATP) [1] 0.51 (ATP, crude cell extracts [13]) [13] 0.57 (5-phospho-a-d-ribose 1-diphosphate, crude cell extracts [13]) [13] 0.6 (ATP, recombinant enzyme [13]) [13] 37 (5-phospho-a-d-ribose 1-diphosphate, crude cell extracts [13]) [13] 89 (ATP, crude cell extracts [13]) [13] pH-Optimum 7.5 ( assay at [10,11]) [10, 11] 8.5 ( assay at [1,3-6,12,13]) [1, 3-6, 12, 13] Temperature optimum ( C) 25 ( assay at [3,5,6,10,11]) [3, 5, 6, 10, 11] 28 ( assay at [4]) [4] 30 ( assay at [13]) [13]

4 Enzyme Structure Molecular weight 44600 ( calculated mass from amino acid sequence [13]) [13] 44800 ( calculated mass from amino acid sequence [13]) [13] 186000 ( dynamic light scattering in the presence of AMP or histidine [14]) [14] 210000-221000 ( sedimentation, equilibrium centrifugation with meniscus depletion method [4]) [4] 216000 ( ultracentrifugation analysis [9]) [9]

176

2.4.2.17


Subunits dimer ( active form [15]) [15] hexamer ( 6 * 36000, viscometric methods, equilibrium centrifugation with meniscus depletion method after dialysis against 5.0 M guanidine-HCl and 0.143 M 2-mercaptoethanol [4]; 6 * 33000, SDS-disc gel electrophoresis [9]; 6 * 33367, dimers arranged in a hexamer, in the presence of AMP [14]; inactive form, in complex with histidine [15]) [4, 9, 14, 15]

5 Isolation/Preparation/Mutation/Application Purification (ammonium sulfate precipitation and DEAE-cellulose chromatography [1]; chromatography on Sephadex G-75 [3]; Mn2+ precipitation, DEAE-cellulose and ammonium sulfate precipitation, later on Sephadex G-150 [4]; first purification by heating the crude extract, ammonium sulfate precipitation and selective histidine-dependent ammonium sulfate precipitation [9]) [1, 3, 4-6, 9, 10, 11] (centrifugation and DEAE-Sephacel anion-exchange column [14]) [14] (the recombinant enzymes are produced as fused proteins with a maltose-binding protein, and the purification is made by a two step amylose resin column followed by a digestion with Factor Xa [13]) [13] (nickel-nitrilotriacetic acid column, Sephadex G-200 later on [15]) [15] Crystallization (vapour diffusion method, trigonal prisms are obtained using 1.3 M sodium tartrate, 50-200 mM magnesium chloride, 100 mM citrate buffer pH 5.6 and enzyme in the presence of 2 mM AMP, round shaped crystals are obtained with 1.36-1.44 M ammonium sulfate, 0-0.3 M sodium chloride, 100 mM HEPES buffer pH 7.5 and enzyme in the presence of 2 mM AMP [14]) [14] (hanging drop vapour diffusion method at 16 C, the apocrystals are obtained using 0.1 M buffer MES pH 6.5 and magnesium sulfate as precipitant, crystals in the presence of AMP and histidine are obtained using 0.1 M sodium citrate pH 5.6, 0.5 M ammonium sulfate and 1M lithium sulfate with 5 mM AMP and 0.1 mM histidine [15]) [15] Cloning [12] (expressed in Escherichia coli [15]) [15] (expression in Escherichia coli [13]) [13]

177


2.4.2.17

6 Stability pH-Stability Additional information ( overview: stability at various pH-values [9]) [9] Temperature stability 45 ( very sensitive to heat inactivation, 0.4 mM histidine stabilizes the enzyme to inactivation by heat [1]) [1] 47.5 ( 1.9 mg enzyme/ml, 80 min, 75% loss of activity without addition of AMP, with 0.05 mM AMP about 60% loss of activity, with 0.5 mM AMP about 40% loss of activity, with 5 mM AMP about 25% loss of activity [8]) [8] 48 ( 3 mg enzyme/ml, 10 min, 15% remaining activity without addition of histidine, with 0.000086 mM histidine about 15% remaining activity after 15 min, with 0.00069 mM histidine about 70% remaining activity after 80 min, with 0.00345 mM histidine about 80% remaining activity after 80 min, with 0.0069 mM histidine about 55% remaining activity after 50 min [8]) [8] Additional information ( heat inactivation depends on protein concentration and inhibitors [8]) [8] General stability information , l-histidine, 0.4 mM, stabilizes against heat inactivation, 0.04 mM does not stabilize against heat inactivation, at 1.33 mM and higher heat inactivation is greater than the control [1] , NaCl and 2-mercaptoethanol stabilize the very labile enzyme [4] , overview, stability of the enzyme at various pH-values, salt concentrations and histidine concentrations [9] , slight stabilization by 10 mM MgCl2 or CaCl2 by 1 mM MnCl2 and by 1 mM histidine at pH-values above 7 [9] , histidine or AMP stabilizes the enzyme with respect to thermal inactivation [8] Storage stability , -15 C, several months [3] , 4 C, 0.01 M Tris, 0.10 M NaCl, 0.4 mM histidine, 2.8 mM 2-mercaptoethanol, 0.5 mM EDTA, pH 7.5, 50% loss of activity after several days [4] , 4 C, HEPES buffer pH 7.5, final ammonium sulfate microcrystalline enzyme in 60% saturated ammonium sulfate, 0.1 M NaCl, 0.01 M Tris, 1.5 mM EDTA, 10 mM dithiothreitol, stable for 1 month, greater than 95% activity retained [9] , storage in liquid nitrogen of quick-frozen enzyme in HEPES buffer pH 7.5, 0.1 M NaCl, 0.01 M Tris, 0.5 mM EDTA, 1 mM dithiothreitol, 1 mM histidine, indefinitely stable, preserves 100% activity. The critical factor for stability seems to be the maintenance of a sulfhydryl-reducing environment,

178

2.4.2.17


high dithiothreitol concentrations has no adverse effect at either 0 C or 37 C [9] , -20 C, 50 mM Tris-HCl, pH 7.5, 0.4 mM DTT and one protease inhibitor per litre, 50% v/v glycerol, long term storage [14]

References [1] Martin, R.G.: The first enzyme in histidine biosynthesis: the nature of feedback inhibition by histidine. J. Biol. Chem., 238, 257-268 (1963) [2] Tebar, A.R.; Ballesteros, A.O.: Kinetic properties of ATP phosphoribosyltransferase of Escherichia coli. Mol. Cell. Biochem., 11, 131-136 (1976) [3] Ames, B.N.; Martin, R.G.; Garry, B.J.: The first step in histidine biosynthesis. J. Biol. Chem., 236, 2019-2026 (1961) [4] Voll, M.J.; Appella, E.; Martin, R.G.: Purification and composition studies of phosphoribosyladenosine triphosphate:pyrophosphate phosphoribosyltransferase, the first enzyme of histidine biosynthesis. J. Biol. Chem., 242, 1760-1767 (1967) [5] Morton, D.P.; Parsons, S.M.: Synergistic inhibition of ATP phosphoribosyltransferase by guanosine tetraphosphate and histidine. Biochem. Biophys. Res. Commun., 74, 172-177 (1977) [6] Morton, D.P.; Parsons, S.M.: Inhibition of ATP phosphoribosyltransferase by AMP and ADP in the absence and presence of histidine. Arch. Biochem. Biophys., 181, 643-648 (1977) [7] Dall-Larsen, T.; Kryvi, H.; Klungsoyr, L.: Dinitrophenol, dicoumarol and pentachlorophenol as inhibitors and parasite substrates in the ATP phosphoribosyltransferase reaction. Eur. J. Biochem., 66, 443-446 (1976) [8] Kryvi, H.: Thermal stability of phosphoribosyladenosine triphosphate synthetase as reflected in its circular dichroism and activity properties. Effect of inhibitors. Biochim. Biophys. Acta, 317, 123-130 (1973) [9] Parsons, S.M.; Koshland, D.E.: A rapid isolation of phosphoribosyladenosine triphosphate synthetase and comparison to native enzyme. J. Biol. Chem., 249, 4104-4109 (1974) [10] Morton, D.P.; Parsons, S.M.: Biosynthetic direction substrate kinetics and product inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase. Arch. Biochem. Biophys., 175, 677-686 (1976) [11] Kleeman, J.E.; Parsons, S.M.: Reverse direction substrate kinetics and inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase. Arch. Biochem. Biophys., 175, 687693 (1976) [12] Mizukami, T.; Hamu, A.; Ideka, M.; Oka, T.; Katsumata, R.: Cloning of the ATP phosphoribosyl transferase gene of Corynebacterium glutamicum and application of the gene to l-histidine production. Biosci. Biotechnol. Biochem., 58, 635-638 (1994) [13] Ohta, D.; Fujimori, K.; Mizutani, M.; Nakayama, Y.; Kunpaisal-Hashimoto, R.; Munzer, S.; Kozaki, A.: Molecular cloning and characterization of ATP179


2.4.2.17

phosphoribosyl transferase from Arabidopsis, a key enzyme in the histidine biosynthetic pathway. Plant Physiol., 122, 907-914 (2000) [14] Lohkamp, B.; Coggins, J.R.; Lapthorn, A.J.: Purification, crystallization and preliminary X-ray crystallographic analysis of ATP-phosphoribosyltransferase from Escherichia coli. Acta Crystallogr. Sect. D, D56, 1488-1491 (2000) [15] Cho, Y.; Sharma, V.; Sacchettini, J.C.: Crystal structure of ATP phosphoribosyltransferase from Mycobacterium tuberculosis. J. Biol. Chem., 278, 8333-8339 (2003)

180

Anthranilate phosphoribosyltransferase

2.4.2.18

1 Nomenclature EC number 2.4.2.18 Systematic name N-(5-phospho-d-ribosyl)-anthranilate:diphosphate transferase

phospho-a-d-ribosyl-

Recommended name anthranilate phosphoribosyltransferase Synonyms PR transferase PRT TrpD anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase anthranilate phosphoribosylpyrophosphate phosphoribosyltransferase anthranilate-5-phosphoribosylphosphate phosphoribosyltransferase anthranilate-PP-ribose-P phosphoribosyltransferase phosphoribosyl-anthranilate pyrophosphorylase phosphoribosylanthranilate pyrophosphorylase phosphoribosylanthranilate transferase phosphoribosyltransferase, anthranilate Additional information ( in some organisms, this enzyme is part of a multifunctional protein together with one or more other components of the system for biosynthesis of tryptophan (EC 4.1.1.48, EC 4.1.3.27, EC 4.2.1.20, EC 5.3.1.24) [11-13]) [11-13] CAS registry number 9059-35-2

2 Source Organism

Enterobacter cloacae [11] Enterobacter hafniae [11] Enterobacter liquefaciens [11] Citrobacter freundii [11] Citrobacter ballerupensis [11] Proteus vulgaris [11] Proteus morganii [11]

181


2.4.2.18

Serratia marinorubra [11] Aeromonas formicans [11] Erwinia carotovora [11, 15] Erwinia dissolvens [11] Aerobacter aerogenes [12] Hafnia alvei [14] Escherichia coli (anthranilate synthetase complex consisting of 2 separate subunits: component I and II [1]; K-12 [12]) [1, 5, 12] Neurospora crassa [2] Salmonella typhimurium (strains: trp+, TAX6trpR782, trpA703trpR782 and trpAB1653trpR782 [3]) [3, 8, 9, 13] Saccharomyces cerevisiae [4] Hansenula henricii [6, 7] Serratia marcescens [10, 11] Sulfolobus solfataricus [16] Pectobacterium carotovorum [17] Corynebacterium glutamicum (wild-type and 5-methyltryptophan resistant mutant [18]) [18]

3 Reaction and Specificity Catalyzed reaction N-(5-phospho-d-ribosyl)-anthranilate + diphosphate = anthranilate + 5phospho-a-d-ribose 1-diphosphate ( reaction mechanism [7]) Reaction type pentosyl group transfer Natural substrates and products S anthranilate + 5-phospho-a-d-ribose 1-diphosphate ( enzyme of tryptophan biosynthesis [1-18]) [1-18] P N-(5-phospho-d-ribosyl)-anthranilate + diphosphate Substrates and products S anthranilate + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ? [1-18]) [1-18] P N-(5-phospho-d-ribosyl)-anthranilate + diphosphate [1-3] Inhibitors 3-hydroxyanthranilate ( competitive [7]) [7] 5-methyltryptophan ( 0.32 mM [18]) [18] l-tryptophan ( transferase activity of component II is only inhibitable by l-tryptophan when the component is in the complex, this inhibition does not appear to depend upon the feedback-sensitive site of complex I [1]; when phosphoribosyltransferase is not an aggregate with anthranilate synthase, it is not subject to tryptophan inhibition, inhibitor site is on the anthranilate synthase component [12]; 0.83 mM [18]) [1, 3, 12, 18] 182

2.4.2.18


N-(5-phospho-d-ribosyl)-anthranilate [3] anthranilate ( substrate inhibition above 0.008 mM [7]) [7] sodium diphosphate [3] Metals, ions Mg2+ ( required [3, 12]; activates [7]; Km : 0.056 mM [7]; dependent on [4]) [3, 4, 7, 12] Turnover number (min±1) 0.84 (anthranilate, at 25 C [16]) [16] 2.1 (anthranilate, at 40 C [16]) [16] 4.02 (anthranilate, at 50 C [16]) [16] 6.3 (anthranilate, at 60 C [16]) [16] 20.4 (anthranilate, cosubstrate: 5-phospho-a-d-ribose 1-diphosphate [6]) [6] 24 (anthranilate, at 87 C [16]) [16] 174 (anthranilate, cosubstrate: 5-phospho-a-d-ribose 1-diphosphate [4]) [4] 246 (anthranilate, cosubstrate: 5-phospho-a-d-ribose 1-diphosphate [8,9]) [8, 9] 264 (5-phospho-a-d-ribose 1-diphosphate) [5] 264 (anthranilate) [5] Specific activity (U/mg) 0.049 ( wild-type, crude extract [18]) [18] 0.4 [7] 0.5 ( strain TAX6trpR782 and trpAB165trpR782 [3]) [3] 1.54 [13] 1.58 [4] 2.7 ( strain trpA703trpR782 [3]) [3] 3.2 ( strain trp+ [3]) [3] 16 [2] 17.6 ( strain trpAB1653 [3]) [3] 24.5 [15] 734 [12] 1540 [13] 4090 [10] Additional information [3] Km-Value (mM) 0.000005 (anthranilate, at 87 C, Km decreases at lower temperatures [16]) [16] 0.0016 (anthranilate) [4] 0.003 (anthranilate, TAX6trpR782 [3]) [3] 0.004 (anthranilate, trpAB1653trpR782 [3]) [3] 0.0046 (anthranilate) [7] 0.01 (5-phospho-a-d-ribose 1-diphosphate) [16] 0.013 (5-phospho-a-d-ribose 1-diphosphate, TAX6trpR782 [3]) [3] 183


2.4.2.18

0.0224 (5-phospho-a-d-ribose 1-diphosphate) [4] 0.06 (5-phospho-a-d-ribose 1-diphosphate, trpAB1653trpR782 [3]) [3] 0.1 (5-phospho-a-d-ribose 1-diphosphate, complex [1]) [1] 0.2 (5-phospho-a-d-ribose 1-diphosphate, component II [1]) [1] 0.88 (5-phospho-a-d-ribose 1-diphosphate) [7] Ki-Value (mM) 0.012 (3-hydroxyanthranilate) [7] 0.03 (anthranilate) [7] pH-Optimum 6.9 [3] 7 ( assay at [3]) [3] 7.4-7.7 [7] 7.5 ( assay at [4]) [4] Temperature optimum ( C) 25 ( assay at [4]) [4] 37 ( assay at [7]) [7]

4 Enzyme Structure Molecular weight 45000 ( gel filtration [10,11]) [10, 11] 67000 ( gel filtration [11,15]) [11, 15] 70000 ( gel filtration [7,14]; and 150000, disc gel electrophoresis [13]) [7, 13, 14] 73300 ( gel filtration [16]) [16] 83000 ( sedimentation equilibrium [4]) [4] 90000 ( tryptophan auxotroph with no anthranilate synthase activity, sucrose density gradient sedimentation [12]) [12] 150000 ( and 70000, disc gel electrophoresis [13]) [13] 170000 ( sucrose density gradient sedimentation, complex with anthranilate synthase activity [12]) [12] 220000 ( and larger than 1000000, strain trpAB1653trpR782, gel filtration [3]) [3] 320000 ( strain TAX6trpR782, gel filtration [3]) [3] Subunits ? ( x * 72000, strain trpAB1653trpR782, SDS-PAGE [3]; enzyme exists in both monomeric and dimeric forms, SDS-PAGE [13]) [3, 13] dimer ( 2 * 40000, SDS-PAGE [15]; 2 * 37000, SDS-PAGE [14]; 2 * 42000, SDS-PAGE [4]; homodimer, each monomer consists of 2 subdomains a and a/b [17]) [4, 14, 15, 17] monomer ( 1 * 43000, SDS-PAGE [10]) [10] Additional information ( in some organisms, this enzyme is part of a multifunctional protein together with one or more 184

2.4.2.18


other components of the system for biosynthesis of tryptophan: EC 4.1.1.48, EC 4.1.3.27, EC 4.2.1.20, EC 5.3.1.24 [11-13]; no aggregation with other enzymes [7]; anthranilate synthase-phosphoribosylanthranilate transferase complex exists in Citrobacter species in all other bacteria examined phosphoribosylanthranilate transferase and anthranilate synthase are separate enzyme molecules [11, 12, 13]; enzyme complex is a tetramer composed of 2 molecules each of component II and II [3]) [3, 7, 11-13]

5 Isolation/Preparation/Mutation/Application Purification [15] (partial, enzyme complex [12]) [12] [14] (partial [2]) [2] (enzyme complex, homogeneity [3]; unaggregated form [13]) [3, 13] (95% pure [4]) [4] [7] [10] (95% pure [16]) [16] Renaturation (reconstitution with indole-3-glycerophosphate synthetase [2]) [2] Crystallization [14] (hanging drop vapor diffusion method [16]) [16] (hanging drop vapor diffusion method, complexed with Mn2+ diphosphate [17]) [17] Cloning [16] [17] [18]

6 Stability Temperature stability 4 ( loss of 10% activity in 24 h [4]) [4] 46 ( half-life: strain TAX6trpR782: 1.5 trpAB1653trpR782: 5 min [3]) [3] 70 ( 50% loss of activity after 69 min [16]) [16] 85 ( 50% loss of activity after 35 min [16]) [16]

min,

strain

185


2.4.2.18

General stability information , dilution inactivates [12] , freezing and thawing inactivates [12] , glycerol, 10%, is essential for storage, but it must be replaced by polyethylene glycol to achieve crystals that are not severely temperature dependent and radiation sensitive [14] Storage stability , -15 C, crude extract is stable for 2 years [12] , 4 C, 10% loss of activity after 2 months [12] , addition of 10% glycerol at -70 C and then dropping the solution into liquid N2 , can be thawed and frozen several times without loss of activity [4] , -25 C, 500 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM MgCl2 , 5% loss of activity after 2 weeks [7]

References [1] Ito, J.; Yanofsky, C.: Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: Comparative studies on the complex and the subunits. J. Bacteriol., 97, 734-742 (1969) [2] Wegman, J.; DeMoss, J.A.: The enzymatic conversion of anthranilate to indolylglycerol phosphate in Neurospora crassa. J. Biol. Chem., 240, 37813788 (1965) [3] Grieshaber, M.: On the evolution of a oligocephalic enzyme. Glutaminechorismate-amidotransferase-free anthranilate phosphoribosyltransferases from mutant strains of Salmonella typhimurium. Z. Naturforsch. C, 33c, 235-244 (1978) [4] Hommel, U.; Lustig, A.; Kirschner, K.: Purification and characterization of yeast anthranilate phosphoribosyltransferase. Eur. J. Biochem., 180, 33-40 (1989) [5] Gonzalez, J.E.; Sommerville, R.L.: The anthranilate aggregate of Escherichia coli: kinetics of inhibition by tryptophan of phosphoribosyltransferase. Biochem. Cell Biol., 64, 681-691 (1986) [6] Kane, J.F.; Jensen, R.A.: Metabolic interlock. The influence of histidine on tryptophan biosynthesis in Bacillus subtilis. J. Biol. Chem., 245, 2384-2390 (1970) [7] Bode, R.; Birnbaum, D.: Enzymes of the aromatic amino acid biosynthesis in Hansenula henricii: anthranilate phosphoribosylpyrophosphate-phosphoribosyltransferase (E.C.2.4.2.18). Z. Allg. Mikrobiol., 18, 559-566 (1978) [8] Henderson, E.J.; Zalkin, H.; Hwang, L.H.: The anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase aggregate. Catalytic and regulatory properties of aggregated and unaggregated forms of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase. J. Biol. Chem., 245, 1424-1431 (1970) [9] Grieshaber, M.; Bauerle, R.: Monomeric and dimeric forms of component II of the anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate

186

2.4.2.18

[10]

[11] [12] [13]

[14] [15]

[16]

[17]

[18]


phosphoribosyltransferase complex of Salmonella typhimurium. Implications concerning the mode of assembly of the complex. Biochemistry, 13, 373-383 (1974) Largen, M.; Mills, S.E.; Rowe, J.; Yanofsky, C.: Purification, subunit structure and partial amino-acid sequence of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the enteric bacterium Serratia marcescens. Eur. J. Biochem., 67, 31-36 (1976) Largen, M.; Belser, W.L.: Tryptophan biosynthetic pathway in the Enterobacteriaceae: some physical properties of the enzymes. J. Bacteriol., 121, 239-249 (1975) Egan, A.F.; Gibson, F.: Anthranilate synthase-anthranilate 5-phosphoribosyl 1-pyrophosphate phosphoribosyltransferase from Aerobacter aerogenes. Biochem. J., 130, 847-859 (1972) Marcus, S.L.; Balbinder, E.: Purification of anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase from Salmonella typhimurium using affinity chromatography: resolution of monomeric and dimeric forms. Biochem. Biophys. Res. Commun., 47, 438-444 (1972) Edwards, S.E.; Kraut, J.; Xuong, N.; Ashford, V.; Halloran, T.P.; Mills, S.L.: Crystallization and preliminary X-ray studies of T4 phage b-glucosyltransferase. J. Mol. Biol., 203, 525-526 (1988) Largen, M.; Mills, S.E.; Rowe, J.; Yanofsky, C.: Purification and properties of a third form of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the Enterobacteriaceae. J. Biol. Chem., 253, 409-412 (1978) Ivens, A.; Mayans, O.; Szadkowski, H.; Wilmanns, M.; Kirschner, K.: Purification, characterization and crystallization of thermostable anthranilate phosphoribosyltransferase from Sulfolobus solfataricus. Eur. J. Biochem., 268, 2246-2252 (2001) Kim, C.; Xuong, N.-H.; Edwards, S.; Madhusudan; Yee, M.-C.; Spraggon, G.; Mills, S.E.: The crystal structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum. FEBS Lett., 523, 239-246 (2002) O'Gara, J.P.; Dunican, L.K.: Mutations in the trpD gene of Corynebacterium glutamicum confer 5-methyltryptophan resistance by encoding a feedbackresistant anthranilate phosphoribosyltransferase. Appl. Environ. Microbiol., 61, 4477-4479 (1995)

187

Nicotinate-nucleotide diphosphorylase (carboxylating)

2.4.2.19

1 Nomenclature EC number 2.4.2.19 Systematic name nicotinate-nucleotide:diphosphate phospho-a-d-ribosyltransferase (carboxylating) Recommended name nicotinate-nucleotide diphosphorylase (carboxylating) Synonyms GSP70 NAD pyrophosphorylase NadC QAPRTase QPRTase general stress protein 70 nicotinate mononucleotide pyrophosphorylase (carboxylating) (EC 2.4.2.19) nicotinate-nucleotide pyrophosphorylase (carboxylating) nicotinate-nucleotide:pyrophosphate phospho-a-d-ribosyltransferase (decarboxylating) pyrophosphorylase, nicotinate mononucleotide (carboxylating) quinolinate phosphoribosyltransferase quinolinate phosphoribosyltransferase (decarboxylating) quinolinate phosphoribosyltransferase [decarboxylating] quinolinic acid phosphoribosyltransferase quinolinic phosphoribosyltransferase CAS registry number 37277-74-0

2 Source Organism 188

Pseudomonas sp. [1] Bos taurus [2] Lentinus edodes [3, 4] Nicotiana tabacum [5] Sus scrofa [6, 8, 9, 10, 12, 14, 15, 16, 19] Ricinus communis [7] Salmonella typhimurium [13, 26, 27, 30]

2.4.2.19


Escherichia coli [11] Rattus norvegicus [17] Homo sapiens [18, 28] Alcaligenes eutrophus (nov. subsp. quinolinicus [20]; subsp. quinolinicus [21]) [20, 21] Saccharomyces cerevisiae [22] Helicobacter pylori [23] Burkholderia cepacia (DOB1 and other phthalate-degrading strains have two dissimilar genes for this enzyme, while non-phthalate-degrading strains have only a single gene [24]) [24] Nicotiana rustica [25] Nicotiana tabacum [25] Nicotiana sylvestris [25] Mycobacterium tuberculosis [29]

3 Reaction and Specificity Catalyzed reaction nicotinate d-ribonucleotide + diphosphate + CO2 = pyridine-2,3-dicarboxylate + 5-phospho-a-d-ribose 1-diphosphate (, formation of a ternary complex consisting of the enzyme, pyridine-2,3-dicarboxylate and 5-phospho-a-d-ribose 1-diphosphate [6]; , ordered binding mechanism [11]; , formation of a ternary complex comprising the enzyme, quinolinate and 5-phosphoribosyl-1-diphosphate [20]; , predominantly ordered kinetic mechanism in which productive binding of quinolinate precedes that of 5-phospho-a-d-ribose 1-diphosphate [26]) Reaction type pentosyl group transfer Natural substrates and products S Additional information (, enzyme is probably involved in the regulation of nicotine biosynthesis [5]; , intermediary enzyme in the de novo NAD biosynthetic pathway [9]; , intermediary enzyme in the de novo NAD biosynthetic pathway [14, 15, 16]; , enzyme of the kynurenine pathway [22]; , key enzyme of NAD+ biosynthesis [23]; , the recruitment of this gene for growth on phthalate gives Burkholderia cepacia an advantage over other phthalatedegrading bacteria in the environment [24]; , key enzyme in NAD+ biosynthesis, also plays an important role in ensuring nicotinic acid available for the synthesis of defensive pyridine alkaloids [25]; , key enzyme in catabolism of quinolinate. Quinolinate acts as a most potent endogenous exitotoxin to neurons. Elevation of quinolinate levels in the brain has been linked to the pathogenesis of neurodegenerative disorders [28]; , essential enzyme for the de novo biosynthesis of NAD+ [29]) [5, 9, 14, 15, 16, 22, 23, 24, 25, 28, 29] P ? 189


2.4.2.19

Substrates and products S nicotinic acid + 5-phospho-a-d-ribose 1-diphosphate [2] P ? S pyridine-2,3-dicarboxylate + 5-phospho-a-d-ribose 1-diphosphate (Reversibility: ir [18]; ? [1-17,19-30]) [1-30] P nicotinate d-ribonucleotide + diphosphate + CO2 (, i.e. b-niacin mononucleotide [6]) [1-30] Inhibitors 2-hydroxynicotinate (, 50% at 0.01 M [7]) [7] 2-oxoglutarate (, 1 mM, 60% inhibition [12]; , 1 mM, 55% inhibition [19]) [12, 19] 5-phosphoribosyl-1-diphosphate (, inhibition at alkaline pH and at physiological pH, pH 7.4, but not at an acidic pH, competitive for quinolinate. In presence of 30% glycerol, both the kidney and liver enzyme are inhibited, even at acidic pH [8]) [8] ADP (, 2 mM, 25% inhibition [6]; , 2 mM, 25% inhibition [9]; , 1 mM, 12% inhibition [21]) [6, 9, 21] AMP (, 2 mM, 7% inhibition [9]) [9] ATP (, 2 mM, 82% inhibition [6]; , inhibition is removed by raising Mg2+ concentrations [9]; , inhibition is completely recovered by raising Mg2+ concentration [21]; , 2 mM, 82% inhibition [9]; , 1 mM, 22% inhibition [21]; , inhibition is completely recovered by raising Mg2+ concentration [21]) [6, 9, 21] Ag+ (, 1 mM, complete inhibition [16]) [16] Al3+ (, 1 mM, 82.8% inhibition [16]) [4, 16] Ba2+ (, 1 mM, 38.1% inhibition [16]) [16] CDP (, 2 mM, 20% inhibition [9]; , 1 mM, 13% inhibition [21]) [9, 21] CTP (, 2 mM, 77% inhibition [9]; , 1 mM, 30% inhibition [21]) [9, 21] Ca2+ (, 1 mM, 34.9% inhibition [16]) [16] Cd2+ (, 1 mM, 75.5% inhibition [16]) [16, 21] Cl- [4] Co2+ (, 1 mM, 65.2% inhibition [16]) [16, 21] Cr3+ (, 1 mM, 46.4% inhibition [16]) [16] Cu2+ (, 1 mM, 87% inhibition [6]; , 1 mM, 86.9% inhibition [16]) [6, 16, 21] d-fructose-1,6-diphosphate (, competitive with respect to 5-phosphoribosyl-1-diphosphate and noncompetitive with respect to quinolinate [11]) [11] Fe2+ (, 1 mM, 93% inhibition [6]; , 1 mM, 61.5% inhibition [16]) [4, 6, 16] Fe3+ (, 1 mM, 92.9% inhibition [16]) [4, 16] GDP (, 2 mM, 29% inhibition [9]) [9] GTP (, 2 mM, 77% inhibition [9]; , 1 mM, 17% inhibition [21]) [9, 21]

190

2.4.2.19


H2 PO-4 [20] Hg2+ (, 1 mM, 99.9% inhibition [16]) [16, 21] IDP (, 2 mM, 29% inhibition [9]) [9] IMP (, 2 mM, 6% inhibition [9]) [9] ITP (, 2 mM, 81% inhibition [9]; , 1 mM, 24% inhibition [21]) [9, 21] l-glutamic acid (, 1 mM, 31% inhibition [12]; , 1 mM, 41% inhibition [19]) [12, 19] l-malic acid (, 1 mM, 75% inhibition [12]; , 1 mM, 67% inhibition [19]) [12, 19] Mg2+ (, 1 mM, 7.7% inhibition [16]) [16] Mn2+ (, 1 mM, 80.3% inhibition [16]) [16] NAD+ (, 19% inhibition at 1 mM, 15% inhibition at 10 mM [7]) [7] NEM (, 5 mM, 86% inhibition [9]) [9] NO3- [4] Ni2+ (, 1 mM, 76.5% inhibition [16]) [4, 16, 21] PCMB (, 0.05 mM, complete inhibition [6,9]) [6, 9] Sr2+ (, 47.6% inhibition [16]) [16] Tris [2] UDP (, 2 mM, 32% inhibition [9]) [9] UTP (, 2 mM, 74% inhibition [9]; , 1 mM, 19% inhibition [21]) [9, 21] Zn2+ (, 1 mM, 82.1% inhibition [16]) [4, 16, 21] acetic acid (, 26% inhibition [19]) [19] aspartic acid (, 1 mM, 23% inhibition [12]; , 1 mM, 37% inhibition [19]) [12, 19] citric acid (, 1 mM, 95% inhibition [12]; , 1 mM, 89% inhibition [19]) [12, 19] dTDP (, 1 mM, 14% inhibition [21]) [21] dTTP (, 1 mM, 29% inhibition [21]) [21] diphosphate (, noncompetitive with respect to both 5-phosphoribosyl-1-diphosphate and quinolinate [11]) [11] dipicolinic acid (, 1 mM, 24% inhibition [9]) [9] dithiobis(2-nitrobenzoic acid) (, 10 mM, 88.7% inhibition [9]) [9] formic acid (, 1 mM, 54% inhibition [19]) [19] fumaric acid (, 1 mM, 72% inhibition [12]; , 1 mM, 67% inhibition [19]) [12, 19] glycerol (, inhibition increases as the pH raises [12]; , glycerol markedly activates enzyme activity at pH 6.1 and 6.5, inhibition above pH 7.0, inhibition is strongest at pH 9.0 [21]) [12, 21] isocinchomeric acid (, 1 mM, 15% inhibition [9]) [9] lactic acid (, 1 mM, 29% inhibition [19]) [19] lutidinic acid (, 1 mM, 17% inhibition [9]) [9] maleic acid (, 1 mM, 34% inhibition [12]; , 1 mM, 42% inhibition [19]) [12, 19] methyl-3-amidopyridine-2-carboxylate [7]

191


2.4.2.19

methyl-3-cyanopyridine 2-carboxylate [7] monoiodoacetic acid (, 50 mM, 99.8% inhibition [9]; , 5 mM, 50% inhibition [6]) [6, 9] nicotinate mononucleotide (, competitive with respect to 5phosphoribosyl-1-diphosphate [11]; , 1 mM, 46% inhibition [21]) [11, 21, 26] oxaloacetic acid (, 1 mM, 56% inhibition [12]; , 1 mM, 59% inhibition [19]) [12, 19] phthalic acid (, competitive to quinolinate [6,9]; , dead-end inhibitor, competitive with respect to quinolinate, uncompetitive with respect to 5-phosphoribosyl-1-diphosphate [11]) [6, 9, 11, 17, 24, 26] picolinic acid [7] succinic acid (, 1 mM, 51% inhibition [12]; , 1 mM, 46% inhibition [19]) [12, 19] Cofactors/prosthetic groups NAD+ (, 0.1 mM, 75% inhibition [2]) [2] Activating compounds glycerol (, glycerol markedly activates enzyme activity at pH 6.1 and pH 6.5, inhibition above pH 7.0, inhibition is strongest at pH 9.0 [21]) [21] Metals, ions Cd2+ (, can partially replace Mg2+ [16]) [16] Co2+ (, can partially replace Mg2+ [16]) [16] K+ (, monovalent cation required, K+ , Li+ or NH+4 markedly stimulate [2]) [2] Li+ (, monovalent cation required, K+ , Li+ or NH+4 markedly stimulate [2]) [2] Mg2+ (, maximal activity at 0.1-0.2 mM [2]; , divalent cation required, Km : 0.2 mM [3]; , optimal Mg2+ concentration: 1 mM [6]; , absolute requirement. Optimal concentration is 1 mM in absence of ATP and 7 mM in presence of ATP at 5 mM [9]; , absolute requirement [10]; , required [11]; , divalent cation required, Mg2+ is most effective at 1 mM [16]; , absolute requirement for divalent cations. Mg2+ is most effective. Optimal concentrations are 4, 6 and 10 mM for 20, 40 and 100 mM sodium acetate/acetic acid buffer, pH 5.5 [19]; , absolute requirement for a divalent metal ion, Mg2+ is most effective, optimal activity at 1.5 mM [20]) [2, 3, 6, 9, 10, 11, 16, 19, 20] Mn2+ (, half as effective as Mg2+ [2]; , 0.3 mM, can fully replace Mg2+ [10]; , at 1 mM, 80% of the activation with Mg2+ [20]) [2, 10, 20] NH+4 (, monovalent cation required, K+ , Li+ or NH+4 markedly stimulate [2]) [2] Zn2+ (, can partially replace Mg2+ [16]) [16]

192

2.4.2.19


Turnover number (min±1) Additional information (, turnover rate is decreased by adding glycerol [21]) [21, 26] Specific activity (U/mg) 0.01235 [17] 0.0133 (, enzyme from liver [18]) [18] 0.0187 (, enzyme from brain [18]) [18] 0.0227-0.0919 (, enzyme from kidney [8]) [8] 0.05169 [6] 0.0517 [14, 15] 0.073 [2] 0.091 [19] 0.75 [7] 0.88 [1] 0.9 [13, 27] Additional information [11] Km-Value (mM) 0.0051 (quinolinate) [5] 0.0056 (quinolinate) [18] 0.0064 (quinolinate) [11] 0.01 (nicotinic acid) [2] 0.011 (quinolinate) [3] 0.012 (quinolinate, , enzyme from liver and brain [17]) [7, 17] 0.0156 (5-phospho-a-d-ribose 1-diphosphate) [11] 0.0196 (quinolinate) [13] 0.02 (quinolinate) [27] 0.021 (5-phospho-a-d-ribose 1-diphosphate) [5] 0.022 (5-phospho-a-d-ribose 1-diphosphate, , enzyme from liver [17]) [17] 0.023 (5-phospho-a-d-ribose 1-diphosphate, , enzyme from brain [17]) [3, 17] 0.025 (quinolinate) [26] 0.03 (5-phospho-a-d-ribose 1-diphosphate) [26] 0.032 (5-phospho-a-d-ribose 1-diphosphate) [27] 0.0322 (5-phospho-a-d-ribose 1-diphosphate) [13] 0.04 (quinolinate) [19] 0.045 (5-phospho-a-d-ribose 1-diphosphate) [7] 0.05 (5-phospho-a-d-ribose 1-diphosphate) [2] 0.051 (quinolinic acid, , in presence of 30% glycerol [21]) [21] 0.057 (5-phospho-a-d-ribose 1-diphosphate) [21] 0.06 (quinolinate) [2] 0.091 (quinolinate) [21] 0.111 (5-phospho-a-d-ribose 1-diphosphate) [20] 0.12 (quinolinate) [6, 16] 0.128 (5-phospho-a-d-ribose 1-diphosphate) [21] 0.133 (quinolinate) [20] 193


2.4.2.19

0.14 (5-phospho-a-d-ribose 1-diphosphate) [19] 0.18 (5-phospho-a-d-ribose 1-diphosphate) [6, 16] Ki-Value (mM) 0.0014 (phthalic acid) [17] 0.044 (phthalate) [26] 0.05 (5-phosphoribosyl-1-diphosphate, , enzyme from liver [8]) [8] 0.063 (nicotinate mononucleotide) [26] 0.0838 (nicotinate mononucleotide) [21] 0.17 (phthalic acid) [6, 9] 0.265 (5-phosphoribosyl-1-diphosphate) [21] 0.8 (5-phosphoribosyl-1-diphosphate, , enzyme from kidney [8]) [8] 2.2 (5-phosphoribosyl-1-diphosphate) [26] pH-Optimum 5.5 (, in presence of 1 mM 5-phospho-a-d-ribose 1-diphosphate, enzyme from kidney [8]) [8, 19] 6 (, in presence of 1 mM 5-phospho-a-d-ribose 1-diphosphate, enzyme from liver [8]) [8] 6.1 [6, 16] 6.2 [2] 6.5 [3, 18] 6.5-7 [17] 6.5-7.7 [7] 8.5-9 [20] 9 (, in presence of 0.4 mM 5-phospho-a-d-ribose 1-diphosphate, enzyme from kidney and liver [8]) [8] pH-Range 5.2-7.8 (, half maximal activity at pH 5.2 and pH 7.8 [16]) [16] 6.4-7.3 (, pH 6.4: about 50% of maximal activity, pH 7.3: about 55% of maximal activity [2]) [2] Temperature optimum ( C) 50 [4] 60 [20]

4 Enzyme Structure Molecular weight 68000 (, gel filtration [7]) [7] 70000 (, gel filtration [11]) [11] 72000 (, sucrose density gradient centrifugation [7]; , gel filtration [13,27]) [7, 13, 27] 160000 (, gel filtration [3]; , sucrose density gradient centrifugation, gel filtration [17]) [3, 17] 167000 (, sucrose density gradient centrifugation [18]) [18] 170000 (, gel filtration [18]) [18] 194

2.4.2.19


172000 (, sedimetation velocity method [14,15]) [14, 15] 173000 (, gel filtration [14,15]) [14, 15] 178000 (, calculation from sedimentation and ultracentrifugation data [1]) [1] 202000 (, equilibrium sedimentation [6,10]) [6, 10] 210000 (, gel filtration [6,10]; , gel filtration [20]) [6, 10, 20] Subunits ? (, x * 34000, SDS-PAGE [14]; , x * 34200, equilibrium sedimentation in guanidine HCl [10]; , x * 33500, SDS-PAGE [6,10,15]) [6, 10, 14, 15] dimer (, 2 * 35000, SDS-PAGE [7, 13, 27]; , 2 * 36000, SDS-PAGE [11]) [7, 11, 13, 14, 27] octamer (, 8 * 27500, SDS-PAGE [20]) [20] pentamer (, 5 * 32000, SDS-PAGE [17]; , 5 * 34000, SDSPAGE [18]) [17, 18] Posttranslational modification glycoprotein (, enzyme contains 1% mannose [19]) [19]

5 Isolation/Preparation/Mutation/Application Source/tissue brain [17, 18, 28] cell suspension culture [5] endosperm (, endosperm of etiolated seedlings [7]) [7] kidney [8, 19] leaf [5] liver [2, 6, 8, 9, 10, 12, 15, 16, 17, 18] root (, transcript level increases markedly 12-24 h after damage to aerial tissue [25]) [5, 25] seedling (, endosperm of etiolated seedlings [7]) [7] stem [5] Purification [1] [2] [3, 4] [6, 14, 15, 19] [13, 27] [11] [17] [18] Crystallization [1] [6, 8, 10, 14]

195


2.4.2.19

(hanging-drop vapor-diffusion method, determination of crystal structure of the enzyme with bound quinolinate to 2.8 A resolution and with bound nicotinic acid mononucleotide [30]) [30] (hanging-drop vapor-diffusion method [23]) [23] (hangig-drop vapor diffusion method, X-ray crystal structure of the apoenzyme is determined by multiple isomorphous replacement at 2.4 A resolution, complex with quinolinate, phthalate, nicotinate mononucleotideand ternary complex with phthalate and a substrate analog 5-phosphoribosyl-1(b-methylene)diphosphate [29]) [29] Cloning [14] (subcloned into a T7-based expression system [27]) [13, 27] [11] (expression in Escherichia coli [28]) [28] (expression in Escherichia coli [25]) [25] (expression in Escherichia coli [25]) [25] (overexpression in Escherichia coli [29]) [29]

6 Stability pH-Stability 3-3.5 (, 37 C, denatured abruptly [19]) [19] 4.5-9.5 (, 37 C, 30 min, completely stable [19]) [19] 5.5-10 [6] Temperature stability 55 (, 10 min, 48% loss of activity [7]) [7] 80 (, 3 min, 50% loss of activity [17]) [17] Additional information (, quinolinate at 0.8 mM gives 50% protection against heat inactivation. 22% inhibition in presence of quinolinate [7]) [7] Storage stability , -20 C, enzyme in treated extract, stable [5] , 0-4 C, crystalline enzyme is stable for at least 2 years [19] , -90 C, stable for several months in 0.05 M potassium phosphate buffer, pH 7.0, 50% sucrose w/v and 0.01 M dithiothreitol [7] , 4 C, stable for 1 month [11]

References [1] Packman, P.M.; Jacoby, W.B.: Crystalline quinolinate phosphoribosyltransferase. J. Biol. Chem., 240, PC4107-PC4108 (1965)

196

2.4.2.19


[2] Gholson, R.K.; Ueda, I.; Ogasawara, N.; Henderson, L.M.: The enzymatic conversion of quinolinate to nicotinic acid mononucleotide in mammalian liver. J. Biol. Chem., 239, 1208-1214 (1964) [3] Taguchi, H.; Iwai, K.: Purification and properties of quinolinate phosphoribosyltransferase from the Shiitake mushroom (Lentinus edodes). J. Nutr. Sci. Vitaminol., 20, 269-281 (1974) [4] Taguchi, H.; Iwai, K.: Characteristics of quinolinate phosphoribosyltransferase from the Shiitake mushroom (Lentinus edodes). J. Nutr. Sci. Vitaminol., 20, 283-291 (1974) [5] Wagner, R.; Wagner, K.G.: Determination of quinolinic acid phosphoribosyltransferase in tobacco. Phytochemistry, 23, 1881-1883 (1984) [6] Iwai, K.; Taguchi, H.: Crystallization and properties of quinolinate phosphoribosyltransferase from hog liver. Methods Enzymol., 66, 96-101 (1980) [7] Mann, D.F.; Byerrum, R.U.: Quinolinic acid phosphoribosyltransferase from castor bean endosperm. I. Purification and characterization. J. Biol. Chem., 249, 6817-6823 (1974) [8] Shibata, K.; Iwai, K.: Effect of 5-phosphoribosyl-1-pyrophosphate on crystalline quinolinate phosphoribosyltransferase activities from hog kidney and hog liver. Agric. Biol. Chem., 44, 2785-2791 (1980) [9] Taguchi, H.; Iwai, K.: Inhibition of hog liver crystalline quinolinate phosphoribosyltransferase by nucleotides, quinolinate analogues and sulfhydryl reagents. Agric. Biol. Chem., 40, 385-389 (1976) [10] Musick, W.D.L.: Preliminary crystallographic studies on quinolinate phosphoribosyltransferase. J. Mol. Biol., 117, 1101-1107 (1977) [11] Bhatia, R.; calvo, K.C.: The sequencing, expression, purification, and steady-state kinetic analysis of quinolinate phosphoribosyl transferase from Escherichia coli. Arch. Biochem. Biophys., 325, 270-278 (1996) [12] Iwai, K.; Shibata, K.; Taguchi, H.: Crystalline quinolinate phosphoribosyltransferase from hog liver: the molecular weight and some enzymic properties. Agric. Biol. Chem., 43, 351-355 (1979) [13] Hughes, K.T.; Dessen, A.; Gray, J.P.; Grubmeyer, C.: The Salmonella typhimurium nadC gene: sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyltransferase. J. Bacteriol., 175, 479-486 (1993) [14] Iwai, K.; Taguchi, H.: Purification and crystallization of quinolinate phosphoribosyltransferase from hog liver. Biochem. Biophys. Res. Commun., 56, 884-891 (1974) [15] Taguchi, H.; Iwai, K.: The isolation and physicochemical properties of crystalline quinolinate phosphoribosyltransferase from hog liver. Agric. Biol. Chem., 39, 1493-1500 (1975) [16] Taguchi, H.; Iwai, K.: Properties of the crystalline quinolinate phosphoribosyltransferase from hog liver. Agric. Biol. Chem., 39, 1599-1604 (1975) [17] Okuno, E.; Schwarcz, R.: Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain. Biochim. Biophys. Acta, 841, 112-119 (1985)

197


2.4.2.19

[18] Okuno, E.; White, R.J.; Schwarcz, R.: Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain. J. Biochem., 103, 1054-1059 (1988) [19] Shibata, K.; Iwai, K.: Isolation and properties of crystalline quinolinate phosphoribosyltransferase from hog kidney. Biochim. Biophys. Acta, 611, 280-288 (1980) [20] Iwai, K.; Shibata, K.; Taguchi, H.; Itakura, T.: Properties of crystalline quinolinate phosphoribosyltransferase from Alcaligenes eutrophus nov. Subsp. Quinolinicus. Agric. Biol. Chem., 43, 345-350 (1979) [21] Shibata, K.; Iwai, K.: Crystalline quinolinate phosphoribosyltransferase from Alcaligenes eutrophus subsp. quinolinicus: effects of metal ions, phosphorous-compounds, niacin nucleotides and glycerol. Agric. Biol. Chem., 44, 119-123 (1980) [22] Panozzo, C.; Nawara, M.; Suski, C.; Kucharczyka, R.; Skoneczny, M.; Becam, A.-M.; Rytka, J.; Herbert, C.J.: Aerobic and anaerobic NAD+ metabolism in Saccharomyces cerevisiae. FEBS Lett., 517, 97-102 (2002) [23] Kim, M.K.; Kim, Y.S.; Rho, S.H.; Im, Y.J.; Lee, J.H.; Kang, G.B.; Eom, S.H.: Crystallization and preliminary X-ray crystallographic analysis of quinolinate phosphoribosyltransferase of Helicobacter pylori. Acta Crystallogr. Sect. D, 59, 1265-1266 (2003) [24] Chang, H.-K.; Zylstra, G.J.: Role of quinolinate phosphoribosyltransferase in degradation of phthalate by Burkholderia cepacia DBO1. J. Bacteriol., 181, 3069-3075 (1999) [25] Sinclair, S.J.; Murphy, K.J.; Birch, C.D.; Hamill, J.D.: Molecular characterization of quinolinate phosphoribosyltransferase (QPRTase) in Nicotiana. Plant Mol. Biol., 44, 603-617 (2000) [26] Cao, H.; Pietrak, B.L.; Grubmeyer, C.: Quinolinate phosphoribosyltransferase: kinetic mechanism for a type II PRTase. Biochemistry, 41, 3520-3528 (2002) [27] Hughes, K.T.; Dessen, A.; Gray, J.P.; Grubmeyer, C.: The Salmonella typhimurium nadC gene: Sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyltransferase. J. Bacteriol., 175, 479486 (1993) [28] Fukuoka, S.-I.; Nyaruhucha, C.M.; Shibata, K.: Characterization and functional expression of the cDNA encoding human brain quinolinate phosphoribosyltransferase. Biochim. Biophys. Acta, 1395, 192-201 (1998) [29] Sharma, V.; Grubmeyer, C.; Sacchettini, J.C.: Crystal structure of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis: a potential TB drug target. Structure, 6, 1587-1599 (1998) [30] Eads, J.C.; Ozturk, D.; Wexler, T.B.; Grubmeyer, C.; Sacchettini, J.C.: A new function for a common fold: the crystal structure of quinolinic acid phosphoribosyltransferase. Structure, 5, 47-58 (1997)

198

Dioxotetrahydropyrimidine phosphoribosyltransferase

2.4.2.20

1 Nomenclature EC number 2.4.2.20 Systematic name 2,4-dioxotetrahydropyrimidine-nucleotide:diphosphate phospho-a-d-ribosyltransferase Recommended name dioxotetrahydropyrimidine phosphoribosyltransferase Synonyms 2,4-dioxotetrahydropyrimidine-nucleotide:pyrophosphate phospho-a-d-ribosyltransferase dioxotetrahydropyrimidine phosphoribosyl transferase dioxotetrahydropyrimidine ribonucleotide pyrophosphorylase dioxotetrahydropyrimidine-ribonucleotide pyrophosphorylase phosphoribosyltransferase, dioxotetrahydropyrimidine uracil pyrophosphorylase CAS registry number 37277-75-1

2 Source Organism Bos taurus [1]

3 Reaction and Specificity Catalyzed reaction a 2,4-dioxotetrahydropyrimidine d-ribonucleotide + diphosphate = a 2,4-dioxotetrahydropyrimidine + 5-phospho-a-d-ribose 1-diphosphate Reaction type pentosyl group transfer Substrates and products S uric acid + diphosphate (Reversibility: ? [1]) [1] P 3-ribosyluric acid 5-phosphate + ? [1] S xanthine + diphosphate (Reversibility: ? [1]) [1]

199

Dioxotetrahydropyrimidine phosphoribosyltransferase

2.4.2.20

P 3-ribosylxanthine 5'-phosphate + ? [1] S Additional information ( enzyme also synthesizes a number of pyrimidine ribonucleotides from the corresponding base, e.g. uracil, orotic acid, thymine, 6-azathymine, 6-azauracil, 5-fluorouracil or 5-iodouracil and diphosphorylribose phosphate [1]) [1] P ? Inhibitors 3-ribosylxanthine 5'-phosphate ( xanthine and uracil diphosphorylase [1]) [1] UMP ( xanthine and uracil pyrophosphorylase [1]) [1] Metals, ions Mg2+ ( required at 2 mM or higher for maximal activity [1]) [1] Specific activity (U/mg) 0.497 [1] pH-Optimum 6 ( for uric acid reaction [1]) [1] 7.6-8.6 ( for xanthine reaction [1]) [1] 8-9.2 ( for orotic acid reaction [1]) [1] 9.5 ( for uracil reaction [1]) [1] Temperature optimum ( C) 37 (assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue erythrocyte [1] Purification (purified 5400fold, ammonium sulfate fractionation, chromatography on DEAE-cellulose, treatment with Alumina CY Gel, calcium phosphate gel adsorption and elution and purification with hydroxylapatite [1]) [1]

6 Stability Temperature stability 50 ( pH 6.0, 40 min, 12-22% loss of activity [1]) [1]

References [1] Hatfield, D.; Wyngaarden, J.B.: 3-Ribosylpurines. I.Synthesis of (3-ribosyluric acid) 5Âphosphate and (3-ribosylxanthine) 5'-phosphate by a pyrimidine ribonucleotide pyrophosphorylase. J. Biol. Chem., 239, 2580-2586 (1964) 200

Nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase

2.4.2.21

1 Nomenclature EC number 2.4.2.21 Systematic name nicotinate-nucleotide:5,6-dimethylbenzimidazole phospho-d-ribosyltransferase Recommended name nicotinate-nucleotide-dimethylbenzimidazole phosphoribosyltransferase Synonyms N(1)-a-phosphoribosyltransferase N1 -a-phosphoribosyltransferase NN:DBI PRT nicotinate mononucleotide-dimethylbenzimidazole phosphoribosyltransferase nicotinate ribonucleotide:benzimidazole (adenine) phosphoribosyltransferase phosphoribosyltransferase, nicotinate mononucleotide-dimethylbenzimidazole CAS registry number 37277-76-2

2 Source Organism

Propionibacterium shermanii [1] Clostridium sticklandii [2, 3] Pseudomonas denitrificans [4] Salmonella typhimurium [5, 8, 9] Salmonella enterica [6, 7]

3 Reaction and Specificity Catalyzed reaction b-nicotinate d-ribonucleotide + 5,6-dimethylbenzimidazole = nicotinate + aribazole 5'-phosphate ( single displacement mechanism [2]; mechanism [6,7])

201


2.4.2.21

Reaction type pentosyl group transfer Natural substrates and products S nicotinic acid mononucleotide + benzimidazole (Reversibility: ? [6]) [6] P 1-a-d-ribofuranosylbenzimidazole 5'-phosphate + nicotinate ( a-ribazole 5'-phosphate; intermediate for the lower ligand of cobalamin [6]) [6] Substrates and products S NMN + benzimidazole (Reversibility: ir [1]) [1] P ? S b-nicotinate d-ribonucleotide + 5(6)-nitrobenzimidazole (Reversibility: ? [2]) [2] P nicotinate + N1 -(5-phospho-a-d-ribosyl)-5(6)-nitrobenzimidazole S b-nicotinate d-ribonucleotide + 5,6-dichlorobenzimidazole (Reversibility: ? [2]) [2] P nicotinate + N1 -(5-phospho-a-d-ribosyl)-5,6-dichlorobenzimidazole S b-nicotinate d-ribonucleotide + 5,6-dimethylbenzimidazole (Reversibility: ir [1]; ? [2-5]) [1-5] P nicotinate + N1 -(5-phospho-a-d-ribosyl)-5,6-dimethylbenzimidazole [1, 5] S nicotinamide nucleoside + benzimidazole (Reversibility: ir [1]) [1] P ? S nicotinic acid mononucleotide + 4,5-dimethyl-1,2-phenylenediamine (Reversibility: ? [5]) [5] P nicotinate + ? S nicotinic acid mononucleotide + adenine (Reversibility: ? [2,3]) [2, 3] P 7-a-d-ribofuranosyladenine 5'-phosphate + nicotinate [2, 3] S nicotinic acid mononucleotide + adenine (Reversibility: ? [5]) [5] P nicotinate + ? S nicotinic acid mononucleotide + benzimidazole (Reversibility: ? [5]) [5] P nicotinate + N1 -(5-phospho-a-d-ribosyl)-benzimidazole S nicotinic acid mononucleotide + benzimidazole ( best substrate [1]; highly specific for nicotinic acid mononucleotide [2]) (Reversibility: ir [1]; ? [2-6]) [1, 2, 4-6] P 1-a-d-ribofuranosylbenzimidazole 5'-phosphate + nicotinate ( a-ribazole 5'-phosphate [4]) [1, 2] S nicotinic acid mononucleotide + guanine (Reversibility: ? [5]) [5] P nicotinate + ? S nicotinic acid mononucleotide + histidine (Reversibility: ? [5]) [5] 202

2.4.2.21


P nicotinate + ? S nicotinic acid mononucleotide + imidazole (Reversibility: ? [5]) [5] P nicotinate + ? S nicotinic acid nucleoside + benzimidazole (Reversibility: ir [1]) [1] P ? S Additional information ( not substrate: NAD, ribose 1-phosphate, ribose 5-phosphate, 2-methylbenzimidazole [1]) [1] P ? Specific activity (U/mg) 2.3 [2] 11.6 [1] Additional information [4] Km-Value (mM) 0.000016 (dimethylbenzimidazole) [4] 0.01 (adenine) [2] 0.01 (dimethylbenzimidazole, or less [5]) [5] 0.083 (nicotinic acid mononucleotide) [4] 0.3 (nicotinic acid mononucleotide, cosubstrate adenine [2]) [2] 0.5 (benzimidazole) [2] 0.68 (nicotinic acid mononucleotide) [5] 0.7 (nicotinic acid mononucleotide, cosubstrate benzimidazole [2]) [2] 2 (benzimidazole) [1] pH-Optimum 8.5-9.4 [1] 8.6 ( assay at [2]) [2] 8.6-9.2 ( nicotinic acid mononucleotide + adenine or benzimidazole [2]) [2] 10 [5] pH-Range 7.3-9.7 ( pH 7.3: about 60% of maximum activity, pH 9.7: about 90% of maximum activity [1]) [1] Temperature optimum ( C) 30 ( assay at [1,4]) [1, 4] 37 ( assay at [2]) [2] 45 [5]

4 Enzyme Structure Molecular weight 71000 ( gel filtration [4]) [4]

203


2.4.2.21

Subunits dimer ( 2 * 35000, SDS-PAGE [4]; 2 * a, Glu317 is at catalytic site, x-ray structure [8]) [4, 8]

5 Isolation/Preparation/Mutation/Application Purification (partial [1]) [1] [2] [4] Crystallization (in complex with substrate and with reaction product [8]) [8] (in complex with different substrate analogs, reaction pathway [6,7]) [6, 7] Cloning [4] (gene cobT, comparison with homologues [9]) [9]

6 Stability Storage stability , -15 C, 0.01 M potassium phosphate buffer, pH 6.8, 0.005 M DTT, 10% ethylene glycol, several months [2]

References [1] Friedmann, H.C.: Partial purification and properties of a single displacement trans-N-glycoside. J. Biol. Chem., 240, 413-418 (1965) [2] Fyfe, J.A.; Friedmann, H.C.: Vitamin B12 biosynthesis. Enzyme studies on the formation of the a-glycosidic nucleotide precursor. J. Biol. Chem., 244, 16591666 (1969) [3] Friedmann, H.C.; Fyfe, J.A.: Pseudovitamin B12 biosynthesis. Enzymatic formation of a new adenylic acid, 7-a-d-ribofuranosyladenine 5-phosphate. J. Biol. Chem., 244, 1667-1671 (1969) [4] Cameron, B.; Blanche, F.; Rouyez, M.C.; Bisch, D.; Famechon, A.; Couder, M.; Cauchois, L.; Thibaut, D.; Debussche, L.; Crouzet, J.: Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5-phosphate) synthase. J. Bacteriol., 173, 60666073 (1991) [5] Trzebiatowski, J.R.; Escalante-Semerena, J.C.: Purification and characterization of CobT, the nicotinate-mononucleotide:5,6-dimethylbenzimidazole

204

2.4.2.21


phosphoribosyltransferase enzyme from Salmonella typhimurium LT2. J. Biol. Chem., 272, 17662-17667 (1997) [6] Cheong, C.G.; Escalante-Semerena, J.C.; Rayment, I.: Capture of a labile substrate by expulsion of water molecules from the active site of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica. J. Biol. Chem., 277, 41120-41127 (2002) [7] Cheong, C.G.; Escalante-Semerena, J.C.; Rayment, I.: Structural investigation of the biosynthesis of alternative lower ligands for cobamides by nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase from Salmonella enterica. J. Biol. Chem., 276, 37612-37620 (2001) [8] Cheong, C.G.; Escalante-Semerena, J.C.; Rayment, I.: The three-dimensional structures of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethylbenzimidazole and its reaction products determined to 1.9 ANG. resolution. Biochemistry, 38, 16125-16135 (1999) [9] Trzebiatowski, J.R.; O'Toole, G.A.; Escalante-Semerena, J.C.: The cobT gene of Salmonella typhimurium encodes the NaMN:5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1 -(5-phosphoa-d-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin. J. Bacteriol., 176, 3568-3575 (1994)

205

Xanthine phosphoribosyltransferase

2.4.2.22

1 Nomenclature EC number 2.4.2.22 Systematic name XMP:diphosphate 5-phospho-a-d-ribosyltransferase Recommended name xanthine phosphoribosyltransferase Synonyms 5-phospho-a-d-ribose-1-diphosphate:xanthine phospho-d-ribosyltransferase XGPRT [Swissprot] XMP pyrophosphorylase XPRT XPRTase Xan phosphoribosyltransferase phosphoribosyltransferase, xanthine xanthosine 5'-phosphate pyrophosphorylase xanthylate pyrophosphorylase xanthylic pyrophosphorylase CAS registry number 9023-10-3

2 Source Organism

206

Lactobacillus casei [1] Escherichia coli [1, 5] Leishmania mexicana [2] Leishmania donovani [2, 3, 8] Leishmania braziliensis [2] Leishmania tarentolae [2] Streptococcus faecalis [4] Saccharomyces cerevisiae [6] Toxoplasma gondii [7]

2.4.2.22


3 Reaction and Specificity Catalyzed reaction XMP + diphosphate = 5-phospho-a-d-ribose 1-diphosphate + xanthine Reaction type pentosyl group transfer Substrates and products S 5-phospho-a-d-ribose 1-diphosphate + 4,6-dihydroxypyrazolo[3,4-d]pyrimidine (, at 0.064% of the activity with xanthine [4]) (Reversibility: ? [4]) [4] P ? S 5-phospho-a-d-ribose 1-diphosphate + 5,7-dihydroxy-v-triazolo[4,5d]pyrimidine (, at 16.8% of the activity with xanthine [4]) (Reversibility: ? [4]) [4] P ? S 5-phospho-a-d-ribose 1-diphosphate + adenine (, at 0.25% of the activity with xanthine [4]; , less than 1% of the activity with xanthine [7]) (Reversibility: ? [4,7]) [4, 7] P AMP + diphosphate S 5-phospho-a-d-ribose 1-diphosphate + guanine (Reversibility: ? [2, 5, 7, 8]) [2, 5, 7, 8] P GMP + diphosphate S 5-phospho-a-d-ribose 1-diphosphate + hypoxanthine (, at 0.19% of the activity with xanthine [4]) (Reversibility: ? [2, 4, 5, 6, 7, 8]) [2, 4, 5, 6, 7, 8] P IMP + diphosphate S 5-phospho-a-d-ribose 1-diphosphate + xanthine (, the enzymes catalyzing the transribosylation from 5-phospho-a-dribose 1-diphosphate to guanine, hypoxanthine and xanthine are inseparable [2]) (Reversibility: ? [1, 2, 3, 4, 5, 6, 7, 8]) [1, 2, 3, 4, 5, 6, 7, 8] P 9-(5-phospho-b-d-ribosyl)xanthine + diphosphate [1, 2, 3, 4] Inhibitors 1-methylxanthine [4] 2,4-dihydroxy-5,6-tetramethylenepyrimidine [4] 2,4-dihydroxypteridine [4] 2,4-dihydroxypyrido[2,3-d]pyrimidine [4] 2,4-dihydroxypyrido[3,2-d]pyrimidine [4] 2,4-dihydroxypyrido[3,4-d]pyrimidine [4] 2,4-dihydroxypyrrolo[2,3-d]pyrimidine [4] 2,6-diaminopurine [7] 2,6-dichloropurine [7] 2,6-dioxo-1,3,7-trimethylpurine [7]

207


2,6-dioxo-1,3,9-trimethylpurine [7] 2,6-dioxo-3-isobutyl-1-methylpurine [7] 2,6-dioxo-7-(b-hydroxypropyl)-1,3-dimethylpurine [7] 2,6-dithiopurine [7] 2,6-dithioxanthine [4] 2-amino-1-methylhypoxanthine [7] 2-amino-1-methylpurine [7] 2-amino-2-aminohypoxanthine [7] 2-amino-6-chloropurine [7] 2-amino-9-deazahypoxanthine [7] 2-amino-9-methylhypoxanthine [7] 2-aminohypoxanthine [7] 2-aminopurine [7] 2-aminopurine-6-thione [7] 2-aza-3-deazahypoxanthine [7] 2-aza-6-amino-3-deazapurine [7] 2-aza-6-thio-3-deazapurine [7] 2-hydroxy-6-methylthiopurine [4] 2-hydroxypurine [4] 2-mercaptopurine [4] 2-methylhypoxanthine [4] 2-methylthio-6-hydroxypurine [4] 2-oxo-1-methylhypoxanthine [7] 2-oxo-3-methylhypoxanthine [7] 2-oxo-6-thiopurine [7] 2-oxohypoxanthine [7] 2-oxohypoxanthine-N3 -oxide [7] 2-oxopurine [7] 2-thiohypoxanthine [7] 2-thioxanthine [4] 3-methylxanthine [4] 4,6-dihydroxypyrazolo[3,4-d]-pyrimidine [4] 4-aminoimidazole-5-carboxamide [4] 4-hydroxypyrazolo[3,4-d]pyrimidine [4] 4-mercapto-6-hydroxypyrazolo[3,4-d]pyrimidine [4] 4-mercaptopyrazolo[3,4-d]pyrimidine [4] 4-oxopyrimidine [7] 5(4)-amino-4-imidazolecarboxamide [7] 5,7-dihydroxy(1,2,5)thiadiazolo[3,4-d]pyrimidine [4] 5,7-dihydroxy-v-triazolo[4,5-d]pyrimidine [4] 5,7-dihydroxypyrazolo[4,3-d]pyrimidine [4] 5-acetamidouracil [4] 5-aminouracil [4] 5-anilinouracil [4] 5-fluorocytosine [7] 5-formamidouracil [4] 6-amino-1-methylpurine [7] 208

2.4.2.22

2.4.2.22


6-amino-2,8-diaza-3-deazapurine [7] 6-amino-2-chloropurine [7] 6-amino-2-methylpurine [7] 6-amino-2-oxopurine [7] 6-amino-7-deazapurine [7] 6-benzylaminopurine [7] 6-chloropurine [7] 6-hydroxymethylpterin [7] 6-mercaptopurine [4] 6-methoxypurine [7] 6-methylaminopurine [7] 6-thiopurine [7] 6-thioxanthine [4] 7-methylxanthine [4] 8-aza-1,3-dideazapurine [7] 8-aza-1-nitro-1,3-dideazapurine [7] 8-aza-2,6-diaminopurine [7] 8-aza-7-deaza-6-aminopurine [7] 8-aza-7-deaza-6-thiopurine [7] 8-aza-7-deazahypoxanthine [7] 8-azahypoxanthine [7] 8-bromo-2-aminohypoxanthine [7] 8-mercaptoxanthine [4] 8-methylxanthine [4] 8-oxohypoxanthine [7] 8-thiohypoxanthine [7] 9-methylxanthine [4] ATP (, 1 mM, 11% inhibition [4]) [4] CDP (, 1 mM, 10% inhibition [4]) [4] CTP (, 1 mM, 27% inhibition [4]) [4] GDP (, 1 mM, 93 inhibition [4]) [4] GMP (, 1 mM, 89% inhibition [4]) [4] GTP (, 1 mM, 91% inhibition [4]) [4] TDP (, 1 mM, 18% inhibition [4]) [4] TTP (, 1 mM, 13% inhibition [4]) [4] UDP (, 1 mM, 15% inhibition [4]) [4] UTP (, 1 mM, 17% inhibition [4]) [4] XDP (, 1 mM, 89% inhibition [4]) [4] XMP (, 1 mM, 92% inhibition [4]) [4] XTP (, 1 mM, 92% inhibition [4]) [4] adenine [4, 7] benzonitrile [7] cytosine [7] guanine [1, 4, 7] hypoxanthine [4, 7] isocytosine [7] isoguanine [4] 209


2.4.2.22

purine [4] uracil [4, 7] uric acid [4] xanthine [4, 7] Metals, ions Co2+ (, efficiency is equal to Mn2+ [2]; , less efficient in activation than Mn2+ [2]) [2] Mg2+ (, very low activation [2]; , magnesium and sulfate can bind together in the active site, in the absence of other products or substrates. The magnesium interacts with a highly conserved aspartate residue [5]) [2, 5] Mn2+ (, efficient activation [2]) [2] Zn2+ (, very low activation [2]) [2] Turnover number (min±1) 156 (hypoxanthine) [8] 210 (xanthine) [8] 3288 (hypoxanthine) [5] 6720 (5-phospho-a-d-ribose 1-diphosphate) [5] 6720 (guanine) [5] 9006 (xanthine) [5] Specific activity (U/mg) 3.65 [4] Km-Value (mM) 0.001 (xanthine) [1] 0.0012 (hypoxanthine) [7] 0.0013 (guanine) [7] 0.0029 (adenine) [7] 0.003 (xanthine) [7] 0.0043 (guanine) [5] 0.0071 (xanthine) [8] 0.019 (5,7-dihydroxy-v-triazolo[4,5-d]pyrimidine) [4] 0.02 (xanthine) [4] 0.0305 (xanthine) [5] 0.053 (5-phospho-a-d-ribose 1-diphosphate) [4] 0.0908 (hypoxanthine) [5] 0.139 (5-phospho-a-d-ribose 1-diphosphate) [5] 0.448 (hypoxanthine) [8] Ki-Value (mM) 0.002 (2-aminopurine-2-thione) [7] 0.0022 (5,7-dihydroxypyrazolo[4,3-d]pyrimidine) [4] 0.0035 (hypoxanthine) [7] 0.0041 (2,4-dihydroxypyrido[3,2-d]pyrimidine) [4] 0.006 (6-thiopurine) [7] 0.0079 (5(4)-amino-4-imidazolecarboxamide) [7]

210

2.4.2.22


0.012 (2-aminohypoxanthine) [7] 0.014 (2-amino-9-deazahypoxanthine) [7] 0.014 (2-oxohypoxanthine) [7] 0.021 (5-phospho-a-d-ribose 1-diphosphate) [4] 0.022 (2-aza-3-deazahypoxanthine) [7] 0.024 (2,4-dihydroxypteridine) [4] 0.024 (xanthine) [4] 0.026 (2-amino-7-deazahypoxanthine) [7] 0.03 (2-aza-6-thio-3-deazapurine) [7] 0.032 (5,7-dihydroxy(1,2,5)thiadiazolo[3,4-d]pyrimidine) [4] 0.035 (8-aza-7-deazahypoxanthine) [7] 0.04 (2-amino 1-methylhypoxanthine) [7] 0.053 (6-amino-2-oxopurine) [7] 0.067 (2-aminopurine) [7] 0.068 (2-thiohypoxanthine) [7] 0.081 (8-aza-7-deaza-6-aminopurine) [7] 0.1 (6-chloropurine) [7] 0.1 (guanine) [1] 0.11 (5,7-dihydroxy-v-triazolo[4,5-d]pyrimidine) [4] 0.15 (2,6-dithioxanthine) [4] 0.17 (2-hydroxy-6-methylthiopurine) [4] 0.2 (4-mercapto-6-hydroxypyrazolo[3,4-d]pyrimidine) [4] 0.23 (2-amino-6-chloropurine) [7] 0.23 (2-amino-9-methylhypoxanthine) [7] 0.231 (2-aza-6-amino-3-deazapurine) [7] 0.246 (2-oxo-6-thiopurine) [7] 0.286 (cytosine) [7] 0.29 (2-amino 1-methylpurine) [7] 0.31 (4,6-dihydroxypyrazolo[3,4-d]-pyrimidine) [4] 0.31 (6-amino 1-methylpurine) [7] 0.34 (6-thioxanthine) [4] 0.37 (6-amino-2-methylpurine) [7] 0.379 (4-oxopyrimidine) [7] 0.38 (2-thioxanthine) [4] 0.38 (6-methoxypurine) [7] 0.39 (8-aza-1-nitro-1,3-dideazapurine) [7] 0.39 (8-mercaptoxanthine) [4] 0.41 (2,6-dichloropurine) [7] 0.42 (2,6-dithiopurine) [7] 0.42 (8-thiohypoxanthine) [7] 0.437 (6-amino-2,8-diaza-3-deazapurine) [7] 0.54 (8-bromo-2-aminohypoxanthine) [7] 0.57 (2-oxopurine) [7] 0.62 (3-methylxanthine) [4] 0.657 (5-fluorocytosine) [7] 0.75 (6-amino-8-bromopurine) [7] 0.808 (uracil) [7] 211


2.4.2.22

0.85 (8-aza-2,6-diaminopurine) [7] 0.87 (benzonitrile) [7] 0.88 (8-aza-7-deaza 6-thiopurine) [7] 0.97 (6-methylaminopurine) [7] 1 (2,6-dioxo-1,3,7-trimethylpurine) [7] 1.06 (isocytosine) [7] 1.1 (6-benzylaminopurine) [7] 1.3 (2-oxohypoxanthine-N3 -oxide) [7] 1.4 (2-oxo 1-methylhypoxanthine) [7] 1.5 (2,6-dioxo 3-isobutyl-1-methylpurine) [7] 1.7 (6-amino-7-deazapurine) [7] 1.9 (2,6-dioxo-7-(b-hydroxypropyl)-1,3-dimethylpurine) [7] 1.9 (2-oxo 7-methylhypoxanthine) [7] 2 (8-azahypoxanthine) [7] 2.2 (6-amino-2-chloropurine) [7] 2.5 (8-aza-6-aminopurine) [7] 2.6 (8-aza-2-aminohypoxanthine) [7] 3.1 (2-amino-7-methylhypoxanthine) [7] 3.4 (6-hydroxymethylpterin) [7] 4.1 (8-oxohypoxanthine) [7] 5.2 (8-aza-1,3-dideazapurine) [7] 5.3 (2,6-dioxo-1,3,9-trimethylpurine) [7] pH-Optimum 7.4-8.8 [4] 7.5 (, reaction with hypoxanthine and guanine [7]) [7] 7.8 (, reaction with xanthine [7]) [7] 8-9.5 (, reaction with adenine [7]) [7] pH-Range 6.9-9.1 (, 80% of maximal activity at pH 6.9 and 9.1 [4]) [4]

4 Enzyme Structure Molecular weight 42000 (, gel filtration [4]) [4] 54000 (, in absence of 5-phospho-a-d-ribose 1-diphosphate, gel filtration [3]) [3] 62000 (, in presence of 5-phospho-a-d-ribose 1-diphosphate, gel filtration [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue promastigote [2]

212

2.4.2.22


Purification [8] [4] Crystallization (hanging drop vapor diffusion method [5]) [5] Cloning (expression in Escherichia coli [8]) [8] (isolation and characterization of the XPT1 gene [6]) [6] Application medicine (, the enzyme lacks a mammalian counterpart and is, therefore, a potential target for antiparasitic therapy [8]) [8]

6 Stability pH-Stability 5.6-10 (, stable [4]) [4] Temperature stability 25 (, pH 5.6-10, stable for 30 min [4]) [4] General stability information , increase in potassium phosphate buffer concentration from 1-50 mM increases stability [4] , stabilizing compounds are: 5-phospho-a-d-ribose 1-diphosphate, XMP, 6-oxo-substituted purine nucleotides [4] Storage stability , -70 C, stable for 6 months in intacts cells [4]

References [1] Krenitsky, T.A.; Neil, S.M.; Miller, R.L.: Guanine and xanthine phosphoribosyltransfer activities of Lactobacillus casei and Escherichia coli. Their relationship to hypoxanthine and adenine phosphoribosyltransfer activities. J. Biol. Chem., 245, 2605-2611 (1970) [2] Kidder, G.W.; Nolan, L.L.: Xanthine phosphoribosyltransferase in Leishmania: divalent cation activation. J. Protozool., 29, 405-409 (1982) [3] Tuttle, J.V.; Krenitsky, T.A.: Purine phosphoribosyltransferases from Leishmania donovani. J. Biol. Chem., 255, 909-916 (1980) [4] Miller, R.L.; Adamczyk, D.L.; Fyfe, J.A.; Elion, G.B.: Xanthine phosphoribosyltransferase from Streptococcus faecalis. Properties and specificity. Arch. Biochem. Biophys., 165, 349-358 (1974) [5] Vos, S.; de Jersey, J.; Martin, J.L.: Crystal structure of Escherichia coli xanthine phosphoribosyltransferase. Biochemistry, 36, 4125-4134 (1997)

213


2.4.2.22

[6] Guetsova, M.L.; Crother, T.R.; Taylor, M.W.; Daignan-Fornier, B.: Isolation and characterization of the Saccharomyces cerevisiae XPT1 gene encoding xanthine phosphoribosyl transferase. J. Bacteriol., 181, 2984-2986 (1999) [7] Naguib, F.N.M.; Iltzsch, M.H.; el Kouni, M.M.; Panzica, R.P.; el Kouni, M.H.: Structure-activity relationships for the binding of ligands to xanthine or guanine phosphoribosyltransferase from Toxoplasma gondii. Biochem. Pharmacol., 50, 1685-1693 (1995) [8] Jardim, A.; Bergeson, S.E.; Shih, S.; Carter, N.; Lucas, R.W.; Merlin, G.; Myler, P.J.; Stuart, K.; Ullman, B.: Xanthine phosphoribosyltransferase from Leishmania donovani. Molecular cloning, biochemical characterization, and genetic analysis. J. Biol. Chem., 274, 34403-34410 (1999)

214

Deoxyuridine phosphorylase

2.4.2.23

1 Nomenclature EC number 2.4.2.23 Systematic name 2'-deoxyuridine:phosphate 2-deoxy-a-d-ribosyltransferase Recommended name deoxyuridine phosphorylase Synonyms deoxyuridine:orthophosphate deoxy-d-ribosyltransferase phosphorylase, deoxyuridine CAS registry number 37277-77-3

2 Source Organism Mus musculus [1]

3 Reaction and Specificity Catalyzed reaction 2'-deoxyuridine + phosphate = uracil + 2-deoxy-d-ribose 1-phosphate Reaction type pentosyl group transfer Substrates and products S deoxyuridine + phosphate (Reversibility: r [1]) [1] P uracil + deoxy-d-ribose 1-phosphate [1] Inhibitors 5-azauracil ( reaction is inhibited in the direction of deoxyuridine synthesis more than in the direction of their cleavage at the same concentration of 5-azauracil [1]) [1]

215

Deoxyuridine phosphorylase

2.4.2.23

5 Isolation/Preparation/Mutation/Application Source/tissue liver [1]

References [1] Cihak, A.; Sorm, F.: Inhibition by 5-azauracil of the uridine phosphorylase and deoxyuridine phosphorylase activities in a cell-free extract of mouse liver. Biochim. Biophys. Acta, 80, 672-674 (1964)

216

1,4-b-D-Xylan synthase

2.4.2.24

1 Nomenclature EC number 2.4.2.24 Systematic name UDP-d-xylose:1,4-b-d-xylan 4-b-d-xylosyltransferase Recommended name 1,4-b-d-xylan synthase Synonyms 1,4-b-xylan synthase EC 2.4.1.72 (formerly) synthase, 1,4-b-xylan xylan synthase xylan synthetase xylosyltransferase, uridine diphosphoxylose-1,4-b-xylan CAS registry number 37277-73-9

2 Source Organism

Pisum sativum (pea [9]) [9, 10] Acer pseudoplatanus (sycamore [7]) [7, 8] Zea mays (corn [1]) [1] Zinnia elegans [2] Avena sativa (oat [3]) [3] Phaseolus vulgaris (L., cv. Canadian wonder [6]) [4, 6, 11, 12] Phaseolus mungo (mung bean [5]) [5] Populus robusta (poplar [8]) [8]

3 Reaction and Specificity Catalyzed reaction UDP-d-xylose + (1,4-b-d-xylan)n = UDP + (1,4-b-d-xylan)n+1 Reaction type pentosyl group transfer

217


2.4.2.24

Natural substrates and products S UDP-d-xylose + (1,4-b-d-xylan)n ( formation of hemicellulose of cell wall [6]; catalyzes synthesis of the xylan main chain during the biogenesis of the plant cell wall [7, 8]) [6-8] P UDP + (1,4-b-d-xylan)n+1 Substrates and products S UDP-d-xylose + (1,4-b-d-xylan)n (Reversibility: ? [1-3, 5-9]) [1-3, 5-9] P UDP + (1,4-b-d-xylan)n+1 ( with high substrate concentration or prolonged incubation, three polysaccharides other than neutral xylan are synthesized, one of these is apparently a glycolipid, and the other two are apparently glucuronoxylans [3]) [1, 3, 7] Inhibitors ADP [8] AMP [1] CDPcholine [8] CMP [8] GMP [1, 8] UDP [1, 8] UDP-d-glucuronic acid [9] UMP [1, 8] UTP [1, 8] Activating compounds Additional information ( no requirement for detergent [7]) [7] Metals, ions Mg2+ ( stimulates, Mg2+ more effective than Mn2+ [7]) [7] Mn2+ ( stimulates, Mg2+ more effective than Mn2+ [7]) [7] Additional information ( no requirement for divalent metal ion [5]) [5] Km-Value (mM) 0.4 (UDP-d-xylose) [7] pH-Optimum 6.5-7.2 ( crude extract [1]) [1] Temperature optimum ( C) 25 ( assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue cambium [8] cell suspension culture [6] cob ( immature [1]) [1]

218

2.4.2.24


epicotyl ( etiolated plants [10]) [9, 10] mesophyll ( differentiating into tracheary elements [2]) [2] seedling [3] shoot [5] xylem ( differentiated [7, 8]; differentiating [8]; developing [11]) [7, 8, 11] Localization Golgi apparatus [10, 11] membrane ( bound [6,8]; associated [7]) [6-8] Application biotechnology ( enzyme activity is hardly affected by addition of organic solvents [12]) [12]

6 Stability General stability information , sucrose, 0.4 M, stabilizes enzyme in crude extracts [1] Storage stability , -15 C, 50% inactivation of particulate system after 4-5 days [7] , 0 C, inactivation of particulate system after 2 days [7] , storage in liquid N2 keeps activity of particulate system constant over a period of 30 days [7]

References [1] Bailey, R.W.; Hassid, W.Z.: Xylan synthesis from uridine-diphosphate-d-xylose by particulate preparations from immature corncobs. Proc. Natl. Acad. Sci. USA, 56, 1586-1593 (1966) [2] Suzuki, K.; Ingold, E.; Suguyama, M.; Komamine, A.: Xylan synthase activity in isolated mesophyll cells of Zinnia elegans durind differentiation to treachery elements. Plant Cell Physiol., 32, 303-306 (1991) [3] Ben-Arie, R.; Ordin, L.; Kindinger, J.I.: Cell-free xylan synthesizing enzyme system from Avena sativa. Plant Cell Physiol., 14, 427-434 (1973) [4] Bolwell, G.P.; Northcote, D.H.: Arabinan synthase and xylan synthase activities of Phaseolus vulgaris. Subcellular localization and possible mechanism of action. Biochem. J., 210, 497-507 (1983) [5] Odzuck, W.; Kauss, H.: Biosynthesis of pure araban and xylan. Phytochemistry, 11, 2489-2494 (1972) [6] Bolwell, G.P.; Northcote, D.H.: Induction by growth factors of polysaccharide synthases in bean cell suspension cultures. Biochem. J., 210, 509-515 (1983)

219


2.4.2.24

[7] Dalessandro, G.; Northcote, D.H.: Xylan synthetase activity in differentiated xylem cells of sycamore trees (Acer pseudoplatasnus). Planta, 151, 53-60 (1981) [8] Dalessandro, G.; Northcote, D.H.: Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees. Planta, 151, 61-67 (1981) [9] Baydoun, E. A.H.; Waldron, K.W.; Brett, C.T.: The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls. Biochem. J., 257, 853-858 (1989) [10] Baydoun, E.A.H.; Brett, C.T.: Distribution of xylosyltransferases and glucuronyltransferase within the Golgi apparatus in etiolated pea (Pisum sativum L.) epicotyls. J. Exp. Bot., 48, 1209-1214 (1997) [11] Gregory, A.C.E.; Smith, C.; Kerry, M.E.; Wheatley, E.R.; Bolwell, G.P.: Comparative subcellular immunolocation of polypeptides associated with xylan and callose synthases in French bean (Phaseolus vulgaris) during secondary wall formation. Phytochemistry, 59, 249-259 (2002) [12] Kerry, M.E.; Gregory, A.C.E.; Bolwell, G.P.: Differential behaviour of four plant polysaccharide synthases in the presence of organic solvents. Phytochemistry, 57, 1055-1060 (2001)

220

Flavone apiosyltransferase

2.4.2.25

1 Nomenclature EC number 2.4.2.25 Systematic name UDP-apiose:5,4'-dihydroxyflavone 7-O-b-d-glucoside 2''-O±b-d-apiofuranosyltransferase Recommended name flavone apiosyltransferase Synonyms UDPapiose:7-O-(b-d-glucosyl)-flavone apiosyltransferase apiosyltransferase, uridine diphosphoapiose-flavone CAS registry number 37332-49-3

2 Source Organism Petroselinum hortense [1]

3 Reaction and Specificity Catalyzed reaction UDP-apiose + 5,7,4'-trihydroxyflavone 7-O-b-d-glucoside = UDP + 5,7,4'-trihydroxyflavone 7-O-[b-d-apiosyl-(1!2)-b-d-glucoside) Reaction type pentosyl group transfer Natural substrates and products S UDPapiose + 7-O-(b-d-glucosyl)-apigenin ( biosynthesis of apiin [1]) [1] P ? Substrates and products S UDPapiose + 7-O-(b-d-glucosyl)-apigenin ( specific for UDPapiose as glycosyl donor [1]) (Reversibility: ? [1]) [1] P UDP + 7-O-b-d-apiofuranosyl-1,2-b-d-glucosyl-apigenin ( i.e. apiin [1]) [1]

221


2.4.2.25

S UDPapiose + biochanin A ( i.e. 5,7-dihydroxy-4'-methoxyisoflavone-7-glucoside [1]) (Reversibility: ? [1]) [1] P ? S Additional information ( overview: 7-O-b-glucosides of a number of flavonoids and of 4-substituted phenols can act as acceptors, not: flavonol-3-glucoside, flavonol-7-glucoside, apigenin-8-C-glucoside, aglycones of flavonoids, glucose [1]) [1] P ? Inhibitors Ca2+ [1] Co2+ [1] Mg2+ [1] Mn2+ [1] UDP [1] 7-O-(b-d-glucosyl)-apigenin [1] apiin [1] 7-O-(b-d-glucosyl)-chrysoeriol [1] iodoacetamide ( at 1 mM causes an inhibition of 55%, reversed by cysteine [1]) [1] p-chloromercuribenzoate ( at 1 mM inhibits the reaction completely [1]) [1] Additional information ( no effect: EDTA, NH+4 , K+ [1]) [1] Activating compounds dithioerythritol ( stimulates enzyme activity up to 35%, optimum concentration: 6 mM [1]) [1] Specific activity (U/mg) Additional information [1] Km-Value (mM) 0.066 (apigenin-7-glucoside) [1] pH-Optimum 7 ( higher apiin yield in Tris-HCl than in phosphate buffer [1]) [1] Temperature optimum ( C) 30 ( assay at [1]) [1]

4 Enzyme Structure Molecular weight 50000 ( gel filtration [1]) [1]

222

2.4.2.25


5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture [1] Localization soluble [1] Purification (purified 123fold by protamine sulfate and ammonium sulfate precipitation and chromatography on DEAE-cellulose, SephadexG-100 and hydroxylapatite [1]) [1]

6 Stability pH-Stability 6-8 ( highest stability [1]) [1] General stability information , albumin, 0.1 mg, significant stabilization [1] , solution of enzyme after hydroxylapatite chromatography in sodium phosphate buffer, pH 7.0, rapidly loses activity upon freezing [1] Storage stability , -20 C, Tris-HCl, dithiothreitol, protein concentration 0.5-1 mg/ml [1]

References [1] Ortmann, R.; Sutter, A.; Grisebach, H.: Purification and properties of UDPapiose: 7-O-(b-d-glucosyl)-flavone apiosyltransferase from cell suspension cultures of parsley. Biochim. Biophys. Acta, 289, 293-302 (1972)

223

Protein xylosyltransferase

2.4.2.26

1 Nomenclature EC number 2.4.2.26 Systematic name UDP-d-xylose:protein b-d-xylosyltransferase Recommended name protein xylosyltransferase Synonyms UDP-d-xylose:core protein b-d-xylosyltransferase UDP-d-xylose:core protein xylosyltransferase UDP-d-xylose:proteoglycan core protein b-d-xylosyltransferase UDP-xylose-core protein b-d-xylosyltransferase XT ( 2 isoforms XT-I and XT-II [10]) [6, 7, 9-11] uridine diphosphoxylose-core protein b-xylosyltransferase uridine diphosphoxylose-protein xylosyltransferase xylosyltransferase, uridine diphosphoxylose-core protein bAdditional information ( the enzyme competes for the substrate UDP-d-xylose with glycogenin, EC 2.4.1.186, which utilizes UDP-d-xylose as an alternative substrate to UDP-d-glucose [13]) [13] CAS registry number 55576-38-0

2 Source Organism Gallus gallus (hen [13]) [1-4, 13] Rattus norvegicus (2 isoforms XT-I and XT-II [10]; Lewis1WR1 [7]) [5, 7, 8, 10, 13] Homo sapiens (2 isoforms XT-I and XT-II [10]) [6, 7, 9-13] Mus musculus [13] Bos taurus [13]

224

2.4.2.26


3 Reaction and Specificity Catalyzed reaction transfers a b-d-xylosyl residue from UDP-d-xylose to the serine hydroxy group of an acceptor protein substrate ( isoform XT-I contains the DxD motif at position 183, which has shown to be essential for binding of nucleotide sugars in glycosyltransferases [10]; chondroitin sulfate attachment site, determination of the consensus sequence for the recognition signal of the enzyme [6]; most probable mechanism: ordered single displacement with UDPxylose as the leading substrate and the xylosylated peptide as the first product released [5]) Reaction type pentosyl group transfer Natural substrates and products S UDP-d-xylose + acceptor protein substrate ( enzyme is associated with large chondroitin sulfate-containing proteoglycans [12]; enzyme initiates the biosynthesis of glycosaminoglycan lateral chains in proteoglycans by transfer of xylose from UDP-xylose to specific serine residues of the core protein [9, 11-13]; enzyme may play a role in maintaining the haemostatic potential of the follicular fluid [12]; initiation of chondroitin sulfate biosynthesis [6]) [1, 3-6, 9, 11-13] P UDP + acceptor protein substrate with xyloserine S Additional information ( enzyme activity in seminal plasma of infertile men is significantly reduced [11]; involved in the biosynthesis of the linkage region of proteochondroitin sulfate [1]) [1, 11] P ? Substrates and products S UDP-d-xylose + CDEASGIGPEVPDDRD ( synthetic peptide [5]) (Reversibility: ? [5]) [5] P UDP + CDEAS(-d-xylose)GIGPEVPDDRD S UDP-d-xylose + FMLEDEASGIGP ( synthetic peptide [5]) (Reversibility: ? [5]) [5] P UDP + FMLEDEAS(-d-xylose)GIGP S UDP-d-xylose + GVEGSADFLK ( derived from collagen X [6]) (Reversibility: ? [6]) [6] P UDP + GVEGS(-d-xylose)ADFLK [6] S UDP-d-xylose + KKDSGPY (Reversibility: ? [6]) [6] P UDP + KKDS(-d-xylose)GPY [6] S UDP-d-xylose + KTKGSGFFVF (Reversibility: ? [6]) [6] P UDP + KTKGS(-d-xylose)GFFVF [6] S UDP-d-xylose + NFDEIDRSGFGFN (Reversibility: ? [6]) [6] P UDP + NFDEIDRS(-d-xylose)GFGFN [6] S UDP-d-xylose + PLVSSGEDEPK ( derived from neurocan protein [6]) (Reversibility: ? [6]) [6]

225


2.4.2.26

P UDP + PLVSSGEDEPK ( d-xylose bound to a serine residue [6]) [6] S UDP-d-xylose + QEEEGSGGGOK ( best substrate [6]; peptide derived from bikunin [6]) (Reversibility: ? [6]) [6] P UDP + QEEEGS(-d-xylose)GGGOK [6] S UDP-d-xylose + QEEEGSGGGQGG ( peptide derived from bikunin [8]) (Reversibility: ? [8]) [8] P UDP + QEEEGS(-d-xylose)GGGQGG [8] S UDP-d-xylose + QEEEGSGGGQKK ( peptide derived from bikunin [8]) (Reversibility: ? [8]) [8] P UDP + QEEEGS(-d-xylose)GGGQKK [8] S UDP-d-xylose + SDDYSGSGSG ( synthetic peptide [5]) (Reversibility: ? [5]) [5] P UDP + SDDYS(-d-xylose)GSGSG S UDP-d-xylose + SENEGSGMAEQK ( synthetic leukocyte-derived amyloid precursor-like protein homologous peptide [7]) (Reversibility: ? [7]) [7] P UDP + SENEGS(-d-xylose)GMAEQK [7] S UDP-d-xylose + Ser-Gly-Ala-Gly-Ala-Gly ( silk sequence hexapeptide [3]) (Reversibility: ? [3]) [3] P UDP + xylosyl-Ser-Gly-Ala-Gly-Ala-Gly S UDP-d-xylose + TENEGSGLTNIK ( synthetic leukocyte-derived b-A4-amyloid protein precursor homologous peptide [7]) (Reversibility: ? [7]) [7] P UDP + TENEGS(-d-xylose)GLTNIK [7] S UDP-d-xylose + VCRSGSGLVGK ( derived from apolipoprotein J [6]) (Reversibility: ? [6]) [6] P UDP + VCRSGSGLVGK ( d-xylose bound to a serine residue [6]) [6] S UDP-d-xylose + WAGGDASGE (Reversibility: ? [6]) [6] P UDP + WAGGDAS(-d-xylose)GE [6] S UDP-d-xylose + acceptor protein substrate ( transfers a b-dxylosyl residue to specific serine residues dependent on the consensus signal sequence [6-8]; transfers a b-d-xylosyl residue from UDPd-xylose to the serine hydroxyl group of an acceptor protein substrate [1, 3-13]; exogenous protein acceptor obtained by Smith degradation of bovine chondroitin sulfate-protein complex [1, 13]; tryptic and chymotryptic fragments from fibroin [3]) (Reversibility: ? [1-13]) [113] P UDP + acceptor protein substrate proteoglycan core protein [6-13] S UDP-d-xylose + bikunin ( recombinant bikunin [11]; recombinant mutant bikunin [7, 12]; recombinant wild-type from Chang liver hepatocytes and mutant, expressed in Escherichia coli [6]) (Reversibility: ? [6,7,11,12]) [6, 7, 11, 12] P UDP + xylosyl-bikunin ( bound to serine residue [6, 7, 11, 12]) [6, 7, 11, 12]

226

2.4.2.26


S UDP-d-xylose + bikunin-derived aminoterminus homologous peptide (Reversibility: ? [7]) [7] P UDP + xylosyl-serine bikunin-derived aminoterminus homologous peptide [7] S UDP-d-xylose + cartilage chondroitin sulfate proteoglycan ( degraded by HF (hydrofluoric acid) or TFMS (trifluoromethanesulfonic acid) [6]; deglycosylation by Staphylococcus aureus V8 protease decreases the activity with the resulting peptides [5]; HF-deglycosylated acceptor [5]; the consensus sequence, acidic-acidic-Xxx-SerGly-Xxx-Gly, in the acceptor proteoglycan is important [5]) (Reversibility: ? [5,6]) [5, 6] P UDP + cartilage chondroitin sulfate proteoglycan with xylosylserine S UDP-d-xylose + cartilage proteoglycan ( modified substrate degradation method [4]; Smith-degraded or HF-treated [3,4]) (Reversibility: ? [3,4]) [3, 4] P UDP + cartilage proteoglycan with xylosylserine S UDP-d-xylose + silk fibroin ( acceptor substrate contains repetitive sequence Gly-Ser-Gly-Ala-Gly-Ala [6]; acceptor substrate from Bombyx mori consists in large part of the repeating hexapeptide: Ser-Gly-Ala-Gly-Ala-Gly [3]) (Reversibility: ? [3, 6, 13]) [3, 6, 13] P UDP + silk fibroin with xylosylserine [13] S Additional information ( substrate preparation and specificity [13]; substrate specificity [8]; tripeptide SGG is a poor substrate [13]; very low activity with tripeptide SGG and peptide LNFSTGW [6]) [6, 8, 13] P ? Inhibitors Zn2+ ( strong [8]) [8] glycosaminoglycans ( associate with the enzyme [12]) [12] heparin [10, 12] Additional information ( no inhibition by CDP [13]; competitive inhibition of the acceptor substrates [5]) [5, 13] Metals, ions Ca2+ ( essentially required, Mg2+ and Mn2+ are equally effective [8]) [8] Mg2+ ( essentially required, Ca2+ and Mn2+ are equally effective [8]) [8] Mn2+ ( essentially required, Mg2+ and Ca2+ are equally effective [8]) [8] Specific activity (U/mg) 0.0285 ( purified enzyme [9]) [9] 0.419 ( purified enzyme [8]) [8] Additional information ( activity in several tissues, overview [7]; activity in several human cell lines, overview [7]; enzyme assay [4]) [1, 4, 7, 11, 12]

227


2.4.2.26

Km-Value (mM) 0.0006 (bikunin, recombinant mutant bikunin [6,7]) [6, 7] 0.0009 (bikunin, recombinant wild-type bikunin [6]) [6] 0.0065 (UDP-d-xylose) [8] 0.008 (QEEEGSGGGQGG) [8] 0.019 (SENEGSGMAEQK) [7] 0.019 (UDP-d-xylose, Smith-degraded proteoglycan, value on serine basis [4]) [4] 0.02 (TENEGSGLTNIK) [7] 0.022 (QEEEGSGGGOK) [6] 0.022 (bikunin-derived aminoterminus homologous peptide) [7] 0.025 (UDP-d-xylose) [1] 0.062 (CDEASGIGPEVPDDRD) [5] 0.093 (QEEEGSGGGQKK) [8] 0.11 (cartilage chondroitin sulfate proteoglycan) [5] 0.12 (FMLEDEASGIGP) [5] 0.13 (NFDEIDRSGFGFN) [6] 0.155 (cartilage chondroitin sulfate proteoglycan, TFMS-degraded substrate [6]) [6] 0.18 (UDP-d-xylose) [5] 0.19 (cartilage chondroitin sulfate proteoglycan, HF-degraded substrate [6]) [6] 0.39 (PLVSSGEDEPK) [6] 0.42 (WAGGDASGE) [6] 0.545 (silk fibroin) [6] 0.79 (SDDYSGSGSG) [5] 0.94 (VCRSGSGLVGK) [6] 2.67 (GVEGSADFLK) [6] 3.625 (KTKGSGFFVF) [6] 8.27 (KKDSGPY) [6] Additional information [5, 8] Ki-Value (mM) Additional information ( competitive inhibition of the acceptor substrates [5]) [5] pH-Optimum 6.5 ( assay at [4-7,11,12]) [4-7, 11, 12] 6.5-7 [1] 7 ( depends on buffer system [8]) [8] pH-Range 5.7-7.5 ( half-maximal activity at pH 5.7 and pH 7.5 [1]) [1] Temperature optimum ( C) 34 ( assay at [6]) [6] 37 ( assay at [1, 4, 5, 7, 11, 12]) [1, 4, 5, 7, 8, 11, 12]

228

2.4.2.26


4 Enzyme Structure Molecular weight 71000 ( gel filtration [8]) [8] 95000-100000 ( gel filtration [2]) [2] 110000-120000 ( gel filtration [1]) [1] 120000 ( gel filtration, amino acid sequence determination [9,10]) [9, 10] Additional information ( enzyme activity is also detected in a peak of MW 500000 [9]) [9] Subunits ? ( x * 91000, isoform XT-I, amino acid sequence determination [10]; x * 97000, isoform XT-II, amino acid sequence determination [10]) [10] monomer ( 1 * 120000, SDS-PAGE [9,10]; 1 * 78000, SDSPAGE [8]) [8-10] tetramer ( 2 * 23000 + 2 * 27000, 2 pairs of dissimilar subunits, SDSPAGE [2]) [2] Posttranslational modification glycoprotein ( 3 potential N-glycosylation sites, isoforms XT-I and XT-II [10]; N-glycosylated [9]) [9, 10]

5 Isolation/Preparation/Mutation/Application Source/tissue EFO-27 cell ( low activity [12]) [12] HeLa cell ( low activity [12]) [12] JAR cell ( choriocarcinoma cell line [9,10]) [9, 10, 12] brain ( low content of isoform XT-II, and very low content of isoform XT-I [10]; 8% activity compared to epiphyseal cartilage [1]) [1, 7, 10] cartilage ( from ear [8]; sternal, from heart [6,7]; embryonic [1-4,13]; epiphyseal [1,4]) [1-4, 6-8, 13] cerebrospinal fluid [7] chondrocyte ( from heart sternal cartilage [6,7]; cell culture supernatant [6,7]) [6, 7, 13] chondrosarcoma cell [5, 13] choriocarcinoma cell [9, 10] ear ( cartilage [8]) [8] embryo [2-4, 13] epiphysis ( cartilage [1,4]; highest activity [1]; embryonic [4]) [1, 4] granulosa cell ( cell culture [12]) [12]

229


2.4.2.26

heart ( isoform XT-II, low content [10]; sternal cartilage [6,7]) [6, 7, 10] kidney ( isoforms XT-I and XT-II [10]) [7, 10, 13] liver ( isoform XT-II [10]) [10, 13] lung ( isoform XT-II, and low content of isoform XT-I [10]) [7, 10] mastocytoma [13] ovarian follicle ( mainly produced by granulosa-lutein cells [12]) [12] ovary ( low activity [7]) [7] oviduct [13] pancreas ( isoforms XT-I and XT-II [10]) [10] placenta ( isoform XT-I and XT-II [10]) [10] seminal plasma ( of healthy and infertile men with oligo-, asthenoor teratozoospermia, and of men after vasectomy [11]) [11] seminal vesicle ( highest activity [11]) [11] serum ( of healthy and infertile men with oligo-, astheno- or teratozoospermia, and of men after vasectomy [11]; from heart blood [7]) [7, 11] skeletal muscle ( isoform XT-II, low content [10]) [10] spleen [7] testis [7] thyroid gland [7] Additional information ( no activity in EFO-21 cells [12]; enzyme activity in seminal plasma of infertile men is significantly reduced [11]; activity in several human cell lines, culture supernatant, overview [7]; no activity in liver and intestine with an exogenous receptor [1]) [1, 7, 11, 12] Localization cytosol [1, 7] extracellular ( isoform XT-I [10]; in cell culture supernatant [6,9,10,12]; human cell cultures [7]) [6, 7, 9, 10, 12] membrane ( isoform XT-II, type II transmembrane protein [10]) [10] Purification (partial [1]) [1-3] (affinity chromatography on QEEEGSGGGQGG-resin [8]) [8] (wild-type, and recombinant from CHO-K1 cells [10]; partial [6,7,12]; from cell culture supernatant [9,12]) [6, 7, 9, 10, 12] Cloning (DNA sequence determination and analysis of isoforms XT-I and XT-II, chromosome mapping [10]) [10] (DNA sequence determination and analysis, chromosome mapping, functional expression of isoforms XT-I and XT-II in CHO-K1 cells, no activity when XT-II is expressed fused to the aminoterminal peptide tag, 8% of the

230

2.4.2.26


recombinant activity is located in the cytosol and membranes, 92% recombinant activity is secreted into the medium [10]) [10] Application medicine ( monitoring of the seminal plasma for enzyme activity is proposed to be an advantegeous additional biochemical parameter to improve in vitro fertilization methods, because the activity is reduced in seminal plasma of infertile men [11]) [11]

6 Stability Storage stability , -20 C or -80 C, Tris-HCl 50 mM, pH 7.0, NaCl 50 mM, stable for at least 15 weeks [8] , storage at 4 C, 25 C or 37 C lead to rapid loss of activity [8]

References [1] Stoolmiller, A.C.; Horwitz, A.L.; Dorfman, A.: Biosynthesis of the chondroitin sulfate proteoglycan. Purification and properties of xylosyltransferase. J. Biol. Chem., 247, 3525-3532 (1972) [2] Schwartz, N.B.; Roden, L.: Biosynthesis of chondroitin sulfate. Purification of UDP-d-xylose:core protein b-d-xylosyltransferase by affinity chromatography. Carbohydr. Res., 37, 167-180 (1974) [3] Campbell, P.; Jacobsson, I.; Benzing-Purdie, L.; Roden, L.; Fessler, J.H.: Silk± a new substrate for UDP-d-xylose:proteoglycan core protein b-d-xylosyltransferase. Anal. Biochem., 137, 505-516 (1984) [4] Sandy, J.D.: The assay of xylosyltransferase in cartilage extracts. A modified procedure for preparation of Smith-degraded proteoglycan. Biochem. J., 177, 569-574 (1979) [5] Kearns, A.E.; Campbell, S.C.; Westley, J.; Schwartz, N.B.: Initiation of chondroitin sulfate biosynthesis: a kinetic analysis of UDP-d-xylose:core protein b-d-xylosyltransferase. Biochemistry, 30, 7477-7483 (1991) [6] Brinkmann, T.; Weilke, C.; Kleesiek, K.: Recognition of acceptor proteins by UDP-d-xylose proteoglycan core protein b-d-xylosyltransferase. J. Biol. Chem., 272, 11171-11175 (1997) [7] Götting, C.; Kuhn, J.; Brinkmann, T.; Kleesiek, K.: Xylosylation of alternatively spliced isoforms of Alzheimer APP by xylosyltransferase. J. Protein Chem., 17, 295-302 (1998) [8] Pfeil, U.; Wenzel, K.-W.: Purification and some properties of UDP-xylosyltransferase of rat ear cartilage. Glycobiology, 10, 803-807 (2000) [9] Kuhn, J.; Götting, C.; Schnolzer, M.; Kempf, T.; Brinkmann, T.; Kleesiek, K.: First isolation of human UDP-d-xylose:proteoglycan core protein b-d-xylosyltransferase secreted from cultured JAR choriocarcinoma cells. J. Biol. Chem., 276, 4940-4947 (2001)

231


2.4.2.26

[10] Götting, C.; Kuhn, J.; Zahn, R.; Brinkmann, T.; Kleesiek, K.: Molecular cloning and expression of human UDP-d-xylose:proteoglycan core protein b-dxylosyltransferase and its first isoform XT-II. J. Mol. Biol., 304, 517-528 (2000) [11] Götting, C.; Kuhn, J.; Brinkmann, T.; Kleesiek, K.: Xylosyltransferase activity in seminal plasma of infertile men. Clin. Chim. Acta, 317, 199-202 (2002) [12] Götting, C.; Kuhn, J.; Tinneberg, H.-R.; Brinkmann, T.; Kleesiek, K.: High xylosyltransferase activities in human follicular fluid and cultured granulosa-lutein cells. Mol. Hum. Reprod., 8, 1079-1086 (2002) [13] Meezan, E.; Manzella, S.; Roden, L.: Menage a trois: glycogenin, proteoglycan core protein xylosyltransferase and UDP-xylose. Trends Glycosci. Glycotechnol., 7, 303-332 (1995)

232

dTDP-dihydrostreptose-streptidine6-phosphate dihydrostreptosyltransferase

2.4.2.27

1 Nomenclature EC number 2.4.2.27 Systematic name dTDP-l-dihydrostreptose:streptidine-6-phosphate dihydrostreptosyltransferase Recommended name dTDP-dihydrostreptose-streptidine-6-phosphate dihydrostreptosyltransferase Synonyms dihydrostreptosyltransferase, thymidine diphosphodihydrostreptose-streptidine 6-phosphate CAS registry number 73699-20-4

2 Source Organism Streptomyces griseus [1]

3 Reaction and Specificity Catalyzed reaction dTDP-l-dihydrostreptose + streptidine 6-phosphate = dTDP + O-1,4-a-l-dihydrostreptosyl-streptidine 6-phosphate Reaction type pentosyl group transfer Natural substrates and products S dTDP-l-dihydrostreptose + streptidine 6-phosphate ( biosynthesis of streptomycin [1]) [1] P ? Substrates and products S dTDP-l-dihydrostreptose + streptidine 6-phosphate (Reversibility: ? [1]) [1] P dTDP + O-a-l-dihydrostreptosyl-1,4-streptidine 6-phosphate [1]

233

dTDP-dihydrostreptose-streptidine-6-phosphate dihydrostreptosyltransferase

2.4.2.27

S Additional information ( not: streptidine, 2-deoxystreptamine, 4deoxystreptamine [1]) [1] P ? Inhibitors dTDP [1] dTTP [1] Metals, ions Co2+ ( as effective as Mg2+ [1]) [1] Mg2+ ( Mn2+ or Mg2+ required, maximal activity with 10 mM Mg2+ [1]) [1] Mn2+ ( Mn2+ or Mg2+ required, maximal activity with 3 mM Mn2+ [1]) [1] Specific activity (U/mg) Additional information [1] pH-Optimum 8.2 ( glycine/NaOH buffer [1]) [1] Additional information ( no distinct pH-optimum observed in TrisHCl buffer between pH 7.3 and 9 [1]) [1] Temperature optimum ( C) 0 ( assay at [1]) [1]

4 Enzyme Structure Molecular weight 63000 ( gel filtration [1]) [1] Subunits dimer ( 2 * 35000, SDS-PAGE in presence of 2-mercaptoethanol [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification (ammonium sulfate precipitation, Sephadex G-200, chromatography on DEAE-cellulose, Ultrogel AcA44 column, DEAE-Sepharose column [1]) [1]

6 Stability General stability information , addition of streptidine is important for stabilization of enzyme activity during purification, purified transferase is also stable in absence of streptidine [1] 234

2.4.2.27

dTDP-dihydrostreptose-streptidine-6-phosphate dihydrostreptosyltransferase

Storage stability , 4 C, 50 mM Tris-HCl, pH 7.8 at 0 C, containing 5 mM mercaptoethanol, 1 mM EDTA, 2.5 mM streptidine, 10% v/v glycerol, stable for at least 4 months [1]

References [1] Kniep, B.; Grisebach, H.: Biosynthesis of streptomycin. Purification and properties of a dTDP-l-dihydrostreptose:streptidine-6-phosphate dihydrostreptosyltransferase from Streptomyces griseus. Eur. J. Biochem., 105, 139144 (1980)

235

S-Methyl-5'-thioadenosine phosphorylase

2.4.2.28

1 Nomenclature EC number 2.4.2.28 Systematic name S-methyl-5-thioadenosine:phosphate S-methyl-5-thio-a-d-ribosyl-transferase Recommended name S-methyl-5'-thioadenosine phosphorylase Synonyms 5'-deoxy-5'-methylthioadenosine phosphorylase 5'-methylthioadenosine nucleosidase 5'-methylthioadenosine phosphorylase MTA phosphorylase MTAPase MeSAdo phosphorylase MeSAdo/Ado phosphorylase methylthioadenosine nucleoside phosphorylase methylthioadenosine phosphorylase phosphorylase, methylthioadenosine CAS registry number 61970-06-7

2 Source Organism

236

Trypanosoma brucei brucei [8, 25] Leishmania donovani [9] Rattus norvegicus (Wistar [27]) [1, 3, 4, 16, 27] Caldariella acidophila [2, 14] Sulfolobus solfataricus [5, 19, 24, 29, 30] Homo sapiens [6, 7, 10, 15, 17, 20-23, 26-28, 32, 33] Mus musculus [7, 12] Drosophila melanogaster [11, 13] Bos taurus [18] Sus scrofa [27] Pyrococcus furiosus [31]

2.4.2.28


3 Reaction and Specificity Catalyzed reaction 5'-methylthioadenosine + phosphate = adenine + 5-methyl-5-thio-a-d-ribose 1-phosphate (also acts on 5'-deoxyadenosine and other analogues having 5'deoxy groups; active and substrate-binding sites, three-dimensional structures [26, 30]; subunit structure, model [30]; catalytic mechanism [26, 30]; 3 binding sites between substrate and enzyme [15]; sequential mechanism [5, 14]; phosphorolytic mechanism [1-3, 20]; equilibrium-ordered reaction, 5'-methylthioadenosine is the first substrate to bind and 5-methylthioribose 1-phosphate is the first product to be released [3]; ordered bisubstrate biproduct reaction with methylthioadenosine the first substrate to add and adenine the last product to leave the enzyme [11]) Reaction type pentosyl group transfer Natural substrates and products S 5'-deoxy-5'-(hydroxyethylthio)adenosine + phosphate ( trypanocidal substrate analogue [25]; preincubation of cells with the substrate lead to 22-37% inhibition of spermidine synthesis from ornithine and 2-7fold increased cytosolic levels of S-adenosyl-l-methionine and Sadenosyl-l-homocysteine, and up to 8fold increased cytosolic level of 5'methyladenosine [25]) [25] P adenine + 5-hydroxyethylthio-d-ribose 1-phosphate S 5'-deoxy-5'-methylthioadenosine + phosphate ( involved in cardiac purine breakdown during ischemia [27]; salvage reaction [26]; substrate is a by-product of polyamine biosynthesis, which is essential for cell growth and proliferation [26]) [26, 27] P adenine + 5-methylthio-d-ribose 1-phosphate S 5'-methylthioadenosine + phosphate ( important for the salvage of adenine and methionine [23]; physiological significance of 5'-methylthioadenosine cleavage is probably related to removal of the thioether which in turn exerts a significant inhibition on methyl transfer reactions [2, 14]; an antiproliferative effect on stimulated human lymphocytes and virally transformed mouse fibroblasts [14]; involved in salvage of adenine and methionine from 5'-methylthioadenosine [2,7,14]) [2, 7, 14, 19, 23, 28] P adenine + 5-methylthio-d-ribose 1-phosphate S adenosine + phosphate [9, 19] P adenine + d-ribose 1-phosphate S guanosine + phosphate [19] P guanine + d-ribose 1-phosphate S inosine + phosphate [19] P hypoxanthine + d-ribose 1-phosphate

237


2.4.2.28

S Additional information ( inverse correlation between enzyme expression and progression of melanocytic tumors, important role of enzyme inactivation in the development of melanomas [32]; enzyme is involved in the methionine dependent tumor cell growth [28]; metabolic pathway, overview [25]; enzyme plays a role in purine and polyamine metabolism and in the regulation of transmethylation reactions [22]; involved in the catabolism of 5'-methylthioadenosine, adenosine, guanosine and inosine [19]) [19, 22, 25, 28, 32] P ? Substrates and products S 2',3'-dideoxyadenosine + phosphate ( substrate for trypanosomal but not for mammalian enzyme [8]) (Reversibility: ? [8]) [8] P adenine + 2,3-deoxy-d-ribose 1-phosphate S 2'-deoxyadenosine + phosphate ( low activity [20]; substrate for trypanosomal but not for mammalian enzyme [8]) (Reversibility: r [20]; ? [8,9]) [8, 9, 20] P adenine + 2-deoxy-d-ribose 1-phosphate S 2-amino-5'-deoxy-5'-(hydroxyethylthio)adenosine + phosphate ( 28% activity compared to 5'-methyladenosine or 5'-deoxy-5'-(hydroxyethylthio)adenosine [25]) (Reversibility: ? [25]) [25] P 2-aminoadenosine + 5-hydroxyethylthio-d-ribose 1-phosphate S 2-chloro-2'-deoxyadenosine + phosphate (Reversibility: r [20]) [20] P 2-chloroadenine + 2-deoxy-d-ribose 1-phosphate S 2-chloro-5'-O-methyl-2'-deoxyadenosine + phosphate (Reversibility: r [20]) [20] P 2-chloroadenine + 5-O-methyl-2-deoxy-d-ribose 1-phosphate S 2-chloroadenosine + phosphate (Reversibility: r [20]) [20] P 2-chloroadenine + d-ribose 1-phosphate S 2-fluoro-5'-deoxy-5'-(hydroxyethylthio)adenosine + phosphate ( 149% activity compared to 5'-methyladenosine or 5'-deoxy-5'-(hydroxyethylthio)adenosine [25]) (Reversibility: ? [25]) [25] P 2-fluoroadenosine + 5-hydroxyethylthio-d-ribose 1-phosphate S 3'-deoxyadenosine + phosphate ( substrate for trypanosomal but not for mammalian enzyme [8]) (Reversibility: ? [8]) [8] P adenine + 3-deoxy-d-ribose 1-phosphate S 5'-deoxy-5'-(hydroxyethylthio)adenosine + phosphate ( trypanocidal substrate analogue [25]) (Reversibility: ? [25]) [25] P adenine + 5-hydroxyethylthio-d-ribose 1-phosphate S 5'-deoxy-5'-methylthioadenosine + phosphate ( the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates [30]; substrate-induced conformational change involving Glu163, which is located at the interface between subunits and swings in toward the active site upon nucleoside binding [30]; sugar specificity [26]) (Reversibility: r [12, 26]; ? [4, 10, 18, 22, 27, 30]) [4, 10, 12, 18, 22, 26, 27, 30]

238

2.4.2.28


P adenine + 5-methylthio-d-ribose 1-phosphate [26] S 5'-deoxyadenosine + phosphate ( phosphate-dependent [12]) (Reversibility: r [12,20]; ? [9]) [9, 12, 20] P adenine + 5-deoxy-d-ribose 1-phosphate [12] S 5'-ethylthioadenosine + phosphate ( 60% of the activity with 5'-methylthioadenosine [15]) (Reversibility: ? [15,16]) [15, 16] P adenine + 5-ethylthio-d-ribose 1-phosphate S 5'-iodo-5'-deoxyadenosine + phosphate (Reversibility: r [20]) [20] P adenine + 5-iodo-5-deoxy-d-ribose 1-phosphate S 5'-isobutylthioadenosine + phosphate ( 97% of the reaction with 5'-methylthioadenosine [2,14]) (Reversibility: ? [2,14]) [2, 14] P adenine + 5-isobutylthio-d-ribose 1-phosphate S 5'-isobutylthioinosine + phosphate ( 8.1% of the reaction with 5'-methylthioadenosine [2,14]) (Reversibility: ? [2,14]) [2, 14] P hypoxanthine + 5-isobutylthio-d-ribose 1-phosphate S 5'-methylselenoadenosine + phosphate ( 95% of the activity with 5'-methylthioadenosine [15]) (Reversibility: ? [15]) [15] P adenine + 5-methylseleno-d-ribose 1-phosphate S 5'-methylthioadenosine + phosphate ( binding of phosphate and 5-methylthioribose 1-phosphate to the enzyme induces a conformational transition that stabilizes the folded structure of the enzyme [29]; 5'-methylthioadenosine and adenine form ternary complexes with the enzyme only in presence of phosphate and methylthioribose 1phosphate, respectively [29]; completely dependent on phosphate [1, 15, 25]; 5'-methylthioadenosine is the best substrate [9]; preferred direction of reaction is temperature-dependent [9]) (Reversibility: r [6, 8, 9, 12, 13, 17, 20]; ? [1-3, 11, 14-16, 18, 19, 21-25, 28, 29, 31, 32]) [1-3, 8, 9, 11-25, 28, 29, 31, 32] P adenine + 5-methylthio-d-ribose 1-phosphate [2, 3, 12, 13, 15-19, 21, 29] S 5'-methylthioinosine + phosphate ( 8.8% of the reaction with 5'-methylthioadenosine [2]; 8.9% of the reaction with 5'-methylthioadenosine [15]) (Reversibility: ? [2, 14, 15]) [2, 14, 15] P hypoxanthine + 5-isobutylthio-d-ribose 1-phosphate S 5'-n-butylthioadenosine + phosphate ( 93.3% of the reaction with 5'-methylthioadenosine [2, 14]) (Reversibility: ? [2, 14]) [2, 14] P adenine + 5-n-butylthio-d-ribose 1-phosphate S 5'-n-propylthioadenosine + phosphate (Reversibility: ? [16]) [16] P adenine + 5-n-propylthio-d-ribose 1-phosphate S 6-methylpurine 2'-deoxyribonucleoside + phosphate ( substrate for trypanosomal but not for mammalian enzyme [8]) (Reversibility: ? [8]) [8] P 6-methylpurine + 2-deoxy-d-ribose 1-phosphate S adenosine + phosphate (Reversibility: r [9, 20]; ? [19, 27, 31]) [9, 19, 20, 27, 31] 239


2.4.2.28

P S P S

adenine + d-ribose 1-phosphate guanosine + phosphate (Reversibility: ? [19, 31]) [19, 31] guanine + d-ribose 1-phosphate inosine + phosphate ( most effective substrate [19]) (Reversibility: ? [19, 31]) [19, 31] P hypoxanthine + d-ribose 1-phosphate S Additional information ( 6-amino purine nucleosides are the preferred substrates, substrate specificity [31]; fluorine substitution at the C-2 position of the purine ring increases activity by 50%, whereas substitution with an amino group reduces activity to about onethird of the control [25]; replacement of the 6-amino group by an OH- group and of N-7 by a methinic radical results in almost complete loss of activity [15]; specificity in both directions of nucleoside cleavage and nucleoside synthesis [6]; substrate specificity [9, 15, 19]; no activity with S-adenosyl-l-methionine [19]; no activity with S-adenosylhomocysteine [4, 19]) [4, 6, 9, 15, 19, 25, 31] P ? Inhibitors 2'-deoxyguanosine [9] 2'-deoxyinosine ( weak, competitive [9]) [9] 2-bromo-9-(1,3-dihydroxy-2-propoxymethyl)adenine ( strong [7]) [7] 2-bromo-9-(2-hydroxyethoxymethyl)adenine [7] 2-chloro-9-(1,3-dihydroxy-2-propoxymethyl)adenine ( strong [7]) [7] 2-chloro-9-(2-hydroxyethoxymethyl)adenine [7] 2-deoxyribose 1-phosphate [3] 2-iodo-9-(1,3-dihydroxy-2-propoxymethyl)adenine ( strong [7]) [7] 2-iodo-9-(2-hydroxyethoxymethyl)adenine [7] 5'-chloroformycin [21] 5'-deoxy-5'-chloroformycin ( competitive [12]) [12] 5'-deoxy-5'-methylthiotubercidin [10] 5'-deoxy-adenosine ( and analogues [20]; strong, competitive [20]) [20] 5'-dimethylthioadenosine ( weak [16]; sulfonium salt, noncompetitive [15]) [15, 16] 5'-ethylthioadenosine [16] 5'-methylthiotubercidin ( competitive [15]) [10, 15, 16, 21] 5'-n-propylthioadenosine [16] 9-(phosphonoalkyl)adenine [7] 9-(phosphonoheptyl)adenine [7] 9-[(1-hydroxy-3-iodo-2-propoxy)methyl]adenine ( competitive [7]) [7] DTNB [17] l-methionine ( weak [3]) [3]

240

2.4.2.28


N-ethylmaleimide ( 5'-methylthioadenosine partly protects [17]) [17] S-adenosyl-l-homocysteine ( no inhibition [19]; weak [16]) [16] SH-group blocking compounds ( inactivation [4,17]) [4, 17] adenine ( strong [16,20]; competitive [20]) [3, 10, 16] adenine arabinoside ( weak, competitive [9]) [9] adenosine ( strong, competitive [20]) [20] dithiothreitol ( 0.8 M, reduction of thermostability [31]) [31] d-fructose 1-phosphate [3] guanidine hydrochloride ( only recombinant enzyme [24]) [24] guanine ( weak [20]) [3, 20] guanosine ( weak, competitive [9]) [9] inosine ( weak, competitive [9]) [9] iodoacetamide ( only recombinant enzyme [24]; no inhibition [17]; reversal by dithiothreitol [15]) [15, 24] iodoacetate ( only recombinant enzyme [24]; incorporation of 12 mol iodoactate per mol of enzyme [19]; no inhibition [17]) [15, 19, 24] p-chloromercuribenzoic acid ( strong, partially reversed by dithiothreitol [15]) [15, 17] proteinase K ( recombinant enzyme, 10% remaining activity after 4 h at 37 C, phosphate protects [29]) [29] d-ribose 1-phosphate [3] subtilisin ( recombinant enzyme, 24% remaining activity after 4 h at 37 C, phosphate protects [29]) [29] Additional information ( promotor hypermethylation strongly reduces enzyme expression [32]; no inhibition by S-adenosyl-l-methionine [19]; activity is not affected by alkylating, mercaptide-forming and oxidizing thiol reagents [19]; no effect by alkylating, mercaptideforming or oxidizing thiol reagents [2]; no inhibition by EDTA, putrescine, cadaverine [3]) [2, 3, 19, 32] Activating compounds SH-group reducing agents ( requirement [4,15,17]) [4, 15, 17] dithiothreitol ( other thiols are less effective [15]; required [15,17]) [15, 17] putrescine ( increases activity [4]; no effect [3]) [4] spermidine ( increases activity [4]) [4] spermine ( increases activity [4]) [4] Metals, ions Additional information ( no requirement for metal ion, e.g. Mg2+ , Mn2+ or Ca2+ [1,3]) [1, 3] Turnover number (min±1) 438.6 (guanosine) [31] 562.8 (inosine) [31]

241


2.4.2.28

1367 (adenosine) [31] 1468 (5'-methylthioadenosine) [31] Specific activity (U/mg) 0.023 [16] 0.0234 ( purified enzyme [16]) [16] 0.0283 ( partially purified enzyme [1]) [1] 0.048 ( substrate 2-amino-5'-deoxy-5'-(hydroxyethylthio)adenosine [25]) [25] 0.092 ( partially purified enzyme, forward reaction, substrate 5'methylthioadenosine [20]) [20] 0.131 ( purified enzyme [15]) [15] 0.17 ( substrate 5'-methyladenosine or 5'-deoxy-5'-(hydroxyethylthio)adenosine [25]) [25] 0.25 ( substrate 2-fluoro-5'-deoxy-5'-(hydroxyethylthio)adenosine [25]) [25] 0.26 ( substrate 5'-deoxyadenosine, forward reaction [20]) [20] 0.365 ( substrate 5'-deoxy-5'-(hydroxyethylthio)adenosine, preincubation with substrate [25]) [25] 0.67 ( purified enzyme [6]) [6] 2.12 ( purified recombinant enzyme [24]; purified enzyme [19]) [19, 24] 2.46 ( purified enzyme [14]) [14] 2.48 ( purified enzyme [2]) [2] 6.35 ( purified enzyme [31]) [31] 9.5 ( purified recombinant enzyme [21]) [21] 10.2 ( purified enzyme [17]) [17] 10.3 [18] Additional information ( kinetics [3]; substrate specificity [20]) [3, 20, 27] Km-Value (mM) 0.00001 (5'-deoxy-5'-methylthioadenosine) [4] 0.00047 (5'-methylthioadenosine) [16] 0.004 (5'-deoxy-5'-methylthioadenosine) [12] 0.004 (5'-methylthioadenosine, recombinant enzyme [21]) [21] 0.0043 (5'-deoxyadenosine) [20] 0.005 (5'-methylthioadenosine) [17] 0.0055 (5'-methylthioadenosine) [11, 17] 0.008 (5-methylthioribose 1-phosphate) [17] 0.013 (5'-methylthioadenosine) [20] 0.014 (5'-deoxy-5'-methylthioadenosine) [27] 0.023 (5'-deoxyadenosine) [10] 0.023 (adenine) [17] 0.024 (5'-methylthioadenosine, recombinant enzyme [24]) [19, 24] 0.025 (5'-methylthioadenosine) [15] 0.026 (5'-deoxy-5'-methylthioadenosine) [10] 0.095 (5'-methylthioadenosine) [2, 14] 242

2.4.2.28


0.109 (adenosine) [31] 0.123 (phosphate) [19] 0.125 (phosphate, recombinant enzyme [24]) [24] 0.147 (5'-methylthioadenosine) [31] 0.184 (adenosine) [27] 0.2 (phosphate) [16] 0.3 (5'-methylthioadenosine) [1] 0.32 (phosphate) [17] 0.6 (phosphate) [1] 0.75 (phosphate, recombinant enzyme [21]) [21] 0.916 (guanosine) [31] 0.963 (inosine) [31] 3.5 (phosphate) [12] 6.1 (phosphate) [2, 13, 14] 7.5 (phosphate) [10] 13.5 (phosphate) [11] Additional information ( nonlinear kinetics with 5'-methylthioadenosine, 2 Km -values, probably due to 2 enzyme activities or 2 interconvertible forms [13]) [13, 20] Ki-Value (mM) 0.0002-0.0007 (2-bromo-9-(1,3-dihydroxy-2-propoxymethyl)adenine) [7] 0.0002-0.0007 (2-chloro-9-(1,3-dihydroxy-2-propoxymethyl)adenine) [7] 0.0002-0.0007 (2-iodo-9-(1,3-dihydroxy-2-propoxymethyl)adenine) [7] 0.001-0.01 (2-bromo-9-(2-hydroxyethoxymethyl)adenine) [7] 0.001-0.01 (2-chloro-9-(2-hydroxyethoxymethyl)adenine) [7] 0.001-0.01 (2-iodo-9-(2-hydroxyethoxymethyl)adenine) [7] 0.015 (9-(phosphonoheptyl)adenine, low phosphate concentration of 3.5 mM [7]) [7] 0.031 (5'-methylthiotubercidin) [10] 0.172 (adenine) [10] pH-Optimum 7 ( assay at [25]) [25] 7.1-7.6 [17] 7.2 [2, 4] 7.4 ( assay at [21,29,31]) [19, 21, 29, 31] 7.4-8 [3] 7.5 ( assay at [16]) [1, 16] Additional information ( pI: 5.7 [19]; pI: 5.5 [17]; pI: 5.2 [2]) [2, 17, 19] pH-Range 5-10 [19] 6-9 ( 50% of activity maximum at pH 6.0 and pH 9.0 [2]) [2] 6.2-8.6 ( 50% of activity maximum at pH 6.2 and pH 8.6 [17]) [17] 6.5-8.5 ( considerable decrease of activity below pH 6.5 and above pH 8.5 [1]) [1]

243


2.4.2.28

Temperature optimum ( C) 37 ( assay at [1, 3, 16-18, 21, 25, 27]) [1, 3, 1618, 21, 25, 27] 47 [10] 70 ( assay at [2, 14, 19, 24]) [2, 4, 14, 19, 24] 80 ( assay at [31]) [31] 95 [2, 14] 100 ( recombinant enzyme [24]) [24] 120 ( recombinant enzyme [30]) [5, 19, 30] 125 [31] Temperature range ( C) 20-60 [9] 30-120 ( recombinant enzyme [24]) [24] 30-150 [19] 70-160 [31] 86-95 ( 2 different activation energy-dependent processes occur below and above 100 C [31]; 86 C: 50% of activity maximum, 95 C: activity maximum, no activity at 40 C [2,14]) [2, 14, 31]

4 Enzyme Structure Molecular weight 54000 ( gel filtration [20]) [20] 55000 ( gel filtration [6]) [6] 64000 ( gel filtration [4]) [4] 89000 ( gel filtration [9]) [9] 90000 ( recombinant enzyme, gel filtration [21]; gel filtration [16]) [16, 21] 98000 ( sedimentation equilibrium [18]; gel filtration [17,18]) [17, 18] 160000 ( recombinant enzyme [24]; gel filtration [19,24]) [19, 24] 180000 ( gel filtration [31]) [31] Subunits dimer ( 2 * ?, SDS-PAGE [4]; 2 * 30300, SDS-PAGE [6]) [4, 6] hexamer ( 6 * 30000, SDS-PAGE [31]; 6 * 27000, recombinant enzyme, crystal structure analysis [29]; 6 * 27000, recombinant enzyme, SDS-PAGE [24]; 6 * 27000, SDS-PAGE [19]) [19, 24, 29, 31] trimer ( 3 * 30000, recombinant enzyme, SDS-PAGE [21]; 3 * 32500, SDS-PAGE with addition of 8 M urea and 1% 2-mercaptoethanol [17]; 3 * 32000, SDS-PAGE [18]) [17, 18, 21] Additional information ( enzyme is formed by a trimer of dimers with three symmetric intersubunit disulfide bonds linking the dimers to one another, each monomer contains one active site, which is located near a dimer interface [30]; base- and methylribose-binding sites [26]; over244

2.4.2.28


all and active site structure, trimer of 3 identical subunits [26]; secondary structure by circular dichroism spectra [19]; presence of 6 disulfide bonds, organisation in 2 trimers [19]) [19, 26, 30]

5 Isolation/Preparation/Mutation/Application Source/tissue CCRF S-180 II cell [7, 12] DHL-9 cell ( diffuse histiocytic lymphoma cell line, possesses no enzyme activity, but intact gene [33]) [33] HL-60 cell [7] HMB-2 cell ( cell line derived from metastases of malignant melanomas [32]) [32] HeLa cell [28] J-82 cell ( tumor-derived cell line, bladder carcinoma [28]) [28] MOLT-4 cell [23] MRC-5 cell ( lung fibroblast cell line [28]) [28] Mel Ei cell ( cell line derived from primary cutaneous melanoma [32]) [32] Mel Im cell ( cell line derived from metastases of malignant melanomas [32]) [32] Mel Ju cell ( cell line derived from metastases of malignant melanomas [32]) [32] Mel Wei cell ( cell line derived from primary cutaneous melanoma [32]) [32] Mel-HO cell ( low content [32]; cell line derived from primary cutaneous melanoma [32]) [32] Mel-Juso cell ( low content [32]; cell line derived from primary cutaneous melanoma [32]) [32] RAJI cell ( tumor-derived cell line, Burkitt's lymphoma [28]) [28] SK-N-SH cell ( tumor-derived cell line, neuroblastoma [28]) [28] heart ( left ventricular specimen [27]; low activity [3]) [3, 27] hematopoetic stem cell ( and progenitor cells [23]) [23] kidney [3] liver [3, 6, 16, 18, 20] lung [3] lymphocyte ( peripheral blood [10]) [10] lymphoma cell line ( diffuse, histiocytic [33]) [33] melanocyte ( primary [32]) [32] melanoma cell ( reduced activity due to hypermethylation of the promotor [32]) [32] melanoma cell line ( SK-Ml-28 cell line derived from metastases of malignant melanomas [32]) [32] mononuclear cell [22] peripheral blood [10, 22, 23] 245


2.4.2.28

peripheral blood mononuclear cell ( CD34+ erythroid burst-forming unit, granulocyte/monocyte colony-forming unit, granulocyte/erythrocyte/ macrophage/megakaryocyte colony-forming unit progenitors and primitive high proliferative potential colony-forming cells in the purified CD34+ cells are cultured [23]) [23] placenta [17] promastigote [9] prostate gland [1, 15] skin fibroblast cell line ( FC1010 skin fibroblast cell line [28]) [28] spleen [3, 4] testis [3] Additional information ( strongly reduced expression in all melanoma cell lines due to promotor hypermethylation, no enzyme content and expression in melanoma cell line HTZ19d [32]; enzyme defect is associated with but not responsible for methionine-dependent tumor cell growth [28]; enzyme is deficient in patients with T-cell acute lymphoblastic leukemia, no activity in enzyme-deficient CEM cells [23]; enzyme deficiency in malignant cells, partial or total deletions in the gene of leukemia cell lines of patients with T-cell acute lymphoblastic leukemia [22]; wide tissue distribution [3]) [3, 22, 28, 32] Localization cytosol ( exclusively [19]) [1, 3, 5] Purification [9] (partial [1,3]; from lung [3]) [1, 3, 4, 16] [2, 14] (recombinant from Escherichia coli [24, 29, 30]) [5, 19, 24, 29, 30] (recombinant His-tagged enzyme from Escherichia coli [26]; recombinant from Escherichia coli [21]; partial [10, 20]) [6, 10, 15, 17, 20, 21, 26] (partial [13]) [13] [18] [31] Crystallization (from recombinant enzyme, hanging drop-vapour diffusion method, protein solution, 7-10 mg/ml, 18 C, reservoir solution for native crystals: Tris-HCl 10 mM, pH 7.4, 28-30% dioxane, 12% 2-methyl-2,4-pentanediol, 0.12 M MgCl2 , 0,04 M NaCl, for crystals of enzyme complexed with substrates or sulfate and phosphate ions, substrates are added and NaCl is exchanged for MgSO4 or NH4 Cl and KH2 PO4, respectively, X-ray structure determination and analysis [29]) [29] (from recombinant enzyme, hanging drop-vapour diffusion method, protein 25 mg/ml, 10 mM Tris-HCl, pH 7.4, 5 mM DTT, 0.4 M NaCl against reservoir solution of 80 mM MES, pH 5.9, 5 mM DTT, 12% w/v polyethylene glycol 6000, 25% v/v ethylene glycol, in presence of substrates adenine, phosphate and sulfate, structure analysis by X-ray diffraction at light scattering [26]) [26]

246

2.4.2.28


Cloning (overexpression in Escherichia coli strain RB791, amino acid determination, incorrect positioning of disulfide bonds, the recombinant enzyme is less thermostable and thermophilic than the native enzyme [24,29]) [24, 29] (wild-type and promotor deletion mutant, expression in HeLa cells, luciferase reporter assay [33]; quantitative PCR, RNA mutational analysis, analysis of methylation status of the enzymes promotor [32]; re-expression in Mel Im cell line [32]; functional expression in enzyme deficient cell line MCF-7 [28]; expression of His-tagged protein in Escherichia coli BL21(DE3) cells [26]; from cells of patients with T-cell acute lymphoblastic leukemia, DNA sequence analysis, 9p21 chromosome, topologic map [22]; the gene maps on the 9p21 chromosome, strictly linked to the tumor suppressor gene p16INK4A, functional expression in Escherichia coli strain JM105 [21]) [21, 22, 26, 28, 32, 33] Engineering V56I ( natural polymorphism present in 7 of 9 melanoma cell lines, not in SK-Mel-28 and in HTZ19d [32]) [32] Additional information ( cell line DHL-9 contains a promotor sequence with a deletion of 14 bases, the deletion mutant allele is widespread throughout the japanese population and not responsible for the enzyme deficiency in this cell line [33]; melanoma cell line HTZ19d is a deletion mutant without enzyme expression and activity [32]) [32, 33] Application medicine ( enzyme is a potential chemotherapeutic target [26]; possibility of targeting the enzyme in the design of an enzyme-selective therapy for patients with T-cell acute lymphoblastic leukemia and other enzymedeficient malignancies [23]; gene is linked to the important tumor suppressor gene p16INK4A, enzyme is of key importance in defining the chromosomal area homozygously deleted in a large number of human tumors, absence of enzyme activity only in malignant cells is of great value in developing selective antitumor therapeutic strategies [21]) [21, 23, 26]

6 Stability Temperature stability 40-55 ( 15 min, stable [17]) [17] 65 ( rapid inactivation [15]) [15] 70 ( recombinant enzyme, 0.8 M DTT, stable [24]; 15 min, complete loss of activity [17]) [17, 24] 90 ( recombinant enzyme, 0.8 M DTT, 2 h, loss of 38% activity [24]) [24] 100 ( 5 h, 98% remaining activity [31]; 1 h, stable [2,14]; recombinant enzyme, 1 h, 95% remainig activity in presence of 100 mM phosphate [29]; recombinant enzyme, 1 h, 85% remaining activity [24]; stable for at least 2 h [5,19]) [2, 5, 14, 19, 24, 29, 31] 247


2.4.2.28

110 ( recombinant enzyme, 2 h, stable [30]; recombinant enzyme, 10 min, 50% remaining activity in absence and 90% remaining activity in presence of 100 mM phosphate [29]) [29] 111 ( recombinant enzyme, melting temperature [24]) [24] 118 ( recombinant enzyme in presence of 100 mM phosphate, melting temperature [24]) [24] 120 ( recombinant enzyme, 10 min, no activity in absence and 50% remaining activity in presence of 100 mM phosphate [29]) [29, 30] 130 ( half-life: 43 min [31]; half-life: 15 min [5,19]) [5, 19, 31] 132 ( 10 min, melting temperature [5,19]) [5, 19] 137 ( melting temperature [31]) [31] 139 ( melting temperature in presence of 100 mM phosphate [31]) [31] 140 ( half-life: 13 min [31]; half-life: 5 min [5,19]) [5, 19, 31] 145 ( half-life: 5 min [31]) [31] Additional information ( disulfide linkages play a key role in thermal stability [30]; the recombinant enzyme, expressed in Escherichia coli, is less thermostable and thermophilic than the native enzyme due to incorrect positioning of disulfide bonds [24]; high degree of thermal stability [4]; resistance to thermal inactivation is increased remarkably by addition of 5'-methylthioadenosine or phosphate [17]) [4, 17, 24, 30] Oxidation stability , O2 partially inactivates [15] Organic solvent stability 1-propanol ( at 50%: 88% remaining activity after 60 min at 50 C, 58% remaining activity after 60 min at 70 C, 22% remaining activity after 60 min at 90 C [31]; no loss of activity after 24 h at room temperature in presence of 50% methanol, loss of 90% activity at 90 C after 1 h [19]) [19, 31] acetonitrile ( at 50%: stable at 90 C for 30 min, loss of 62% activity after 60 min [31]; loss of 89% activity at 70 C after 1 h [19]) [19, 31] dimethylformamide ( at 50%: 48% remaining activity after 60 min at 50 C, 5% remaining activity after 60 min at 70 C, no activity at 90 C [31]; no loss of activity after 24 h at room temperature in presence of 50% dimethylformamide, loss of 21% activity at 90 C after 1 h [19]) [19, 31] ethanol ( at 50%: 90% remaining activity after 60 min at 50 C, 65% remaining activity after 60 min at 70 C, 30% remaining activity after 60 min at 90 C [31]; no loss of activity after 24 h at room temperature in presence of 50% ethanol, loss of 88% activity at 90 C after 1 h [19]) [19, 31] methanol ( at 50%: stable at 90 C for 30 min, loss of 77% activity after 60 min [31]; no loss of activity after 24 h at room temperature in presence of 50% methanol, loss of 80% activity at 90 C after 1 h [19]) [19, 31] 248

2.4.2.28


tetrahydrofuran ( at 50%: 77% remaining activity after 60 min at 50 C, 50% remaining activity after 60 min at 70 C, 3% remaining activity after 60 min at 90 C [31]; loss of 76% activity at 70 C after 30 min, complete loss of activity after 1 h at 70 C [19]) [19, 31] General stability information , no loss of activity after 24 h at room temperature in presence of 9 M urea, 4 M guanidine hydrochloride, 0.075% SDS, 50% methanol, 50% ethanol, 50% dimethylformamide, 1 M NaCl, and 1% Triton X-100 [19] , no loss of activity after treatment with thermolysin, trypsin, and chymotrypsin for 24 h at 37 C [19] , phosphate, and less efficiently also arsenate and sulfate, stabilize the recombinant enzyme against inactiviation by temperature, SDS, urea, and proteolytic enzymes [29] , recombinant enzyme, 90 C in 2% SDS, 30 min, loss of 60% activity [29] , recombinant enzyme, 90 C in 8 M urea, 30 min, loss of 70% activity [29] , rapid inactivation in absence of reducing agents: 50% inactivation within 24 h at both 4 C and -20 C [17] , resistance to thermal inactivation is increased remarkably by addition of 5'-methylthioadenosine or phosphate [17] , stable to freeze-thawing [15] , dithiothreitol 0.8 M, reduction of thermostability [31] , enzyme is extremely stable to proteolytic cleavage [31] Storage stability , -20 C, stable for at least 3 weeks [1] , -70 C, stable for at least 4 months [3] , -20 C, stable for several months [14] , -20 C, purified enzyme, Tris-HCl 10 mM, pH 7.4, 1 mM EDTA, stable at least 1 year [19] , -20 C, 5 mM DTT, 50 mM potassium phosphate, pH 7.4, less than 10% loss of activity after 1 month [17] , 20 C, Tris-HCl 10 mM, pH 7.4, purified enzyme, stable for at least 1 year [31]

References [1] Pegg, A.E.; Williams-Ashman, H.G.: Phosphate-stimulated breakdown of 5'methylthioadenosine by rat ventral prostate. Biochem. J., 115, 241-247 (1969) [2] Carteni'-Farina, M.; Oliva, A.; Romeo, G.; Napolitano, G.; De Rosa, M.; Gambacorta, A.; Zappia, V.: 5'-Methylthioadenosine phosphorylase from Caldariella acidophila. Purification and properties. Eur. J. Biochem., 101, 317-324 (1979)

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[3] Garbers, D.L.: Demonstration of 5'-methylthioadenosine phosphorylase activity in various rat tissues. Some properties of the enzyme from rat lung. Biochim. Biophys. Acta, 523, 82-93 (1978) [4] Lee, S.H.; Cho, Y.D.: Purification and properties of 5'-deoxy-5'methylthioadenosine phosphorylase from rat spleen. Korean Biochem. J., 26, 433-439 (1993) [5] Cacciapuoti, G.; Porcelli, M.; Bertoldo, C.; Zappia, V.: Thermophilicity and thermostability of 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus. Life Chem. Rep., 10, 75-81 (1992) [6] Toorchen, D.; Miller, R.L.: Purification and characterization of 5'-deoxy-5'methylthioadenosine (MTA) phosphorylase from human liver. Biochem. Pharmacol., 41, 2023-2030 (1991) [7] Savarese, T.M.; Harrington, S.; Nakamura, C.; Chen, Z.H.; Kumar, P.; Mikkilineni, A.; Abushanab, E.; Chu, S.H.; Parks, R.E.: 5'-Deoxy-5'-methylthioadenosine phosphorylase. V. Acycloadenosine derivatives as inhibitors of the enzyme. Biochem. Pharmacol., 40, 2465-2471 (1990) [8] Ghoda, L.Y.; Savarese, T.M.; Northup, C.H.; Parks, R.E.; Garofalo, J.; Katz, L.; Ellenbogen, B.B.; Bacchi, C.J.: Substrate specificities of 5'-deoxy-5'methylthioadenosine phosphorylase from Trypanosoma brucei brucei and mammalian cells. Mol. Biochem. Parasitol., 27, 109-118 (1988) [9] Koszalka, G.W.; Krenitsky, T.A.: 5'-Methyladenosine (MTA) phosphorylase from promastigotes of Leishmania donovani. Adv. Exp. Med. Biol., 195B, 559-563 (1986) [10] White, M.W.; Vandenbark, A.A.; Barney, C.L.; Ferro, A.J.: Structural analogs of 5'-methylthioadenosine as substrates and inhibitors of 5'-methylthioadenosine phosphorylase and as inhibitors of human lymphocyte transformation. Biochem. Pharmacol., 31, 503-507 (1982) [11] Shugart, L.; Mahoney, L.; Chastain, B.: Kinetic studies of Drosophila melanogaster methylthioadenosine nucleoside phosphorylase. Int. J. Biochem., 13, 559-564 (1981) [12] Savarese, T.M.; Crabtree, G.W.; Parks, R.E.: 5'-Methylthioadenosine phosphorylase. I. Substrate activity of 5'-deoxyadenosine with the enzyme from Sarcoma 180 cells. Biochem. Pharmacol., 30, 189-199 (1981) [13] Shugart, L.; Tancer, M.; Moore, J.: Methylthioadenosine nucleoside phosphorylase activity in Drosophila melangoaster. Int. J. Biochem., 10, 901904 (1979) [14] Zappia, V.; Carteni-Farina, M.; Romeo, G.; De Rosa, M.; Gambacorta, A.: Purification and properties of 5'-methyladenosine phosphorylase from Caldariella acidophila. Methods Enzymol., 94, 355-361 (1983) [15] Zappia, V.; Oliva, A.; Cacciapuoti, G.; Galletti, P.; Mignucci, G.; Carteni-Farina, M.: Substrate specificity of 5'-methylthioadenosine phosphorylase from human prostate. Biochem. J., 175, 1043-1050 (1978) [16] Ferro, A.J.; Wrobel, N.C.; Nicolette, J.A.: 5-Methylthioribose 1-phosphate: a product of partially purified, rat liver 5'-methylthioadenosine phosphorylase activity. Biochim. Biophys. Acta, 570, 65-73 (1979) [17] Della Ragione, F.; Carteni-Farina, M.; Gragnaniello, V.; Schettino, M.I.; Zappia, V.: Purification and characterization of 5'-deoxy-5'-methylthioadeno250

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[18]

[19]

[20] [21]

[22]

[23]

[24]

[25] [26] [27] [28] [29]


sine phosphorylase from human placenta. J. Biol. Chem., 261, 12324-12329 (1986) Della Ragione, F.; Oliva, A.; Gragnaniello, V.; Russo, G.L.; Palumbo, R.; Zappia, V.: Physicochemical and immunological studies on mammalian 5'deoxy-5'-methylthioadenosine phosphorylase. J. Biol. Chem., 265, 62416246 (1990) Cacciapuoti, G.; Porcelli, M.; Bertoldo, C.; De Rosa, M.; Zappia, V.: Purification and characterization of extremely thermophilic and thermostable 5'methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus. Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds. J. Biol. Chem., 269, 24762-24769 (1994) Fabianowska-Majewska, K.; Duley, J.; Fairbanks, L.; Simmonds, A.; Wasiak, T.: Substrate specificity of methylthioadenosine phosphorylase from human liver. Acta Biochim. Pol., 41, 391-395 (1994) Della Ragione, F.; Takabayashi, K.; Mastropietro, S.; Mercurio, C.; Oliva, A.; Russo, G.L.; Della Pietra, V.; Borriello, A.; Nobori, T.; et al.: Purification and characterization of recombinant human 5'-methylthioadenosine phosphorylase: definite identification coding cDNA. Biochem. Biophys. Res. Commun., 223, 514-519 (1996) Nobori, T.; Takabayashi, K.; Tran, P.; Orvis, L.; Batova, A.; Yu, A.L.; Carson, D.A.: Genomic cloning of methylthioadenosine phosphorylase: a purine metabolic enzyme deficient in multiple different cancers. Proc. Natl. Acad. Sci. USA, 93, 6203-6208 (1996) Yu, J.; Batova, A.; Shao, L.-e.; Carrera, C.J.; Yu, A.L.: Presence of methylthioadenosine phosphorylase (MTAP) in hematopoietic stem/progenitor cells: its therapeutic implication for MTAP (-) malignancies. Clin. Cancer Res., 3, 433-438 (1997) Cacciapuoti, G.; Fusco, S.; Caiazzo, N.; Zappia, V.; Porcelli, M.: Heterologous expression of 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus: characterization of the recombinant protein and involvement of disulfide bonds in thermophilicity and thermostability. Protein Expr. Purif., 16, 125-135 (1999) Bacchi, C.J.; Goldberg, B.; Rattendi, D.; Gorrell, T.E.; Spiess, A.J.; Sufrin, J.R.: Metabolic effects of a methylthioadenosine phosphorylase substrate analog on african trypanosomes. Biochem. Pharmacol., 57, 89-96 (1999) Appleby, T.C.; Erion, M.D.; Ealick, S.E.: The structure of human 5'-deoxy5'-methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis. Structure, 7, 629-641 (1999) Slominska, E.M.; Kalsi, K.K.; Yacoub, M.H.; Smolenski, R.T.: The role of 5'deoxy-5'-methyl thioadenosine phosphorylase in cardiac adenosine breakdown and adenine production. Adv. Exp. Med. Biol., 486, 159-162 (2000) Tang, B.; Li, Y.N.; Kruger, W.D.: Defects in methylthioadenosine phosphorylase are associated with but not responsible for methionine-dependent tumor cell growth. Cancer Res., 60, 5543-5547 (2000) Cacciapuoti, G.; Servillo, L.; Moretti, M.A.; Porcelli, M.: Conformational changes and stabilization induced by phosphate binding to 5'-methylthio-

251


[30]

[31]

[32]

[33]

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adenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus. Extremophiles, 5, 295-302 (2001) Appleby, T.C.; Mathews, I.I.; Porcelli, M.; Cacciapuoti, G.; Ealick, S.E.: Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'methylthioadenosine phosphorylase from Sulfolobus solfataricus. J. Biol. Chem., 276, 39232-39242 (2001) Cacciapuoti, G.; Bertoldo, C.; Brio, A.; Zappia, V.; Porcelli, M.: Purification and characterization of 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus. Substrate specificity and primary structure analysis. Extremophiles, 7, 159-168 (2003) Behrmann, I.; Wallner, S.; Komyod, W.; Heinrich, P.C.; Schuierer, M.; Buettner, R.; Bosserhoff, A.K.: Characterization of methylthioadenosin phosphorylase (MTAP) expression in malignant melanoma. Am. J. Pathol., 163, 683-690 (2003) Kadariya, Y.; Nishioka, J.; Nakatani, K.; Nakashima, K.; Nobori, T.: Deletion of dinucleotide repeat (D14 allele) in the methylthioadenosine phosphorylase (MTAP) promoter and the allelotype of MTAP promoter in the Japanese population. Jpn. J. Cancer Res., 93, 369-373 (2002)

Queuine tRNA-ribosyltransferase

2.4.2.29

1 Nomenclature EC number 2.4.2.29 Systematic name tRNA-guanine:queuine tRNA-d-ribosyltransferase Recommended name queuine tRNA-ribosyltransferase Synonyms Q-insertase TGT guanine insertion enzyme guanine, queuine-tRNA transglycosylase queuine insertase queuine tRNA ribosyltransferase ribosyltransferase, queuine transfer ribonucleate tRNA guanine transglycosidase tRNA guanine transglycosylase tRNA transglycosylase tRNA-guanine transglycosylase transfer ribonucleate glycosyltransferase virulence-associated protein VACC [Swissprot] CAS registry number 72162-89-1

2 Source Organism

Escherichia coli [1, 4, 10, 15, 16, 17, 18, 19, 21, 22, 23, 24] Oryctolagus cuniculus [2, 5, 20] Salmonella typhimurium [3] Xenopus laevis [6, 8] Triticum aestivum [7] Rattus norvegicus [9] Mus musculus [11] Homo sapiens [11, 13] Shigella flexneri [11, 12, 23] Haemophilus influenza [11]

253


2.4.2.29

Helicobacter pylori [11] Synechocystis sp. [11] Thermotoga maritima [11] Caenorhabditis elegans [11] Methanococcus jannaschii [11] Archaeoglobus fulgidus [11] Methanobacterium thermoautotrophicum [11] Zymomonas mobilis [14, 24] Pyrococcus horikoshii [15]

3 Reaction and Specificity Catalyzed reaction tRNA guanine + queuine = tRNA queuine + guanine (, ping-pong kinetic mechanism [23]; , double-displacement mechanism that involves the formation of a covalent enzyme-RNA intermediate [24]) Reaction type pentosyl group transfer Natural substrates and products S 7-(aminomethyl)-7-deazaguanine + tRNAguanine (, the enzyme catalyzes the posttranscriptional base exchange of the queuosine precursor 7-(aminomethyl)-7-deazaguanine with the genetically encoded guanine in tRNAASp , tRNAAsn , tRNAHis and tRNATyr [18]; , the enzyme is involved in the hypermodification of cognate tRNAs, leading to the exchange of G34 by 7-methylamino-7-deazaguanine at the wobble position in the anticodon loop [12]; , the enzyme is responsible for the posttranscriptional modification of specific tRNAs (Asn, Asp, His and Tyr) with queuine. The enzyme catalyzes base exchange of guanosine34 with 7-aminomethyl-7-deazaguanine [4]; , the enzyme exchanges the genetically encoded guanine at position 34 with a queuine precursor 7(aminomethyl)-7-deazaguanine [21]) (Reversibility: ? [4, 12, 18, 21]) [4, 12, 18, 21] P guanine + tRNA(7-(aminomethyl)-7-deazaguanine) [18] S Additional information (, post-translational modification of tRNA [1,2]; , formation of queuosine [1]; , key enzyme involved in the incorporation of the modified base queuine into tRNA [10]; , higher expression of the subunit TGT60KD in cancer cells than in normal cells. The expression levels of the TGT60KD subunit regulate enzyme activity and the level of queuosine. TGT60KD plays significant roles in carcinogenesis [13]; , the enzyme is required for virulence [23]) [1, 2, 10, 13, 23] P ?

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Substrates and products S 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one + tRNAguanine (, 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)one appears to partition between: 1. normal turnover, 2. inactivation, 3. an alternative processing to an unidentrified fluoride-released product [19]) (Reversibility: ? [19]) [19] P guanine + tRNA2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)one S 2-thiohypoxanthine + tRNAguanine (Reversibility: ? [9]) [9] P guanine + tRNA2-thiohypoxanthine S 6-thioguanine + tRNAguanine (Reversibility: ? [9]) [9] P guanine + tRNA6-thioguanine S 7-(aminomethyl)-7-deazaguanine + tRNAguanine (, E. coli preQ0-tRNATyr , containing7-(cyano)-7-deazaguanine in the anticodon and E. coli guaninetRNATyr containing guanosine in the anticodon are good acceptors [1]; , poor substrate [5]; , no activity [7]) (Reversibility: ir [1, 5]; ? [4, 9, 12, 14, 18, 21]) [1, 4, 5, 9, 12, 14, 18, 21] P guanine + tRNA(7-(aminomethyl)-7-deazaguanine) [1] S 7-(cyano)-7-deazaguanine + tRNAguanine (Reversibility: ? [1, 9]) [1, 9] P guanine + tRNA(7-(cyano)-7-deazaguanine) (, inserted 7-(cyano)-7-deazaguanine is located in the first position of the anticodon [1]) [1] S 7-deazaguanine + tRNAguanine (Reversibility: ir [5]) [5] P guanine + tRNA(7-deazaguanine) [5] S 8-azaguanine + tRNAguanine (Reversibility: r [5,9]) [5, 9] P guanine + tRNA(8-azaguanine) S dihydroqueuine + tRNAguanine (Reversibility: ir [5]; ? [6]) [5, 6] P guanine + tRNAdihydroqueuine S guanine + tRNAguanine (, yeast tRNAAsp [2]; , yeast tRNA [3]) (Reversibility: r [1, 5, 7]; ? [2, 3, 9]) [1, 2, 3, 5, 7, 9] P tRNAguanine + guanine S queuine + tRNAguanine (, nucleosides in position 36, 37 and 38 influence the efficiency of conversion of G-34 to queuosine-34 [6]; , tRNATyr , tRNAHis , tRNAAsn , or tRNAASp from an E. coli mutant or rat ascites hepatoma cells [9]) (Reversibility: ir [5]; ? [6, 7, 9]) [5, 6, 7, 9] P guanine + tRNAqueuine Inhibitors 2,4-diamino-6-hydroxypyrimidine (, 1.2 mM, 52% inhibition [5]) [5] 2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one (, inactivation and competitive inhibition. 2-amino-5-(fluoromethyl)pyrrolo[2,3d]pyrimidin-4(3H)-one appears to partition between: 1. normal turnover, 2.

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inactivation, 3. an alternative processing to an unidentrified fluoride-released product [19]) [19] 3-deazaguanine (, 0.25 mM, 50% inhibition [5]) [5] 6-methylmercaptoguanine (, 0.05 mM, 65% inhibition [5]) [5] 6-thioguanine (, 0.05 mM, 74% inhibition [5]) [5] 7-deazaguanine (, 0.06 mM, 27% inhibition [5]) [5] 7-methylguanine (, 0.05 mM, 94% inhibition [5]) [5, 23] 8-azaguanine (, 0.05 mM, complete inhibition [5]) [5] 8-bromoguanine (, 0.05 mM, 86% inhibition [5]) [5] 8-dimethylaminomethyl-7-deazaguanine (, 0.05 mM, 32% inhibition [5]) [5] Cd2+ (, 10 mM [7]) [7] Co2+ (, 10 mM [7]) [7] Cu2+ (, 10 mM [7]) [7] Mn2+ (, 10 mM [7]) [7] Ni2+ (, 10 mM [7]) [7] Pb2+ (, 10 mM [7]) [7] Zn2+ (, 10 mM [7]) [7] adenine (, 0.05 mM, 14% inhibition [5]) [5] biopterin (, 0.02 mM, 39% inhibition [5]) [5] dimethylsuberimidate (, inactivates by cross-linking, tRNA protects [4]) [4] ethylacetimidate [4] folic acid (, 1.2 mM, complete inhibition [5]) [5] isocytosine (, 1.2 mM, 52% inhibition [5]) [5] lumazine (, 1.2 mM, 18% inhibition [5]) [5] neplanocin A (, 0.1 mM, 45% inhibition [5]) [5] pterin (, 0.0048 mM, 82% inhibition [5]) [5] pterin-6-carboxylic acid (, 1.2 mM, 57% inhibition [5]) [5] queuine (, 0.01 mM, 93% inhibition [5]) [5] sepiapterin (, 0.13 mM, 33% inhibition [5]) [5] tetrahydrobiopterin (, 0.02 mM, 82% inhibition [5]) [5] Metals, ions Ba2+ (, 1 mM, less effective than Mg2+ in meeting the divalent ion requirement [7]) [7] Ca2+ (, 1 mM, less effective than Mg2+ in meeting the divalent ion requirement [7]) [7] Cs+ (, less effective than Na+ in meeting the monovalent ion requirement [7]) [7] K+ (, less effective than Na+ in meeting the monovalent ion requirement [7]) [7] Li+ (, less effective than Na+ in meeting the monovalent ion requirement [7]) [7]

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Mg2+ (, stimulates [3]; , the enzyme requires a monovalent or a divalent cation. Mg2+ is most effective in meeting the divalent cation requirement. Optimal Mg2+ concentration is 5.0 mM [7]) [3, 7] Na+ (, the enzyme requires a monovalent or a divalent cation. Na+ is most effective in meeting the monovalent requirement. Optimal concentration is 114 mM [7]) [7] Rb+ (, less effective than Na+ in meeting the monovalent ion requirement [7]) [7] Sr2+ (, 1 mM, less effective than Mg2+ in meeting the divalent ion requirement [7]) [7] Turnover number (min±1) 27.66 (2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3 H)-one) [19] 35.4 (guanine, , histidine-tagged mutant enzyme D89E, pH 7.3 [17]) [17] 42.6 (tRNA, , histidine-tagged mutant enzyme D89E, pH 7.3 [17]) [17] 45 (guanine, , histidine-tagged mutant enzyme D89E, pH 8.5 [17]) [17] 55.8 (tRNA, , histidine-tagged mutant enzyme D89E, pH 8.5 [17]) [17] 72.6 (guanine, , histidine-tagged wild-type enzyme, pH 7.3 [17]) [17] 72.6 (tRNA, , histidine-tagged wild-type enzyme, pH 7.3 [17]) [17] 351 (tRNA, , histidine-tagged wild-type enzyme, pH 8.5 [17]) [17] 454.2 (guanine, , histidine-tagged wild-type enzyme, pH 8.5 [17]) [17] Additional information (, turnover-numbers for deoxyuracil-DNA analogues [15]; , turnover-numbers for mutants of the E. coli tRNATyr minihelix [21]; , turnover-numbers for several tRNA analogs. Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TY C arm of in vitro-transcribed yeast tRNAPhe [22]) [15, 2, 22, 24] Specific activity (U/mg) Additional information [2, 4, 9] Km-Value (mM) 0.0000033 (tRNAAsp , , yeast tRNAAsp [2]) [2] 0.000014 (7-(aminomethyl)-7-deazaguanine) [1] 0.000053 (guanine) [1] 0.00006 (guanine) [7] 0.0001 (guanine, , histidine-tagged wild-type enzyme, pH 7.3 [17]) [17] 0.0001 (guanine, , wild-type enzyme [24]) [24] 0.00011 (tRNA, , histidine-tagged mutant enzyme D89E, pH 7.3 [17,24]) [17, 24]

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0.00012 (tRNA, , histidine-tagged wild-type enzyme, pH 7.3 [17]) [17] 0.00012 (tRNA, , wild-type enzyme [24]) [24] 0.00015 (guanine) [2] 0.0003 (guanine, , mutant enzyme D264E [24]) [24] 0.00032 (tRNATyr , , wild-type enzyme [18]) [18] 0.00039 (tRNA, , histidine-tagged wild-type enzyme, pH 8.5 [17]) [17] 0.00046 (tRNA, , mutant enzyme D264E [24]) [24] 0.00051 (tRNA, , histidine-tagged mutant enzyme D89E, pH 8.5 [17]) [17] 0.00057 (guanine, , histidine-tagged wild-type enzyme, pH 8.5 [17]) [17] 0.00083 (guanine) [9] 0.0011 (guanine, , mutant enzyme S90A [18]) [18] 0.00165 (guanine) [3] 0.0021 (7-(aminomethyl)-7-deazaguanine) [9] 0.00261 (guanine, , histidine-tagged mutant enzyme D89E, pH 7.3 [17]) [17, 24] 0.003 (guanine, , wild-type enzyme [18]) [18] 0.00632 (guanine, , histidine-tagged mutant enzyme D89E, pH 8.5 [17]) [17] 0.0097 (tRNATyr , , mutant enzyme S90C [18]) [18] 0.152 (2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3H)-one) [19] Additional information (, Km for yeast tRNA is 0.014 mg [3]; , Km -values for several queuine-cognate tRNAs [10]; , Km -values for deoxyuracil-DNA analogues [15]; , Km -values for several tRNA analogues at 20 C and at 37 C [16]; , Km -values for mutants of the E. coli tRNATyr minihelix [21]; , Km -values for several tRNA analogs. Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TY C arm of in vitro-transcribed yeast tRNAPhe [22]) [3, 10, 11, 15, 16, 21, 22] Ki-Value (mM) 2.3e-005 (8-azaguanine) [5] 9e-005 (pterin) [5] 9.5e-005 (queuine) [7] 0.001 (7-methylguanine) [5] 0.011 (folic acid) [5] 0.054 (7-deazaguanine) [5] 0.1 (neplanocin A) [5] 0.114 (2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3 H)-one, , competitive inhibition [19]) [19] 0.136 (2-amino-5-(fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3 H)-one, , inactivation [19]) [19] 0.25 (3-deazaguanine) [5]

258

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pH-Optimum 7.6 [7] 8 [3] Temperature optimum ( C) 37 [3]

4 Enzyme Structure Molecular weight 104000 (, gel filtration [2]) [2] 140000 (, gel filtration [7]) [7] Additional information (, sequence identities between prokaryotic, eukaryotic and archaebacterial enzymes [11]) [11] Subunits ? (, x * 44000, histidine-tagged wild-type enzyme [24]) [24] dimer (, 1 * 60000 + 1 * 43000, denaturing PAGE in presence of urea [2]; , 2 * 68000, SDS-PAGE [7]) [2, 7] Posttranslational modification Additional information (, one subunit has a MW of 60000-61000 Da as determined by SDS-PAGE and 55921 Da calculated from nucleotide sequence, this suggests that the protein is posstranslationally modified [20]) [20]

5 Isolation/Preparation/Mutation/Application Source/tissue HPB-ALL cell [13] HUT-78 cell [13] Jurkat [13] Molt-4F cell [13] Morris hepatomy 7316A cell [9] PEER [13] erythrocyte [2] germ [7] liver [9] oocyte [6, 8] reticulocyte [5, 20] Localization cytosol [2] Purification (wild-type and mutant enzymes D89E, D89D, D89C and D89A [17]; mutant enzyme S90F, S90C and S90A [18]) [4, 17, 18]

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[2] [7] [9] Crystallization (mutant enzyme D280E [24]) [14, 24] [15] Cloning (overexpression of D89E, D89D, D89C and D89A in Escherichia coli [17]; mutant enzymes S90F and S90C [18]) [4, 17, 18] (cDNA sequence of the 60000 Da subunit, 55921 Da calculated from nucleotide sequence [20]) [20] Engineering D264A (, mutant shows no catalytic activity [24]) [24] D264E (, mutant enzyme is capable of forming an enzyme-RNA covalent intermediate, however , unlike wild-type enzyme, only hydroxylamine is capable of cleaving the enzyme-RNA covalent complex [24]) [24] D264H (, mutant shows no catalytic activity [24]) [24] D264K (, mutant shows no catalytic activity [24]) [24] D264N (, mutant shows no catalytic activity [24]) [24] D264Q (, mutant shows no catalytic activity [24]) [24] D89A (, less than 1% of the activity of the histidine-tagged wild-type enzyme [17]) [17] D89C (, less than 1% of the activity of the histidine-tagged wild-type enzyme [17]) [17] D89E (, about 50% of the activity of the histidine-tagged wild-type enzyme [17]) [17] D89N (, less than 1% of the activity of the histidine-tagged wildtype enzyme [17]) [17] S90A (, activity of the mutant enzyme is to low to determine Vmax and Km -value [18]) [18] S90C (, 30fold increase in Km -value for tRNATyr and 4fold increase in Km -value for guanine [18]) [18] S90F (, mutant enzyme has no detectable solubility and reduced solubility [18]) [18] Application medicine (, the enzyme is required for pathogenicity in Shigella flexneri and therefor is a potentially novel antibacterial target [23]) [23]

6 Stability General stability information , linear KCl gradient to elute the enzyme from phosphocellulose results in complete loss of activity [2]

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Storage stability , -80 C, 10% glycerol, 25% loss of activity per month [2]

References [1] Okada, N.; Noguchi, S.; Kasai, H.; Shindo-Okada, N.; Ohgi, T.; Goto, T.; Nishimura, S.: Novel mechanism of post-transcriptional modification of tRNA. Insertion of bases of Q precursors into tRNA by a specific tRNA transglycosylase reaction. J. Biol. Chem., 254, 3067-3073 (1979) [2] Howes, N.K.; Farkas, W.R.: Studies with a homogeneous enzyme from rabbit erythrocytes catalyzing the insertion of guanine into tRNA. J. Biol. Chem., 253, 9082-9087 (1978) [3] Gunduz, U.; Kacar, Y.: Transfer -RNA guanine transglycosylase from Salmonella typhimurium. Period. Biol., 90, 321-326 (1988) [4] Garcia, G.A.; Koch, K.A.; Chong, S.: tRNA-guanine transglycosylase from Escherichia coli. Overexpression, purification and quaternary structure. J. Mol. Biol., 231, 489-497 (1993) [5] Farkas, W.R.; Jacobson, K.B.; Katze, J.R.: Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase. Biochim. Biophys. Acta, 781, 64-75 (1984) [6] Haumont, E.; Droogmans, L.; Grosjean, H.: Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence. Eur. J. Biochem., 168, 219-225 (1987) [7] Walden, T.L.; Howes, N.; Farkas, W.R.: Purification and properties of guanine, queuine-tRNA transglycosylase from wheat germ. J. Biol. Chem., 257, 13218-13222 (1982) [8] Carbon, P.; Haumont, E.; Fournier, M.; de Henau, S.; Grosjean, H.: Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity. EMBO J., 2, 1093-1097 (1983) [9] Shindo-Okada, N.; Okada, N.; Ohgi, T.; Goto, T.; Nishimura, S.: Transfer ribonucleic acid guanine transglycosylase isolated from rat liver. Biochemistry, 19, 395-400 (1980) [10] Kung, F.-L.; Garcia, G.A.: tRNA-guanine transglycosylase from Escherichia coli: recognition of full-length 'queuine-cognate' tRNAs. FEBS Lett., 431, 427-432 (1998) [11] Romier, C.; Meyer, J.E.W.; Suck, D.: Slight sequence variations of a common fold explain the substrate specificities of tRNA-guanine transglycosylases from the three kingdoms. FEBS Lett., 416, 93-98 (1997) [12] Brenk, R.; Naerum, L.; Graedler, U.; Gerber, H.-D.; Garcia, G.A.; Reuter, K.; Stubbs, M.T.; Klebe, G.: Virtual screening for submicromolar leads of tRNA-guanine transglycosylase based on a new unexpected binding mode detected by crystal structure analysis. J. Med. Chem., 46, 1133-1143 (2003) [13] Ishiwata, S.; Katayama, J.; Shindo, H.; Ozawa, Y.; Itoh, K.; Mizugaki, M.: Increased expression of queuosine synthesizing enzyme, tRNA-guanine 261


[14] [15] [16] [17] [18] [19]

[20]

[21] [22]

[23] [24]

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transglycosylase, and queuosine levels in tRNA of leukemic cells. J. Biochem., 129, 13-17 (2001) Romier, C.; Reuter, K.; Suck, D.; Ficner, R.: Crystal structure of tRNA-guanine transglycosylase: RNA modification by base exchange. EMBO J., 15, 2850-2857 (1996) Nonekowski, S.T.; Kung, F.-L.; Garcia, G.A.: The Escherichia coli tRNA-guanine transglycosylase can recognize and modify DNA. J. Biol. Chem., 277, 7178-7182 (2002) Curnow, A.W.; Garcia, G.A.: tRNA-guanine transglycosylase from Escherichia coli. Minimal tRNA structure and sequence requirements for recognition. J. Biol. Chem., 270, 17264-17267 (1995) Kittendorf, J.D.; Barcomb, L.M.; Nonekowski, S.T.; Garcia, G.A.: tRNA-guanine transglycosylase from Escherichia coli: Molecular mechanism and role of aspartate 89. Biochemistry, 40, 14123-14133 (2001) Reuter, K.; Chong, S.; Ullrich, F.; Kersten, H.; Garcia, G.A.: Serine 90 is required for enzymic activity by tRNA-guanine transglycosylase from Escherichia coli. Biochemistry, 33, 7041-7046 (1994) Hoops, G.C.; Townsend, L.B.; Garcia, G.A.: Mechanism-based inactivation of tRNA-guanine transglycosylase from Escherichia coli by 2-amino-5Biochemistry, 34, (fluoromethyl)pyrrolo[2,3-d]pyrimidin-4(3 H)-one. 15539-15544 (1995) Deshpande, K.L.; Seubert, P.H.; Tillman, D.M.; Farkas, W.R.; Katze, J.R.: Cloning and characterization of cDNA encoding the rabbit tRNA-guanine transglycosylase 60-kilodalton subunit. Arch. Biochem. Biophys., 326, 1-7 (1996) Nonekowski, S.T.; Garcia, G.A.: tRNA recognition by tRNA-guanine transglycosylase from escherichia coli: the role of U33 in U-G-U sequence recognition. RNA, 7, 1432-1441 (2001) Kung, F.-L.; Nonekowski, S.; Garcia, G.A.: tRNA-guanine transglycosylase from Escherichia coli: recognition of noncognate-cognate chimeric tRNA and discovery of a novel recognition site within the TyC arm of tRNAPhe . RNA, 6, 233-244 (2000) Goodenough-Lashua, D.M.; Garcia, G.A.: tRNA-guanine transglycosylase from E. coli: a ping-pong kinetic mechanism is consistent with nucleophilic catalysis. Bioorg. Chem., 31, 331-344 (2003) Kittendorf, J.D.; Sgraja, T.; Reuter, K.; Klebe, G.; Garcia, G.A.: An essential role for aspartate 264 in catalysis by tRNA-guanine transglycosylase from Escherichia coli. J. Biol. Chem., 278, 42369-42376 (2003)

NAD+ ADP-ribosyltransferase

2.4.2.30

1 Nomenclature EC number 2.4.2.30 Systematic name NAD+ :poly(adenine-diphosphate-d-ribosyl)-acceptor ADP-d-ribosyltransferase Recommended name NAD+ ADP-ribosyltransferase Synonyms (adenosine diphosphoribose)transferase, nicotinamide adenine dinucleotideprotein ADP-ribosyltransferase ADP-ribosyltransferase (polymerizing) ADPRT [Swissprot] C3 exoenzyme NAD(+) ADP-ribosyltransferase NAD+ :ADP-ribosyltransferase (polymerizing) NAD-protein ADP-ribosyltransferase PARP PARP-related/IaI-related H5/proline-rich PH5P TANK1 TANK2 TRF1-interacting ankyrin-related ADP-ribose polymerase tankyrase-like protein tankyrase-related protein VPARP adenosine diphosphate ribosyltransferase exoenzyme C3 exoenzyme S msPARP pADPRT poly(ADP-ribose) polymerase poly(ADP-ribose) polymerase poly(ADP-ribose) synthase poly(ADP-ribose) transferase poly(ADP-ribosyl)transferase poly[ADP-ribose] synthetase

263


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CAS registry number 58319-92-9

2 Source Organism

Homo sapiens [1, 6, 10] Helix pomatia [2] Dictyostelium discoideum [3] Clostridium botulinum (type C [4]) [4] Mesocricetus auratus [5] Bos taurus (calf [11]) [7, 9, 11] Cryptothecodinium cohnii [8]

3 Reaction and Specificity Catalyzed reaction NAD+ + (ADP-d-ribosyl)n -acceptor = nicotinamide + (ADP-d-ribosyl)n+1 acceptor Reaction type pentosyl group transfer Natural substrates and products S Additional information (, the enzyme modifies eukaryotic 21000-24000 Da GTP-binding proteins [4]; , ADP-ribosylation seems to be involved in regulation of differentiation, the enzyme may be centrally involved in tumorigenic cell transformation, the enzyme appears to be a central controller of cell processes: higher activities shift the cell towards proliferation, low activities shift the cell towards differentiation, role of the enzyme in DNA repair [6]; , cuts produced in vivo on DNA during DNA repair activate the enzyme, which then synthesizes poly(ADP-ribose) on histone H1, in particular, and contributes to the opening of the 25 nm chromatin fiber, resulting in the increased accessibility of DNA to excision repair enzymes [9]; , role of the enzyme in DNA repair, the unmodified polymerase molecules bind tightly to DNA strand breaks: auto-poly(ADP-ribosyl)ation of the protein then effects ist release and allows access to lesions for DNA repair enzymes [10]) [4, 6, 9, 10] P ? Substrates and products S NAD+ + (ADP-d-ribosyl)n -acceptor (, catalyzes poly(ADP-ribosyl)ation of the synthetase itself, automodification [1]; , poly(ADP-ribosyl)ation of histone H1 [1, 3, 9]; , poly(ADP-ribosyl)ation of histone [2, 6, 8]; , poly(ADP-ribosyl)ation of 21000-24000 Da platelet membrane proteins [4]; , poly(ADP-ribosyl)ation of DNA li-

264

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gase [6]; , poly(ADP-ribosyl)ation of topoisomerase I [6]; , poly(ADP-ribosyl)ation of topoisomerase II [6]; , poly(ADP-ribosyl)ation of ADP-ribosyltransferase [6]; , poly(ADP-ribosyl)ation of high mobility group proteins [6]; , poly(ADP-ribosyl)ation of endonuclease [6]; , poly(ADP-ribosyl)ation of terminal deoxynucleotidyltransferase [6]; , auto-poly(ADP-ribosyl)ation [10]) (Reversibility: ? [111]) [1-11] P nicotinamide + (ADP-d-ribosyl)n+1 -acceptor Inhibitors 1(2H)-phthalazinone (, IC50: 0.012 mM [7]) [7] 1,10-phenathroline (, activates in presence of Mg2+ , inhibits in absence of Mg2+ [7]) [7] 1,2-benzopyrone (, IC50: 2.8 mM [7]) [7] 1,3-benzodiazine (, IC50: 2.0 mM [7]) [7] 1,3-dihydroxynaphthalene (, IC50: 1.3 mM [7]) [7] 1,4-benzoquinone (, IC50: 0.4 mM [7]) [7] 1,4-naphthalenedione (, IC50: 0.25 mM [7]) [7] 1,5-dihydroxy-4-phthalazione (, 0.001 mM, 95% inhibition of the 116000 Da enzyme [3]) [3] 1,5-dihydroxyisoquinoline (, IC50: 0.00039 mM [7]) [7] 1,8-naphthalimide (, IC50: 0.0014 mM [7]) [7] 1-hydroxy-2-methyl-4-aminonaphthalene (, IC50: 1.3 mM [7]) [7] 1-hydroxyisoquinoline (, IC50: 0.007 mM [7]) [7] 1-indanone (, IC50: 0.81 mM [7]) [7] 1-methylnicotinamide chloride (, IC50: 3.8 mM [7]; , IC50: 1.7 mM [11]) [7, 11] 2,3-benzodiazine (, IC50: 0.15 mM [7]) [7] 2,3-dichloro-1,4-naphthoquinone (, IC50: 0.26 mM [7]) [7] 2,3-dihydro-1,4-phthalazinedione (, IC50: 0.03 mM [7]) [7] 2,4(1H,3H)-quinazolinedione (, IC50: 0.0081 mM [7]) [7] 2,6-difluorobenzamide (, IC50: 0.18 mM [7]) [7] 2-acetamidobenzamide (, IC50: 1.0 mM [7]) [7] 2-amino-3-chloro-1,4-naphthoquinone (, IC50: 0.82 mM [7]) [7] 2-aminobenzamide (, IC50: 0.65 mM [7]; , IC50: 0.1 mM [11]) [7, 11] 2-bromobenzamide (, IC50: 2.9 mM [7]) [7] 2-chlorobenzamide (, IC50: 1.0 mM [7]) [7] 2-fluorobenzamide (, IC50: 0.12 mM [7]) [7] 2-hydroxy-1,4-naphthoquinone (, IC50: 0.33 mM [7]) [7] 2-hydroxybenzamide (, IC50: 0.82 mM [7]) [7] 2-mercapto-4(3H)-quinazolinone (, IC50: 0.044 mM [7]) [7] 2-methoxybenzamide (, IC50: 0.2 mM [7]) [7] 2-methyl-1,4-benzopyrone (, IC50: 0.045 mM [7]) [7] 2-methyl-1,4-naphthoquinone (, IC50: 0.42 mM [7]) [7] 2-methyl-3-phytyl-1,4-naphthoquinone (, IC50: 0.52 mM [7]) [7] 2-methyl-4(3H)-quinazolinone (, IC50: 0.056 mM [7]) [7]

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2-methylbenzamide (, IC50: 1.5 mM [7]) [7] 2-methylchromone (, IC50: 0.045 mM [7]) [7] 2-nitro-6(5H)-phenanthridione (, IC50: 0.00035 mM [7]) [7] 2-phenylchromone (, IC50: 0.022 mM [7]) [7] 2-trichloromethyl-4(3H)-quinazolinone (, IC50: 2.2 mM [7]) [7] 2H-benz[c]isoquinolin-1-one (, IC50: 0.0003 mM [7]) [7] 2H-benz[de]isoquinoline-1,3-dione (, IC50: 0.0014 mM [7]) [7] 3,4-dihydro-1(2H)-naphthalenone (, IC50: 0.31 mM [7]) [7] 3,5-dibromosalicylamide (, IC50: 0.56 mM [7]) [7] 3,5-dimethoxybenzamide (, IC50: 1.2 mM [7]) [7] 3,5-dinitrobenzamide (, IC50: 2.5 mM [7]) [7] 3-(N,N-dimethylamino)benzamide (, IC50: 0.12 mM [7]) [7] 3-acetamidobenzamide (, IC50: 0.012 mM [7]) [7] 3-acetamidosalicylamide (, IC50: 2.0 mM [7]) [7] 3-aminobenzamide (, 1 mM, 99% inhibition [1]; , 1 mM, 98% inhibition [2]; , IC50: 0.33 mM [7]; , IC50: 0.0054 [11]) [1, 2, 3, 7, 11] 3-aminobenzoic acid (, 1 mM, 12% inhibition [1]) [1] 3-aminophthalhydrazide (, 0.1 mM, 98% inhibition of the 116000 Da enzyme [3]; , IC50: 0.023 mM [7]) [3, 7] 3-bromobenzamide (, IC50: 0.055 mM [7]) [7] 3-chlorobenzamide (, IC50: 0.22 mM [7]) [7] 3-fluorobenzamide (, IC50: 0.2 mM [7]) [7] 3-guanidinobenzamide (, 0.1 mM, 71% inhibition of the 116000 Da enzyme [3]) [3] 3-hydroxybenzamide (, 0.1 mM, 89% inhibition of the 116000 Da enzyme [3]; , IC50: 0.0091 mM [7]) [3, 7] 3-isobutyl-1-methylxanthine (, IC50: 3.1 mM [11]) [11] 3-methoxybenzamide (, 1 mM, 98% inhibition [2]; , 0.01 mM, 85% inhibition of the 116000 Da enzyme, 84% inhibition of the 90000 Da enzyme [3]; , 1 mM, 96% inhibition of the 116000 Da enzyme, 95% inhibition of the 90000 Da enzyme [3]; , IC50: 0.017 mM [7]; , IC50: 0.0034 mM [11]) [2, 3, 7, 11] 3-methylbenzamide (, IC50: 0.19 mM [7]) [7] 3-nitrobenzamide (, IC50: 0.16 mM [7]) [7] 3-nitrophthalhydrazide (, IC50: 0.072 mM [7]) [7] 3-nitrosalicylamide (, IC50: 1.6 mM [7]) [7] 4,8-dihydroxy-2-quinolinecarboxylic acid (, IC50: 0.19 mM [7]) [7] 4-amino-1,8-naphthalimide (, IC50: 0.00018 mM [7]) [7] 4-aminobenzamide (, IC50: 1.8 mM [7]; , IC50: 0.4 mM [11]) [7, 11] 4-aminophthalhydrazide (, IC50: 0.29 mM [7]) [7] 4-bromobenzamide (, IC50: 2.2 mM [7]) [7] 4-chlorobenzamide (, IC50: 0.3 mM [7]) [7] 4-chromanone (, IC50: 0.72 mM [7]) [7] 4-fluorobenzamide (, IC50: 0.2 mM [7]) [7] 4-hydroxy-2-methylquinoline (, IC50: 0.074 mM [7]) [7] 4-hydroxy-2-quinolinecarboxylic acid (, IC50: 0.67 mM [7]) [7] 266

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4-hydroxybenzamide (, IC50: 0.28 mM [7]) [7] 4-hydroxycoumarin (, IC50: 0.57 mM [7]) [7] 4-hydroxypyridine (, IC50: 2.3 mM [7]) [7] 4-hydroxyquinazoline (, IC50: 0.0095 mM [7]) [7] 4-hydroxyquinoline (, IC50: 0.08 mM [7]) [7] 4-methoxybenzamide (, IC50: 1.1 mM [7]) [7] 4-methylbenzamide (, IC50: 1.8 mM [7]) [7] 4-nitrophthalhydrazide (, IC50: 0.51 mM [7]) [7] 5-acetamidosalicylamide (, IC50: 0.045 mM [7]) [7] 5-aminosalicylamide (, IC50: 0.1 mM [7]) [7] 5-bromodeoxyuridine (, IC50: 0.015 mM [11]) [11] 5-bromouracil (, IC50: 0.16 mM [7]) [7] 5-bromouridine (, IC50: 0.21 mM [7]) [7] 5-chlorosalicylamide (, IC50: 0.19 mM [7]) [7] 5-chlorouracil (, IC50: 0.27 mM [7]) [7] 5-hydroxy-1,4-naphthoquinone (, IC50: 0.25 mM [7]) [7] 5-hydroxy-2-methyl-1,4-naphthoquinone (, IC50: 0.7 mM [7]) [7] 5-iodouracil (, IC50: 0.071 mM [7]) [7] 5-iodouridine (, IC50: 0.043 mM [7]) [7] 5-methylnicotinamide (, IC50: 0.35 mM [7]; , 0.15 mM, 31% inhibition [8]; , IC50: 0.07 [11]) [7, 8, 11] 5-methyluracil (, IC50: 0.29 mM [7]) [7] 5-nitrouracil (, IC50: 0.43 mM [7]) [7] 6(5H)-phenanthridinone (, IC50: 0.0003 mM [7]) [7] 6-aminocoumarin (, IC50: 0.85 mM [7]) [7] 6-aminonicotinamide (, IC50: 1.1 mM [7]) [7] 8-acetamidocarsalam (, IC50: 1.4 mM [7]) [7] 8-methylnicotinamide (, IC50: 7.8 mM [11]) [11] Cu2+ [1] EDTA (, 5 mM, 41% inhibition in presence of Mg2+ , 2% inhibition in absence of Mg2+ [7]) [7] GTP(gS) (, in presence of Mg2+ [3]) [3] Hg2+ [1] KCl (, 100 mM, 85% inhibition of the 90000 Da enzyme [3]) [3] N-(2-chloroethyl)1,8-naphthalamide (, IC50: above 1.8 mM [7]) [7] N-hydroxynaphthalimide sodium salt (, IC50: 0.45 mM [7]) [7] NEM (, 0.5 mM [5]) [5] PCMB (, complete [1]) [1] Zn2+ (, ZnCl2 , IC50: 0.077 mM [11]) [1, 11] acetophenone (, IC50: 2.3 mM [7]) [7] all-trans-retinal (, IC50: 0.45 mM [7]) [7] a-NAD+ (, 0.5 mM, 40% inhibition of the 116000 Da enzyme, 44% inhibition of the 90000 Da enzyme [3]) [3] a-picolinamide (, IC50: 0.25 mM [7]) [7] arachidonic acid [7] benzamide (, IC50: 0.22 mM [7]; , 0.15 mM, 48% inhibition [8]; , IC50: 0.0033 mM [11]) [7, 8, 11] 267


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benzoyleneurea (, IC50: 0.0081 mM [7]) [7] caffeine (, 1 mM, 23% inhibition [2]; , 1 mM, 86% inhibition of the 116000 Da enzyme, 57% inhibition of the 90000 Da enzyme [3]; , IC50: 1.4 mM [11]) [2, 3, 11] carbonylsalicylamide (, IC50: 0.46 mM [7]) [7] carsalam (, 5 mM, 88% inhibition in presence of Mg2+ , 68% inhibition in absence of Mg2+ [7]) [7] chlorthenoxazin (, IC50: 0.0085 mM [7]) [7] chromone-2-carboxylic acid (, IC50: 0.56 mM [7]) [7] cyclohexanecarboxamide (, IC50: 0.62 mM [7]) [7] flavone (, IC50: 0.022 mM [7]) [7] g-linolenic acid (, IC50: 0.12 mM [7]) [7] hypoxanthine (, IC50: 1.7 mM [11]) [11] isonicotinamide (, IC50: 0.99 mM [7]) [7] isonicotinate hydrazide (, IC50: 4.8 MM [11]) [11] isoquinoline (, 5 mM, 47% inhibition in presence of Mg2+ , 34% inhibition in absence of Mg2+ [7]) [7] linoleic acid (, IC50: 0.048 mM [7]) [7] linolenic acid (, IC50: 0.11 mM [7]) [7] m-acetamidoacetophenone (, IC50: 0.93 mM [7]) [7] m-aminoacetophenone (, IC50: 1.9 mM [7]) [7] m-hydroxyacetophenone (, IC50: 0.6 mM [7]) [7] m-phthalamide (, IC50: 0.05 mM [7]) [7] menadione sodium bisulfite (, IC50: 0.72 mM [7]) [7] nicotinamide (, 1 mM, 93% inhibition [1]; , 1 mM, 91% inhibition [2]; , 1 mM, 96% inhibition of the 116000 Da enzyme, 95% inhibition of the 90000 Da enzyme [3]; , IC50: 0.21 mM [7]; , 0.15 mM, 25% inhibition [8]; , IC50: 0.031 mM [11]) [1, 2, 3, 5, 7, 8, 11] norharman (, IC50: 4.7 mM [7]) [7] novobiocin (, IC50: 2.2 mM, 5 mM, 90% inhibition in presence of Mg2+ , 59% inhibition in absence of Mg2+ [7]) [7] oleic acid (, IC50: 0.082 mM [7]) [7] palmitoleic acid (, IC50: 0.095 mM [7]) [7] phthalamide (, IC50: 1.0 mM [7]) [7] phthalazine (, 5 mM, 91% inhibition in presence of Mg2+ , 79% inhibition in absence of Mg2+ [7]) [7] pyrazinamide (, IC50: 0.13 mM [11]) [11] quinazoline (, 5 mM, 63% inhibition in presence of Mg2+ , 50% inhibition in absence of Mg2+ [7]) [7] reserpine (, IC50: 0.79 mM [7]) [7] theobromine (, 1 mM, 76% inhibition [2]; , IC50: 0.11 mM [11]) [2, 11] theophylline (, 1 mM, 72% inhibition [2]; , 1 mM, 68% inhibition of the 116000 Da enzyme, 39% inhibition of the 90000 Da enzyme [3]; , 0.15 mM, 62% inhibition [8]; , IC50: 0.0046 [11]) [2, 3, 8, 11] thiobenzamide (, IC50: 0.62 mM [7]) [7]

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thionicotinamide (, IC50: 1.8 mM [7]) [7] thymidine (, 1 mM, 70% inhibition [2]; , 1 mM, 94% inhibition of the 116000 Da enzyme, 88% inhibition of the 90000 Da enzyme [3]; , IC50: 0.18 mM [7]; , 0.15 mM, 39% inhibition [8]; , IC50: 0.043 [11]) [2, 3, 5, 7, 8, 11] trans-decahydro-1-naphthalenone (, IC50: 4.3 mM [7]) [7] vitamin K1 (, IC50: 0.0019 mM [7]) [7] vitamin K3 (, IC50: 0.42 mM [7]) [7] xanthurenic acid (, 5 mM, 88% inhibition in presence of Mg2+ , 65% inhibition in absence of Mg2+ [7]) [7] Activating compounds 1,10-phenanthroline (, activates in presence of Mg2+ , inhibits in absence of Mg2+ [7]) [7] ATP (, 5-10 mM, 20-30% stimulation [5]) [5] DNA (, absolute requirement [1,3]; , required [8]; , the enzyme is completely dependent on the presence of DNA containing single or double stranded breaks. Activation results in a decondensation of chromatin superstructure in vitro, which is caused mainly by hyper(ADP-ribosyl)ation of histone H1 [9]; , enzyme has an N-terminal binding domain [10]) [1, 3, 8, 9, 10] GDP (, increases activity in absence of Mg2+ [4]) [4] GTP (, increases activity in absence of Mg2+ [4]) [4] GTP(gS) (, increases activity in absence of Mg2+ [4]) [4] harmaline hydrochloride (, activates more strongly in absence of Mg2+ than in presence of Mg2+ [7]) [7] phthalic acid (, activates more strongly in absence of Mg2+ than in presence of Mg2+ [7]) [7] Metals, ions Ba2+ (, enhances activity [1]) [1] Mg2+ (, enhances both the automodification and poly(ADP-ribosyl)ation of histone H1 [1]; , divalent cation required, Mg2+ preferred to Mn2+ , optimal concentration 5 mM [5]; , activates [7]) [1, 5, 7] Mn2+ (, divalent cation required, Mg2+ preferred to Mn2+ [5]) [5] Sr2+ (, enhances activity [1]) [1] Specific activity (U/mg) 0.0017 [3] 0.0545 [2] 0.325 [6] 1.02 [1] Km-Value (mM) 0.002 (NAD+ ) [4] 0.02 (NAD+, , 90000 Da protein [3]) [3] 0.0267 (NAD+ ) [2]

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0.077 (NAD+, , 116000 Da protein [3]) [3] Additional information (, kinetics for NAD+ utilization are not Michaelis-Menten type [5]) [5] pH-Optimum 8 (, in presence of DNA and histone H1 [3]) [2, 3, 5] 8.5 (, reaction with histone in presence of DNA, 0.1 M glycine-NaOH buffer [1]) [1] 8.7 (, reaction with histone in presence of DNA, 0.1 M Tris-HCl buffer [1]) [1] Temperature optimum ( C) 5 (, 116000 Da enzyme [3]) [3] 10 (, 90000 Da enzyme [3]) [3] 18 [2] 25 [5]

4 Enzyme Structure Subunits ? (, x * 25000, SDS-PAGE [4]; , 1 * 90000, two active proteins are detected: MW 90000 Da and 116000 Da, SDS-PAGE [3]; , 1 * 116000, two active proteins are detected: MW 90000 Da and 116000 Da, SDS-PAGE [3]) [3, 4] monomer (, 1 * 116000, SDS-PAGE [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue BHK-21 cell [5] foot [2] placenta [1] thymus [7, , 11] Localization cytosol (, 10% of the activity [5]) [5] nucleus (, 90% of the activity, chromatin-bound [5]) [5, 6, 9] polysome [6] Purification [1, 6] (partial [2]) [2] [3] [4] [8]

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6 Stability General stability information , unstable in absence of Mg2+ [5]

References [1] Ushiro, H.; Yokoyama, Y.; Shizuta, Y.: Purification and characterization of poly (ADP-ribose) synthetase from human placenta. J. Biol. Chem., 262, 2352-2357 (1987) [2] Burtscher, H.J.; Klocker, H.; Schneider, R.; Auer, B.; Hirsch-Kauffmann, M.; Schweiger, M.: ADP-ribosyltransferase from Helix pomatia. Purification and characterization. Biochem. J., 248, 859-864 (1987) [3] Kofler, B.; Wallraff, E.; Herzog, H.; Schneider, R.; Auer, B.; Schweiger, M.: Purification and characterization of NAD+ :ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum. Biochem. J., 293, 275-281 (1993) [4] Aktories, K.; Roesener, S.; Blaschke, U.; Chhatwal, G.S.: Botulinum ADPribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes. Eur. J. Biochem., 172, 445-450 (1988) [5] Furneaux, H.M.; Pearson, C.K.: Adenosine diphosphate ribose transferase from baby-hamster kidney cells (BHK-21/C13). Characterization of the reaction and product. Biochem. J., 187, 91-103 (1980) [6] Schweiger, M.; Auer, B.; Burtscher, H.J.; Hirsch-Kauffmann, M.; Klocker, H.; Schneider, R.: The Fritz-Lipmann lecture. DNA repair in human cells. Biochemistry of the hereditary diseases Fanconis anaemia and Cockayne syndrome. Eur. J. Biochem., 165, 235-242 (1987) [7] Banasik, M.; Komura, H.; Shimoyama, M.; Ueda, K.: Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase. J. Biol. Chem., 267, 1569-1575 (1992) [8] Werner, E.; Sohst, S.; Gropp, F.; Simon, D.; Wagner, H.; Kroeger, H.: Presence of poly (ADP-ribose) polymerase and poly (ADP-ribose) glycohydrolase in the dinoflagellate Crypthecodinium cohnii. Eur. J. Biochem., 139, 8186 (1984) [9] De Murcia, G.; Huletsky, A.; Poirier, G.G.: Review: Modulation of chromatin structure by poly(ADP-ribosyl)ation. Biochem. Cell Biol., 66, 626-635 (1987) [10] Satoh, M.S.; Lindahl, T.: Role of poly(ADP-ribose) formation in DNA repair. Nature, 356, 356-358 (1992) [11] Rankin, P.W.; Jacobson, E.L.; benjamin, R.C.; Moss, J.; Jacobson, M.K.: Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo. J. Biol. Chem., 264, 4321-4317 (1989)

271

NAD(P)+ -Arginine ADP-ribosyltransferase

2.4.2.31

1 Nomenclature EC number 2.4.2.31 Systematic name NAD(P)+ :l-arginine ADP-d-ribosyltransferase Recommended name NAD(P)+ -arginine ADP-ribosyltransferase Synonyms (adenosine diphosphoribose)transferase, nicotinamide adenine dinucleotidearginine ADP-ribosyltransferase ADPRT ART AT1 AT2 Dombrock blood group carrier molecule NAD(P)+ -arginine ADP-ribosyltransferase NAD+ :l-arginine ADP-d-ribosyltransferase NAD+ :arginine ADP-ribosyltransferase NAD+ :arginine ecto-mono(ADP-ribosyl)transferase NAD-arginine ADP-ribosyltransferase NAD-arginine mono-ADP-ribosyltransferase B NAD-dependent ADPribosyltransferase NAD:arginine ADP-ribosyltransferase B RT6 RT6.1 RT6.2 T-cell surface protein Rt6.1 T-cell surface protein Rt6.2 alloantigen Rt6.1 alloantigen Rt6.2 arginine specific ADP-ribosyltransferase arginine specific mono-ADP-ribosyltransferase arginine-specific mono-ADP-ribosyltransferase mono(ADP-ribosyl)transferase Additional information (cf. EC 2.4.2.36)

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NAD(P)+-Arginine ADP-ribosyltransferase

CAS registry number 81457-93-4

2 Source Organism Meleagris gallopavo [1, 2, 3, 4, 6, 7, 8, 9, 15] Gallus gallus (2 enzyme forms: AT1 and AT2 [17]) [5, 17, 22, 25, 26, 28, 30, 33] Oryctolagus cuniculus (2 enzyme forms: enzyme type a and enzyme type b [11]) [10, 11, 12, 15, 16, 22, 31, 33] phage T4 (E. coli B/r infected with phage T4 [13]) [13, 14] Sus scrofa [16] Rattus norvegicus [16, 18, 19, 27] Canis sp. [16] Mus musculus [16, 18, 19, 20, 22, 27, 29, 31, 32] Corturnix sp. [16] Pseudomonas aeruginosa [21] Homo sapiens [22, 23, 24, 27, 31] Bos taurus [23]

3 Reaction and Specificity Catalyzed reaction NAD+ + l-arginine = nicotinamide + N2 -(ADP-d-ribosyl)-l-arginine (, rapid equilibrium random sequential mechanism [8]) Reaction type pentosyl group transfer Natural substrates and products S FGF-2 + NAD+ (, ADP-ribosylation of FGF-2 may provide an additional level of control of FGF-2 activity [23]) [23] P ? S actin + NAD+ (, the ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration [5]) [5] P nicotinamide + ? S Additional information (, the enzyme might be involved in regulating T cell and immune system activity [19]; , regulation of cytotoxic T cell functions [29]; , enzyme is involved in posttranslational modification of proteins [31]; , involved in immune regulation [31]) [19, 29, 31] P ?

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Substrates and products S A1 peptide of cholera toxin + NAD+ (, four mol of ADP-ribose are incorporated into 1 mol of A1 peptide. Modification of the A1 peptide increases its enzymatic activity up to 4fold [26]) (Reversibility: ? [26]) [26] P nicotinamide + ? S DNase I + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S P33 + NAD+ (, preferential endogenous acceptor [33]; , no activity with the enzyme from skeletal muscle sarcoplasmic reticulum [33]) (Reversibility: ? [33]) [33] P nicotinamide + ? S RNA polymerase + NAD+ (, a-subunit of RNA polymerase [13]; , E. coli RNA polymerase [14]) (Reversibility: ir [13]) [13, 14] P Additional information (, the amino acid carrying the ADP-ribosyl residue appears to be arginine [13]) [13] S Ras + NAD+ (, ADP-ribosylation of Ras at Arg41 and Arg 128. The double mutant RasR141K/R128K is ribosylated at an alternative site Arg135 [21]) (Reversibility: ? [21]) [21] P nicotinamide + ? S actin + NAD+ (, non-muscle b/g actin, skeletal muscle a actin, smooth muscle g actin [5]; , a-actin and b/g-actin [33]; , no activity with the enzyme from skeletal muscle sarcoplasmic reticulum [33]) (Reversibility: ? [5,33]) [5, 33] P nicotinamide + ? S agmatine + NAD+ (Reversibility: ? [1, 7, 8, 24]) [1, 7, 8, 22, 24] P nicotinamide + ? S arginine + NAD+ (Reversibility: ? [1]) [1] P nicotinamide + ADP-ribose-l-arginine S arginine methyl ester + NAD+ (Reversibility: ? [1,11]) [1, 11] P nicotinamide + N2 -(ADP-d-ribosyl)-l-arginine [1] S arginine methyl ester + NADP+ (Reversibility: ? [1,2,3,4]) [1, 2, 3, 4] P nicotinamide phosphate + N2 -(ADP-d-ribosyl)-l-arginine S basic fibroblast growth factor FGF-2 + NAD+ (Reversibility: ? [23]) [23] P nicotinamide + ? S b-lactoglobulin + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S bovine plasma albumin + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S bovine serum albumin + NAD+ (, poor ADP-ribose acceptor [3]) (Reversibility: ? [3]) [3] P nicotinamide + ? S casein + NAD+ (Reversibility: ? [33]) [33] P nicoitinamide + ? 274

2.4.2.31


S diethylamino-(benzylidineamino)guanidine + NAD+ (Reversibility: ? [24]) [24] P nicotinamide + ? S guanidine + NAD+ (Reversibility: ? [1]) [1] P nicotinamide + ? S guanidinobutyrate + NAD+ (Reversibility: ? [1]) [1] P nicotinamide + ? S guanidinopropionate + NAD+ (Reversibility: ? [1]) [1] P nicotinamide + ? S histone + NAD+ (Reversibility: ? [3, 19]) [3, 19, 28] P nicotinamide + ? S histone f1 + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S histone f2a + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S histone f2b + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S histone f3 + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S human a-globulin + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S human b-globulin + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S human g-globulin + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S lysozyme + NAD+ (Reversibility: ? [3, 6, 14]) [3, 6, 14] P nicotinamide + ? S ovalbumin + NAD+ (, poor ADP-ribose acceptor [3]) (Reversibility: ? [3,6]) [3, 6] P nicotinamide + ? S p-nitrobenzylidine aminoguanidine + NAD+ (Reversibility: ? [15]) [15] P nicotinamide + mono-ADP-ribosylated p-nitrobenzylidine aminoguanidine [15] S polyarginine + NAD+ (Reversibility: ir [13]; ? [3, 6, 28, 33]) [3, 6, 13, 28, 33] P nicotinamide + ? S polylysine + NAD+ (, poor ADP-ribose acceptor [3]) (Reversibility: ? [3]) [3] P nicotinamide + ? S trypsin inhibitor + NAD+ (Reversibility: ? [6]) [6] P nicotinamide + ? S Additional information (, enzyme catalyzes auto-ADP ribosylation [19]; , in contrast to Yac-1, the Yac-2 enzyme has significant NAD glycohydrolase activity and may preferentially hydrolyze NAD+ [31]) [19, 31] P ? 275


2.4.2.31

Inhibitors 1,2-naphthoquinone [28] 1,4-naphthoquinone [28] 2-mercaptoethanol (, marked decrease in activity, NAD+ and dithiothreitol protects [11]) [11] 4-amino-1-naphthol [28] 5,8-dihydroxy-1,4-naphthoquinone [28] AMP (, 7.8 mM, decreases activity to 78% of the uninhibited control [13]) [13] K2 HPO4 (, IC50: 50 mM [33]; , IC50: 40 mM [33]) [33] NEM (, inhibition of RT6.1, no affect on Rt6.2 and Glu207 mutant of RR6.1 [32]) [32] NaCl (, IC50: 50 mM [33]; , IC50: 100 mM [33]; , inhibits AT1 transferase [17]) [17, 33] dithiothreitol (, 20% inhibition by 5 mM, 25% inhibition by 10 mM [10]) [10] glycerol [1] nicotinamide [2, 8, 14] novobiocin (, IC50: 0.2 mM [33]; , IC50: 0.4 mM [33]) [33] theophylline [2] thymidine [2] Cofactors/prosthetic groups NAD+ (, NAD+ is preferred over NADP+, NAD:arginine ADP-ribosyltransferase A [4]) [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] NADP+ (, NAD+ is preferred over NADP+, NAD:arginine ADP-ribosyltransferase A [4]) [1, 4] Activating compounds 2-mercaptoethanol (, required by AT1 transferase, activates AT2 transferase [17]) [17] ATP (, half-maximal stimulation with 2.5 mM [6]) [6] App(NH)p (, 10 mM, stimulates [6]) [6] Br- (, activates NAD:arginine ADP-ribosyltransferase A [4]) [4] CHAPS (, activates [7]) [7] Cl- (, activates NAD:arginine ADP-ribosyltransferase A, NaCl is maximally effective at 250 mM [4]) [4] DNA (, enhances activity [33]) [33] F- (, activates NAD:arginine ADP-ribosyltransferase A [4]) [4] GTP (, 10 mM, stimulates [6]) [6] NAD+ (, excess [23]) [23] PO34- (, activates NAD:arginine ADP-ribosyltransferase A [4]) [4] SCN- (, activates NAD:arginine ADP-ribosyltransferase A, NaSCN is maximally effective at 100 mM [4]) [4] Triton X-100 (, enhances activity [7]) [7] Triton X-114 (, enhances activity [7]) [7]

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Triton X-305 (, enhances activity [7]) [7] Tween 20 (, enhances activity [7]) [7] dithiothreitol (, activity of RT6.1 increases fivefold, no effect on activity of RT6.2. Cys201 confers thiol sensitivity [32]) [32] heparin [23] histone (, increases activity in absence of salt [4]) [4] lysolecithin (, stimulates 4-6fold in the order of declining effectiveness: C14 , C12 , C10 , C8 [7]) [7] lysophosphatidylcholine (, increases activity in the order of declining effectiveness C18 , C16 , C14 , C12 , C10 , C8 , NAD:arginine ADP-ribosyltransferase A [4]) [4] tetrapolyphosphate (, 10 mM, stimulates [6]) [6] tripolyphosphate (, 10 mM, stimulates [6]) [6] Additional information (, more than 85% of the enzyme activity is lost within 1 min Vortex-mixing at room temperature of dilute enzyme. When the less-active form of the enzyme is treated with 10 mM dithiothreitol plus 0.2 M NaCl under anaerobic conditions, more than 50% of the enzyme activity is restored [25]) [25] Metals, ions NaCl (, activates AT2 transferase [17]) [17] Specific activity (U/mg) 1.32 [33] 7.6 (, NAD:arginine ADP-ribosyltransferase C [9]) [9] 14 [12] 53 (, NAD:arginine ADP-ribosyltransferase B [4]) [3, 4] 353 (, NAD:arginine ADP-ribosyltransferase A [4]) [2, 4] Additional information (, easy assay procedure [15]) [1, 13, 15] Km-Value (mM) 0.0025 (a-actin) [5] 0.007 (NAD+ ) [8] 0.01 (g-actin) [5] 0.014 (NAD+, , in presence of lysolecithin [7]) [7] 0.0143 (NAD+ ) [13] 0.015 (NAD+, , NAD:arginine ADP-ribosyltransferase A [4]; , NAD:arginine ADP-ribosyltransferase C [9]) [4, 9] 0.015 (b/g-actin) [5] 0.02 (NAD+, , reaction with g-actin [5]) [5] 0.025 (NAD+, , in presence of NaCl [7]) [7] 0.03 (NAD+, , reaction with b/g-actin [5]) [1, 5] 0.035 (NAD+, , reaction with a-actin [5]) [5] 0.036 (NAD+, , NAD:arginine ADP-ribosyltransferase B [4]) [3, 4] 0.1 (NAD+ ) [10, 24] 0.118 (NAD, , reaction with 20 mM agmatine, enzyme Yac-1 [31]) [31]

277


2.4.2.31

0.13 (NAD+ ) [28] 0.142 (NAD+, , reaction with 20 mM agmatine, enzyme Yac-2 [31]) [31] 0.2 (NAD+ ) [33] 0.26 (agamatine) [8] 0.33 (NAD+ ) [16] 1 (agmatine) [7] 1.3 (arginine methyl ester, , in presence of 250 mM NaCl, NAD:arginine ADP-ribosyltransferase A [4]) [4] 2 (agmatine, , NAD:arginine ADP-ribosyltransferase A [9]) [9] 3 (arginine methyl ester, , NAD:arginine ADP-ribosyltransferase B [4]) [3, 4] 9.4 (agmatine, , enzyme Yac-1 [31]) [31] 15 (agamatine, , enzyme Yac-2 [31]) [31] Additional information (, Km value for poly(L-arginine) is 0.5 mg/ ml [33]; , Km -value for poly(L-arginine) is 0.04 mg/ml [33]) [33] Ki-Value (mM) 0.0049 (1,4-naphthoquinone) [28] 0.37 (nicotinamide) [8] 4 (nicotinamide) [13] pH-Optimum 7-7.5 [16] 7-8.5 [16] 7.5 [13] 8 [28] 8.5 [5, 6] 9 [33] pH-Range 6-9 (, pH 6: about 50% of maximal activity, pH 9: about 80% of maximal activity [13]) [13] Temperature optimum ( C) 20 [13] Temperature range ( C) 20-37 (, 20 C: optimum, 37 C: about 35% of maximal activity [13]) [13]

278

2.4.2.31


4 Enzyme Structure Molecular weight 25300 (, NAD:arginine ADP-ribosyltransferase A, gel filtration [4]) [4] 25500 (, NAD:arginine ADP-ribosyltransferase A, gel filtration [9]) [9] 26000 (, NAD:arginine ADP-ribosyltransferase C, gel filtration [9]; , gel filtration [13]) [9, 13] 27500 (, gel filtration [28]) [28] 32000 (, gel filtration [3]) [3] Subunits ? (, x * 27500, SDS-PAGE [33]; , x * 28300, SDS-PAGE [2]; , x * 32000, AT1 transferase, SDS-PAGE [17]; , x * 32000, NAD:arginine ADP-ribosyltransferase B, SDS-PAGE [4]; , x * 34000, AT2 transferase, SDS-PAGE [17]; , x * 35318, AT1 transferase, calculation from nucleotide sequence [17]; , x * 36134, calculation from nucleotide sequence [12]; , x * 38500, enzyme type b, SDS-PAGE [11]; , x * 39000, enzyme type a, SDS-PSGE [11]; , x * 40000, SDS-PAGE [33]; , x * 42000, SDSPAGE under non-reducing conditions [30]; , x * 44000, SDS-PAGE under reducing conditions [30]) [2, 4, 11, 17, 30, 33] monomer (, 1 * 28000, SDS-PAGE [28]; , 1 * 28000, NAD:arginine ADP-ribosyltransferase A, SDS-PAGE [4]; , x * 32000, SDS-PAGE [3]) [3, 4, 28] Posttranslational modification glycoprotein (, N-glycosyl modification [30]) [30]

5 Isolation/Preparation/Mutation/Application Source/tissue T-lymphocyte (, surface of mature T-lymphocytes [18]) [18] aortic endothelium ( aortic arch endothelium [23]) [23] bone marrow [17] erythroblast [28] erythrocyte (, very low activity [17]) [1, 2, 3, 4, 6, 7, 8, 9, 15, 17, 22, 28] fetus (, low level [20]) [20] granulocyte ( peripheral polymorphonuclear [25,33]) [25, 33] heart (, low activity [17]) [16, 17, 20] hepatoma cell [23] intestine [18] leukocyte (, high activity [17]) [17] liver (, low level [20]) [17, 20] lung (, low level [20]) [17, 18]

279


2.4.2.31

lymphocyte [31] lymphoma cell [22, 27] polymorphonuclear leucocyte [24, 26] skeletal muscle (, low activity [17]) [10, 11, 12, 15, 17, 20, 27, 31, 33] spleen (, low level [20]) [17, 18, 27, 30] splenocyte (, from BALB/c mouse [32]) [32] testis [27] thymus [18] Localization membrane (, associated with [12]; , anchored to membranes by glycosylphosphatidylinositol [16, 30, 31, 32]; , Yac-1 enzyme is anchored to membranes by glycosylphosphatidylinositol [31]; , glycosylphosphatidylinositol-anchored membrane protein [22]; , Yac-2 enzyme is membrane-bound but appears not to be glycosylphosphatidylinositol-anchored [22, 31]; , the enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol [24]) [12, 16, 20, 22, 30, 31, 32] microsome [11] nucleus (, 98% of NAD:arginine ADP-ribosyltransferase A is located to the nucleus [9]) [9] plasma membrane (, NAD:arginine ADP-ribosyltransferase C is enriched 11fold in membranes over nuclei [9]) [9] sarcoplasmic reticulum [10, 33] Additional information (, Yac-1 enzyme may have a signal peptide and may be secreted [22]; , the enzyme displays a hydrophobic amino terminus consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus suggesting that the protein is destined for export [27]) [22, 27] Purification (NAD:arginine ADP-ribosyltransferase B [3, 4]; NAD:arginine ADP-ribosyltransferase A [4,9]; NAD:arginine ADP-ribosyltransferase C [9]) [2, 3, 4, 9, 10] [25, 28, 30] (partial [10]; enzyme type a and enzyme type b [11]) [10, 11, 12, 33] [13, 14] Cloning (in COS 7 cells transiently transfected with AT1 cDNA activity is detected in the culture medium. In COS 7 cells transfected with AT2 CDNA activity is found in both culture medium and cell lysate [17]; expression in Escherichia coli [28]) [17, 28] (expression in Escherichia coli [12]) [12, 31]

280

2.4.2.31


(At6-1 and RT6-2 cDNA [18]; expression of Art 1 in 293T cells as a recombinant fusion protein with the Fc portion of human IgG1 [20]; ART5 [27]; Yac-1 and Yac-2 enzymes are expressed as glutathione S-transferase fusion proteins in Escherichia coli [22]) [18, 20, 22, 27, 31, 32] [31] Engineering C201F (, mutant enzyme of RT6.1, mutant enzyme loses thiol-dependency [32]) [32] C80S (, mutant enzyme of RT6.1 remains thiol-dependent [32]) [32]

6 Stability General stability information , both propylene glycol and NaCl stabilize [2] , highly unstable both in routine storage and in assay, can be stabilized by addition of 30-50% glycerol to the storage buffer and by addition of nontransferase protein to the assay [1] , more than 85% of the enzyme activity is lost within 1 min Vortex-mixing at room temperature of dilute enzyme. When the less-active form of the enzyme is treated with 10 mM dithiothreitol plus 0.2 M NaCl under anaerobic conditions, more than 50% of the enzyme activity is restored [25] , unstable during dialysis, loss of activity can be strongly reduced by using buffers containing 40% glycerol [13] Storage stability , 0-4 C, 50 mM sodium phosphate, pH 7.1, 100 mM NaCl, NAD:arginine ADP-ribosyltransferase B, stable [4]

References [1] Moss, J.; Stanley, S.J.; Oppenheimer, N.J.: Substrate specificity and partial purification of a stereospecific NAD- and guanidine-dependent ADP-ribosyltransferase from avian erythrocytes. J. Biol. Chem., 254, 8891-8894 (1979) [2] Moss, J.; Stanley, S.J.; Watkins, P.A.: Isolation and properties of an NADand guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes. J. Biol. Chem., 255, 5838-5840 (1980) [3] Yost, D.A.; Moss, J.: Amino acid-specific ADP-ribosylation. Evidence for two distinct NAD:arginine ADP-ribosyltransferases in turkey erythrocytes. J. Biol. Chem., 258, 4926-4929 (1983) [4] Moss, J.; Vaughan, M.: NAD: arginine mono-ADP-ribosyltransferases from animal cells. Methods Enzymol., 106, 430-437 (1984) [5] Terashima, M.; Mishima, K.; Yamada, K.; Wakutani, T.; Shimoyama, M.: ADP-ribosylation of actins by arginine-specific ADP-ribosyltransferase purified from chicken heterophils. Eur. J. Biochem., 204, 305-311 (1992) 281


2.4.2.31

[6] Watkins, P.A.; Moss, J.: Effects of nucleotides on activity of a purified ADPribosyltransferase from turkey erythrocytes. Arch. Biochem. Biophys., 216, 74-80 (1982) [7] Moss, J.; Osborne, J.C.; Stanley, S.J.: Activation of an erythrocyte NAD:arginine ADP-ribosyltransferase by lysolecithin and nonionic and zwitterionic detergents. Biochemistry, 23, 1353-1357 (1984) [8] Osborne, J.C.; Stanley, S.J.; Moss, J.: Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: the soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cholerae. Biochemistry, 24, 5235-5240 (1985) [9] West, R.E.; Moss, J.: Amino acid specific ADP-ribosylation: specific NAD: arginine mono-ADP-ribosyltransferases associated with turkey erythrocyte nuclei and plasma membranes. Biochemistry, 25, 8057-8062 (1986) [10] Taniguchi, M.; Tanigawa, Y.; Tsuchiya, M.; Mishima, K.; Obara, S.; Yamada, K.; Shimoyama, M.: Arginine-specific ADP-ribosyltransferase from rabbit skeletal muscle sarcoplasmic reticulum is solubilized as the active form with trypsin: partial purification and characterization. Biochem. Biophys. Res. Commun., 164, 128-133 (1989) [11] Peterson, J.E.; Larew, J.S.-A.; Graves, D.J.: Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes. J. Biol. Chem., 265, 17062-17069 (1990) [12] Zolkiewska, A.; Nightingale M.S.; Moss, J.: Molecular characterization of NAD:arginine ADP-ribosyltransferase from rabbit skeletal muscle. Proc. Natl. Acad. Sci. USA, 89, 11352-11356 (1992) [13] Skorko, R.; Zillig, W.; Rohrer, H.; Mailhammer, R.: Purification and properties of the NAD+ : protein ADP-ribosyltransferase responsible for the T4phage-induced modification of the a subunit of DNA-dependent RNA polymerase of Escherichia coli. Eur. J. Biochem., 79, 55-66 (1977) [14] Goff, C.G.: Coliphage-induced ADP-ribosylation of Escherichia coli RNA polymerase. Methods Enzymol., 106, 418-429 (1984) [15] Soman, G.; Miller, J.F.; Graves, D.: Use of guanylhydrazones as substrates for guanidine-specific mono-ADP-ribosyltransferases. Methods Enzymol., 106, 403-410 (1984) [16] McMahon, K.K.; Piron, K.J.; Ha, V.T.; Fullerton, A.T.: Developmental and biochemical characteristics of the cardiac membrane-bound arginine-specific mono-ADP-ribosyltransferase. Biochem. J., 293, 789-793 (1993) [17] Shimoyama, M.; Tsuchiya, M.; Hara, N.; Yamada, K.; Osago, H.: Molecular cloning and characterization of arginine-specific ADP-ribosyltransferases from chicken bone marrow cells. Adv. Exp. Med. Biol., 419, 137-144 (1997) [18] Kanaitsuka, T.; Bortell, R.; Stevens, L.A.; Moss, J.; Sardinha, D.; Rajan, T.V.; Zipris, D.; Mordes, J.P.; Greiner, D.L.; Rossini, A.A.: Expression in BALB/c and C57BL/6 mice of Rt6-1 and Rt6-2 ADP-ribosyltransferases that differ in enzymic activity. J. Immunol., 159, 2741-2749 (1997) [19] Rigby, M.R.; Bortell, R.; Stevens, L.A.; Moss, J.; Kanaitsuka, T.; Shigeta, H.; Mordes, J.P.; Greiner, D.L.; Rossini, A.A.: Rat RT6.2 and mouse Rt6 locus 1 are NAD+ :arginine ADP ribosyltransferases with auto-ADP ribosylation activity. J. Immunol., 156, 4259-4265 (1996) 282

2.4.2.31


[20] Braren, R.; Glowacki, G.; Nissen, M.; Haag, F.; Koch-Nolte, F.: Molecular characterization and expression of the gene for mouse NAD+ :arginine ecto-mono(ADP-ribosyl)transferase, Art1. Biochem. J., 336, 561-568 (1998) [21] Ganesan, A.K.; Mende-Mueller, L.; Selzer, J.; Barbieri, J.T.: Pseudomonas aeruginosa exoenzyme S, a double ADP-ribosyltransferase, resembles vertebrate mono-ADP-ribosyltransferases. J. Biol. Chem., 274, 9503-9508 (1999) [22] Okazaki, I.J.; Kim, H.-J.; Moss, J.: Cloning and characterization of a novel membrane-associated lymphocyte NAD:arginine ADP-ribosyltransferase. J. Biol. Chem., 271, 22052-22057 (1996) [23] Jones, E.M.; Baird, A.: Cell-surface ADP-ribosylation of fibroblast growth factor-2 by an arginine-specific ADP-ribosyltransferase. Biochem. J., 323, 173-177 (1997) [24] Donnelly, L.E.; Rendell, N.B.; Murray, S.; Allport, J.R.; Lo, G.; Kefalas, P.; Taylor, G.W.; MacDermot, J.: Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leukocytes. Biochem. J., 315, 635-641 (1996) [25] Ohno, T.; Badruzzaman, M.; Nishikori, Y.; Tsuchiya, M.; Jidoi, J.; Shimoyama, M.: vortex-mixing-induced inactivation of arginine-specific ADP-ribosyltransferase activity and re-activation of the less-active form by dithiothreitol plus NaCl under anaerobic conditions. Biochem. Mol. Biol. Int., 32, 213-220 (1994) [26] Terashima, M.; Shimoyama, M.: ADP-ribosylation of A1 peptide of cholera toxin by chicken arginine-specific ADP-ribosyltransferase with a concomitant increase in ADP-ribosyltransferase activity of the peptide. Biomed. Res., 14, 329-335 (1993) [27] Moss, J.; Balducci, E.; Cavanaugh, E.; Kim, H.J.; Konczalik, P.; Lesma, E.A.; Okazaki, I.J.; Park, M.; Shoemaker, M.; Stevens, L.A.; Zolkiewska, A.: Characterization of NAD:arginine ADP-ribosyltransferases. Mol. Cell. Biochem., 193, 109-113 (1999) [28] Davis, T.; Sabir, J.S.M.; Tavassoli, M.; Shall, S.: Purification, characterization, and molecular cloning of a chicken erythroblast mono(ADP-ribosyl)transferase. Adv. Exp. Med. Biol., 419, 145-154 (1997) [29] Wang, J.; Nemoto, E.; Dennert, G.: Regulation of cytotoxic T cell functions by a GPI-anchored ecto-ADP-ribosyltransferase. Adv. Exp. Med. Biol., 419, 191-201 (1997) [30] Tsuchiya, M.; Osago, H.; Yamada, K.; Shimoyama, M.: A newly identified glycosylphosphatidylinositol-anchored arginine-specific ADP-ribosyltransferase in chicken spleen. Adv. Exp. Med. Biol., 419, 245-248 (1997) [31] Okazaki, I.J.; Kim, H.-J.; Moss, J.: Molecular cloning and characterization of lymphocyte and muscle ADP-ribosyltransferases. Adv. Exp. Med. Biol., 419, 129-136 (1997) [32] Hara, N.; Badruzzaman, M.; Sugae, T.; Shimoyama, M.; Tsuchiya, M.: Mouse Rt6.1 is a thiol-dependent arginine-specific ADP-ribosyltransferase cysteine 201 confers thiol sensitivity on the enzyme. Eur. J. Biochem., 259, 289-294 (1999)

283


2.4.2.31

[33] Taniguchi, M.; Tsuchiya, M.; Shimoyama, M.: Comparison of acceptor protein specificities on the formation of ADP-ribose.acceptor adducts by arginine-specific ADP-ribosyltransferase from rabbit skeletal muscle sarcoplasmic reticulum with those of the enzyme from chicken peripheral polymorphonuclear cells. Biochim. Biophys. Acta, 1161, 265-271 (1993)

284

Dolichyl-phosphate D-xylosyltransferase

2.4.2.32

1 Nomenclature EC number 2.4.2.32 Systematic name UDP-d-xylose:dolichyl-phosphate d-xylosyltransferase Recommended name dolichyl-phosphate d-xylosyltransferase

2 Source Organism Gallus gallus (hen [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-d-xylose + dolichyl phosphate = UDP + dolichyl d-xylosyl phosphate Reaction type pentosyl group transfer Natural substrates and products S UDP-d-xylose + dolichyl phosphate ( involved in glycoprotein biosynthesis [1]) (Reversibility: ? [1]) [1] P GDP + dolichyl d-xylosyl phosphate [1] Substrates and products S UDP-d-xylose + dolichyl phosphate (Reversibility: ? [1]) [1] P GDP + dolichyl d-xylosyl phosphate [1] Inhibitors EDTA ( 10 mM, strong [1]) [1] UDP [1] Metals, ions Mn2+ ( requirement [1]) [1] Km-Value (mM) 0.00055 (UDP-d-xylose) [1]

285

Dolichyl-phosphate D-xylosyltransferase

2.4.2.32

pH-Optimum 6-7 [1] pH-Range 5.2-7.8 ( about half-maximal activity at pH 5.2 and 7.8 [1]) [1] Temperature optimum ( C) 37 ( assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue oviduct [1] Localization membrane [1]

References [1] Waechter, C.J.; Lucas, J.J.; Lennarz, W.J.: Evidence for xylosyl lipids as intermediates in xylosyl transfers in hen oviduct membranes. Biochem. Biophys. Res. Commun., 56, 343-350 (1974)

286

Dolichyl-xylosyl-phosphate-protein xylosyltransferase

2.4.2.33

1 Nomenclature EC number 2.4.2.33 Systematic name dolichyl-d-xylosyl-phosphate:protein d-xylosyltransferase Recommended name dolichyl-xylosyl-phosphate-protein xylosyltransferase

2 Source Organism Gallus gallus (hen [1]) [1]

3 Reaction and Specificity Catalyzed reaction dolichyl d-xylosyl phosphate + protein = dolichyl phosphate + d-xylosylprotein Reaction type pentosyl group transfer Natural substrates and products S dolichyl d-xylosyl phosphate + protein ( involved in glycoprotein biosynthesis [1]) [1] P ? Substrates and products S dolichyl d-xylosyl phosphate + protein (Reversibility: ? [1]) [1] P dolichyl phosphate + d-xylosylprotein [1] Inhibitors Additional information ( no inhibition by EDTA [1]) [1] Metals, ions Mn2+ ( requirement [1]) [1] Temperature range ( C) 37 ( assay at [1]) [1]

287

Dolichyl-xylosyl-phosphate-protein xylosyltransferase

2.4.2.33

5 Isolation/Preparation/Mutation/Application Source/tissue oviduct [1] Localization membrane [1]

References [1] Waechter, C.J.; Lucas, J.J.; Lennarz, W.J.: Evidence for xylosyl lipids as intermediates in xylosyl transfers in hen oviduct membranes. Biochem. Biophys. Res. Commun., 56, 343-350 (1974)

288

Indolylacetylinositol arabinosyltransferase

2.4.2.34

1 Nomenclature EC number 2.4.2.34 Systematic name UDP-l-arabinose:indol-3-ylacetyl-myo-inositol l-arabinosyltransferase Recommended name indolylacetylinositol arabinosyltransferase Synonyms IAA-myo-inositol-arabinosyl synthase UDP-arabinose:indol-3-ylacetyl-myo-inositol arabinosyl transferase arabinosylindolylacetylinositol synthase arabinosyltransferase, uridine diphosphoarabinose-indolylacetylinositol CAS registry number 84720-96-7

2 Source Organism Zea mays (sweet corn [1]) [1]

3 Reaction and Specificity Catalyzed reaction UDP-l-arabinose + indol-3-ylacetyl-myo-inositol = UDP + indol-3-ylacetylmyo-inositol l-arabinoside Reaction type pentosyl group transfer Natural substrates and products S UDP-l-arabinose + indol-3-ylacetyl-myo-inositol ( involved in biosynthesis of low molecular weight esters of indol-3-ylacetic acid in maize kernels [1]) (Reversibility: ? [1]) [1] P UDP + indol-3-ylacetyl-myo-inositol l-arabinoside [1] Substrates and products S UDP-l-arabinose + indol-3-ylacetyl-myo-inositol (Reversibility: ? [1]) [1] P UDP + indol-3-ylacetyl-myo-inositol l-arabinoside [1] 289

Indolylacetylinositol arabinosyltransferase

2.4.2.34

Temperature optimum ( C) 37 ( assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue kernel ( immature [1]) [1]

References [1] Corcuera, L.J.; Bandurski, R.S.: Biosynthesis of indol-3-yl-acetyl-myoinositol arabinoside in kernels of Zea mays L.. Plant Physiol., 70, 1664-1666 (1982)

290

Flavonol-3-O-glycoside xylosyltransferase

2.4.2.35

1 Nomenclature EC number 2.4.2.35 Systematic name UDP-d-xylose:flavonol-3-O-glycoside 2''-O-b-d-xylosyltransferase Recommended name flavonol-3-O-glycoside xylosyltransferase Synonyms UDP-xylose:flavonol 3-glycoside xylosyltransferase uridine diphosphoxylose-flavonol 3-glycoside xylosyltransferase CAS registry number 83380-90-9

2 Source Organism Tulipa sp. (tulip, cv. Apeldoorn [1]) [1] Euonymus alatus (f. ciliato-dentatus [2]) [2]

3 Reaction and Specificity Catalyzed reaction UDP-d-xylose + a flavonol 3-O-glycoside = UDP + a flavonol 3-[-d-xylosyl(1!2)-b-d-glycoside] Reaction type pentosyl group transfer Natural substrates and products S UDP-d-xylose + flavonol 3-O-glycoside ( involved in flavonoid metabolism, pathway of flavonol 3-O-triglycoside biosynthesis [1]) [1] P UDP + flavonol 3-O-d-xylosylglycoside Substrates and products S UDP-d-xylose + 3,5,7-trihydroxyflavanone ( i.e. pinobanksin [2]) (Reversibility: ? [2]) [2] P UDP + flavonol 3-xyloside-5,7-dihydroxyflavanone

291


S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P S P 292

2.4.2.35

UDP-d-xylose + 7,4'-dihydroxyflavonol (Reversibility: ? [2]) [2] UDP + kaempferol 3-O-xyloside-7,4'-dihydroxyflavonol UDP-d-xylose + dihydrokaempferol (Reversibility: ? [2]) [2] UDP + dihydrokaempferol 3-O-xylosylglucoside UDP-d-xylose + dihydroquercetin ( i.e. taxifolin, poor substrate [2]) (Reversibility: ? [2]) [2] UDP + dihydroquercetin 3-O-xyloside UDP-d-xylose + fisetin (Reversibility: ? [2]) [2] UDP + fisetin 3-O-xyloside UDP-d-xylose + flavonol 3-O-diglycoside (Reversibility: ? [1]) [1] UDP + flavonol 3-O-triglycoside UDP-d-xylose + flavonol 3-O-glycoside (Reversibility: ? [1]) [1] UDP + flavonol 3-O-d-xylosylglycoside [1] UDP-d-xylose + isorhamnetin (Reversibility: ? [2]) [2] UDP + isorhamnetin 3-O-xyloside UDP-d-xylose + isorhamnetin 3,7-O-diglucoside ( poor substrate [1]) (Reversibility: ? [1]) [1] ? UDP-d-xylose + isorhamnetin 3-O-glucoside (Reversibility: ? [1]) [1] UDP + isorhamnetin 3-O-xylosylglucoside UDP-d-xylose + kaempferol ( best substrate tested [2]) (Reversibility: ? [2]) [2] UDP + kaempferol 3-O-xyloside [2] UDP-d-xylose + kaempferol 3-O-glucoside (Reversibility: ? [1]) [1] UDP + kaempferol 3-O-xylosylglucoside UDP-d-xylose + kaempferol 3-O-rhamnoside ( poor substrate [1]) (Reversibility: ? [1]) [1] UDP + kaempferol 3-O-xylosylrhamnoside UDP-d-xylose + kaempferol 4'-monomethyl ether (Reversibility: ? [2]) [2] UDP + kaempferol 3-O-xyloside-4'-monomethyl ether UDP-d-xylose + kaempferol 5,7,4'-trimethyl ether (Reversibility: ? [2]) [2] UDP + kaempferol 3-O-xyloside-5,7,4'-trimethyl ether UDP-d-xylose + myricetin (Reversibility: ? [2]) [2] UDP + myricetin 3-O-xyloside UDP-d-xylose + myricetin 3-O-rhamnoside ( poor substrate [1]) (Reversibility: ? [1]) [1] UDP + myricetin 3-O-xylosylrhamnoside UDP-d-xylose + quercetin (Reversibility: ? [2]) [2] UDP + quercetin 3-O-xyloside UDP-d-xylose + quercetin 3-O-arabinoglucoside ( poor substrate [1]) (Reversibility: ? [1]) [1] UDP + quercetin 3-O-xylosylarabinoglucoside

2.4.2.35


S UDP-d-xylose + quercetin 3-O-galactoside ( best substrate tested [1]) (Reversibility: ? [1]) [1] P UDP + quercetin 3-O-xylosylgalactoside S UDP-d-xylose + quercetin 3-O-glucoside ( poor substrate [1]) (Reversibility: ? [1]) [1] P UDP + quercetin 3-O-d-xylosylglucoside S UDP-d-xylose + quercetin 3-O-glucoside ( i.e. isoquercitrin, poor substrate [2]) (Reversibility: ? [1]; ? [2]) [1, 2] P UDP + quercetin 3-O-xylosylglucoside S UDP-d-xylose + quercetin 3-O-rhamnoside ( poor substrate [1]) (Reversibility: ? [1]) [1] P UDP + quercetin 3-O-xylosylrhamnoside [1] S UDP-d-xylose + quercetin 3-O-rhamnosylglucoside ( i.e. rutin, poor substrate [1]) (Reversibility: ? [1]) [1] P UDP + quercetin 3-O-d-xylosylrhamnosylglucoside S UDP-d-xylose + quercetin 5-monomethyl ether (Reversibility: ? [2]) [2] P UDP + quercetin 3-O-xyloside-5-monomethyl ether S UDP-d-xylose + quercetin 7-O-glucoside ( i.e. quercimeritrin, poor substrate [2]) (Reversibility: ? [2]) [2] P UDP + quercimeritrin 3-O-xylosyl-7-O-glucoside S UDP-d-xylose + rhamnetin (Reversibility: ? [2]) [2] P UDP + rhamnetin 3-O-xyloside S Additional information ( aglycones are no substrates [1]; only UDP-d-xylose can function as sugar donor [2]) [1, 2] P ? Inhibitors Cu2+ ( high inhibition when applied in high concentrations [1]) [1, 2] Mn2+ [1] Zn2+ ( high inhibition when applied in high concentrations [1]) [1, 2] p-chloromercuribenzoate [1] Activating compounds 2-mercaptoethanol ( slight stimulation [1]; strong stimulation even in the presence of Mg2+ [2]) [1, 2] NH+4 ( high activation [1]) [1] dithioerythritol ( slight stimulation [1]) [1] glutathione (slight stimulation [1]) [1] sucrose ( slight stimulation [1]) [1] Additional information ( no stimulation by bovine serum albumin, not affected by anions like chloride and sulfate [1]) [1] Metals, ions Ca2+ ( high activation [1]) [1] Additional information ( no effect by Mg2+ [1]) [1]

293


2.4.2.35

Km-Value (mM) 0.00083 (kaempferol) [2] 0.00119 (quercetin) [2] 0.025 (UDP-xylose, kaempferol as xylosyl acceptor [2]) [2] pH-Optimum 7 ( Tris-HCl buffer [2]) [2] 7.5 ( imidazole-HCl buffer [2]) [2] 8.5-9 [1]

4 Enzyme Structure Molecular weight 30000 ( gel filtration [1]) [1] 48000 ( gel filtration [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue anther [1] leaf [2] pollen ( at the stage of middle postmeiotic pollen ripening [1]) [1] tapetum [1] Purification (partial by a method that includes Sephadex G-200 chromatography, Sephadex G-15 chromatography, isoelectric focusing and DEAE-Sephadex chromatography [1]) [1] (partial by a method that includes Sephadex G-25 chromatography and DEAE-cellulose chromatography [2]) [2]

6 Stability Storage stability , -20 C, 20mM Tris-HCl buffer, pH 7.5, 14 mM 2-mercaptoethanol and 10% glycerol, two days, 11% loss of activity, 55% loss after 8 days, 84% loss after 40 days [2]

294

2.4.2.35


References [1] Kleinehollenhorst, G.; Behrens, H.; Pegels, G.; Srunk, N.; Wiermann, R.: Formation of flavonol 3-O-diglycosides and flavonol 3-O-triglycosides by enzyme extracts from anters of Tulipa cv. Apeldoorn. Z. Naturforsch. C, 37C, 587-599 (1982) [2] Ishikura, N.; Yang, Z.Q.: UDP-d-xylose: flavonol 3-O-xylosyltransferase from young leaves of Euonymus alatus f. ciliato-dentatus. Z. Naturforsch. C, 46C, 1003-1010 (1991)

295

NAD+ -diphthamide ADP-ribosyltransferase

2.4.2.36

1 Nomenclature EC number 2.4.2.36 Systematic name NAD+ :peptide-diphthamide N-(ADP-d-ribosyl)transferase Recommended name NAD+ -diphthamide ADP-ribosyltransferase Synonyms (adenosine diphosphoribose)transferase, nicotinamide adenine dinucleotideelongation factor 2 ADP-ribosyltransferase NAD(+)-diphthamide ADP-ribosyltransferase NAD-diphthamide ADP-ribosyltransferase NAD-diphthamide ADP-ribosyltransferase NAD-elongation factor 2 ADP-ribosyltransferase NAD:elongation factor 2-adenosine diphosphate ribose-transferase mono(ADPribosyl)transferase Additional information (cf. EC 2.4.2.31) CAS registry number 52933-21-8

2 Source Organism Pseudomonas aeruginosa [1-3] Mesocricetus auratus (baby [1]) [1] Bos taurus [1]

3 Reaction and Specificity Catalyzed reaction NAD+ + peptide diphthamide = nicotinamide + peptide N-(ADP-d-ribosyl)diphthamide Reaction type pentosyl group transfer

296

2.4.2.36

NAD+-diphthamide ADP-ribosyltransferase

Natural substrates and products S NAD+ + elongation factor 2 ( i.e. elongation factor 2 of baby hamster kidney cells, forward ADP-ribosylation reaction is reversed by fragment A from Pseudomonas aeruginosa [1]; liver enzyme [1]; NAD-glycohydrolase activity without acceptor substrate [3]) (Reversibility: r [1]; ? [1]) [1-3] P nicotinamide + ADPribose-elongation factor 2 [1-3] Substrates and products S NAD+ + elongation factor 2 ( i.e. elongation factor 2 of baby hamster kidney cells, forward ADP-ribosylation reaction is reversed by fragment A from Pseudomonas aeruginosa [1]; liver enzyme [1]; NAD-glycohydrolase activity without acceptor substrate [3]) (Reversibility: r [1]; ? [1]) [1-3] P nicotinamide + ADPribose-elongation factor 2 [1-3] Inhibitors cytoplasmic extract of pyBHK-cells ( not fragment A [1]) [1] elastase ( inactivation follows pseudo-first order kinetic [2]) [2] histamine ( 250 mM, almost complete inhibition [1]) [1] Additional information ( cellular ADPribosyltransferase is not inhibited by anti-fragment A-antiserum [1]; fragment A and toxin A are not inhibited by histamine [1]) [1] Activating compounds 2-mercaptoethanol ( activation, together with SDS or guanidine hydrochloride [3]) [3] SDS ( enhances activity together with dithiothreitol, cysteine, 2mercaptoethanol or sulfite [3]) [3] cysteine ( activation, together with SDS or guanidine hydrochloride [3]) [3] dithiothreitol ( enhances activity together with urea [3]) [3] guanidine hydrochloride ( enhances activity together with dithiothreitol, cysteine, 2-mercaptoethanol or sulfite [3]) [3] sulfite ( activation, together with SDS or guanidine hydrochloride [3]) [3] urea ( enhances activity together with dithiothreitol [3]) [3] Additional information ( exotoxin is synthesized in a catalytically inactive, proenzyme form, unfolding of the toxin molecule may cause activation [3]) [3] pH-Optimum 6.6 ( assay at, nicotinamide + ADPribose-elongation factor 2 [1]) [1] 8 ( assay at, NAD+ + elongation factor 2 [1,2]) [1, 2] Temperature optimum ( C) 22 ( assay at [1]) [1] 25 ( assay at [2]) [2]

297

NAD+-diphthamide ADP-ribosyltransferase

2.4.2.36

4 Enzyme Structure Subunits monomer ( 1 * 72000, exotoxin A, SDS-PAGE [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue culture supernatant [2, 3] kidney ( polyoma virus transformed baby hamster kidney cells, i.e. pyBHK-cells [1]) [1] liver [1] Localization extracellular ( exotoxin A [2,3]; fragment A of diphtheria toxin [1]) [1-3] Purification (exotoxin A [2]) [2]

References [1] Lee, H.; Iglewski, W.J.: Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A. Proc. Natl. Acad. Sci. USA, 81, 2703-2707 (1984) [2] Sanai, Y.; Morihara, K.; Tsuzuki, H.; Homma, J.Y.; Kato, I.: Proteolytic cleavage of exotoxin A from Pseudomonas aeruginosa: formation of an ADP-ribosyltransferase active fragment by the action of Pseudomonas elastase. FEBS Lett., 120, 131-134 (1980) [3] Leppla, S.H.; Martin, O.C.; Muehl, L.A.: The exotoxin P. aeruginosa: a proenzyme having an unusual mode of activation. Biochem. Biophys. Res. Commun., 81, 532-538 (1978)

298

NAD+ -dinitrogen-reductase ADP-Dribosyltransferase

2.4.2.37

1 Nomenclature EC number 2.4.2.37 Systematic name NAD+ :[dinitrogen reductase] (ADP-d-ribosyl)transferase Recommended name NAD+ -dinitrogen-reductase ADP-d-ribosyltransferase Synonyms (adenosine diphosphoribose)transferase, nicotinamide adenine dinucleotideazoferredoxin ADP-ribosyltransferase DRAT NAD-azoferredoxin (ADPribose)transferase NAD-dinitrogen-reductase ADP-d-ribosyltransferase azoferredoxin ADP-ribosyltransferase dinitrogenase reductase ADP-ribosyltransferase CAS registry number 117590-45-1

2 Source Organism Azospirillum brasilense (strain Sp7 [6]) [6, 7, 9] Azospirillum lipoferum [5] Rhodospirillum rubrum (strain UR2 [2]) [1-4, 6-16]

3 Reaction and Specificity Catalyzed reaction NAD+ + [dinitrogen reductase] = nicotinamide + ADP-d-ribosyl-[dinitrogen reductase] ( regulation of nitrogenase by enzyme and dinitrogenase reductase activating glycohydrolase [8,12]; regulation of enzyme, signal pathways [9]) Reaction type pentosyl group transfer

299

NAD+-dinitrogen-reductase ADP-D-ribosyltransferase

2.4.2.37

Natural substrates and products S NAD+ + [dinitrogen reductase] ( involved in regulation of nitrogenase EC 1.18.6.1 activity, [1, 2]; involved in regulation of nitrogenase EC 1.18.6.1 through reversible ADP-ribosylation of one of the two identical subunits of dinitrogenase reductase (i.e. component II or iron protein) [2]) (Reversibility: ? [1, 2]) [1, 2] P ? Substrates and products S NAD+ + [dinitrogen reductase] ( high specificity for acceptor substrate, the only acceptor is dinitrogen reductase [1-3,5]) (Reversibility: ? [1-7]) [1-7] P nicotinamide + ADP-d-ribosyl-[dinitrogen reductase] [1] S etheno-NAD+ + [dinitrogen reductase] (Reversibility: ? [1]) [1] P ? S nicotinamide guanine dinucleotide + [dinitrogen reductase] (Reversibility: ? [1]) [1] P ? S nicotinamide hypoxanthine dinucleotide + [dinitrogen reductase] (Reversibility: ? [1]) [1] P ? Inhibitors 3-aminobenzamide [1] ATP ( in the presence of ADP, 63% inhibition at 1,5 mM [3]) [3] KCl ( reversible [1]) [1] MgATP2- ( in the presence of Mg-ADP [3]) [3] NADH [1] NADP+ [1] NaBr ( strong [1]) [1] NaCl ( reversible [1]) [1, 11] nicotinamide [1] Cofactors/prosthetic groups 2'-deoxy-ADP ( activation [3]) [3] ADP ( activation [6]) [6] Activating compounds 2'-deoxy-ADP ( activation [3]) [3] ADP-b-S ( i.e. adenosine 5'-O-(2-thiodiphosphate), activation [3]) [3] MgADP- ( stimulation by binding to dinitrogen reductase (not from Azotobacter vinelandii), no activation by ATP, GDP, IDP, etheno-ADP, AMPCH2 -P, 8-bromo-ADP [3]) [3] Metals, ions Mg2+ ( activation [6]) [6]

300

2.4.2.37


Specific activity (U/mg) 0.004-0.015 [3] 0.077 [1] Km-Value (mM) 0.7 (NAD+, plus ADP-d-ribosyl-[dinitrogen reductase] from Azotobacter vinelandii, in the absence of MgADP- [3]) [3] 2 (NAD+, plus native ADP-d-ribosyl-[dinitrogen reductase], in the presence of MgADP- [3]) [1, 3] 2.5 (NAD+, plus ADP-d-ribosyl-[dinitrogen reductase] from Klebsiella pneumoniae, in the absence of MgADP- [3]) [3] Additional information ( activity of enzyme depends on the redox status of the Fe4S41+/2+ cluster of nitrogenase Fe protein [10]) [10] pH-Optimum 7 [1] Temperature optimum ( C) 30 ( assay at [1,3,6]) [1, 3, 6]

4 Enzyme Structure Molecular weight 30900 ( gel filtration [1]) [1] Subunits monomer ( 1 * 29000, SDS-PAGE [1]) [1] Additional information ( chemical cross-linking of enzyme dinitrogenase reductase substrate [11]) [11]

5 Isolation/Preparation/Mutation/Application Localization soluble [1] Purification (to near homogeneity [1]) [1] Cloning [2, 4, 5, 7] Engineering Additional information ( enzyme mutants with altered substrate recognition and with altered posttranslational regulation of enzyme activity [14]; random PCR mutagenesis, mutants with altered regulatory properties [16]) [16]

301


2.4.2.37

6 Stability General stability information , ADP and NaCl stabilize during purification and storage [1] , DTT stabilizes during purification [1] , bovine serum albumin increases stability towards freeze thawing in the absence of ADP [3] , desalting procedures during purification inactivate [1] , extremely unstable in crude extract or partially purified preparation [1] , one freeze-thawing cycle leads to 50% loss of activity, ADP stabilizes [1] Storage stability , -80 C, in liquid N2 [1] , 0 C, in 0.2 M NaCl and 1 mM ADP, 18 h [1]

References [1] Lowery, R.G.; Ludden, P.W.: Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum. J. Biol. Chem., 263, 16714-16719 (1988) [2] Fitzmaurice, W.P.; Saari, L.L.; Lowery, R.G.; Ludden, P.W.; Roberts, G.P.: Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum. Mol. Gen. Genet., 218, 340-347 (1989) [3] Lowery, R.G.; Ludden, P.W.: Effect of nucleotides on the activity of dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum. Biochemistry, 28, 4956-4961 (1989) [4] Fu, H.A.; Wirt, H.J.; Burris, R.H.; Roberts, G.P.: Functional expression of a Rhodospirillum rubrum gene encoding dinitrogenase reductase ADP-ribosyltransferase in enteric bacteria. Gene, 85, 153-160 (1989) [5] Fu, H.A.; Fitzmaurice, W.P.; Roberts, G.P.; Burris, R.H.: Cloning and expression of draTG genes from Azospirillum lipoferum. Gene, 86, 95-98 (1990) [6] Fu, H.A.; Hartman, A.; Lowery, R.G.; Fitzmaurice, W.P.; Roberts, G.P.; Burris, R.H.: Posttranslational regulatory system for nitrogenase activity in Azospirillum spp. J. Bacteriol., 171, 4679-4685 (1989) [7] Zhang, Y.; Burris, R.H.; Roberts, G.P.: Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense. J. Bacteriol., 174, 3364-3369 (1992) [8] Grunwald, S.K.; Lies, D.P.; Roberts, G.P.; Ludden, P.W.: Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-Ribosyltransferase and dinitrogenase reductase activating glycohydrolase. J. Bacteriol., 177, 628-635 (1995) [9] Zhang, Y.; Burris, R.H.; Ludden, P.W.; Roberts, G.P.: Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase

302

2.4.2.37

[10]

[11] [12]

[13] [14] [15]

[16]


activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense. J. Bacteriol., 177, 2354-2359 (1995) Halbleib, C.M.; Zhang, Y.; Ludden, P.W.: Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein. J. Biol. Chem., 275, 3493-3500 (2000) Grunwald, S.K.; Ludden, P.W.: NAD-dependent crosslinking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum. J. Bacteriol., 179, 3277-3283 (1997) Zhang, Y.; Pohlmann, E.L.; Halbleib, C.M.; Ludden, P.W.; Roberts, G.P.: Effect of PII and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae. J. Bacteriol., 183, 1610-1620 (2001) Ma, Y.; Ludden, P.W.: Role of the dinitrogenase reductase arginine 101 residue in dinitrogenase reductase ADP-ribosyltransferase binding, NAD binding, and cleavage. J. Bacteriol., 183, 250-256 (2001) Kim, K.; Zhang, Y.; Roberts, G.P.: Correlation of activity regulation and substrate recognition of the ADP-ribosyltransferase that regulates nitrogenase activity in Rhodospirillum rubrum. J. Bacteriol., 181, 1698-1702 (1999) Grunwald, S.K.; Ryle, M.J.; Lanzilotta, W.N.; Ludden, P.W.: ADP-ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase. J. Bacteriol., 182, 2597-2603 (2000) Zhang, Y.; Kim, K.; Ludden, P.W.; Roberts, G.P.: Isolation and characterization of drat mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum. Microbiology, 147, 193-202 (2001)

303

Glycoprotein 2-b-D-xylosyltransferase

2.4.2.38

1 Nomenclature EC number 2.4.2.38 Systematic name UDP-d-xylose:glycoprotein (d-xylose to the 3,6-disubstituted mannose of 4N-{N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!3)-[N-acetyl-b-dglucosaminyl-(1!2)-a-d-mannosyl-(1!6)]-b-d-mannosyl-(1!4)-N-acetylb-d-glucosaminyl-(1!4)-N-acetyl-b-d-glucosaminyl}asparagine) 2-b-d-xylosyltransferase Recommended name glycoprotein 2-b-d-xylosyltransferase Synonyms XylT b-1,2-xylosyltransferase b-1,4-mannosylglycoprotein b-1,2-xylosyltransferase

2 Source Organism

Lymnaea stagnalis [1] Acer pseudoplatanus [2] Arabidopsis thaliana [3] Arabidopsis thaliana [3] Glycine max [4]

3 Reaction and Specificity Catalyzed reaction UDP-d-xylose + 4-N-{N-acetyl-b-d-glucosaminyl-(1!]2)-a-d-mannosyl(1!3)-[N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!6)]-b-d-mannosyl-(1!4)-N-acetyl-b-d-glucosaminyl-(1!4)-N-acetyl-b-d-glucosaminyl}asparagine = UDP + 4-N-{N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!3)-[N-acetyl-b-d-glucosaminyl-(1!2)-a-d-mannosyl-(1!6)]-[b-dxylosyl-(1!2)]-b-d-mannosyl-(1!4)-N-acetyl-b-d-glucosaminyl-(1!4)-Nacetyl-b-d-glucosaminyl} asparagine Reaction type transfer of glycosyl group 304

2.4.2.38


Natural substrates and products S Additional information ( probably involved in the biosynthesis of the inner part of hemocyanin glycans [1]) [1] P ? Substrates and products S UDP-d-xylose + GlcNAcb1-2Mana1-3Manb1-O-(CH2 )7 -CH3 (Reversibility: ? [1]) [1] P UDP + GlcNAcb1-2Mana1-3(Xylb1-2)Manb1-O-(CH2 )7 -CH3 [1] S UDP-d-xylose + GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-2Mana1-3) Manb1-4GlcNAc (Reversibility: ? [1]) [1] P UDP + GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-2Mana1-3)(Xylb1-2) Manb1-4GlcNAc [1] S UDP-d-xylose + GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-2Mana1-3) Manb1-O-(CH2 )7 -CH3 (Reversibility: ? [1]) [1] P UDP + GlcNAcb1-2Mana1-6(Galb1-4GlcNAcb1-2Mana1-3)(Xylb1-2) Manb1-O-(CH2 )7 -CH3 [1] S UDP-d-xylose + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4(Fuca1-6)GlcNAc1-O-(N-benzyloxycarbonyl)-Tyr (Reversibility: ? [4]) [4] P UDP + GlcNAcb1 -2Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb14GlcNAcb1-4(Fuca1-6)GlcNAc1-O-(N-benzyloxycarbonyl)-Tyr [4] S UDP-d-xylose + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAc (Reversibility: ? [1]) [1] P UDP + GlcNAcb1-2Mana1 -6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb14GlcNAcb1-4GlcNAc [1] S UDP-d-xylose + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAc-Asn (Reversibility: ? [1]) [1] P UDP + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb14GlcNAcb1-4GlcNAc-Asn [1] S UDP-d-xylose + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAc1-O-(N-benzyloxycarbonyl)-Tyr (Reversibility: ? [4]) [4] P UDP + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb14GlcNAcb1-4GlcNAc1-O-(N-benzyloxycarbonyl)-Tyr [4] S UDP-d-xylose + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb14GlcNAcb1-4GlcNAc1-O-(pyrid-2-yl)amine (Reversibility: ? [2, 3]) [2, 3] P UDP + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb14GlcNAcb1-4GlcNAc1-O-(pyrid-2-yl)amine [2, 3] S UDP-d-xylose + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)Manb1-O(CH2 )7 -CH3 (Reversibility: ? [1]) [1] P UDP + GlcNAcb1-2Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb1-O(CH2 )7 -CH3 [1] S UDP-d-xylose + Mana1-6(GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb14GlcNAc1-O-(N-benzyloxycarbonyl)-Tyr (Reversibility: ? [4]) [4]

305


2.4.2.38

P UDP + Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb1-4GlcNAcb14GlcNAc1-O-(N-benzyloxycarbonyl)-Tyr [4] S UDP-d-xylose + Mana1-6(GlcNAcb1-2Mana1-3)Manb1-O-(CH2 )7 -CH3 (Reversibility: ? [1]) [1] P UDP + Mana1-6(GlcNAcb1-2Mana1-3)(Xylb1-2)Manb1-O-(CH2 )7 -CH3 [1] Inhibitors Additional information ( substrate inhibition [1]) [1] Metals, ions Mn2+ ( 6.25-12.5 mM, stimulation [1]) [1] Specific activity (U/mg) 7e-006 [4] pH-Optimum 7 [1, 4]

4 Enzyme Structure Molecular weight 55000-59000 ( gel filtration, SDS-PAGE [4]) [4] 62000 ( calculation from sequence [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue central nervous system [1] Localization Golgi apparatus [2] microsome [4] Purification [3, 4] Cloning (expression in insect cells [3]) [3]

6 Stability pH-Stability 6.5-7.5 ( sensitive to changes in pH of storage solution [4]) [4] Storage stability , 4 C to -20 C, HEPES-buffer, pH 7.0, 10% glycerol, 0.2% Triton X-100, 1 mM dithiothreitol, 90 days, 20% [4] 306

2.4.2.38


References [1] Mulder, H.; Dideberg, F.; Schachter, H.; Spronk, B.A.; De Jong-Brink, M.; Kamerling, J.P.; Vliegenthart, J.F.G.: In the biosynthesis of N-glycans in connective tissue of the snail Lymnaea stagnalis of incorporation GlcNAc by b 2GlcNAc-transferase I is an essential prerequisite for the action of b 2GlcNAc-transferase II and b 2Xyl-transferase. Eur. J. Biochem., 232, 272283 (1995) [2] Tezuka, K.; Hayashi, M.; Ishihara, H.; Akazawa, T.; Takahashi, N.: Studies on synthetic pathway of xylose-containing N-linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.). Eur. J. Biochem., 203, 401-413 (1992) [3] Strasser, R.; Mucha, J.; Mach, L.; Altmann, F.; Wilson, I.B.H.; Glossl, J.; Steinkellner, H.: Molecular cloning and functional expression of b 1,2-xylosyltransferase cDNA from Arabidopsis thaliana. FEBS Lett., 472, 105-108 (2000) [4] Zeng, Y.; Bannon, G.; Thomas, V.H.; Rice, K.; Drake, R.; Elbein, A.: Purification and specificity of b 1,2-xylosyltransferase, an enzyme that contributes to the allergenicity of some plant proteins. J. Biol. Chem., 272, 31340-31347 (1997)

307

Xyloglucan 6-xylosyltransferase

2.4.2.39

1 Nomenclature EC number 2.4.2.39 Systematic name UDP-d-xylose:xyloglucan 1,6-a-d-xylosyltransferase Recommended name xyloglucan 6-xylosyltransferase Synonyms EC 2.4.1.169 (formerly) xyloglucan 6-a-d-xylosyltransferase xylosyltransferase, uridine diphosphoxylose-xyloglucan 6aCAS registry number 80238-01-3

2 Source Organism

Glycine max (soy bean [1]) [1, 2, 4] Pisum sativum (pea, cv. Alaska or Caprice [3]) [3] Phaseolus vulgaris (dwarf-french-bean, cv. Canadian Wonder [5]) [5] Tamarindus indica (tamarind [6]) [6]

3 Reaction and Specificity Catalyzed reaction transfers an a-d-xylosyl residue from UDP-d-xylose to a glucose residue in xyloglucan, forming an a-1,6-d-xylosyl-d-glucose linkage Reaction type hexosyl group transfer Natural substrates and products S UDP-d-xylose + xyloglucan ( involved in xyloglucan biosynthesis of higher plants [2]; responsible for xyloglucan side-chain formation [1]) (Reversibility: ? [1, 2]) [1, 2] P ?

308

2.4.2.39


Substrates and products S UDP-d-xylose + (glucosyl)xyloglucan ( transfers an a-d-xylosyl residue from UDP-d-xylose to a glucose residue in xyloglucan [1-4]; xylosyl-transfer is closely linked to glucosyl-transfer (EC 2.4.1.168) [4]; GDP-d-glucose or GDP-d-mannose cannot replace UDPxylose [5]; other xylosyl-acceptors are b-1,3-glucan and xylan [1]; no acceptors are cello-oligosaccharides and fragment oligosaccharides from endoglucanase digest [1]; standard oligosaccharides for enzyme assay [6]) (Reversibility: ? [1-6]) [1-6] P UDP + (xylosyl-glucosyl)xyloglucan ( forms an a-1,6-d-xylosyl-d-glucose linkage [1]) [1-5] Inhibitors Cu2+ [5] EDTA ( weak [1]) [1] GDPglucose ( weak [4]) [4] Tris-HCl buffer [1] detergents ( no solubilization possible due to this inhibition [1]) [1] phosphate buffer [1] Additional information ( no inhibition by tunicamycin, ATP or GTP [1]) [1] Activating compounds GDP-d-glucose ( activation [1, 5]; about half as effective as UDPglucose, in concentrations exceeding UDPxylose [1]; protection [5]) [1, 5] GDP-d-mannose ( activation, protection [5]) [5] TDPglucose ( activation, as effective as UDPglucose, in concentrations exceeding UDPxylose [1]) [1] UDPglucose ( activation, xylose is effectively incorporated in the presence of UDPglucose, the transfer must be preceded by elongation of the b-1,4-glucan-backbone, because xylosyl residues constitute the side chains, in concentrations exceeding UDPxylose, no activation by CDPglucose or ADPglucose [1]; 2 mM [5]) [1, 2, 5] Metals, ions Ca2+ ( activation, can replace Mn2+ to some extent [5]) [5] Co2+ ( activation, can replace Mn2+ to some extent [5]) [5] Mg2+ ( activation, can replace Mn2+ to some extent [1,5]) [1, 5] Mn2+ ( activation [1, 3, 5]; 10 mM [1]) [1, 3, 5] Km-Value (mM) Additional information ( kinetic study [1]) [1] pH-Optimum 6 ( incorporation of xylosyl residues into polymeric acceptors [1]) [1] 6.5-7 ( UDPglucose + UDPxylose [1]) [1] 7 ( broad [5]) [5]

309


2.4.2.39

Temperature optimum ( C) 35 [1]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture [1, 4, 5] hypocotyl ( elongation region [2]) [2] seedling [3] Localization Golgi membrane [2, 3] membrane [1, 4, 5]

6 Stability General stability information , DTT, 1 mM, EDTA, 1 mM, sucrose, 0.4 M, bovine serum albumin, 0.1%, stabilize [1] Storage stability , 0 C, crude membrane-bound enzyme preparation, 1 day [4]

References [1] Hayashi, T.; Matsuda, K.: Biosynthesis of xyloglucan in suspension-cultured soybean cells. Occurrence and some properties of xyloglucan 4-b-d-glucosyltransferase and 6-a-d-xylosyltransferase. J. Biol. Chem., 256, 1111711122 (1981) [2] Hayashi, T.; Koyama, T.; Matsuda, K.: Formation of UDP-xylose and xyloglucan in soybean Golgi membranes. Plant Physiol., 87, 341-345 (1988) [3] White, A.R.; Xin, Y.; Pezeshk, V.: Xyloglucan glucosyltransferase in Golgi membranes from Pisum sativum (pea). Biochem. J., 294, 231-238 (1993) [4] Hayashi, T.; Matsuda, K.: Biosynthesis of xyloglucan in suspension-cultured soybean cells. Evidence that the enzyme system of xyloglucan synthesis does not contain b-1,4-glucan 4-b-glucosyltransferase activity (EC 2.4.1.12). Plant Cell Physiol., 22, 1571-1584 (1981) [5] Campbell, R.E.; Brett, C.T.; Hillman, J.R.: A xylosyltransferase involved in the synthesis of a protein-associated xyloglucan in suspension-cultured dwarfFrench-bean (Phaseolus vulgaris) cells and its interaction with a glucosyltransferase. Biochem. J., 253, 795-800 (1988) [6] Marry, M.; Cavalier, D.M.; Schnurr, J.K.; Netland, J.; Yang, Z.; Pezeshk, V.; York, W.S.; Pauly, M.; White, A.R.: Structural characterization of chemically and enzymatically derived standard oligosaccharides isolated from partially purified tamarind xyloglucan. Carbohydr. Polym., 51, 347-356 (2002) 310

Zeatin O-b-D-xylosyltransferase

2.4.2.40

1 Nomenclature EC number 2.4.2.40 Systematic name UDP-d-xylose:zeatin O-b-d-xylosyltransferase Recommended name zeatin O-b-d-xylosyltransferase Synonyms uridine diphosphoxylose-zeatin xylosyltransferase xylosyltransferase, uridine diphosphoxylose-zeatin zeatin O-xylosyltransferase Additional information (cf. EC 2.4.1.203) CAS registry number 110541-22-5

2 Source Organism no activity in Phaseolus lunatus [1] Phaseolus vulgaris (cv. Great Northern [1-4]; gene ZOX1 [4]) [1-5] Phaseolus vulgaris x Phaseolus coccineus [2]

3 Reaction and Specificity Catalyzed reaction UDP-d-xylose + zeatin = UDP + O-b-d-xylosylzeatin (does not act on UDPglucose; localisation of site determining the substrate specificity for the UDP-sugar donors [5]) Reaction type pentosyl group transfer Natural substrates and products S UDP-d-xylose + trans-zeatin ( may be involved in the nuclear-cytoplasmic transport of cytokinins and related molecules or possibly, with chromatin of rapidly dividing cells [2]) [2] P ?

311


2.4.2.40

Substrates and products S UDP-d-xylose + dihydrozeatin (Reversibility: ? [1,5]) [1, 5] P UDP + O-b-d-xylosyldihydrozeatin [5] S UDP-d-xylose + zeatin ( substrate specificity [5]; absolutely specific for UDP-d-xylose [1, 4, 5]; trans-zeatin [1, 2]; cis-zeatin and ribosylzeatin are no substrates [1]) (Reversibility: ? [1-5]) [1-5] P UDP + O-b-d-xylosylzeatin [1-5] Inhibitors UDP-glucose [5] Specific activity (U/mg) 0.0049 [1] Km-Value (mM) 0.002 (trans-zeatin) [1] 0.003 (UDP-xylose) [1] 0.01 (dihydrozeatin) [1] pH-Optimum 8 ( assay at [5]) [5] 8-8.5 [1] Temperature optimum ( C) 25 ( assay at [1]) [1] 27 ( assay at [3,4]) [3, 4]

4 Enzyme Structure Molecular weight 50000 ( gel filtration [1]) [1, 3] 51000 ( DNA sequence determination [4]) [4] Additional information ( an inactive form of MW about 70 kDa is detected by immunostaining in vegetative tissue prior to posttranslational processing of the protein [3]) [3] Subunits monomer ( 1 * 50000, SDS-PAGE [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue cotyledon [2] embryo ( immature [1]) [1] endosperm ( callus [2]) [2, 3] seed ( immature [2,4]) [2-4]

312

2.4.2.40


Additional information ( very low expression level in vegetative tissues [4]; no activity in vegetative tissues, activity is apparent only in reproductive tissue, endosperm callus represents a unique form inbetween [2,4]) [2, 4] Localization cytosol [1-3] nucleus [2, 3] Purification (partial [1]) [1] Cloning (ZOX1 gene, DNA sequence determination and analysis [4]; in vitro transcription and translation, posttranslational processing by bean endosperm extract [3]; expression in Escherichia coli and in Spodoptera frugiperda Sf21 cells, via baculovirus infection, produced an insoluble protein, not successful [3]) [3, 4] Engineering Additional information ( construction of chimeric recombinant enzyme mutants, exchange of the C-terminus with zeatin O-b-d-glucosyltransferase, EC 2.4.1.203, gene ZOG1, determination of the site determining the substrate specificity [5]; construction of transgenic tobacco plants via Agrobacterium tumefaciens infection, functional expression under control of the CaMV35S promotor in seedlings and leaves, transgenic plants are more sensitive to auxin than wild-type plants, phenotypic alterations are leaf chlorosis, restriction of root elongation and eventual cessation of growth [3]) [3, 5]

References [1] Turner, J.E.; Mok, D.W.S.; Mok, M.C.; Shaw, G.: Isolation and partial purification of an enzyme catalyzing the formation of O-xylosylzeatin in Phaseolus vulgaris embryos. Proc. Natl. Acad. Sci. USA, 84, 3714-3717 (1987) [2] Martin, R.C.; Mok, M.C.; Mok, D.W.S.: Cytolocalization of zeatin O-xylosyltransferase in Phaseolus. Proc. Natl. Acad. Sci. USA, 90, 953-957 (1993) [3] Martin, R.C.; Mok, M.C.; Mok, D.W.S.: Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O-xylosyltransferase from Phaseolus vulgaris. Plant J., 12, 305-312 (1997) [4] Martin, R.C.; Mok, M.C.; Mok, D.W.S.: A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris. Plant Physiol., 120, 553-557 (1999) [5] Martin, R.C.; Cloud, K.A.; Mok, M.C.; Mok, D.W.S.: Substrate specificity and domain analyses of zeatin O-glycosyltransferases. Plant Growth Regul., 32, 289-293 (2000)

313

b-Galactoside a-2,6-sialyltransferase

2.4.99.1

1 Nomenclature EC number 2.4.99.1 Systematic name CMP-N-acetylneuraminate:b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine a2,6-N-acetylneuraminyltransferase Recommended name b-galactoside a-2,6-sialyltransferase Synonyms B-cell antigen CD75 CMP-N-acetylneuraminic acid-glycoprotein sialyltransferase CMP-acetylneuraminate-galactosylglycoprotein sialyltransferase CMP-acetylneuraminate-glycoprotein sialyltransferase Galb1,4-GlcNAc a2,6 sialyltransferase [3, 17] a 2,6-ST a2-6 sialyltransferase antigens, CD75 cytidine monophosphoacetylneuraminate-galactosylglycoprotein sialyltransferase sialotransferase sialyltransferase sialyltransferase, cytidine monophosphoacetylneuraminate-galactosylglycoprotein CAS registry number 9075-81-4

2 Source Organism Rattus norvegicus (commercial product [25, 28, 31]; purified enzyme [11, 17, 22]; Sprague-Dawley [1, 22]) [1, 3-5, 11, 14, 17-19, 22, 25, 27-29, 31] Bos taurus (calf [2]) [2, 7-9, 14, 15] Cavia porcellus [6] Capra hircus [7] Homo sapiens (recombinant [28]) [7, 10, 12, 13, 16, 19, 25, 26, 28-31] Mus musculus [20] Rattus norvegicus [21] Photobacterium damselae (JT0160 [23,24]) [23, 24] 314

2.4.99.1


3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine = CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine (The terminal b-d-galactosyl residue of the oligosaccharide of glycoproteins, as well as lactose, can act as acceptor; Val220 participates in binding the common cytidine moiety in substrate CMP-NeuAc and inhibitor CDP [21]; mechanism [9]) Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-R ( regulation of enzyme expression [18]; R: glycoprotein or glycopeptide [2, 18]; one of a group of glycosyltransferases which act to assemble the carbohydrate units of thyroglobulin [2]; final step in synthesis of serum-type glycoproteins [7]) [2, 7, 18] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-R Substrates and products S CMP-9-O-acetyl-N-acetylneuraminate + asialo-a1 -acid glycoprotein ( comparable or even higher transfer rates than with CMP-Nacetylneuraminate [14]) (Reversibility: ? [14]) [14] P ? S CMP-9-acetamido-N-acetylneuraminate + asialo-a1 -acid glycoprotein (Reversibility: ? [11]) [11] P ? S CMP-9-amino-N-acetylneuraminate + asialo-a1 -acid glycoprotein (Reversibility: ? [11]) [11] P ? S CMP-9-azido-N-acetylneuraminate + asialo-a1 -acid glycoprotein (Reversibility: ? [11]) [11] P ? S CMP-9-benzamido-N-acetylneuraminate + asialo-a1 -acid glycoprotein (Reversibility: ? [11]) [11] P ? S CMP-9-hexanoylamido-N-acetylneuraminate + asialo-a1 -acid glycoprotein (Reversibility: ? [11]) [11] P ? S CMP-N-acetylneuraminate + 4-nitrophenyl-d-galactoside (Reversibility: ? [12]) [12] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-(4-nitro)phenol S CMP-N-acetylneuraminate + Neu5Aca-2,3-Galb-1,4-Glc ( also pyridylaminated [24]) (Reversibility: ? [24]) [24] P CMP + a-N-acetylneuraminyl-2,6-(Neu5Aca-2,3-)Galb-1,4-Glc

315


2.4.99.1

S CMP-N-acetylneuraminate + asialo-Tamm-Horsfall glycoprotein (Reversibility: ? [6]) [6] P CMP + Tamm-Horsfall glycoprotein S CMP-N-acetylneuraminate + asialo-agalacto-prothrombin (Reversibility: ? [3]) [3] P CMP + agalacto-prothrombin S CMP-N-acetylneuraminate + asialo-apoceruloplasmin ( best substrate [1]) (Reversibility: ? [1]) [1] P CMP + apoceruloplasmin S CMP-N-acetylneuraminate + asialo-ceruloplasmin (Reversibility: ? [1,7,11]) [1, 7, 11] P CMP + ceruloplasmin S CMP-N-acetylneuraminate + asialo-chorionic gonadotropin ( acceptor protein from human source [1]) (Reversibility: ? [1]) [1] P CMP + chorionic gonadotropin S CMP-N-acetylneuraminate + asialo-fetuin ( best substrate [10]) (Reversibility: ? [1, 2, 5-7, 9, 10, 28, 30, 31]) [1, 2, 5-7, 9, 10, 28, 30, 31] P CMP + fetuin [1, 28] S CMP-N-acetylneuraminate + asialo-fibrinogen (Reversibility: ? [1]) [1] P CMP + fibrinogen S CMP-N-acetylneuraminate + asialo-immunoglobulin G (Reversibility: ? [9]) [9] P CMP + immunoglobulin G S CMP-N-acetylneuraminate + asialo-immunoglobulin M (Reversibility: ? [9]) [9] P CMP + immunoglobulin M S CMP-N-acetylneuraminate + asialo-orosomucoid ( i.e. asialo-a1 acid glycoprotein [1, 3, 4, 6, 7, 9, 10, 12, 14, 21, 22, 25, 29, 30]; best substrate [7, 9]; about half as effective as asialofetuin [10]; CMP-N-acetylneuraminate can be replaced with comparable or even higher transfer rates by CMP-N-glycolylneuraminate, CMP-9-Oacetyl-N-acetylneuraminate [14]) (Reversibility: ? [1, 3, 4, 6-12, 14, 21, 22, 25, 29, 30]) [1, 3, 4, 6-12, 14, 21, 22, 25, 29, 30] P CMP + orosomucoid S CMP-N-acetylneuraminate + asialo-proteose peptone (Reversibility: ? [7]) [7] P CMP + proteose peptone S CMP-N-acetylneuraminate + asialo-prothrombin (Reversibility: ? [3,6,7]) [3, 6, 7] P CMP + prothrombin S CMP-N-acetylneuraminate + asialo-thyroglobulin ( low activity [7]) (Reversibility: ? [1,7]) [1, 7] P CMP + thyroglobulin S CMP-N-acetylneuraminate + asialo-thyroglobulin glycopeptide (Reversibility: ? [2]) [2] 316

2.4.99.1


P CMP + thyroglobulin glycopeptide S CMP-N-acetylneuraminate + asialo-transferrin ( poor substrate [1]) (Reversibility: ? [1, 3, 7, 15, 25, 30]) [1, 3, 7, 15, 25, 30] P CMP + transferrin S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine ( i.e. N-acetyllactosamine [3, 7, 9, 10, 20, 23, 24, 26]; most active disaccharide substrate [7]; best substrate [9]; poor substrate [10]) (Reversibility: ? [3, 4, 7, 9, 10, 20, 22-24, 26]) [3, 7, 9, 10, 20, 22-24, 26] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine [3, 7, 9, 23, 24] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,2-a-mannosyl-1,6-b-mannosyl-1,4-N-acetylglucosamine (Reversibility: ? [13]) [13] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,2-a-mannosyl-1,6-b-mannosyl-1,4-N-acetylglucosamine [13] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,4-b-d-galactosyl-1,4-b-d-glucosyl-b-1-ceramide ( i.e. paragloboside [16]) (Reversibility: ? [16]) [16] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,4-b-d-galactosyl-1,4-b-d-glucosyl-b-1-ceramide ( i.e. a-2,6-sialylparagloboside [16]) [16] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-R ( R: glycoprotein or glycopeptide [1, 2, 9]; CMP-N-acetylneuraminate analogues can serve as donor substrates, i.e. formyl-, trifluoroacetyl-, benzyloxycarbonyl-, and amino acetyl-N-neuraminates [29]; CMP-N-acetylneuraminate analogues can serve as donor substrates, i.e. the 5''-amino-, the 5''-N-pentanoylamino-, the 5''-glycylamino-, and the 5''-ethoxycarbonylamino derivative and diammonium (cytidin-5'-yl) [(3-deoxy-d-glycero-b-d-galacto-2-nonulpyranosyl)onate] phosphate [27]; synthetic disaccharides [22]; substrate specificity for several synthetic trisaccharides [19]; strictly specific for CMP-N-acetylneuraminate, forms only a2,6-linkages [9, 23, 24]; transfers sialic acid to the terminal galactose residue of asialo-gyloproteins and peptides [2]; the non-reducing terminal galactosyl residue is essential for activity [1]; transfers sialic acid to terminal positions on N-linked glycans [3, 4, 11, 13, 14]; high molecular weight substrates are more efficient acceptors than low molecular weight substrates [2, 7, 10]; donor substrate specificity [11, 27]; acceptor substrate specificity [2, 3, 7, 9, 11, 15, 22, 24, 25, 30]; sialic acid is linked to C-6 of the galactose residue [1-31]) (Reversibility: ? [1-31]) [1-31] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-R [1-31]

317


2.4.99.1

S CMP-N-acetylneuraminate + fetuin ( no activity [7]; glycopeptides [2]; about 30% as effective as asialofetuin [10]) (Reversibility: ? [2,9,10]) [2, 9, 10] P ? S CMP-N-acetylneuraminate + fucosyl-a-1,2-d-galactosyl-b-1,4-d-glucose ( also pyridylaminated [24]) (Reversibility: ? [24]) [24] P CMP + a-N-acetylneuraminyl-2,6-fucosyl-a-1,2-d-galactosyl-b-1,4-d-glucose [24] S CMP-N-acetylneuraminate + lacto-N-neotetraose (Reversibility: ? [3,20]) [3, 20] P ? S CMP-N-acetylneuraminate + lactose ( poor substrate [3, 9]; no activity [2]) (Reversibility: ? [3, 4, 7, 9, 23, 24]) [3, 4, 7, 9, 23, 24] P CMP + 6'-sialyllactose [3, 7, 23, 24] S CMP-N-acetylneuraminate + methyl-b-d-galactopyranoside (Reversibility: ? [23,24]) [23, 24] P CMP + a-N-acetylneuraminyl-2,6-b-d-methylgalactopyranoside S CMP-N-acetylneuraminate + pyridylamino-lactose ( recombinant enzyme [25]) (Reversibility: ? [25]) [25] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-b-d-glucosyl-pyridylaminoside S CMP-N-acetylneuraminate + submaxillary asialo-mucin ( poor substrates, no activity with ovine acceptor [7]) (Reversibility: ? [7]) [7] P CMP + submaxillary mucin S CMP-N-a-neuraminate + ganglioside nLc4Cer (Reversibility: ? [25]) [25] P ? S CMP-N-a-neuraminate + ganglioside nLc6Cer (Reversibility: ? [25]) [25] P ? S CMP-N-a-neuraminate + pyridylaminated ganglioside nLc4Cer (Reversibility: ? [25]) [25] P ? S CMP-N-aminoacetylneuraminate + asialo-a1 -acid glycoprotein (Reversibility: ? [29]) [29] P ? S CMP-N-formylacetylneuraminate + asialo-a1 -acid glycoprotein (Reversibility: ? [29]) [29] P ? S CMP-N-glycolylneuraminate + asialo-a1 -acid glycoprotein ( comparable or even higher transfer rates than with CMP-N-acetylneuraminate [14]) (Reversibility: ? [14]) [14] P ? S Additional information ( the catalytic domain can proceed with sialic acid transfer with increased efficiency until 80 amino acid are 318

2.4.99.1


deleted [30]; no activity with the CMP-NeuAc analogues disodium[(5-acetamido-3,5-dideoxy-d-glycero-b-d-galacto-2-nonulopyranosyl)onate] (methyl-b-d-ribofuranosid-5-yl) phosphate and bis(triethylammonium) [(5-acetamido-3,5-dideoxy-d-glycero-b-d-galacto-2-nonulopyranosyl)onate] [(resorcin-4-yl) (1-deoxy-b-d-ribofuranosid-5-yl)] phosphate [27]; no activity with Lc4Cer and Lc3Cer [25]; enzyme does not recognize type I oligosaccharides [24]; low activity with several methylated substrates, no activity with UDP-Gal, UDP-GalNAc and UDP-Glc as donor substrates [23]; no activity with galactosylhydroxylysine and its derivatives, or galactose-containing oligosaccharides linked to Ser/Thr-glycopeptide of earthworm cuticle collagen [2]; no activity with ovine submaxillary asialo-mucin [7,9]; no activity with submaxillary asialo-mucin [1-4,6]; very low activity with prothrombin and native a1 -acid glycoprotein, no activity with transferrin, asialo-agalacto-a1 -acid glycoprotein [3]; no activity with b-methyll-arabinopyranoside [9]; no activity with antifreeze glycoprotein [3,4,9]; no transfer to terminal N-acetyl-d-galactosamine [2]; no activity with N-acetyl-d-galactosamine or N-acetyl-d-glucosamine, and asialo-agalacto-fetuin, and galactose [10]; galactose-free glycopeptides are poor substrates [2]; no activity with galactose, lactose and N-acetyllactose [2]; thyroglobin is no substrate [1]; poor substrates are the b-1,3- or b-1,6-derivatives of b-d-galactosyl-1,4-N-acetyl-b-d-glucosamine [9]) [1-4, 6, 7, 9, 10, 23, 24, 27, 30] P ? Inhibitors Ba2+ [6] CDP ( wild-type enzyme and mutant V220A [21]; competitive [21]; Mn2+ restores activity [3]) [3, 10, 21] CMP ( competitive, less effective than CDP or CTP [10]) [10] CTP ( also decreases the stimulation by the natural cytosolic rat colon factor [22]) [3, 10, 22] Cu2+ ( weak [10]) [6, 10] EDTA ( weak [3,10]; no inhibition [2]) [3, 10] Hg2+ [6] Mn2+ ( above 10 mM [10]) [10] N-acetyllactosamine ( with asialo-a1 acid glycoprotein as substrate [9]) [9] PCMB ( weak [2]) [2] Pb2+ [6] UTP ( 0.25 mM, inhibition decreases at higher concentrations [10]) [10] aflatoxins ( aflatoxins B1 , B2a , G1 , slightly by aflatoxins B2 and G2 , inhibition is probably due to membrane disruption through binding of the aflatoxins and change in enzyme conformation [5]) [5]

319


2.4.99.1

cycloheximide ( reduces the enzyme expression 3fold in normal hepatocytes and 16 to 17fold in Zaidela ascitic hepatoma, but not in cells isolated from the tumor and cultured for 48 h [18]) [18] deoxycholate ( 9fold stimulation at 0.25%, inactivation at 0.5% [1]) [1] endo F ( inactivation due to deglycosylation of the enzyme, best in methanol or ethanol, less efficient than glycanase [17]) [17] glycanase ( inactivation due to deglycosylation of the enzyme, best in methanol or ethanol [17]) [17] long chain fatty acids ( oleic acid, stearic acid, less efficient: palmitic acid, lauric acid, capric acid, caprylic acid or caproic acid, no inhibition by linoleic acid or linolenic acid [1]) [1] Additional information ( inhibitory capacity of several synthetic trisaccharides [19]; not affected by versenate [7]; no inhibition by linoleic acid or linolenic acid [1]; no inhibition by N-ethylmaleimide [10]) [1, 7, 10, 19] Activating compounds EDTA ( activation [1]; no activation [2]) [1] Triton Cf-54/Tween 80 ( activation [4]) [4, 25] Triton X-100 ( activation [10]) [10, 19, 22, 28] deoxycholate ( 9fold stimulation at 0.25%, inhibition at 0.5% [1]) [1] natural factor from rat colon ( stimulation is decreased by CTP, no activation at 0.75 mM CTP [22]; partially purified [22]; may also be present in rat brain and kidney [22]; specific, 4fold stimulation by decreasing the Km value for the substrates and increasing Vmax, present in the cytosol, temperature dependent, protease sensitive, independent of metal ions or detergent, unaffceted by monosaccharides [22]) [22] Metals, ions MgCl2 ( no stimulation [2,7]; slight stimulation at 5 mM [1]) [1] Additional information ( no metal ion requirement [1-3,6,7]) [13, 6, 7, 10] Specific activity (U/mg) 0.00000022 ( hepatocyte [18]) [18] 0.0000008 ( Ehrlich ascite carcinoma cells, adherent or from tissue culture [20]) [20] 0.00083 ( partially purified enzyme [1]) [1] 0.0009 ( partially purified enzyme [7]) [7] 0.0035 ( partially purified enzyme [7]) [7] 1.6 ( purified, recombinant from Sf9 insect cells [31]) [31] 5.5 ( purified enzyme [23]) [23] 8.2 ( purified enzyme [4]) [4] 26-28 ( purified enzyme [8]) [8]

320

2.4.99.1


Additional information ( assay method development, biosensor-based method using immobilized Sambucus nigra agglutinin [28]; activity of stimulated and unstimulated enzyme with different substrates [22]; kinetics [11]) [1-3, 11, 16, 19, 22, 28, 29] Km-Value (mM) 0.003 (CMP-N-acetylneuraminate) [6] 0.0053 (CMP-N-acetylneuraminate) [3] 0.0064 (asialo-fetuin, expressed as concentration of terminal galactosyl residues [10]) [10] 0.016 (CMP-N-acetylneuraminate) [3] 0.0205 (CMP-N-acetylneuraminate) [10] 0.03 (CMP-9-benzamido-N-acetylneuraminate) [11] 0.035 (CMP-N-acetylneuraminate) [28] 0.035 (asialo-fetuin) [6] 0.046 (CMP-N-acetylneuraminate) [27] 0.047 (CMP-N-acetylneuraminate) [1] 0.05 (CMP-N-acetylneuraminate, wild-type enzyme [21]; with asialo-a1 -acid glycoprotein [11,21]) [11, 21, 25] 0.078 (asialo-ceruloplasmin, expressed as concentration of acceptor sites [1]) [1] 0.08 (CMP-N-acetylneuraminate, with lactose [7]) [7] 0.12 (CMP-9-acetamido-N-acetylneuraminate) [11] 0.14 (CMP-N-acetylneuraminate, with stimulation by the natural cytosolic crat colon factor [22]) [22] 0.14 (asialo-a1 -acid glycoprotein) [3] 0.15 (CMP-N-acetylneuraminate, recombinant enzyme [26]) [26] 0.16 (asialo-transferrin, recombinant enzyme [25]) [25] 0.2 (asialo-a1 -acid glycoprotein, recombinant enzyme [25]) [25] 0.21 (asialo-transferrin) [3] 0.23 (asialo-a1 -acid glycoprotein, with stimulation by the natural cytosolic crat colon factor [22]) [22] 0.24 (CMP-N-acetylneuraminate, without stimulation by the natural cytosolic crat colon factor [22]) [22] 0.27 (CMP-N-acetylneuraminate) [2] 0.28 (asialo-a1 -acid glycoprotein, recombinant enzyme [25]) [25] 0.3 (CMP-N-acetylneuraminate, with N-acetyllactosamine [7]) [7] 0.32 (CMP-N-acetylneuraminate) [23] 0.33 (asialo-a1 -acid glycoprotein, wild-type enzyme [21]) [21] 0.38 (CMP-N-acetylneuraminate) [31] 0.53 (asialo-a1 -acid glycoprotein, without stimulation by the natural cytosolic crat colon factor [22]) [22] 0.555 (CMP-N-acetylneuraminate, recombinant enzyme [31]) [31] 0.59 (asialo-thyroglobulin glycopeptides) [2] 0.72 (CMP-9-amino-N-acetylneuraminate) [11] 0.91 (nLc4Cer, recombinant enzyme [25]) [25]

321


2.4.99.1

1.3 (pyridylamino-N-acetyllactosamine, recombinant enzyme [25]) [25] 1.5 (pyridylamino-nLc4Cer, recombinant enzyme [25]) [25] 1.62 (N-acetyllactosamine) [3] 1.67 (lacto-N-neotetraose) [3] 2.3 (N-acetyllactosamine, recombinant enzyme [26]) [26] 6.82 (lactose) [23] 8.1 (Neu5Aca-2,3-Galb-1,4-Glc) [24] 8.95 (N-acetyllactosaminide) [23] 12 (N-acetyllactosamine) [9] 13.6 (fucosyl-a-1,2-d-galactosyl-b-1,4-d-glucose) [24] 102 (pyridylamino-lactose, recombinant enzyme [25]) [25] 129 (lactose) [3] 174 (methyl-b-d-galactopyranoside) [23] Additional information ( several recombinant truncated mutants [30]; Km -values for synthetic CMP-N-acetylneuraminate analogues [27]; kinetics, several synthetic trisaccharides used as substrates [19]; kinetic data [3,6]; kinetic mechanism [9]) [3, 6, 7, 9, 19, 25, 27, 30] Ki-Value (mM) 0.0047 (CDP, wild-type enzyme [21]) [21] 0.016 (CDP, mutant V220A [21]) [21] 0.18 (CMP) [10] Additional information [6] pH-Optimum 5 [23] 6 ( assay at [21]; broad [10]) [2, 10, 21] 6-6.5 ( 2-(N-morpholino)ethane sulfonic acid buffer [1]) [1] 6.3 [6] 6.4-7.2 ( broad, optimal buffer concentration: 0.05-0.15 M, decreasing activity above 0.15 M [7]) [7] 6.5 ( assay at [14, 19, 20, 28, 31]) [14, 19, 20, 28, 31] 6.8 ( assay at [22]) [22] 7.5 ( Tris-HCl buffer [1]) [1] Additional information ( pI: 4.6 [23]) [23] pH-Range 5-9.2 ( about half-maximal activity at pH 5 and 9.2 [2]) [2] 5-9.7 ( about 65% of maximal activity at pH 5 and about half-maximal activity at pH 9.7 [1]) [1] 5.2-7.4 ( about half-maximal activity at pH 5.2 and 7.4 [10]) [10] 5.4-8 ( about half-maximal activity at pH 5.4 and 8, goat [7]) [7] Temperature optimum ( C) 28 [10] 30 [23] 37 ( assay at [1-4, 7-15, 19-22, 26, 28, 31]) [1-4, 7-15, 19-22, 26, 28, 31] 322

2.4.99.1


4 Enzyme Structure Molecular weight 42900 ( low molecular weight form, sedimentation equilibrium centrifugation, two enzyme forms differing in molecular weight when submitted to gel filtration, the smaller one is probably a degradation product of the larger one [8]) [8] 45000 ( low molecular weight form, gel filtration, two enzyme forms differing in molecular weight when submitted to gel filtration, the smaller one is probably a degradation product of the larger one [8]) [8] 57900 ( high molecular weight form, sedimentation equilibrium centrifugation, two enzyme forms differing in molecular weight when submitted to gel filtration, the smaller one is probably a degradation product of the larger one [8]) [8] 64000 ( gel filtration [23]) [23] 80000 ( high molecular weight form, gel filtration, two enzyme forms differing in molecular weight when submitted to gel filtration, the smaller one is probably a degradation product of the larger one [8]) [8] Subunits monomer ( 1 * 61000, SDS-PAGE [23]; 1 * 40500, SDSPAGE under reducing conditions [4]; 1 * 41500, low molecular weight form, SDS-PAGE under non-reducing conditions [8]; 1 * 44000, low molecular weight form, SDS-PAGE under reducing conditions [8]; 1 * 53500, high molecular weight form, SDS-PAGE under non-reducing conditions [8]; 1 * 56000, high molecular weight form, SDS-PAGE under reducing conditions [8]) [4, 8, 23] Posttranslational modification glycoprotein ( 2times N-glycosylated [26]; catalytic activity of the enzyme depends on the presence of oligosaccharide chains [17]; contains at least 2 N-linked carbohydate chains of the complex type [17]) [17, 26] Additional information ( deglycosylation of the enzyme is much more efficient in presence of methanol or ethanol [17]) [17]

5 Isolation/Preparation/Mutation/Application Source/tissue B-16 cell [16] Burkitt lymphoma cell ( Daudi cell line [25]) [25] Ehrlich ascites carcinoma cell ( high activity in cells grown in tissue culture or adherent to extracellular matrices, low activity in cells not adherent nor grown in tissue culture [20]) [20] U-266 cell ( myeloma cell line [25]) [25] cerebral cortex [6]

323


2.4.99.1

cervical epithelium [10] colostrum [7-9, 14, 15] commercial preparation [25, 28, 31] hepatocyte [18] hepatoma ascites cell line ( cultured cells isolated from the tumor behave as normal hepatocytes [18]; no enzyme activity in Zaidela ascitic hepatoma, but 3.6fold reduced mRNA content of a larger transcript size compared to normal liver cells [18]) [18] jejunum [22] liver [1, 3-5, 11, 13, 17-19, 22, 25, 27-29, 31] melanoma cell [16] myeloma cell line [25] placenta [13] platelet [12] thyroid gland [2] Localization Golgi apparatus ( membrane-bound at the luminal side, trans compartment [17]) [3, 4, 17, 20, 22] Golgi trans face [17] cytosol [7, 8] membrane [1-6, 17, 20, 22] microsome [1, 5, 13, 22] synaptosome [6] Additional information ( deletion of the transmembrane fragment results in loss of acceptor preference [30]; enzyme is not catalytically active in the endoplasmic reticulum and only achieves full activity when it locates itself to the trans compartment of the Golgi system [17]) [17, 30] Purification (further purification of commercial rat liver enzyme [25]; partial [1,22]; CDP-hexanolamine-agarose affinity chromatography [4]) [1, 4, 22, 25] (partial [2,7]; solubilized by ultrasonic treatment [2]; CDP-hexanolamine-agarose affinity chromatography [8]) [2, 7, 8] (partial [6]) [6] (partial [7]) [7] (recombinant from cell culture medium after expression in insect cells [31]; recombinant from COS-1 cells [25]; partial, solubilized with Triton X100 [12]) [12, 25, 31] [23] Cloning (enzyme expression analysis in Zaidela ascitic hepatoma cell lines and normal liver cells [18]) [18] (high level functional expression in Spodoptera frugiperda Sf9 insect cells via baculovirus infection using a vector that fuses the enzyme to the mouse IgM signal peptide and the IgG binding domain of the Staphylococcus aureus protein A at the N-terminus for secretion of the recombinant protein

324

2.4.99.1


into the culture medium [31]; DNA sequence determination, expression of various truncated forms lacking the transmembrane fragment as FLAGtagged proteins, the chimeric mutant, and the membrane-bound enzyme form in CHO-K1 cells [30]; expression in Pichia pastoris, entry in the secretory pathway, i.e. excretion of the soluble enzyme to the medium, by usage of the N-terminal signal sequence of Saccharomyces cerevisiae a-factor, no hyperglycosylation [26,30]; DNA sequence determination, functional overexpression of full length cDNA and fragment of residues 113-1227, the latter fused to staphylococcal protein A, in COS-1 cells [25]; functional expression in Saccharomyces cerevisiae [19]) [19, 25, 26, 30, 31] (expression of wild-type and mutants in COS-1 cells [21]) [21] Engineering D219A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 10.8fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] G221A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 1.6fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] K223A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 6.6fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] L190A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 11.9fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] T224A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 1.3fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] T225A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 3.2fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] V184A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 6.8fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] V220A ( site-directed mutagenesis, mutant of the conserved sialylmotif contained by all members of the sialyltransferase family, 6.0fold increased Km for CMP-NeuAc, only slightly altered Km for the acceptor substrate [21]) [21] Additional information ( construction of a chimeric enzyme with the N-terminal membrane portion of the rat core 2 b1,6-GlcNAc-transferase,

325


2.4.99.1

amino acid residues 1-52, and the residues 1-70 of the human a2,6-sialyltransferase, also FLAG-tagged, functional expression in CHO-K1 cells, broader acceptor substrate specificity than the wild-type [30]) [30] Application biotechnology ( recombinant enzyme may be used for in vitro synthesis of oligosaccharides [26]) [26] synthesis ( recombinant enzyme may be used for in vitro synthesis of oligosaccharides [26]; enzyme can be exploited as biocatalyst in the synthesis of interesting non-natural compounds, especially in view of chemical, regiospecific sialylation, chemo-enzymatic synthesis of modified carbohydrate ligands, and their suitability as probes for studying molecular recognition phenomena [19]) [19, 26]

6 Stability pH-Stability 5.2-5.5 ( most stable [8]) [8] 5.3 ( and below, storage stability decreases with t1=2 : about 3 months [4]) [4] 6 ( full stability for at least 1 year [4]) [4] Temperature stability 40 ( 5 min, 80% remaining activity [23]) [23] 55 ( 5 min, 5% remaining activity [23]) [23] 67 ( 30 s, 90% loss of activity [3]) [3] 100 ( 5 min, complete inactivation [2]) [2] Organic solvent stability methanol ( stable at 10%, 37 C [17]) [17] General stability information , high NaCl concentrations stabilize during storage [4] , glycerol, 50%, stabilizes during storage [8] , high protein concentrations stabilize [8] , stable to lyophilization [2] , use of plastic containers instead of glass stabilizes [8] , fractionation on Ultrogel AcA34 decreases activity, bovine serum albumin restores [12] , freeze-thawing, crude preparation, stable to [10] , glycerol, 20%, v/v, stabilizes [12] Storage stability , -20 C, in 50% glycerol, 35 mM cacodylate, pH 6, 0.45 M NaCl, 0.08% Triton CF-54, at least 1 year [4] , 3 C, microsomal fraction, 2 weeks [1] , -20 C, in 50% glycerol, 12 mM cacodylate, pH 5.3, 2 months [8] , frozen, crude, solubilized enzyme preparation, several months [2]

326

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, -20 C, crude enzyme preparation, at least 3 months [10] , 4 C, partially purified enzyme preparation in 20% glycerol, v/v, at least 1 week [12] , -80 C, 20 mM sodium cacodylate, pH 5.0, stable at least 3 months [23] , 0-4 C, partially purified enzyme preparation, 2-4 weeks [7]

References [1] Hickman, J.; Ashwell, G.; Morell, A.G.; Van den Hamer, C.J.A.; Scheinberg, I.H.: Physical and chemical studies on ceruloplasmin. 8. Preparation of Nacetylneuraminic acid-1-14 C-labeled ceruloplasmin. J. Biol. Chem., 245, 759-766 (1970) [2] Spiro, M.J.; Spiro, R.G.: Glycoprotein biosynthesis: studies on thyroglobulin. Thyroid sialyltransferase. J. Biol. Chem., 243, 6520-6528 (1968) [3] Weinstein, J.; de Souza-e-Silva, U.; Paulson, J.C.: Sialylation of glycoprotein oligosaccharides N-linked to asparagine. Enzymatic characterization of a Galb1!3(4)GlcNAc a2!3 sialyltransferase and a Galb1!4GlcNAc a2!6 sialyltransferase from rat liver. J. Biol. Chem., 257, 13845-13853 (1982) [4] Weinstein, J.; de Souza-e-Silva, U.; Paulson, J.C.: Purification of a Galb1!4GlcNAc a2!6 sialyltransferase and a Galb1!3(4)GlcNAc a2!3 sialyltransferase to homogeneity from rat liver. J. Biol. Chem., 257, 1383513844 (1982) [5] Bernacki, R.J.; Gurtoo, H.L.: Differential inhibition of rat liver sialyltransferase(s) by various aflatoxins and their metabolites. Res. Commun. Chem. Pathol. Pharmacol., 10, 681-692 (1975) [6] Bosmann, H.B.: Synthesis of glycoproteins in brain: identification, purification and properties of a synaptosomal sialyl transferase utilizing endogenous and exogenous acceptors. J. Neurochem., 20, 1037-1049 (1973) [7] Bartholomew, B.A.; Jourdian, G.W.; Roseman, S.: The sialic acids. XV. Transfer of sialic acid to glycoproteins by a sialyltransferase from colostrum. J. Biol. Chem., 248, 5751-5762 (1973) [8] Paulson, J.C.; Beranek, W.E.; Hill, R.L.: Purification of a sialyltransferase from bovine colostrum by affinity chromatography on CDP-agarose. J. Biol. Chem., 252, 2356-2362 (1977) [9] Paulson, J.C.; Rearick, J.I.; Hill, R.L.: Enzymatic properties of b-d-galactoside a2!6 sialytransferase from bovine colostrum. J. Biol. Chem., 252, 2363-2371 (1977) [10] Scudder, P.R.; Chantler, E.N.: Glycosyltransferases of the human cervical epithelium. II. Characterization of a CMP-N-acetylneuraminate: galactosyl-glycoprotein sialyltransferase. Biochim. Biophys. Acta, 660, 136-141 (1981) [11] Gross, H.J.; Rose, U.; Krause, J.M.; Paulson, J.C.; Schmid, K.; Feeney, R.E.; Brossmer, R.: Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases. Biochemistry, 28, 7386-7392 (1989)

327


2.4.99.1

[12] Bauvois, B.; Montreuil, J.; Verbert, A.: Characterization of a sialyl a2-3 transferase and a sialyl a2-6 transferase from human platelets occurring in the sialylation of the N-glycosylproteins. Biochim. Biophys. Acta, 788, 234-240 (1984) [13] Nemansky, M.; Schiphorst, W.E.C.M.; Koeleman, C.A.M.; Van den Eijnden, D.H.: Human liver and human placenta both contain CMP-NeuAc:Galb1!4GlcNAc-R a2!3- as well as a2!6-sialyltransferase activity. FEBS Lett., 312, 31-36 (1992) [14] Higa, H.H.; Paulson, J.C.: Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids. J. Biol. Chem., 260, 8838-8849 (1985) [15] Beyer, T.A.; Rearick, J.I.; Paulson, J.C.; Prieels, J.-P.; Sadler, J.E.; Hill, R.L.: Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences. J. Biol. Chem., 254, 12531-12541 (1979) [16] Tsuchiya, K.; Suzuki, Y.; Mabuchi, K.; Suzuki, T.; Hirabayashi, Y.; Sakiyama, H.: Characterization of sialyltransferase of B16 melanoma cells involved in the formation of melanoma-associated antigen GM3. J. Clin. Biochem. Nutr., 14, 141-149 (1993) [17] Fast, D.G.; Jamieson, J.C.; McCaffrey, G.: The role of the carbohydrate chains of Galb-1,4-GlcNAc a2,6-sialyltransferase for enzyme activity. Biochim. Biophys. Acta, 1202, 325-330 (1993) [18] Jain, N.; Sudhakar, C.; Das, M.R.: Regulation of expression of b-galactoside a2,6-sialyltransferase in a rat tumor, Zajdela ascitic hepatoma. FEBS Lett., 317, 147-151 (1993) [19] Van Dorst, J.A.L.M.; Tikkanen, J.M.; Krezdorn, C.H.; Streiff, M.B.; Berger, E.G.; Van Kuik, J.A.; Kamerling, J.P.; Vliegenthart, J.F.G.: Exploring the substrate specificities of a-2,6- and a-2,3-sialyltransferases using synthetic acceptor analogs. Eur. J. Biochem., 242, 674-681 (1996) [20] Shigeta, S.; Winter, H.C.; Goldstein, I.J.: a-(2!3)- and a-(2!6)-Sialyltransferase activities present in three variants of Ehrlich tumor cells: identification of the products derived from N-acetyllactosamine and b-d-Gal-(1!3)a-d-GalNAc-(1!O)-Bn. Carbohydr. Res., 264, 111-121 (1994) [21] Datta, A.K.; Paulson, J.C.: The sialyltransferase 'sialylmotif ' participates in binding the donor substrate CMP-NeuAc. J. Biol. Chem., 270, 1497-1500 (1995) [22] Nagpurkar, A.; Hunt, D.; Mookerjea, S.: Specific stimulation of a2-6 sialyltransferase activity by a novel cytosolic factor from rat colon. Int. J. Biochem. Cell Biol., 28, 1337-1348 (1996) [23] Yamamoto, T.; Nakashizuka, M.; Kodama, H.; Kajihara, Y.; Terada, I.: Purification and characterization of a marine bacterial b-galactoside a2,6-sialyltransferase from Photobacterium damsela JT0160. J. Biochem., 120, 104110 (1996) [24] Kajihara, Y.; Yamamoto, T.; Nagae, H.; Nakashizuka, M.; Sakakibara, T.; Terada, I.: A novel a-2,6-sialyltransferase: transfer of sialic acid to fucosyl and sialyl trisaccharides. J. Org. Chem., 61, 8632-8635 (1996)

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[25] Nakamura, M.; Tsunoda, A.; Yanagisawa, K.; Furukawa, Y.; Kikuchi, J.; Iwase, S.; Sakai, T.; Larson, G.; Saito, M.: CMP-NeuAc:Galb1!4GlcNAc a2!6sialyltransferase catalyzes NeuAc transfer to glycolipids. J. Lipid Res., 38, 1795-1806 (1997) [26] Malissard, M.; Zeng, S.; Berger, E.G.: Expression of functional soluble forms of human b-1,4-galactosyltransferase I, a-2,6-sialyltransferase, and a-1,3fucosyltransferase VI in the methylotrophic yeast Pichia pastoris. Biochem. Biophys. Res. Commun., 267, 169-173 (2000) [27] Dufner, G.; Schworer, R.; Muller, B.; Schmidt, R.R.: Base- and sugar-modified cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) analogues - synthesis and studies with a(2-6)-sialyltransferase from rat liver. Eur. J. Org. Chem., 8, 1467-1482 (2000) [28] Halliday, J.A.W.; Franks, A.H.; Ramsdale, T.E.; Martin, R.; Palant, E.: A rapid, semi-automated method for detection of Galb1-4GlcNAc a2,6-sialyltransferase (EC 2.4.99.1) activity using the lectin Sambucus nigra agglutinin. Glycobiology, 11, 557-564 (2001) [29] Gross, H.J.; Brossmer, R.: Enzymic transfer of sialic acids modified at C-5 employing four different sialyltransferases. Glycoconjugate J., 12, 739-746 (1995) [30] Legaigneur, P.; Breton, C.; El Battari, A.; Guillemot, J.C.; Auge, C.; Malissard, M.; Berger, E.G.; Ronin, C.: Exploring the acceptor substrate recognition of the human b-galactoside a2,6-sialyltransferase. J. Biol. Chem., 276, 2160821617 (2001) [31] Kim, H.G.; Yang, S.M.; Lee, Y.C.; Do, S.I.; Chung, I.S.; Yang, J.M.: High-level expression of human glycosyltransferases in insect cells as biochemically active form. Biochem. Biophys. Res. Commun., 305, 488-493 (2003)

329

Monosialoganglioside sialyltransferase

2.4.99.2

1 Nomenclature EC number 2.4.99.2 Systematic name CMP-N-acetylneuraminate:d-galactosyl-N-acetyl-d-galactosaminyl-(N-acetylneuraminyl)-d-galactosyl-d-glucosylceramide N-acetylneuraminyltransferase Recommended name monosialoganglioside sialyltransferase Synonyms CMP-N-acetylneuraminate:monosialoganglioside a2!3 sialyltransferase CMP-NANA:GM1 sialyltransferase CMP-NeuAc:GM1 (Galb1,4GalNAc) a2-3 sialyltransferase CMP-NeuAc:GM1 a2-3 sialyltransferase CMP-NeuAc:GM1a2-3-sialyltransferase GD1a synthase GD1a-synthase GD1b-SAT SAT-4 ST-IV ST3GalIV a(2,3)-sialyltransferase IV sialyltransferase IV sialyltransferase, cytidine monophosphoacetylneuraminate-monosialoganglioside CAS registry number 60202-12-2

2 Source Organism Rattus norvegicus (female Wistar [1]; Sprague-Dawley strain [3]; mouse neuroblastoma * rat glioma hybrid cell line [10]) [1-3, 5-7, 8, 9, 10, 11] Homo sapiens [4] cichlidae [9] Mus musculus (mouse neuroblastoma * rat glioma hybrid cell line [10]) [10, 12]

330

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3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + d-galactosyl-N-acetyl-d-galactosaminyl-(Nacetylneuraminyl)-d-galactosyl-d-glucosylceramide = CMP + N-acetylneuraminyl-d-galactosyl-N-acetyl-d-galactosaminyl-(N-acetylneuraminyl)-d-galactosyl-d-glucosylceramide Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + ganglioside GM1 (, involved in ganglioside biosynthesis [2,8]; , the enzyme is crucial for the biosynthesis of functional P-selectin ligands [12]) [2, 8, 12] P CMP + N-acetylneuraminyl-d-galactosyl-N-acetyl-d-galactosaminyl-(Nacetylneuraminyl)-d-galactosyl-d-glucosylceramide Substrates and products S CMP-N-acetylneuraminate + d-galactosyl-N-acetyl-d-galactosaminyl-(Nacetylneuraminyl)-d-galactosyl-d-glucosylceramide (Reversibility: ? [1-12]) [1-12] P CMP + N-acetylneuraminyl-d-galactosyl-N-acetyl-d-galactosaminyl-(Nacetylneuraminyl)-d-galactosyl-d-glucosylceramide (i.e. ganglioside GD1a) [1-12] S CMP-N-acetylneuraminate + asialo-ganglioside GM1 (, sialylated at 61% the rate of GM1 [6]) (Reversibility: ? [6]) [6] P CMP + ? S CMP-N-acetylneuraminate + ganglioside GA1 (Reversibility: ? [2,8]) [2, 8] P CMP + ganglioside GM1b [2, 8] S CMP-N-acetylneuraminate + ganglioside GD1b (, sialylated at 120% the rate of GM1 [6]) (Reversibility: ? [2,6,8]) [2, 6, 8] P CMP + ganglioside GT1b [2, 6, 8] S CMP-N-acetylneuraminate + ganglioside GM1-amide (, better substrate than GM1 [5]) (Reversibility: ? [5]) [5] P CMP + ganglioside GD1a-amide [5] Inhibitors Ba2+ (, 10 mM BaCl2 , 12% inhibition [1]) [1] CMP-N-acetylneuraminate (, above 0.2 M [3]) [3] Ca2+ (, 10 mM CaCl2 , 39% inhibition [1]) [1] Cd2+ (, 10 mM CdCl2 , 85% inhibition [1]) [1] Co2+ (, 10 mM CoCl2 , 44% inhibition [1]) [1] Cu2+ (, 10 mM CuCl2 , complete inhibition [1]) [1] Ni2+ (, 10 mM NiCl2 , 75% inhibition [1]) [1] Zn2+ (, 10 mM ZnCl2 , 10% inhibition [1]) [1] ganglioside GM3 (, in the presence of Triton CF-54 [2]) [2] lactosylceramide (, inhibits reaction with GM1a [2]) [2] 331


2.4.99.2

Activating compounds Triton CF-54 (, activation [2,3,8]; , activates, can partially be replaced by cutscum or Triton X-100, not by sodium taurocholate or deoxycholate, Tween 80 or cholic acid [3]; , the enzyme is most active with a detergent mixture of Triton CF-54 and Tween 80 [4]) [2, 3, 4, 8] Triton X-100 (, activation [1,4]; , slight activation [2]) [1, 2, 4] Tween 80 (, most active with a detergent mixture of Triton CF-54 and Tween 80 [4]) [4] Specific activity (U/mg) 0.00000186 [9] 0.0000033 [9] 0.00118 (, ganglioside GM1 as substrate [5]) [5] 0.00191 (, ganglioside GM1-amide as substrate [5]) [5] 0.0084 [6] Km-Value (mM) 0.000196 (ganglioside GD1b) [7] 0.000538 (ganglioside GM1) [7] 0.025 (ganglioside GM1a, , pH 6.0, 37 C [2]) [2] 0.065 (CMP-N-acetylneuraminate, , pH 6.5, 37 C [6]) [6] 0.093 (ganglioside GM1, , pH 6.6, 37 C [8]) [8] 0.16 (ganglioside GM1a) [2] 0.5 (CMP-N-acetylneuraminate, , pH 6.4, 37 C, reaction with ganglioside GM1 [1]) [1] 0.5 (ganglioside GM1, , pH 6.4, 37 C [1]) [1] 75 (ganglioside GM1, , pH 6.5, 37 C [6]) [6] Ki-Value (mM) Additional information [2] pH-Optimum 6.4 [1] 6.5 (, cacodylate buffer [3]) [3]

4 Enzyme Structure Subunits ? (, x * 44000, SDS-PAGE [6]) [6] Posttranslational modification glycoprotein [6] phosphoprotein (, activation by dephosphorylation, inactivation by phosphorylation [10,11]; , almost exclusively phosphorylated on the serine residue, with less than 2% phosphorylation on the threonine residues [11]) [10, 11]

332

2.4.99.2


5 Isolation/Preparation/Mutation/Application Source/tissue HeLa cell (, strain R [4]) [4] NG 108-15 cell (, mouse neuroblastoma * rat glioma hybrid cell line [10]) [10] T lymphocyte (, CD4, absent in native cells, rapidly up-regulated upon activation [12]) [12] Th1-cell [12] Th2-cell [12] brain [3, 6, 9, 10, 11] liver [1, 2, 7, 8] Localization Golgi apparatus (, distribution [7]) [1, 2, 5, 7, 8] membrane [1, 6] microsome [3, 10] Purification [6, 11]

6 Stability Temperature stability 56 (, t1=2 : 60 s [2]) [2]

References [1] Busam, K.; Decker, K.: Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases. Eur. J. Biochem., 160, 23-30 (1986) [2] Iber, H.; van Echten, G.; Sandhoff, K.: Substrate specificity of a2!3-sialyltransferases in ganglioside biosynthesis of rat liver Golgi. Eur. J. Biochem., 195, 115-120 (1991) [3] Yip, M.C.M.: The enzymic synthesis of disialoganglioside: rat brain cytidine-5-monophospho-N-acetylneuraminic acid: monosialoganglioside (GM1) sialyltransferase. Biochim. Biophys. Acta, 306, 298-306 (1973) [4] Fishman, P.H.; Bradley, R.M.; Henneberry, R.C.: Butyrate-induced glycolipid biosynthesis in HeLa cells: properties of the induced sialyltransferase. Arch. Biochem. Biophys., 172, 618-626 (1976) [5] Klein, D.; Pohlentz, G.; Schwarzmann, G.; Sandhoff, K.: Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver. Eur. J. Biochem., 167, 417-424 (1987) [6] Gu, T.-J.; Gu, X.-B.; Ariga, T.; Yu, R.K.: Purification and characterization of CMP-NeuAc:GM1 (Galb1-4GalNAc) a2-3 sialyltransferase from rat brain. FEBS Lett., 275, 83-86 (1990)

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[7] Trinchera, M.; Pirovano, B.; Ghidoni, R.: Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b a2!8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis. J. Biol. Chem., 265, 18242-18247 (1990) [8] Pohlentz, G.; Klein, D.; Schwarzmann, G.; Schmitz, D.; Sandhoff, K.: Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver. Proc. Natl. Acad. Sci. USA, 85, 7044-7048 (1988) [9] Freischutz, B.; Saito, M.; Rahmann, H.; Yu, R.K.: Activities of five different sialyltransferases in fish and rat brains. J. Neurochem., 62, 1965-1973 (1994) [10] Bieberich, E.; Freischutz, B.; Liour, S.-S.; Yu, R.K.: Regulation of ganglioside metabolism by phosphorylation and dephosphorylation. J. Neurochem., 71, 972-979 (1998) [11] Gu, X.; Preuss, U.; Gu, T.; Yu, R.K.: Regulation of sialyltransferase activities by phosphorylation and dephosphorylation. J. Neurochem., 64, 2295-2302 (1995) [12] Blander, J.M.; Visintin, I.; Janeway, C.A., Jr.; Medzhitov, R.: a(1,3)-Fucosyltransferase VII and a(2,3)-sialyltransferase IV are up-regulated in activated CD4 T cells and maintained after their differentiation into Th1 and migration into inflammatory sites. J. Immunol., 163, 3746-3752 (1999)

334

a-N-Acetylgalactosaminide a-2,6-sialyltransferase

2.4.99.3

1 Nomenclature EC number 2.4.99.3 Systematic name CMP-N-acetylneuraminate:glycano-1,3-(N-acetyl-a-d-galactosaminyl)-glycoprotein a-2,6-N-acetylneuraminyltransferase Recommended name a-N-acetylgalactosaminide a-2,6-sialyltransferase Synonyms CMP-N-acetylneuraminate:a-N-acetylgalactosaminide a2!6 sialyltransferase CMP-Neu5Ac:GalNAc a2,6-sialyltransferase CMP-NeuAc:galactoside (a2-6)-sialyltransferase CMP-sialic acid:a-N-acetylgalactosaminide(R!Galb1!3GalNAca1!O-Ser/ Thr) a2!6 sialyltransferase ST6GalNAc-I ST6GalNAc-II STM ( i.e. human ST6GalNAc-II [14]) [14] a-2,6-ST a-N-acetylgalactosaminylprotein a2!6 sialyltransferase mucin sialyltransferase sialyltransferase 7A [Swissprot] sialyltransferase, cytidine monophosphoacetylneuraminate-a-acetylgalactosaminide a2!6Additional information (not identical with EC 2.4.99.7) CAS registry number 71124-50-0

2 Source Organism

Sus scrofa [1-5, 7, 10] Bos taurus [2, 5] Ovis aries [2, 4, 9, 11] Rattus norvegicus [2] Homo sapiens (ST6GalNAc-II [13]; ST6GalNAc-I [16]) [6, 13, 16] Mus musculus (strain OF1 [8]) [8] Oncorhynchus mykiss (rainbow trout [12]) [12]

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Homo sapiens (STM, i.e. hST6GalNAc-II [14]) [14] Homo sapiens (hST6GalNAc-I [15]) [15]

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + glycano-1,3-(N-acetyl-a-d-galactosaminyl)-glycoprotein = CMP + glycano-(2,6-a-N-acetylneuraminyl)-(N-acetyl-d-galactosaminyl)-glycoprotein ( mechanism [1]) Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + glycano-1,3-(N-acetyl-a-d-galactosaminyl)glycoprotein ( involved in biosynthesis of O-linked oligosaccharide chains of glycoproteins [1,7]; the relative preference of enzyme for a monosaccharide versus a substituted GalNAc may play a role in regulation of chain length during mucin synthesis [2]; pathway of biosynthesis of glycans present in mucins [4]; one of the last steps of the biosynthesis of oligosaccharides of glycoproteins [5]; involved in the biosynthesis of oligosaccharide sequences of glycoproteins [3]; involved in the sialylation of submaxillary gland mucin in vivo [9]; last step in the synthesis of ovine submaxillary mucin, catalyzes the chain-terminating process in oligosaccharide synthesis of glycoproteins, may well be involved in the process of secretion [11]; required for synthesis of polySia side chains in trout egg polysialoglycoproteins, developmental expression pattern of enzyme in cortical vesicles during oogenesis, activity increases during oogenesis [12]; ST6GalNAc-II expression is significantly increased in colorectal carcinomas in cases with metastases to lymph nodes along the vascular trunk, involved in biosynthesis of the TF glycotype [13]; STM catalyzes the synthesis of Oglycans, SIATL1 gene encoding STM is widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cells, but significantly down-regulated or absent in corresponding tumor cell lines [14]; hST6GalNAc-I catalyzes the key step in the biosynthesis of Sialyl-Tn antigen, Neu5Aca(2-6)GalNAca1-O-Ser/Thr [15]; ST6GalNAcI is responsible for the synthesis of Sialyl-Tn antigen, Neu5Aca(2-6)GalNAc, synthesis of Sialyl-Tn structures in mucin O-glycosylation stops further processing and elongation of the carbohydrate chain [16]) [1-5, 7, 9, 11-16] P CMP + glycano-(2,6-a-N-acetylneuraminyl)-(N-acetyl-d-galactosaminyl)glycoprotein

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Substrates and products S CMP-9-O-acetyl-N-acetylneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)-glycoprotein ( poor donor, 10% as effective as CMPN-acetylneuraminate [5]) (Reversibility: ? [5]) [5] P CMP + R-(2,6-9-O-acetyl-N-acetylneuraminyl)-(N-acetyl-d-galactosaminyl)-glycoprotein S CMP-9-acetamidoneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)glycoprotein ( less effective than CMP-N-acetylneuraminate [10]) (Reversibility: ? [10]) [10] P CMP + R-(2,6-9-acetamidoneuraminyl)-(N-acetyl-d-galactosaminyl)-glycoprotein S CMP-9-aminoacetylneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)glycoprotein ( CMP-9-aminoacetylneuraminate can replace CMP-N-acetylneuraminate only with asialofetuin as acceptor [10]) (Reversibility: ? [10]) [10] P CMP + R-(2,6-9-aminoacetylneuraminyl)-(N-acetyl-d-galactosaminyl)glycoprotein S CMP-9-azido-N-acetylneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)-glycoprotein ( CMP-9-azido-N-acetylneuraminate can replace CMP-N-acetylneuraminate with asialofetuin or antifreeze glycoprotein as acceptor [10]) (Reversibility: ? [10]) [10] P CMP + R-(2,6-9-azido-N-acetylneuraminyl)-(N-acetyl-d-galactosaminyl)glycoprotein S CMP-9-benzamidoneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)glycoprotein ( less effective than CMP-N-acetylneuraminate [10]) (Reversibility: ? [10]) [10] P CMP + R-(2,6-9-benzamidoneuraminyl)-(N-acetyl-d-galactosaminyl)-glycoprotein S CMP-9-hexanoylamidoneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)-glycoprotein ( less effective than CMP-N-acetylneuraminate [10]) (Reversibility: ? [10]) [10] P CMP + R-(2,6-9-hexanoylamidoneuraminyl)-(N-acetyl-d-galactosaminyl)-glycoprotein S CMP-N-acetylneuraminate + GTTPSPVPT[GalNAc]TSTTSAP ( hexadecaglycopeptide acceptor, corresponding to the MUC5AC tandem repeat sequence substituted by a GalNAc residue on Thr-9, hST6GalNAcI [15]) (Reversibility: ? [15]) [15] P CMP + GTTPSPVPT[Neu5Aca(2-6)GalNAc]TSTTSAP [15] S CMP-N-acetylneuraminate + GalNAc-R ( R: protein, but not p-nitrophenol [4]; R: protein [9,13]; formation of the TF-related blood group antigen sialosyl-Tn, NeuAca(2-6)GalNAc-R, by the sialyltransferase ST6GalNAc-I [13]) (Reversibility: ? [4,9,13]) [4, 9, 13] P CMP + NeuAca(2-6)GalNAc-R [4, 9, 13] S CMP-N-acetylneuraminate + Galb(1-3)GalNAc-R ( R: protein, but not p-nitrophenol [4,9]; R: p-nitrophenol [8]; R: protein, formation of TF Sia6Core-1, Galb(1-3)(NeuAca(2,6))GalNAc-R, 337


P S

P S

P

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2.4.99.3

or of TF DiSiaCore-1, NeuAca(2-3)Galb(1-3)(NeuAca(2,6))GalNAc-R, by the sialyltransferase ST6GalNAc-II [13]) (Reversibility: ? [4,8,9,13]) [4, 8, 9, 13] CMP + Galb(1-3)(NeuAca(2-6))GalNAc-R [9, 13] CMP-N-acetylneuraminate + NeuAca(2-3)Galb(1-3)GalNAc-R ( R: protein, also p-nitrophenol, but with very low activity possibly due to another factor present in the fraction [4]; R: protein, formation of TF Sia6Core-1, Galb(1-3)(NeuAca(2,6))GalNAc-R, or of TF DiSiaCore-1, NeuAca(2-3)Galb(1-3)(NeuAca(2,6))GalNAc-R, by the sialyltransferase ST6GalNAc-II [13]) (Reversibility: ? [4,13]) [4, 13] CMP + NeuAca(2-3)Galb(1-3)(NeuAca(2-6))GalNAc-R [4, 13] CMP-N-acetylneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)-glycoprotein ( R: H or a b-galactoside [1]; transfers sialic acid to the core region of O-linked glycans [10]; specific for a-N-acetylgalactosamine attached through a-glycosidic linkage to threonine or serine in polypeptide chain [1,11]; acceptor substrate specificity [1, 3, 4, 12, 14]; preference for a monosaccharide versus a disaccharide acceptor [2]; absolute requirement for protein as the aglycon [4]; ST6GalNAc-II demands an acceptor with a protein backbone [13]; Galb(1-3)GalNAc-O-Ser/Thr or Neu5Aca(2-3)Galb(1-3)GalNAc-OSer/Thr acceptor specificity [14]; porcine submaxillary asialo-mucin [1, 4, 11]; ovine submaxillary asialo-mucin, with simple Olinked GalNAca-Thr/Ser [1-5, 11]; asialo-fetuin [1, 2, 6, 8, 10, 11, 13, 14]; native fetuin [11, 14]; bovine submaxillary asialo-mucin [11]; native submaxillary mucin, less effective than asialomucin [11]; asialo-orosomucoid [6]; antifreeze glycoprotein is the best substrate [1]; antifreeze glycoprotein from antarctic fish [3]; antifreeze glycoprotein [1, 5, 10]; sialyl-2,3-antifreeze glycoprotein, better than native form [3]; mucin-type acceptors [9]; erythrocyte hemagglutination inhibitor, milk glycopeptide [11]; native and asialo-polysialoglycoprotein, acceptor carbohydrate unit: Galb(13)GalNAca(1-O)-Ser/Thr, but not GalNAc1 to O-Ser/Thr [12]; CMP-N-glycolylneuraminate can replace CMP-N-acetylneuraminate [5, 11, 12]; CMP-N-glycolylneuraminate has the same efficiency as CMP-N-acetylneuraminate, CMP-9-O-acetyl-N-acetylneuraminate has much lower efficiency than CMP-N-acetylneuraminate [5]; CMP-9acetamidoneuraminate, CMP-9-benzamidoneuraminate and CMP-9-hexanoylamidoneuraminate are less effective, CMP-9-azido-N-acetylneuraminate can replace CMP-N-acetylneuraminate with asialofetuin or antifreeze glycoprotein as acceptor, CMP-9-aminoacetylneuraminate only with asialofetuin as acceptor [10]; hST6GalNAc-I transfers a sialic acid residue in a2,6-linkage to GalNAc-O-Ser/Thr, substrate specificity of hST6GalNAc-I and -II [15]) (Reversibility: r [3]; ir [11]; ? [1, 2, 4-10, 12-16]) [1-16] CMP + R-(2,6-a-N-acetylneuraminyl)-(N-acetyl-d-galactosaminyl)-glycoprotein [1-13]

2.4.99.3


S CMP-N-acetylneuraminate + antifreeze glycoprotein ( antifreeze glycoprotein 8, best acceptor [1]; from antarctic fish [3]) (Reversibility: r [3]; ? [1,5]) [1, 3, 5] P ? S CMP-N-acetylneuraminate + asialo-fetuin ( good substrate [1]; formation of Galb(1-3)(NeuAca(2-6))GalNAc [2]; ST6GalNAc-II [13]; STM, i.e. hST6GalNAc-II, more effective than native fetuin [14]) (Reversibility: ? [1, 2, 6, 8, 11, 13, 14]) [1, 2, 6, 8, 11, 13, 14] P CMP + fetuin S CMP-N-acetylneuraminate + asialo-milk glycopeptide (Reversibility: ? [11]) [11] P CMP + milk glycopeptide S CMP-N-acetylneuraminate + asialo-orosomucoid (Reversibility: ? [6]) [6] P CMP + orosomucoid S CMP-N-acetylneuraminate + asialo-polysialoglycoprotein ( from egg, Sia residue is a-2,6-linked to the proximal GalNAc residue in asialopolysialoglycoprotein, acceptor carbohydrate unit: Galb(1-3)GalNAca(1O)-Ser/Thr, but not GalNAc1 to O-Ser/Thr [12]) (Reversibility: ? [12]) [12] P CMP + a-sialosyl-(2-6)-asialo-polysialoglycoprotein [12] S CMP-N-acetylneuraminate + erythrocyte hemagglutination inhibitor (Reversibility: ? [11]) [11] P ? S CMP-N-acetylneuraminate + lactose ( poor acceptor [2]) (Reversibility: ? [2]) [2] P CMP + 6'-sialyllactose [2] S CMP-N-acetylneuraminate + native fetuin ( STM, i.e. hST6GalNAc-II, 83% as effective as asialo-fetuin [14]) (Reversibility: ? [11,14]) [11, 14] P ? S CMP-N-acetylneuraminate + native polysialoglycoprotein ( from egg, almost identical amount of Neu5Ac is incorporated into native and asialo-polysialoglycoprotein acceptor, acceptor carbohydrate unit: Galb(13)GalNAca(1-O)-Ser/Thr, but not GalNAc1 to O-Ser/Thr [12]) (Reversibility: ? [12]) [12] P ? S CMP-N-acetylneuraminate + p-nitrophenyl-b-d-galactoside (Reversibility: ? [6]) [6] P CMP + a-sialosyl-(2-6)-p-nitrophenyl-b-d-galactoside [6] S CMP-N-acetylneuraminate + submaxillary asialo-mucin ( ovine and porcine submaxillary asialo-mucin are good acceptors [1]; ovine submaxillary asialo-mucin [2,9]; ovine submaxillary asialo-mucin, with simple O-linked GalNAca-Thr/Ser [5]; ovine submaxillary asialo-mucin is the most effective acceptor [11]; porcine sub-

339


P S

P S

P

2.4.99.3

maxillary asialo-mucin [4,11]; bovine submaxillary asialo-mucin [11]) (Reversibility: ir [11]; ? [1, 2, 4, 5, 9]) [1, 2, 4, 5, 9, 11] CMP + submaxillary mucin ( linkage formed is NeuAc(26)GalNAca(1-)O-Thr/Ser [1,5,11]; formation of NeuAca(2-6)GalNAc [2]) [1, 2, 5, 11] CMP-N-glycolylneuraminate + R-1,3-(N-acetyl-a-d-galactosaminyl)-glycoprotein ( CMP-N-glycolylneuraminate can replace CMP-N-acetylneuraminate with the same efficiency [5]) (Reversibility: ? [5, 11, 12]) [5, 11, 12] CMP + R-(2,6-a-N-glycolylneuraminyl)-(N-acetyl-d-galactosaminyl)-glycoprotein Additional information ( no donor: CMP-9-amino-acetylneuraminate [10]; no acceptors: glycoproteins with Asn-linked oligosaccharides, glycosides, mono- and oligosaccharides [1]; very poor acceptor: ovine submaxillary asialo-mucin [2]; no acceptor: lactose [2,11]; no acceptor: human asialo-transferrin [3]; no acceptor: b-galactosyl-1,3-N-acetyl-d-galactosaminyl-p-nitrophenol [4,9]; no acceptor: N-acetyl-d-galactosaminyl-p-nitrophenol [4]; no acceptors: asialo-collacalia mucoid, asialo-proteose peptone, asialo-prothrombin, asialo-thyroglobulin, asialo-transferrin, asialo-chondromucoprotein fractions from cartilage, human blood group substance A or B, various sugars [11]; no acceptor: TF-PAA, i.e. Galb(1-3)GalNAc conjugated to poly[N-(2-hydroxyethyl)acrylamide] [13]; STM contains 2 catalytic domains, the L-sialylmotif, aa 148-194, and S-sialylmotif, aa 303-324, no acceptors: asialo-BSM, a1 acid glycoprotein, glycosphingolipids [14]) [1-4, 9, 11, 13, 14] ?

Inhibitors CDP ( less effective than CTP [7]) [7] CMP ( less effective than CTP [7]) [7] CTP ( competitive to CMP-NeuAc [1]; strong [7]) [1, 7] Ca2+ ( CaCl2 , at 0.004 M: 30% inhibition, at 0.016 M: 60% inhibition [11]) [6, 11] PCMB ( weak [8]) [8] Triton X-100 ( above critical micelle concentration of 0.016%, 15% inhibition, reversible [1]) [1] antifreeze glycoprotein ( competitive to ovine submaxillary asialomucin, non-competitive to CMP-NeuAc [1]) [1] dithioerythritol [6] Additional information ( not inhibited by native ovine submaxillary mucin [11]; not inhibited by EDTA [1,11]; not inhibited by Mg2+ [1,11]; not inhibited by Zn2+ , Mn2+ [1]) [1, 11] Metals, ions Additional information ( no metal ion requirement, e.g. Mn2+ , Mg2+ or Zn2+ [1]; no divalent metal cation requirement [11]; activity decreases with increasing divalent cation concentration [6]) [1, 6, 11] 340

2.4.99.3


Specific activity (U/mg) 0.0003-0.005 ( crude extract, acceptor asialo-fetuin, pH 7.1, 37 C [2]) [2] 0.00435 ( 37 C [5]) [11] 28.3 ( 37 C [5]) [5] 44.6 [1] Km-Value (mM) 0.08 (CMP-9-O-acetyl-N-acetylneuraminate, 37 C [5]) [5] 0.1 (CMP-9-O-acetyl-N-acetylneuraminate, 37 C [5]) [5] 0.15 (CMP-N-acetylneuraminate, 37 C [5]) [5] 0.21 (CMP-N-glycolylneuraminate, 37 C [5]) [5] 0.27 (CMP-N-acetylneuraminate, 37 C [5]) [5] 0.52 (CMP-N-acetylneuraminate, pH 6.5, 37 C, antifreeze glycoprotein 8 as acceptor [1]) [1] 0.52 (CMP-N-glycolylneuraminate, 37 C [5]) [5] 0.57 (CMP-N-acetylneuraminate, 37 C [11]) [11] 0.66 (CMP-N-acetylneuraminate, pH 6.5, 37 C, ovine submaxillary asialo-mucin as acceptor [1]) [1] 1.02 (antifreeze glycoprotein, antifreeze glycoprotein 8, pH 6.5, 37 C [1]) [1] 1.1 (CMP-N-acetylneuraminate) [2] 1.9 (submaxillary asialo-mucin, ovine, calculated in terms of Nacetylgalactosamine acceptor sites, 37 C [11]) [11] 2.2 (CMP-N-acetylneuraminate) [2] 2.7 (CMP-N-acetylneuraminate) [2] 2.8 (CMP-N-acetylneuraminate) [2] 2.93 (submaxillary asialo-mucin, ovine, pH 6.5, 37 C [1]) [1] 4.7 (submaxillary asialo-mucin, ovine, CMP-N-acetylneuraminate as donor, 37 C [5]) [5] Ki-Value (mM) 0.92 (antifreeze glycoprotein, pH 6.5, 37 C [1]) [1] pH-Optimum 5.9 ( acceptor: asialo-fetuin [2]) [2] 6-6.1 ( with cacodylate-acetate buffer [11]) [11] 6-6.5 [7] 6-7 ( acceptor: asialo-polysialoglycoprotein [12]) [12] 7.1 ( acceptor: asialo-fetuin [2]) [2] 7.5 ( acceptor: asialo-fetuin [2]) [2] 7.6 [6] pH-Range 6.2-8.5 ( about half-maximal activity at pH 6.2 and 8.5 [6]) [6] Temperature optimum ( C) 22 ( assay at [12]) [12] 25 ( broad [8]) [8]

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37 ( assay at [1-6, 9, 11, 13-15]; optimal activity at [6]) [1-6, 9, 11, 13-15] Temperature range ( C) 17-38 ( about half-maximal activity at 17 C and 38 C [8]) [8] 20-40 ( about half-maximal activity at 20 C and 40 C [6]) [6]

4 Enzyme Structure Molecular weight 100000-172000 ( enzyme exists in several active molecular weight forms from 100000-172000, gel filtration or PAGE [1]) [1] Subunits ? ( x * 160000, enzyme consists of several electrophoretic forms, 160 kDa: largest one, SDS-PAGE [1]; x * 120000, SDS-PAGE [5]; x * 42000, predicted from the amino acid sequence [14]) [1, 5, 14] Posttranslational modification Additional information ( amino acid sequence of STM has a potential site for casein kinase II phosphorylation and 3 consensus motifs for Nglycosylation at positions Asn-85, Asn-130 and Asn-161 [14]) [14]

5 Isolation/Preparation/Mutation/Application Source/tissue HT-29-MTX cell ( colon cancer cells, hST6GalNAc-I [15]) [15] K-562 cell ( hematopoietic cell line, high ST6GalNAc-I expression [16]) [16] LS174T cell ( human colon cancer cell line, recombinant ST6GalNAc-II [13]) [13] SW-480 cell ( human colon carcinoma cells, recombinant ST6GalNAc-II [13]) [13] blood platelet [6] colorectal adenocarcinoma cell ( ST6GalNAc-II [13]) [13] egg ( unfertilized mature eggs [12]) [12] liver [8] mammary epithelium ( normal human mammary epithelial cell strains 70N, 76N and 81N, STM RNA expression is highest in 76N, followed by 81N and 70N [14]) [14] ovary [12] submaxillary gland [1-5, 7, 9-11] Additional information ( ratio of ST6GalNAc-II expression in colorectal tumor cells versus normal mucosa cells is 1.47 [13]; STM tissue distribution, SIATL1 gene encoding STM is widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cells, but

342

2.4.99.3


significantly down-regulated or absent in corresponding tumor cell lines, STM gene is down-regulated in T-47D, BT-474 and MDA-MB-435 breast tumor cell lines as compared to normal mammary epithelial cell strains, only RNA traces in other tumor cell lines tested, e.g. MDA-231, MDA-468 and MCF-7, only MDA-MB-361 metastatic tumor cell line expresses STM mRNA levels as high as in 76N [14]; no expression of hST6GalNAc-I in MDAMB-231, BT-20, MCF-7 and T47-D cells [15]; low expression of ST6GalNAc-I in gastric carcinoma cell lines MKN45, KATO-III, GP202 and GP220, correlating with very low or absent detection of the Sialyl-Tn antigen [16]) [13-16] Localization Golgi membrane ( less than 4% of sialyltransferase activity [12]) [12] Golgi vesicle ( in ovaries 96% of sialyltransferase activity is found in the Golgi-derived immature cortical vesicles or as soluble enzyme released from the cortical vesicles [12]) [12] membrane ( bound to [3]) [1, 3, 7] microsome [4, 9] mitochondrial outer membrane [8] soluble ( in ovaries 96% of sialyltransferase activity is found in the Golgi-derived immature cortical vesicles or as soluble enzyme released from the cortical vesicles [12]) [12] Purification (solubilized with Triton X-100, CDP-agarose affinity chromatography, 117000fold [1]; partial [7]) [1, 5, 7] (solubilized with Triton X-100, CDP-agarose affinity chromatography, 4500fold [5]) [5] (partial, 44fold [11]) [11] (recombinant ST6GalNAc-II, immobilized metal-affinity chromatography [13]) [13] Cloning (ST6GalNAc-II cDNA is cloned, LS147T and SW-480 human colorectal carcinoma cells are transfected with human ST6GalNAc-II cDNA leading to an increased cell surface expression of Thomsen-Friedenreich-related blood group antigen TF, which depends in SW-480 cells on the ratio of core 2 b-1,6N-acetylglucosaminyltransferase and ST6GalNAc-II [13]) [13] (SIATL1 gene encoding STM is mapped to the long arm of chromosome 17 at q23-qter, full-length cDNA from normal human mammary epithelial cell strain 76N is cloned, sequenced and encodes a protein of 374 amino acids, expression in COS-7 cells [14]) [14] (cDNA encoding the full-length hST6GalNAc-I from HT-29-MTX cells is cloned, stable transfection of MDA-MB-231 breast cancer cells expressing enzyme induces the expression of sialyl-Tn antigen at the cell surface associated with morphological changes, decreased growth and increased migration of the cells [15]) [15]

343


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Application medicine ( St6GalNAc-II expression provides a prognostic factor for patient survival in human colorectal carcinomas, overexpression is related to poor patient survival [13]) [13]

6 Stability pH-Stability 6-6.5 ( maximal stability [7]) [7] Temperature stability 4 ( t1=2 : about 60 min [6]) [6] General stability information , 50 mM NaCl stabilizes during purification [1] , glycerol, 25% w/v, stabilizes during purification [3] , glycerol, 50% w/v, stabilizes during storage [1] , bovine serum albumin, 1 mg/ml, stabilizes purified enzyme [4] Storage stability , 4 C, partially purified, at least 6 months [7] , -20 C, 10 mM sodium cacodylate, pH 6.5, 50 mM NaCl, 50% glycerol, 0.02% NaN3 , at least 6 months, stable [5] , frozen, crude preparation, at least 2 months, stable [11]

References [1] Sadler, J.E.; Rearick, J.I.; Hill, R.L.: Purification to homogeneity and enzymatic characterization of an a-N-acetylgalactosaminide a2!6 sialyltransferase from porcine submaxillary glands. J. Biol. Chem., 254, 5934-5941 (1979) [2] Sherblom, A.P.; Bourassa, C.R.: Specificity of submaxillary gland sialyltransferases. Biochim. Biophys. Acta, 761, 94-102 (1983) [3] Beyer, T.A.; Rearick, J.I.; Paulson, J.C.; Prieels, J.P.; Sadler, J.E.; Hill, R.L.: Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences. J. Biol. Chem., 254, 12531-12541 (1979) [4] Bergh, M.L.E.; van den Eijnden, D.H.: Aglycon specificity of fetal calf liver and ovine and porcine submaxillary gland a-N-acetylgalactosaminide a2!6 sialyltransferase. Eur. J. Biochem., 136, 113-118 (1983) [5] Higa, H.H.; Paulson, J.C.: Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids. J. Biol. Chem., 260, 8838-8849 (1985) [6] Bauvois, B.; Cacan, R.; Fournet, B.; Caen, J.; Montreuil, J.; Verbert, A.: Discrimination between activity of (a2-3)-sialyltransferase and (a2-6)-sialyl-

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[7]

[8] [9]

[10]

[11] [12]

[13]

[14]

[15]

[16]


transferase in human platelets using p-nitrophenyl-b-d-galactoside as acceptor. Eur. J. Biochem., 121, 567-572 (1982) Sadler, J.E.; Rearick, J.I.; Paulson, J.C.; Hill, R.L.: Purification to homogeneity of a b-galactoside a2!3 sialyltransferase and partial purification of an a-N-acetylgalactosaminide a2!6 sialyltransferase from porcine submaxillary glands. J. Biol. Chem., 254, 4434-4443 (1979) Gasnier, F.; Baubichon-Cortay, H.; Louisot, P.; Gateau-Roesch, O.: Sialylation processes in mitochondria: evidence for two distinct sialyltransferases located in the outer membrane. J. Biochem., 110, 702-707 (1991) Bergh, M.L.E.; Koppen, P.L.; Van den Eijnden, D.H.: Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products. Biochem. J., 201, 411-415 (1982) Gross, H.J.; Rose, U.; Krause, J.M.; Paulson, J.C.; Schmid, K.; Feeney, R.E.; Brossmer, R.: Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases. Biochemistry, 28, 7386-7392 (1989) Carlson, D.M.; McGuire, E.J.; Jourdian, G.W.; Roseman, S.: The sialic acids. XVI. Isolation of a mucin sialyltransferase from sheep submaxillary gland. J. Biol. Chem., 248, 5763-5773 (1973) Kitazume, S.; Kitajima, K.; Inoue, S.; Inoue, Y.; Troy, F.A., II: Developmental expression of trout egg polysialoglycoproteins and the prerequisite a2,6-, and a2,8-sialyl and a2,8-polysialyltransferase activities required for their synthesis during oogenesis. J. Biol. Chem., 269, 10330-10340 (1994) Schneider, F.; Kemmner, W.; Haensch, W.; Franke, G.; Gretschel, S.; Karsten, U.; Schlag, P.M.: Overexpression of sialyltransferase CMP-sialic acid:Galb1,3GalNAc-R a6-sialyltransferase is related to poor patient survival in human colorectal carcinomas. Cancer Res., 61, 4605-4611 (2001) Sotiropoulou, G.; Kono, M.; Anisowicz, A.; Stenman, G.; Tsuji, S.; Sager, R.: Identification and functional characterization of a human GalNAc a2,6-sialyltransferase with altered expression in breast cancer. Mol. Med., 8, 42-55 (2002) Julien, S.; Krzewinski-Recchi, M.A.; Harduin-Lepers, A.; Gouyer, V.; Huet, G.; Le Bourhis, X.; Delannoy, P.: Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac:GalNAc a2,6-sialyltransferase (ST6GalNac I) cDNA. Glycoconjugate J., 18, 883-893 (2001) Marcos, N.T.; Cruz, A.; Silva, F.; Almeida, R.; David, L.; Mandel, U.; Clausen, H.; von Mensdorff-Pouilly, S.; Reis, C.A.: Polypeptide GalNAc-transferases, ST6GalNAc-transferase I, and ST3Gal-transferase I expression in gastric carcinoma cell lines. J. Histochem. Cytochem., 51, 761-771 (2003)

345


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1 Nomenclature EC number 2.4.99.4 Systematic name CMP-N-acetylneuraminate:b-d-galactoside a-2,3-N-acetylneuraminyl-transferase Recommended name b-galactoside a-2,3-sialyltransferase Synonyms CMP-N-acetylneuraminate:b-d-galactoside a2-3 sialyltransferase CMP-Neu5Ac:Glb1-3GalNAc a2,3-sialyltransferase CMP-NeuAc:galactoside(a2-3)-sialyltransferase CMP-SA:Galb3GalNAc-R a3-sialyltransferase CMP-sialic acid:Galb1-3GalNAc-R a3-sialyltransferase Gal-NAc6S Gal-b-1,3-GalNAc-a-2,3-sialyltransferase Galb1,3GalNAc a2,3-sialyltransferase NeuAc a-2,3-sialyltransferase SIATFL ST3Gal I ST3GalA.1 ST3GalIA ST3O a 2,3-ST a2 !3 sialyltransferase a3-SA-T a3SA-T b-d-Gal-(1-3)-d-GalNAc a-(2-3)-sialyltransferase b-galactoside a-2,3-sialyltransferase galb1,3galNAca2,3-sialyltransferase sialyltransferase, cytidine monophosphoacetylneuraminate-b-galactoside a2!3Additional information (may be identical with EC 2.4.99.2) CAS registry number 71124-51-1

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2 Source Organism Sus scrofa (enzyme form A and B [1,3,10,11]) [1, 3, 4, 10, 11] Homo sapiens (healthy individuals and Wiscott-Aldrich syndrom patients [9]; ST3Gal II [16]) [7, 9, 13, 14, 15, 16] Mus musculus (ST3Gal I and St3Gal II [2]; ST3GalA.1 and ST3GalA.2 [8]) [2, 5, 8, 16, 17] Ovis aries [6] Gallus gallus (ST3Gal I [12]) [12]

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-R = CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,3-N-acetyl-a-dgalactosaminyl-R Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-R (, enzyme is involved in O-linked glycosylation pathway for synthesis of core 1 oligosaccharides [9]; , enzyme activity is significantly increased in patients with chronic myelogenous leukemia and acute myeloid leukemia [15]) [9, 15] P ? Substrates and products S CMP-N-acetylneuraminate + 3-deoxy-Galb1-3GalNAca-benzyl (Reversibility: ? [15]) [15] P ? S CMP-N-acetylneuraminate + 4-deoxy-Galb1-3GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3(4-deoxy)-Galb1-3GalNAca-benzyl S CMP-N-acetylneuraminate + 6-deoxy-Galb1-3GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3(6-deoxy)-Galb1-3GalNAca-benzyl S CMP-N-acetylneuraminate + Ala-Pro-(Galb1-3GalNAca)-Ser-Ser-Ser (Reversibility: ? [15]) [15] P CMP + NeuAca2-3(Galb1-3GalNAca)-Ser-Ser-Ser S CMP-N-acetylneuraminate + Ala-Pro-(Galb1-3GalNAca)-Thr-Ser-Ser (Reversibility: ? [15]) [15] P CMP + NeuAca2-3(Galb1-3GalNAca)-Thr-Ser-Ser S CMP-N-acetylneuraminate + Galb1-3(2-deoxy)Gala-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3(2-deoxy)Gala-benzyl

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S CMP-N-acetylneuraminate + Galb1-3(4-deoxy)GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3(4-deoxy)GalNAca-benzyl S CMP-N-acetylneuraminate + Galb1-3(6-O-Me)GalNAca-benzyl (Reversibility: ? [4]) [4] P CMP + NeuAca2-4Galb1-3(6-O-Me)GalNAca-benzyl S CMP-N-acetylneuraminate + Galb1-3(6-deoxy)GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3(6-deoxy)GalNAca-benzyl S CMP-N-acetylneuraminate + Galb1-3(6-sulfo)GalNAca-O-allyl (Reversibility: ? [4]) [4] P CMP + NeuAca2-4Galb1-3(6-sulfo)GalNAca-O-allyl S CMP-N-acetylneuraminate + Galb1-3(NeuAca2,6)GalNAca-O-benzyl (Reversibility: ? [4,5]) [4, 5] P CMP + NeuAca2-4Galb1-3(NeuAca2,6)GalNAca-O-benzyl S CMP-N-acetylneuraminate + Galb1-3GalNAc (, best acceptor [1]; , best acceptor for enzyme ST3Gal I and ST3Gal II [2]) (Reversibility: ? [1,2,8,10,12,13]) [1, 2, 8, 10, 12, 13] P CMP + NeuAca2-3Galb1-3GalNAc S CMP-N-acetylneuraminate + Galb1-3GalNAca-8-methoxycarbonyl-octyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3GalNAca-8-methoxycarbonyl-octyl S CMP-N-acetylneuraminate + Galb1-3GalNAca-O-allyl (Reversibility: ? [4]) [4] P CMP + NeuAca2-4Galb1-3GalNAca-O-allyl S CMP-N-acetylneuraminate + Galb1-3GalNAca-O-benzyl (Reversibility: ? [4,13,14,15]) [4, 13, 14, 15] P CMP + NeuAca2-4Galb1-3GalNAca-O-benzyl S CMP-N-acetylneuraminate + Galb1-3GalNAca-Ser (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3GalNAca-Ser S CMP-N-acetylneuraminate + Galb1-3GalNAca-antifreeze glycoprotein (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3GalNAca-antifreeze glycoprotein S CMP-N-acetylneuraminate + Galb1-3GalNAca-o-nitrophenyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3GalNAca-o-nitrophenyl S CMP-N-acetylneuraminate + Galb1-3GalNAca-p-nitrophenyl (Reversibility: ? [9,15]) [9, 15] P CMP + NeuAca2-3Galb1-3GalNAca-p-nitrophenyl S CMP-N-acetylneuraminate + Galb1-3GalNAca-phenyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3Galb1-3GalNAca-phenyl S CMP-N-acetylneuraminate + Galb1-3GlcNAc (, weak acceptor [1]; , very low activity, enzyme ST3Gal I and ST3Gal II [2]) (Reversibility: ? [1,3]) [1, 2] P CMP + NeuAca2-3Galb1-3GlcNAc 348

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S CMP-N-acetylneuraminate + Galb1-3GlcNAcb1-3Galb1-4Glc (, weak, i.e. lactotetraose [1]) (Reversibility: ? [1]) [1] P CMP + NeuAca2-3Galb1-3GlcNAcb1 -3Galb1-4Glc S CMP-N-acetylneuraminate + Galb1-3[6-O-(4,4-azo)pentyl]GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + neuAca2-3Galb1-3[6-O-(4,4-azo)pentyl]GalNAca-benzyl S CMP-N-acetylneuraminate + Galb1-4Glc (, weak acceptor [1]) (Reversibility: ? [1]) [1] P CMP + NeuAca2-3Galb1-4Glc S CMP-N-acetylneuraminate + Galb1-4GlcNAc (, weak acceptor for enzyme B [1]; , no activity [2]) (Reversibility: ? [1]) [1] P CMP + NeuAca2-3Galb1-4GlcNAc S CMP-N-acetylneuraminate + Galb1-6GlcNAc (, weak acceptor for enzyme B [1]) (Reversibility: ? [1]) [1] P CMP + NeuAca2-3Galb1-6GlcNAc S CMP-N-acetylneuraminate + GlcNAcb1-6(D-Fucb1-3)GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3 GlcNAcb1-6(D-Fucb1-3)GalNAca-benzyl S CMP-N-acetylneuraminate + GlcNAcb1-6(Galb1-3)GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3 GlcNAcb1-6(Galb1-3)GalNAca-benzyl S CMP-N-acetylneuraminate + GlcNAcb1-6(GlcNAcb1-6Galb1-3b1-3)GalNAca-benzyl (Reversibility: ? [15]) [15] P CMP + NeuAca2-3GlcNAcb1-6(GlcNAcb1-6Galb1-3b1-3)GalNAca-benzyl S CMP-N-acetylneuraminate + b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-R (, acceptor: Gg4 [12]; , acceptor: antifreeze glycoprotein [1, 3, 15]; , acceptor: asialoglycoprotein [1]; , acceptor glycoprotein [6]; , acceptor: asialo-GM1 [2, 8, 12, 13]; , acceptor: Galb1-3GalNAca-O-allyl/acrylamide copolymer [4]; , acceptor: asialofetuin [7, 8, 12, 13, 16]; , acceptor: GM1 [8, 12, 13, 16]; , acceptor: asialo-bovine-submaxillary mucin, weak activity [12]; , acceptor: GD1b [12,13]; , acceptor: GD1a [13]; , acceptor: asialo-bovine submaxillary mucin [13]; , acceptors are glycosides, glycolipids, or glycoproteins containing Galb1-3GalNAc terminal sequence [13]; , acceptor substrate preference for glycolipid than for Olinked oligosaccharides of glycoproteins [16]; , exhibits transferase activity towards only the disaccharide moiety of Galb1,3GalNAc of glycoproteins and glycolipids: asialo-GM1, GD1a is formed from GM1 and GD1b is formed from GD1b [17]) (Reversibility: ? [1, 3, 4, 6, 7, 8, 12, 13, 15, 16, 17]) [1, 2, 3, 4, 6, 7, 8, 12, 13, 15, 16, 17] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,3-N-acetyl-a-d-galactosaminyl-R

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S Additional information (, no activity with: 3-sulfoGalb1-3GalNAca-O-allyl, 3-sulfoGalb1-3GalNAca-O-benzyl, 3-O-Me-Galb1-3GalNAca-O-Bn, 3-O-Me-Galb1-3(6-O-Me)GalNAca-O-benzyl, 6-sulfoGalb13GalNAca-O-allyl [4]; , no activity with Galb1-4GlcNAc, GalNAcb14GlcNAc or Galb1-4Glc [13]) [4, 13] P ? Inhibitors 3-deoxy-Galb1-3GalNAca-benzyl [15] ATP [12] CDP (, less effective than CTP [3]) [3] CMP [12] CMP (, less effective than CTP [3]) [3] CTP (, competitive to CMP-N-acetylneuraminate [1]; , strong [3]) [1, 3, 10, 12] GM1 (, inhibits reaction with asialoGM1 [8]) [8] GTP [12] Galb1-3(6-deoxy)GalNAca-benzyl [15] Galb1-3GalNAc (, inhibits reaction with asialoGM1 as acceptor [8]) [8] UTP [12] asialoGM1 (, inhibits reaction with Galb1-3GalNAc, asialofetuin or GM1 as acceptor [8]) [8] asialofetuin (, inhibits reaction with asialoGM1 and Galb1-3GalNAc as acceptor [8]) [8] b-d-galactosyl-1,3-N-acetylgalactosylamide (, with porcine submaxillary asialomucin as substrate [1]) [1] lysophosphatidylglycerol (, enzyme forms A and B [10]) [10] lysophosphatidylserine (, enzyme form A [10]) [10] octylglucoside (, irreversible, at higher concentrations [10]) [10] Activating compounds Triton (, activation, enzyme form A only above critical micelle concentration, not form B [10]; , stimulates [11]) [10, 11] lysophosphatidylcholine (, activation, enzyme form A, not B [10]) [10] phospholipid/octylglucoside liposomes (, incorporation into phospholipid/octylglucoside liposomes activates enzyme form A [11]) [11] Additional information (, enzyme form A is incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipid from the membranes of Golgi apparatus from porcine submaxillary glands. Phosphatidylcholine induces the high activity state of the enzyme. Cholesterol has no effect on the activity of the transferase in liposomes. The phospholipids of the Golgi apparatus appear to increase the Km -value for CMPNeuAc. Enzyme form B, lacking the putative lipid-binding domain is not incorporated into phosphatidylcholine liposomes [11]) [11]

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Specific activity (U/mg) 10.6 [3] Additional information [10, 13] Km-Value (mM) 0.00434 (CMP-N-acetylneuraminate, , enzyme form A, with 1% detergent [10]) [10] 0.0044 (CMP-N-acetylneuraminate, , enzyme form A, with 1% Triton [11]) [11] 0.00482 (CMP-N-acetylneuraminate, , enzyme form B [10]) [10] 0.0123 (CMP-N-acetylneuraminate, , enzyme form A, no detergent [10,11]) [10, 11] 0.0137 (CMP-N-acetylneuraminate, , truncated enzyme protein consisting of amino acids 57-340, hST3-D56 [14]) [14] 0.018 (Galb1-3GalNAca-O-benzyl, , truncated enzyme protein consisting of amino acids 26-340, hST3-D25 [14]) [14] 0.02 (CMP-N-acetylneuraminate) [12] 0.021 (CMP-N-acetylneuraminate, , truncated enzyme protein consisting of amino acids 26-340, hST3-D25 [14]) [14] 0.023 (Galb1-3GalNAca-O-benzyl, , truncated enzyme protein consisting of amino acids 57-340, hST3-D56 [14]) [14] 0.03 (CMP-N-acetylneuraminate) [17] 0.03 (Galb1-3GalNAca-benzyl, , enzyme from human placenta [15]) [15] 0.049 (asialofetuin) [13] 0.05 (Galb1-3GalNAc) [12] 0.05 (Galb1-3GalNAca-O-allyl/acrylamide copolymer) [4] 0.1 (asialofetuin, , enzyme ST3GalA.1 [8]) [8] 0.105 (Galb1-3(6-sulfo)GalNAca-O-allyl) [4] 0.11 (Galb1,3GalNAc) [17] 0.15 (GD1b) [17] 0.16 (Galb1,3GalNAc, , enzyme ST3GalA.1 [8]) [8] 0.2 (Galb1-3GalNACa-O-allyl) [4] 0.2 (Galb1-3GalNAc, , enzyme form A [1]) [1] 0.21 (Galb1-3GalNAc, , enzyme form B [1]) [1] 0.26 (Galb1-3GalNAc) [13] 0.32 (b-d-galactosyl-1,3-N-acetylgalactosylamide, , enzyme form A, with 1% Triton X-100 or enzyme form B [10]) [10] 0.35 (Galb1-3GalNAc-Thr antifreeze glycoprotein, , enzyme form A [1]) [1] 0.37 (asialo-GM1) [17] 0.37 (b-d-galactosyl-1,3-N-acetylgalactosylamide, , enzyme form A, with 1% lysophosphatidylcholine or without detergent [10]) [10] 0.39 (Galb1-3GalNAc-Thr antifreeze glycoprotein, , enzyme form B [1]) [1] 0.4 (4-deoxy-Galb1-3GalNAca-benzyl, , enzyme from acute myeloid leukemia [15]) [15]

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0.4 (6-deoxy-Galb1-3GalNAca-benzyl, , enzyme from acute myeloid leukemia [15]) [15] 0.4 (Galb1-3(4-deoxy)GalNAca-benzyl, , enzyme from human placenta [15]) [15] 0.4 (Galb1-3GalNAca-benzyl, , enzyme from acute myeloid leukemia [15]) [15] 0.42 (Galb1-3(NeuAca2,6)GalNAca-O-benzyl) [4] 0.43 (Galb1-3GalNAc) [16] 0.45 (GM1, enzyme ST3GalA.2 [8]) [8] 0.45 (Galb1-3GalNAc) [16] 0.46 (asialo-GM1) [13] 0.48 (asialofetuin, enzyme ST3GalA.2 [8]) [8, 16] 0.5 (4-deoxy-Galb1-3GalNAca-benzyl, , enzyme from human placenta [15]) [15] 0.5 (GM1) [16] 0.5 (Galb1,3GalNAc, , enzyme ST3GalA.2 [8]) [8] 0.5 (Galb1-3[6-O-(4,4-azo)pentyl]GalNAca-benzyl, , enzyme from acute myeloid leukemia [15]) [15] 0.51 (GM1) [16] 0.52 (asialofetuin) [16] 0.56 (asialoGM1) [8] 0.87 (GM1) [13] 1 (GD1b) [12] 1.25 (asialoGM1, , enzyme ST3GalA.1 [8]) [8] 1.3 (GM1) [12] 1.3 (Galb1-3(2-deoxy)Gala-benzyl, , enzyme from acute myeloid leukemia [15]) [15] 1.3 (Galb1-3(4-deoxy)GalNAca-benzyl, , enzyme from acute myeloid leukemia [15]) [15] 1.3 (Galb1-3(6-deoxy)GalNAca-benzyl, , enzyme from acute myeloid leukemia [15]) [15] 1.5 (GM1) [17] 1.6 (Galb1-3(6-deoxy)GalNAca-benzyl, , enzyme from human placenta [15]) [15] 1.67 (GM1, , enzyme ST3GalA.1 [8]) [8] 2.7 (Galb1-3(2deoxy)Gala-benzyl, , enzyme from human placenta [15]) [15] 3.4 (6-deoxy-Galb1-3GalNAca-benzyl, , enzyme from human placenta [15]) [15] 4.7 (Galb1-3GlcNAc, , enzyme form B [1]) [1] 10 (Galb1-3GlcNAc) [12] 15 (Galb1-3GlcNAcb1-3Galb1-4Glc, , enzyme form A [1]) [1] 27 (Galb1-3GlcNAcb1-3Galb1-4Glc, , enzyme form B [1]) [1] 29 (Galb1-6GlcNAc, , enzyme form A [1]) [1] 42 (Galb1-4GlcNAc, , enzyme form B [1]) [1] 65 (Galb1-3GlcNAc, , enzyme form B [1]) [1] 85 (Galb1-3GlcNAc, , enzyme form A [1]) [1] 352

2.4.99.4


130 (Galb1-4Glc, , enzyme form A [1]) [1] 180 (Galb1-4Glc, , enzyme form B [1]) [1] Additional information (, influence of liposome incorporation on the Km -value [11]) [11] Ki-Value (mM) 0.00015 (CTP, , reaction with 1% Triton X-100 or 1% lysophosphatidylcholine, enzyme form A [10]) [10] 0.00016 (CTP, , enzyme form B [10]) [10] 0.00043 (CTP, , reaction with no detergent, enzyme form A [10]) [10] 0.0007 (UTP) [12] 0.001 (CTP) [12] 0.0038 (GTP) [12] 0.011 (ATP) [12] 0.015 (CMP) [12] 0.07 (asialofetuin, , reaction with Galb1-3GalNAc as acceptor, enzyme form ST3GalA.1 [8]) [8] 0.08 (asialofetuin, , reaction with asialoGM1 as acceptor, enzyme form ST3GalA.1 [8]; ,reaction with Galb1-3GalNAc as acceptor, enzyme form ST3GalA.2 [8]) [8] 0.11 (asialoGM1, , reaction with Galb1-3GalNAc as acceptor, enzyme form ST3GalA.2 [8]) [8] 0.15 (Galb1-3GalNac, , reaction with asialoGM1 as acceptor, enzyme form ST3GalA.1 [8]) [8] 0.44 (asialoGM1, , reaction with asialofetuin as substrate, enzyme form ST3GalA.2 [8]) [8] 0.51 (GM1, , reaction with asialoGM1 as acceptor, enzyme form ST3GalA.2 [8]) [8] 0.53 (asialoGM1, , reaction with GM1, enzyme form ST3GalA.2 [8]) [8] 0.8 (3-deoxy-Galb1-3GalNAca-benzyl, , reaction with human placental enzyme [15]) [15] 1.29 (asialoGM1, , reaction with GM1, enzyme form ST3GalA.1 [8]) [8] 3.1 (3-deoxy-Galb1-3GalNAca-benzyl, , reaction with enzyme from acute myeloid leukemia [15]) [15] pH-Optimum 5.5-6.5 [12] 6-6.5 [3] pH-Range 4.5-7.5 (, pH 4.5: about 20% of maximal activity, pH 7.5: about 55% of maximal activity [12]) [12]

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4 Enzyme Structure Molecular weight 44000 (, enzyme form B, sucrose density gradient centrifugation [3]) [3] 50000 (, enzyme form B, gel filtration [3]) [3] 220000 (, enzyme form A, gel filtration [3]) [3] Additional information (, enzyme form A and B are noninterconverting [3]) [3] Subunits ? (, x * 50000, SDS-PAGE [1]; , x * 50000, SDS-PAGE under reducing and nonreducing conditions [3]) [1, 3] Posttranslational modification glycoprotein (, correct glycosylation of the enzyme might play a key role in its folding that may be directly related to the enzymatic activity [7]; , the recombinant hST3Gal I polypeptides transiently expressed in COS-7 cells are glycosylated with complex and high mannose type glycans on each of the five potential N-glycosylation sites [14]) [7, 14]

5 Isolation/Preparation/Mutation/Application Source/tissue CEM cell [13] Ehrlich ascites carcinoma cell (, na-EAT cells are not adherent nor grow in tissue culture, aEAT cells are selected to grow in tissue culture and adhere to extracellular matrices [5]) [5] acute myeloid leukemia cell (, enzyme activity is significantly increased in patients with chronic myelogenous leukemia and acute myeloid leukemia [15]) [15] brain (, ST3GalI gene is weakly expressed, ST3GalII gene is strongly expressed [2]; , embryonic, the gene is expressed in early embryonic stages [12]) [2, 8, 12, 17] colon (, ST3GalII gene is weakly expressed [2]) [2] heart (, ST3GalI gene is weakly expressed [2]; , high expression [13]) [2, 13, 15] hepatoma cell (, slightly expressed in hepatoma and highly expressed in the surrounding tissue [7]) [7] kidney (, ST3GalI gene is weakly expressed [2]; , very low activity [15]) [2, 15] liver (, ST3GalI gene is weakly expressed [2]; , expressed poorly in fetal liver and in adult liver [7]; , high expression [13]) [2, 4, 7, 13] lung (, very low activity [15]) [15]

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lymphocyte (, T-lymphocytes, Epstein-Barr virus immortalized Blymphocytes [9]) [9] lymphoid tissue (, high expression [13]) [13] placenta [15] platelet [9] salivary gland (, ST3GalI gene is strongly expressed, ST3GalII gene is weakly expressed [2]) [2] skeletal muscle (, high expression [13]) [13, 16] spleen (, ST3GalI gene is strongly expressed [2]) [2] submaxillary gland [1, 3, 6, 10, 11] thymus (, ST3GalI gene and ST3GalII gene is weakly expressed [2]) [2] Localization Golgi apparatus (, about 85% of total activity, presumably enzyme form A [10]) [5, 10, 11] membrane (, about 85% of total activity, presumably enzyme form A [10]; , Golgi membrane [14]) [1, 3, 9-11, 14] microsome [6] soluble (, about 15% of total activity, presumably enzyme form B [10]) [10, 13] Purification [3, 10] Cloning (expression in COS-1, Escherichia coli and Pichia pastoris. The recombinant protein expressed in COS-1 can catalyze the transfer of NeuAc from CMPNeuAc to asialo-fetuin. No activity is detected with a 32000 Da protein in Escherichia coli and both 32000 Da and 41000 Da proteins in Pichia pastoris [7]; a soluble form of hST3Gal II is expressed in COS-7 cells [13]; expression in COS-7 cell line of various constructs deleted in the N-terminal portion of the protein sequence. The souble forms of the protein consisting of amino acids 26-340, hST3-D25, and amino acids 57-340, hST3-D56, are efficiently secreted and active. Further deletion of the N-terminal region gives also rise to various polypeptides that are not active within the transfected cells and not secreted in the culture medium [14]; expression in COS cells [16]) [7, 13, 14, 16] (ST3GalI and ST3GalII, expression in COS-7 cells [8]; expression in COS7 cells results in secretion of a catalytically active and soluble form of the enzyme into the medium, primary structure consists of a short NH2 -terminal cytoplasmic domain, a signal-membrabe anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain [17]) [8, 17] (expression in COS-7 cells [12]) [12]

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2.4.99.4

6 Stability pH-Stability 6-6.5 (, maximal stability [3]) [3] Storage stability , -20 C, concentrated enzyme preparation in 50% v/v glycerol, stable for more than 6 months [3]

References [1] Rearick, J.I.; Sadler, J.E.; Paulson, J.C.; Hill, R.L.: Enzymatic characterization of b d-galactoside a2!3 sialyltransferase from porcine submaxillary gland. J. Biol. Chem., 254, 4444-4451 (1979) [2] Kono, M.; Ohyama, Y.; Lee, Y.C.; Hamamoto, T.; Kojima, N.; Tsuji, S.: Mouse b-galactoside a 2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression. Glycobiology, 7, 469-479 (1997) [3] Sadler, J.E.; Rearick, J.I.; Paulson, J.C.; Hill, R.L.: Purification to homogeneity of a b-galactoside a2!3 sialyltransferase and partial purification of an a-N-acetylgalactosaminide a2!6 sialyltransferase from porcine submaxillary glands. J. Biol. Chem., 254, 4434-4443 (1979) [4] Chandrasekaran, E.V.; Jain, R.K.; Larsen, R.D.; Wlasichuk, K.; Matta, K.L.: Selectin ligands and tumor-associated carbohydrate structures: Specificities of a2,3-Sialyltransferases in the assembly of 3'-sialyl-6-sulfo/sialyl Lewis a and x, 3'-sialyl-6'-sulfo Lewis x, and 3'-sialyl-6-sialyl/sulfo blood group Thapten. Biochemistry, 34, 2925-2936 (1995) [5] Shigeta, S.; Winter, H.C.; Goldstein, I.J.: a-(2!3)- and a-(2!6)-sialyltransferase activities present in three variants of Ehrlich tumor cells: identification of the products derived from N-acetyllactosamine and b-d-Gal-(1!3)a-d-GalNAc-(1!O)-Bn. Carbohydr. Res., 264, 111-121 (1994) [6] Bergh, M.L.E.; Koppen, P.L.; Van den Eijnden, D.H.: Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products. Biochem. J., 201, 411-415 (1982) [7] Shang, J.; Qiu, R.; Wang, J.; Liu, J.; Zhou, R.; Ding, H.; Yang, S.; Zhang, S.; Jin, C.: Molecular cloning and expression of Galb1,3GalNAc a2,3-sialyltransferase from human fetal liver. Eur. J. Biochem., 265, 580-588 (1999) [8] Kojima, N.; Lee, Y.C.; Hamamoto, T.; Kurosawa, N.; Tsuji, S.: Kinetic properties and acceptor substrate preferences of two kinds of Galb1,3GalNAc a2,3-sialyltransferase from mouse brain. Biochemistry, 33, 5772-5776 (1994) [9] Higgins, E.A.; Siminovitch, K.A.; Zhuang, D.; Brockhausen, I.; Dennis, J.W.: Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome. J. Biol. Chem., 266, 6280-6290 (1991)

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[10] Westcott, K.R.; Wolf, C.C.; Hill, R.L.: Regulation of b-d-galactoside a2!3 sialyltransferase activity. The effects of detergents and lysophosphatidates. J. Biol. Chem., 260, 13109-13115 (1985) [11] Westcott, K.R.; Hill, R.L.: Reconstitution of a porcine submaxillary gland bd-galactoside a2-3 sialyltransferase into liposomes. J. Biol. Chem., 260, 13116-13121 (1985) [12] Kurosawa, N.; Hamamoto, T.; Inoue, M.; Tsuji, S.: Molecular cloning and expression of chick Galb1,3GalNAc a2,3-sialyltransferase. Biochim. Biophys. Acta, 1244, 216-222 (1995) [13] Giordanengo, V.; Bannwarth, S.; Laffont, C.; Van Miegem, V.; Harduin-Lepers, A.; Delannoy, P.; Lefebvre, J.-C.: Cloning and expression of cDNA for a human Gal(b1-3)GalNAc a2,3-sialyltransferase from the CEM T-cell line. Eur. J. Biochem., 247, 558-566 (1997) [14] Vallejo-Ruiz, V.; Haque, R.; Mir, A.-M.; Schwientek, T.; Mandel, U.; Cacan, R.; Delannoy, P.; Harduin-Lepers, A.: Delineation of the minimal catalytic domain of human Galb1-3GalNAc a2,3-sialyltransferase (hST3Gal I). Biochim. Biophys. Acta, 1549, 161-173 (2001) [15] Kuhns, W.; Rutz, V.; Paulsen, H.; Matta, K.L.; Baker, M.A.; Barner, M.; Granovsky, M.; Brockhausen, I.: Processing O-glycan core 1, Galb1-3GalNAca-R. Specificities of core 2, UDP-GlcNAc: Galb1-3GalNAc-R(GlcNAc to GalNAc) b6-N-acetylglucosaminyltransferase and CMP-sialic acid: Galb1-3GalNAc-R a3-sialyltransferase. Glycoconjugate J., 10, 381-394 (1993) [16] Kim, Y.-J.; Kim, K.-S.; Kim, S.-H.; Kim, C.-H.; Ko, J.H.; Choe, I.-S.; Tsuji, S.; Lee, Y.-C.: Molecular cloning and expression of human Galb1,3GalNAc a2,3-sialyltransferase (hST3Gal II). Biochem. Biophys. Res. Commun., 228, 324-327 (1996) [17] Lee, Y.-C.;Kurosawa, N.; Hamamoto, T.; Nakaoka, T.; Tsuji, S.: Molecular cloning and expression of Galb1,3GalNAca2,3-sialyltransferase from mouse brain. Eur. J. Biochem., 216, 377-385 (1993)

357

Galactosyldiacylglycerol a-2,3-sialyltransferase

2.4.99.5

1 Nomenclature EC number 2.4.99.5 Systematic name CMP-N-acetylneuraminate:1,2-diacyl-3-b-d-galactosyl-sn-glycerol N-acetylneuraminyltransferase Recommended name galactosyldiacylglycerol a-2,3-sialyltransferase Synonyms sialyltransferase, cytidine monophosphoacetylneuraminate-galactosyldiacylglycerol CAS registry number 80237-98-5

2 Source Organism Mus musculus (Swiss-Webster albino [1]) [1] Rattus norvegicus (recombinant enzyme [2,3]) [2, 3]

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + 1,2-diacyl-3-b-d-galactosyl-sn-glycerol = CMP + 1,2-diacyl-3-[3-(a-d-N-acetylneuraminyl)-b-d-galactosyl]-sn-glycerol Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + 1,2-diacyl-3-b-d-galactosyl-sn-glycerol ( i.e. galactosyldiacylglycerol, transfers N-acetylneuraminic acid to position C-3 of the galactosyl residue [1]) (Reversibility: ? [1]) [1] P CMP + 1,2-diacyl-3-[3-(a-d-N-acetylneuraminyl)-b-d-galactosyl]-sn-glycerol ( i.e. sialosylgalactosyldiacylglycerol [1]) [1]

358

2.4.99.5


Substrates and products S CMP-N-acetylneuraminate + 1,2-diacyl-3-b-d-galactosyl-sn-glycerol ( i.e. galactosyldiacylglycerol, transfers N-acetylneuraminic acid to position C-3 of the galactosyl residue [1]) (Reversibility: ? [1]) [1] P CMP + 1,2-diacyl-3-[3-(a-d-N-acetylneuraminyl)-b-d-galactosyl]-sn-glycerol ( i.e. sialosylgalactosyldiacylglycerol [1]) [1] S CMP-N-acetylneuraminate + Galb1-3GlcNAcR ( i.e. rat asialo a1 acid glycoprotein [3]) (Reversibility: ? [3]) [3] P CMP + NeuAca2-3Galb1-3GlcNAcR S CMP-N-acetylneuraminate + Galb1-3GlcNAcb1-3Galb1-4Glc ( i.e. lacto-N-tetraose [3]) (Reversibility: ? [3]) [3] P CMP + ? S CMP-N-acetylneuraminate + Galb1-4GlcNAc ( i.e. N-acetyllactosamine [3]) (Reversibility: ? [3]) [3] P CMP + NeuAca2-3Galb1-4GlcNAc S CMP-N-acetylneuraminate + Galb1-4GlcNAcR ( i.e. human asialo a1 acid glycoprotein [3]) (Reversibility: ? [3]) [3] P CMP + NeuAca2-3Galb1-4GlcNAcR Km-Value (mM) 0.057 (CMP-acetylneuraminate) [3] 0.11 (Galb1-3GlcNAcb1-1Galb1-4Glc) [3] 0.13 (galactosyldiacylglycerol) [1] 0.18 (Galb1-3GlcNAcR) [3] 0.18 (Galb1-4GlcNaAcR) [3] 0.26 (Galb1-4GlcNAcR) [3] 0.78 (CMP-acetylneuraminate) [1] 2.43 (Galb1-4GlcNAc) [3] Additional information ( comparison of kinetic properties of sialyltransferases [3]) [3] pH-Optimum 6.2 [1] Temperature optimum ( C) 37 ( assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue brain [1] liver [3] Localization membrane [1] microsome [1]

359


2.4.99.5

Application biotechnology ( use for chemoenzymatic synthesis of 13 C-labeled sialyloligosaccharide [2]) [2]

References [1] Pieringer, J.; Keech, S.; Pieringer, R.A.: Biosynthesis in vitro of sialosylgalactosyldiacylglycerol by mouse brain microsomes. J. Biol. Chem., 256, 1230612309 (1981) [2] Miyazaki, T.; Sato, H.; Sakakibara, T.; Kajihara, Y.: An approach to the precise chemoenzymatic synthesis of 13C-labeled sialyloligosaccharide on an intact glycoprotein: A novel one-pot [3-13 C]-labeling method for sialic acid analogues by control of the reversible aldolase reaction, enzymatic synthesis of [313 C]-NeuAc-a-(2!3)-[U-13 C]-Gal-b-(1!4)-GlcNAc-b- sequence onto glycoprotein, and its conformational analysis by developed NMR techniques. J. Am. Chem. Soc., 122, 5678-5694 (2000) [3] Williams, M.A.; Kitagawa, H.; Datta, A.K.; Paulson, J.C.; Jamieson, J.C.: Large-scale expression of recombinant sialyltransferases and comparison of their kinetic properties with native enzymes. Glycoconjugate J., 12, 755-761 (1995)

360

N-Acetyllactosaminide a-2,3-sialyltransferase

2.4.99.6

1 Nomenclature EC number 2.4.99.6 Systematic name CMP-N-acetylneuraminate:b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-glycoprotein a-2,3-N-acetylneuraminyltransferase Recommended name N-acetyllactosaminide a-2,3-sialyltransferase Synonyms Gal b-1,3(4) GlcNAc a-2,3 sialyltransferase N-acetyllactosaminide a-2,3-sialyltransferase ST3Gal III [8] ST3N a2!3 sialyltransferase cytidine monophosphoacetylneuraminate-b-galactosyl(1!4)acetylglucosaminide a2!3-sialyltransferase sialyltransferase sialyltransferase, cytidine monophosphoacetylneuraminate-b-galactosyl(1! 4)acetylglucosaminide a2!3Additional information ( the viral enzyme utilizes disaccharides type I-III and also fucosylated Lewis a and Lewis x and represents an enzyme form distinct from the vertebrate enzyme, the enzyme is named v-ST3Gal I within an extra viral nomenclature independent of the nomenclature for vertebrate enzymes [9]) [9] CAS registry number 77537-85-0

2 Source Organism

Bos taurus (calf [1]) [1] Gallus gallus (embryo [1]) [1] Homo sapiens [1-5] Rattus norvegicus [6, 7, 10, 11] Rattus norvegicus [8] Mus musculus [8] myxoma virus (brasilian strain, ATCC VR-115 [9]; from infected european rabbit RK13 cells [9]) [9] 361


2.4.99.6

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosaminylglycoprotein = CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-glycoprotein (acts on b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl termini on glycoprotein) Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosaminylR [1-10] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R Substrates and products S CMP-N-acetylneuraminate + 4-nitrophenyl-d-galactoside (Reversibility: ? [4]) [4] P ? S CMP-N-acetylneuraminate + asialo-a1 acid glycoprotein (Reversibility: ? [1-4]) [1, 4] P ? S CMP-N-acetylneuraminate + b-d-galactosyl-1,3-N-acetyl-d-glucosamine ( best substrate [8]; type II disaccharides are preferred substrates, low activity with glycolipids [8]) (Reversibility: ? [8]) [8] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosamine [8] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosamine ( i.e. N-acetyllactosamine [9]; type II disaccharides are preferred substrates [8]; low activity with glycolipids [8]) (Reversibility: ? [8,9]) [8, 9] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosamine [8, 9] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl1,2-a-mannosyl-1,3-b-mannosyl-1,4-N-acetyl-d-glucosamine (Reversibility: ? [2]) [2] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-1,2-a-mannosyl-1,3-b-mannosyl-1,4-N-acetyl-d-glucosamine [2] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl1,2-a-mannosyl-1,6-b-mannosyl-1,4-N-acetyl-d-glucosamine (Reversibility: ? [2]) [2] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-1,2-a-mannosyl-1,6-b-mannosyl-1,4-N-acetyl-d-glucosamine [2]

362

2.4.99.6


S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl1,O-2-(dimethyloctylsilyl)ethyl (Reversibility: ? [7]) [7] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-1,O-2-(dimethyloctylsilyl)ethyl ( intermediate in the preparative production of gangliosides [7]) [7] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosaminylO(CH2 )8 CO2 CH3 (Reversibility: ? [9]) [9] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-O(CH2 )8 CO2 CH3 [9] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-d-glucosaminylR ( R: glycoprotein or glycopeptide [1-4]; usage of enzyme in the synthesis of P-selectin glycoprotein ligand-1, i.e. PSGL-1, sialylation of a synthetic asialo-precursor, inhibition by sulfated substrate [10]; broad substrate specificity, the viral enzyme utilizes disaccharides type I-III and also fucosylated Lewis a and Lewis x [9]; type II disaccharides are preferred substrates [8,9]; low activity with glycolipids [8]; i.e. type 2 chain, preferred substrate, type 1 chain acceptor is a less effective substrate, oligosaccharides or glycopeptides are better substrates than glycoproteins, preferred structure of branched oligosaccharide substrates are triantennary forms, poor substrates are those with bisected N-acetyl-d-glucosaminyl structures [3]) (Reversibility: ? [1-10]) [1-10] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-R [1-10] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,4-b-d-galactosyl-1,4-b-d-glucosyl-b1-ceramide ( i.e. paragloboside [5]) (Reversibility: ? [5]) [5] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,4-b-d-galactosyl-1,4-b-d-glucosyl-b1-ceramide ( i.e. a-2,3-sialylparagloboside [5]) [5] S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-b-d-N-acetylglucosaminyl-1,6-b-d-galactosyl-1,3-a-d-N-acetylgalactoaminyl-OR (Reversibility: ? [6]) [6] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-b-d-N-acetylglucosaminyl-1,6-b-d-galactosyl-1,3-a-d-N-acetylgalactoaminyl-OR [6] S CMP-N-acetylneuraminate + nLc4Cer ( low activity, at least 1 mM substrate concentration required [8]) (Reversibility: ? [8]) [8] P CMP + Neu-5-NAc-nLc4Cer S Additional information ( type III disaccharides are poor substrates [8]) [8] P ? Inhibitors Triton CF-54 [8] Additional information ( highly sensitive to detergents [3]) [3] Activating compounds Triton X-100 ( activation, 1% w/v [4]) [4] 363


2.4.99.6

Metals, ions Additional information ( assay mixture contains 30 mM MnCl2 [6]) [6] Specific activity (U/mg) 2.7 ( partially purified enzyme [6]) [6] Additional information ( development of a continous coupled spectrophotometric assay method in microtiter plates [11]) [5, 8, 11] Km-Value (mM) 0.11 (b-d-galactosyl-1,4-N-acetyl-d-glucosaminyl-O(CH2 )8 CO2 CH3 ) [9] Additional information [11] pH-Optimum 6 ( assay at [6]) [6] 6.4 [8] 6.7 ( assay at [2]) [2] Temperature optimum ( C) 37 ( assay at [2,4]) [2, 4]

4 Enzyme Structure Subunits Additional information ( sialyl motifs [8,9]; type II transmembrane topology [8,9]) [8, 9]

5 Isolation/Preparation/Mutation/Application Source/tissue B-16 cell [5] brain ( embryonic [1]) [1, 8] colon ( developmental regulation [8]) [8] embryo [1] heart ( developmental regulation [8]) [8] kidney ( developmental regulation [8]) [8] liver ( developmental regulation [8]; fetal, calf [1]) [1, 2, 68, 10, 11] melanoma cell [5] placenta [1-3] platelet [4] spleen [8] Additional information ( expressed in a wide variety of tissues [8]) [8]

364

2.4.99.6


Localization membrane [1] microsome [2] Purification (partial, CDP-ethanolamine-Sepharose affinity chromatography [4]) [4] (recombinant soluble enzyme from the medium of baculovirus transfected Spodoptera frugiperda SF9 cells, partially [7,11]; partially [6]) [6, 7, 11] (recombinant from COS-7 cells [8]) [8] (partially purified from host cell lysate [9]) [9] Cloning (expression as soluble enzyme under control of the late viral polyhedron promotor in Spodoptera frugiperda Sf9 cells via baculovirus infection [11]) [7, 11] (DNA and amino acid sequence determination and analysis, expression in COS-7 cells, fused to staphylococcal protein A [8]) [8] Application synthesis ( combined chemical-enzymic preparation of gangliosides, introduction of a 2-(dimethyloctylsilyl)ethyl lactoside as a versatile intermediate [7]; chemical-enzymatic synthesis of sialyl-Lewis x-containing hexasaccharides found on O-linked glycoproteins, process involves several enzymes of the pathway [6]) [6, 7]

6 Stability General stability information , fractionation on Ultrogel AcA34 decreases activity, bovine serum albumin restores [4] Storage stability , 4 C, partially purified, in 20% v/v glycerol, at least 1 week, after Ultrogel AcA34 fractionation, t1=2 : 24 h [4] , 4 C, concentrated solution of recombinant enzyme, 6 months without loss of activity [11]

References [1] Van den Eijnden, D.H.; Schiphorst, W.E.C.M.: Detection of bgalactosyl(1!4)N-acetylglucosaminide a(2!3)-sialyltransferase activity in fetal calf liver and other tissues. J. Biol. Chem., 256, 3159-3162 (1981) [2] Nemansky, M.; Schiphorst, W.E.C.M.; Koeleman, C.A.M.; Van den Eijnden, D.H.: Human liver and human placenta both contain CMP-NeuAc:Gal b 1!4GlcNAc-R a 2!3- as well as a 2!6-sialyltransferase activity. FEBS Lett., 312, 31-36 (1992) 365


2.4.99.6

[3] Nemansky, M.; Van den Eijnden, D.H.: Enzymic characterization of CMPNeuAc:Gal-b-1,4GlcNAc-R a(2,3)-sialyltransferase from human placenta. Glycoconjugate J., 10, 99-108 (1993) [4] Bauvois, B.; Montreuil, J.; Verbert, A.: Characterization of a sialyl a2-3 transferase and a sialyl a2-6 transferase from human platelets occurring in the sialylation of the N-glycosylproteins. Biochim. Biophys. Acta, 788, 234-240 (1984) [5] Tsuchiya, K.; Suzuki, Y.; Mabuchi, K.; Suzuki, T.; Hirabayashi, Y.; Sakiyama, H.: Characterization of sialyltransferase of B16 melanoma cells involved in the formation of melanoma-associated antigen GM3. J. Clin. Biochem. Nutr., 14, 141-149 (1993) [6] Oehrlein, R.; Hindsgaul, O.; Palcic, M.M.: Use of the core-2-N-acetylglucosaminyltransferase in the chemical-enzymic synthesis of a sialyl-LeX-containing hexasaccharide found on O-linked glycoproteins. Carbohydr. Res., 244, 149-159 (1993) [7] Stangier, P.; Palcic, M.M.; Bundle, D.R.: 2-(Dimethyloctylsilyl)ethyl lactoside: a versatile intermediate for chemical and enzymic ganglioside synthesis. Carbohydr. Res., 267, 153-159 (1995) [8] Kono, M.; Ohyama, Y.; Lee, Y.-C.; Hamamoto, T.; Kojima, N.; Tsuji, S.: Mouse b-galactoside a2,3-sialyltransferases: comparison of in vitro substrate specificities and tissue specific expression. Glycobiology, 7, 469-479 (1997) [9] Sujino, K.; Jackson, R.J.; Chan, N.W.C.; Tsuji, S.; Palcic, M.M.: A novel viral a2 ,3-sialyltransferase (v-ST3Gal I): transfer of sialic acid to fucosylated acceptors. Glycobiology, 10, 313-320 (2000) [10] Koeller, K.M.; Smith, M.E.B.; Wong, C.H.: Chemoenzymatic synthesis of PSGL-1 glycopeptides: sulfation on tyrosine affects glycosyltransferase-catalyzed synthesis of the O-glycan. Bioorg. Med. Chem., 8, 1017-1025 (2000) [11] Gosselin, S.; Alhussaini, M.; Streiff, M.B.; Takabayashi, K.; Palcic, M.M.: A continuous spectrophotometric assay for glycosyltransferases. Anal. Biochem., 220, 92-97 (1994)

366

a-N-Acetylneuraminyl-2,3-b-galactosyl-1,3-Nacetylgalactosaminide a-2,6-sialyltransferase

2.4.99.7

1 Nomenclature EC number 2.4.99.7 Systematic name CMP-N-acetylneuraminate:(a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,3)-Nacetyl-d-galactosaminide a-2,6-N-acetylneuraminyltransferase Recommended name a-N-acetylneuraminyl-2,3-b-galactosyl-1,3-N-acetylgalactosaminide 6-a-sialyltransferase Synonyms NeuAc-a-2,3-Gal-b-1,3-GalNAc-a-2,6-sialyltransferase [Swissprot] sialyltransferase sialyltransferase 3C sialyltransferase 7D sialyltransferase, cytidine monophosphoacetylneuraminate-(a-N-acetylneuraminyl-2,3-b-galactosyl-1,3)-N-acetylgalactosaminide-a-2,6-sialyltransferase Additional information (not identical with EC 2.4.99.3) CAS registry number 129924-24-9

2 Source Organism

Bos taurus (calf [1,3]) [1, 3] Homo sapiens [2, 7] Ovis aries (sheep [1]) [1] Rattus norvegicus [4, 5] Mus musculus [6]

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + N-acetyl-a-neuraminyl(2!3)-b-d-galactosyl(1 !3)-N-acetyl-d-galactosaminyl-R = CMP + N-acetyl-a-neuraminyl-(2!3)b-d-galactosyl-(1!3)-[N-acetyl-a-neuraminyl-(2!6)]-N-acetyl-d-galactosaminyl-R

367

a-N-Acetylneuraminyl-2,3-b-galactosyl-1,3-N-acetylgalactosaminide a-2,6-sialyltransferase

2.4.99.7

Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + a-N-acetylneuraminyl-2,3-b-d-galactosyl1,3-N-acetyl-d-galactosaminyl-R ( pathway in glycoprotein biosynthesis [1-3]) [1-3] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,3-(N-acetylneuraminyl-2,6)-N-acetyl-d-galactosaminyl-R Substrates and products S CMP-N-acetylneuraminate + GlcNac-a1-O-benzyl ( 7.6% of activity with fetuin [7]) (Reversibility: ? [7]) [7] P ? S CMP-N-acetylneuraminate + N-a-acetylneuraminyl-2,3-b-d-galactosyl1,3-N-acetyl-d-galactosaminyl-R ( R can be a protein or p-nitrophenol [3]; attaches sialic acid in a-2,6-linkage to N-acetylgalactosamine only when present in the structure of a-N-acetylneuraminyl-2,3b-galactosyl-1,3-N-acetylgalactosaminyl-R [1]; substrates are sialylated antifreeze glycoprotein as well as N-acetyl-d-galactosamine or b-galactosyl-1,3-N-acetyl-d-galactosamine side chains from ovine submaxillary asialomucin or porcine submaxillary asialo/afucomucin respectively [1]; no substrates are b-d-galactosyl-1,3-N-acetyl-d-galactosaminylR or N-acetylgalactosylaminyl-R [3]) (Reversibility: ? [1-4]) [1-4] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,3-(N-acetylneuraminyl-2,6)-N-acetyl-d-galactosaminyl-R [1-4] S CMP-N-acetylneuraminate + Neu5Aca2-3Galb1 -4GlcNac-a1-O-benzyl ( 224% of activity with fetuin [7]) (Reversibility: ? [7]) [7] P ? S CMP-N-acetylneuraminate + Neu5Aca2-3Galb1-4GlcNac-a1-O-benzyl ( 224% of activity with fetuin [7]) (Reversibility: ? [7]) [7] P ? S CMP-N-acetylneuraminate + Neu5Glca2-3Galb1-3GalNac ( 60.3% of activity with fetuin [7]) (Reversibility: ? [7]) [7] P ? S CMP-N-acetylneuraminate + a1-acid glycoprotein ( acceptor structur: Neu5Aca2-6Galb1-4GlcNac-R, 8.7% of activity with fetuin [7]) (Reversibility: ? [7]) [7] P ? S CMP-N-acetylneuraminate + fetuin ( acceptor structures: Neu5Aca2-3Galb1-3GalNaca1-O-Ser/Thr, Neu5Aca2-3Galb1-3[Neu5Aca26]GalNaca1-O-Ser/Thr and Neu5Aca2-6(3)Galb1-4GlcNac-R [7]) (Reversibility: ? [7]) [7] P ?

368

2.4.99.7


Inhibitors 5,5'-dithiobis-(2-nitrobenzoic acid) ( 0.01 mM, 50% inhibition, 0.1 mM, 97% inhibition [4]) [4] 6,6'-dithiodinicotinic acid carboxypyridine disulfide ( 0.028 mM, 505 inhibition [4]) [4] CMP ( 50 mM, strong inhibition [4]) [4] N-bromosuccinimide ( 5 mM, 98% inhibition [4]) [4] N-ethylmaleimide ( 0.014 mM, 50% non-competitive inhibition, almost complete protection with 0.05 mM CMP-N-acetylneuraminate [4]) [4] mersalyl acid ( 0.025 mM, 50% inhibition [4]) [4] p-chloromercuribenzoic acid ( 0.036 mM, 50% inhibition, 0.1 mM, 97% inhibition [4]) [4] Km-Value (mM) 0.07 (CMP-N-acetylneuraminate) [7] 1.1 (Neu5Gca2-3Galb1-3GalNac) [7] 2.21 (Neu5Aca2-3Galb1-3GalNaca-O-benzyl) [7] pH-Optimum 6 [5] Temperature optimum ( C) 28 [5] 37 ( assay at [3]) [3]

4 Enzyme Structure Subunits ? ( x * 34900, deduced from nucleotide sequence [7]) [7]

5 Isolation/Preparation/Mutation/Application Source/tissue B-cell ( Epstein-Barr virus transformed cell line [2]) [2] T-lymphocyte [2] brain ( activity is 4 times lower in adult than in embryonic brain [5]; constitutive expression of ST6GalNac IV gene [7]) [4, 5, 7] colon ( constitutive expression of ST6GalNac IV gene [7]) [7] heart ( constitutive expression of STGalNac IV gene [7]) [7] kidney ( constitutive expression of ST6GalNac IV gene [7]) [7] liver ( fetal [3]; constitutive expression of ST6GalNac IV gene [7]) [1, 3, 7] lung ( constitutive expression of ST6GalNac IV gene [7]) [7] placenta ( constitutive expression of ST6GalNac IV gene [7]) [7] platelet [2]

369


2.4.99.7

skeletal muscle ( constitutive expression of ST6GalNac IV gene [7]) [7] small intestine ( constitutive expression of ST6GalNac IV gene [7]) [7] spleen ( constitutive expression of ST6GalNac IV gene [7]) [7] submaxillary gland [1] thymus ( constitutive expression of ST6GalNac IV gene [7]) [7] Localization membrane [1, 2, 4] microsome [1-4] Cloning (expression of ST6GalNac IV in COS-7 cells [7]) [7] (ST6GalNac III and IV genes [6]) [6]

References [1] Bergh, M.L.E.; Hooghwinkel, G.J.M.; Van den Eijnden, D.H.: Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an a-N-acetylgalactosaminide a2!6 sialyltransferase with a narrow acceptor specificity in fetal calf liver. J. Biol. Chem., 258, 7430-7436 (1983) [2] Higgins, E.A.; Siminovitch, K.A.; Zhuang, D.; Brockhausen, I.; Dennis, J.W.: Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome. J. Biol. Chem., 266, 62806290 (1991) [3] Bergh, M.L.E.; van den Eijnden, D.H.: Aglycon specificity of fetal calf liver and ovine and porcine submaxillary gland a-N-acetylgalactosaminide a2!6 sialyltransferase. Eur. J. Biochem., 136, 113-118 (1983) [4] Baubichon-Cortay, H.; Broquet, P.; George, P.; Louisot, P.: Different reactivity of two brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAc a23Galb1-3GalNAc a(2-6)sialyltransferase. Glycoconjugate J., 6, 115-127 (1989) [5] Dall'olio, F.: Sialyltransferases of developing rat brain. Glycoconjugate J., 7, 301-310 (1990) [6] Takashima, S.; Kurosawa, N.; Tachida, Y.; Inoue, M.; Tsuji, S.: Comparative analysis of the genomic structures and promoter activities of mouse Siaa2,3Galb1,3GalNAc GalNAca2,6-sialyltransferase genes (ST6GalNAc III and IV): characterization of their Sp1 binding sites. J. Biochem., 127, 399-409 (2000) [7] Harduin-Lepers, A.; Stokes, D.C.; Steelant, W.F.A.; Samyn-Petit, B.; Krzewinski-Recchi, M.-A.; Vallejo-Ruiz, V.; Zanetta, J.-P.; Auge, C.; Delannoy, P.: Cloning, expression and gene organization of a human Neu5Aca2-3Galb1-3GalNAc a2,6-sialyltransferase: hST6GalNAc IV. Biochem. J., 352, 37-48 (2000)

370

a-N-Acetylneuraminate a-2,8sialyltransferase

2.4.99.8

1 Nomenclature EC number 2.4.99.8 Systematic name CMP-N-acetylneuraminate:a-N-acetylneuraminyl-2,3-b-d-galactoside a-2,8N-acetylneuraminyltransferase Recommended name a-N-acetylneuraminate a-2,8-sialyltransferase Synonyms CMP-NAcNeu:GM3 ganglioside sialyltranferase CMP-NeuAc:GD3(a2-8) sialyltranferase CMP-NeuAc:LM1(a2-8) sialyltranferase CMP-sialic acid:GM3 sialyltranferase GD3 synthase SAT-2 ST-II a-2,8-sialyltransferase a-2,8-sialyltransferase 8A ganglioside GD3 synthase ganglioside GD3 synthetase ganglioside GT3 synthase sialyltransferase II sialyltransferase, cytidine monophosphoacetylneuraminate-ganglioside GM3 CAS registry number 67339-00-8

2 Source Organism Gallus gallus (9 days old embryonic brains [7]) [1, 7, 16] Rattus norvegicus (female Wistar [8]; 12-14 days old [2]; Sprague-Dawley [12]) [2-6, 8, 10-12, 15, 17] Homo sapiens [9, 13] Oreochromis mossambicus (cichlid fish [12]) [12] Mus musculus [14, 17]

371

a-N-Acetylneuraminate a-2,8-sialyltransferase

2.4.99.8

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + a-N-acetylneuraminyl-2,3-b-d-galactosyl-R = CMP + a-N-acetylneuraminyl-2,8-a-N-acetylneuraminyl-2,3-b-d-galactosylR Reaction type glycosyl group transfer Natural substrates and products S CMP-a-N-acetylneuraminate + a-N-acetylneuraminyl-2,3-b-d-galactosyl1,4-b-d-glucosylceramide ( branch-point enzyme in ganglioside biosynthetic sequence [5]) (Reversibility: ? [5]) [5] P CMP + a-N-acetylneuraminyl-2,8-a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-b-d-glucosylceramide Substrates and products S CMP-N-acetylneuraminate + N-acyl-lyso-GM3 ( N-acetyl derivative is a better substrate than GM3, detergent-like effect [5]) (Reversibility: ? [5]) [5] P CMP + N-acyl-lyso-GD3 [5] S CMP-N-acetylneuraminate + N-a-acetylneuraminyl-2,3-b-d-galactosyl1,4-b-d-glucosylceramide ( i.e. sialosyllactosylceramide or ganglioside GM3, specificity is determined by the substrate's negative charge and the acyl-residue in amide bond to the amino group of neuraminic acid [5]; poor substrates are GM3 methyl ester, GM3 amide or GM3 methyl amide, no substrates are neuraminyllactosylceramide or Nbiotinylneuraminyllactosylceramide [5]) (Reversibility: ? [1-11]) [1-11] P CMP + a-N-acetylneuraminyl-2,8-a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-b-d-glucosylceramide (i.e. disialosyllactosylceramide or ganglioside GD3) [1-10] S CMP-N-acetylneuraminate + N-butyrylneuraminyl-a-2,3-galactosyl-b1,4-glucosyl-b-1,1-ceramide ( sialylated at about 60% the rate of GM3 [5]) (Reversibility: ? [5]) [5] P CMP + ? S CMP-N-acetylneuraminate + N-glycolylneuraminyl-a-2,3-galactosyl-b1,4-glucosyl-b-1,1-ceramide (Reversibility: ? [5]) [5] P CMP + ? S CMP-N-acetylneuraminate + a-N-acetylneuraminyl-2,3-b-d-galactosyl1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-d-glucosylceramide ( i.e. sialosylneolactotetraosylceramide or ganglioside LM1 [1]) (Reversibility: ? [1]) [1] P CMP + a-N-acetylneuraminyl-2,8-a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-d-glucosylceramide ( i.e. disialosylneolactotetraosylceramide or ganglioside LD1c [1]) [1]

372

2.4.99.8


S CMP-N-acetylneuraminate + disialoganglioside GD1a (Reversibility: ? [7]) [7] P CMP + trisialoganglioside GT1a [7] S CMP-N-acetylneuraminate + trisialoganglioside GT1b (Reversibility: ? [10]) [10] P CMP + ganglioside GQ1b [10] S Additional information ( mouse enzyme is specific toward Nlinked oligosaccharides of glycoproteins [14]) [14] P ? Inhibitors ADP [3] AMP ( less effective than CMP or GMP [3]) [3] ATP [3] Ba2+ ( weak [8]) [8] CDP ( only partially relieved by excess Mg2+ [3]) [3] CMP ( strong [3]) [3] CTP [3] Ca2+ [7, 8] Cd2+ ( strong [8]) [8] Co2+ [8] Cu2+ ( strong [8]) [8, 10] EDTA ( Mg2+ protects [4]; not [7,8,10]) [4, 7, 8, 10] Fe2+ [10] GDP [3] GMP ( as strong as CMP [3]) [3] GTP [3] Mn2+ [10] N-ethylmaleimide [1] Ni2+ [8] TDP [3] TMP ( less effective than AMP [3]) [3] TTP [3] UDP ( weak [3]) [3] UMP ( weak [3]) [3] UTP ( weak [3]) [3] Zn2+ ( weak [8]) [8] ganglioside D1a [6] ganglioside LM1 ( at higher concentrations, substrate inhibition [1]) [1] ganglioside Q1b ( strong [6]) [6] ganglioside T1b [6] lysophospholipids [4] Additional information ( no inhibition by Mg2+ , CMP-N-acetylneuraminate [7]; IAA, 2-mercaptoethanol [8]; no inhibition by ganglioside GM2 [6]) [6-8]

373


2.4.99.8

Activating compounds Histone ( slight activation [7]) [7] Myrj 59 ( activation, most potent activator, can be replaced by the following detergents, descending efficiency: sodium deoxycholate, Triton CF-54, Tween 20, Tween 80/Triton CF-54 ratio 1:2, Triton X-100 or Tween 80 [2]) [2] Nonidet P-40 ( activation [1]) [1] Triton CF-54 ( activation, further enhanced by supplementation with diacyl phospholipids [4]; can be replaced by Triton X-100, Tween 80 or Tween 20 with 25%, 11% or 9% efficiency, respectively [7]) [1, 2, 4, 7, 10, 11] Triton X-100 ( activation [4]) [1, 2, 4, 6-8] Zwittergent 3-10 [1] Zwittergent 3-14 [1] digitonin [4] Additional information ( the presence of detergents is essential for activity, no activation by phosphatidylglycerol [6]) [6] Metals, ions Mg2+ ( slight activation [10]; stimulation [4]) [4, 10] Additional information ( no metal ion requirement [8]) [8, 10] Specific activity (U/mg) 0.00028-0.00055 [5] 0.00105 [4] 0.0189 [2] Km-Value (mM) 0.063 (ganglioside LM1, soluble enzyme preparation [1]) [1] 0.07 (CMP-N-acetylneuraminate) [1] 0.078 (ganglioside GM3) [1] 0.1 (ganglioside GM3) [8] 0.2 (ganglioside GM3) [4, 8] 0.8 (CMP-N-acetylneuraminate) [4] 1 (ganglioside GT1a) [7] pH-Optimum 5.8 [8] 6-7.2 ( trisialoganglioside formation [7]) [7] 6.2 [4] 6.5 ( GM3 or GT1b as substrate [10]) [10] Temperature optimum ( C) 37 ( assay at [1-8,11]) [1-8, 11]

374

2.4.99.8


4 Enzyme Structure Molecular weight 45000 ( SDS-polyacrylamide-gel electrophoresis in the presence of b-mercaptoethanol [16]) [16] 55000 ( SDS-gel electrophoresis [2]) [2] 95000 ( SDS-polyacrylamide-gel electrophoresis in the absence of bmercaptoethanol [16]) [16] Posttranslational modification glycoprotein [4]

5 Isolation/Preparation/Mutation/Application Source/tissue HeLa cell [9] brain [1, 2, 7, 12] cerebellum [15] cerebral cortex [15] heart [2] hippocampus [15] kidney [2] liver [2-6, 8, 10, 11] lung [2] pancreas [2] thalamus [15] Localization Golgi apparatus [3-6, 8, 11, 17] membrane [1-8, 12] Purification (solubilized with Triton X-100, CDP-Sepharose affinity chromatography [2]; homogenized in 0.32M sucrose employing a glass homogenizer with a Teflon pestle [7]; homogenized, centrifuged, resuspended in 25 mM-cacodylate buffer pH 6.5 containing 0.15% Triton X-100, 75mM NaCl and 10 mM MnCl2 [12]) [2, 7, 12] Cloning (expression in CHO-K1 cells [16]) [16] (expression in CHOP cells [15]; expression in neuroblastoma cell line F-11 [17]) [15, 17] (expression of cDNA from melanoma cell line WM266-4 in Namalwa KJM-1 cells [13]) [13] (expression in newborn brain in mouse [14]; expression in neuroblastoma cell line NG108-15 [17]) [14, 17]

375


2.4.99.8

6 Stability Temperature stability 56 ( 20% loss of activity after 120 s [11]) [11]

References [1] Higashi, H.; Basu, M.; Basu, S.: Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain. J. Biol. Chem., 260, 824-828 (1985) [2] Gu, X.-B.; Gu, T.-J.; Yu, R.K.: Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain. Biochem. Biophys. Res. Commun., 166, 387-393 (1990) [3] Eppler, C.M.; MorrØ, D.J.; Keenan, T.W.: Ganglioside biosynthesis in rat liver: alteration of sialyltransferase activities by nucleotides. Biochim. Biophys. Acta, 619, 332-343 (1980) [4] Eppler, C.M.; MorrØ, D.J.; Keenan, T.W.: Ganglioside biosynthesis in rat liver: characterization of cytidine-5-monophospho-n-acetylneuraminic acid:hematoside (GM3) sialyltransferase. Biochim. Biophys. Acta, 619, 318331 (1980) [5] Klein, D.; Pohlentz, G.; Schwarzmann, G.; Sandhoff, K.: Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver. Eur. J. Biochem., 167, 417-424 (1987) [6] Yusuf, H.K.M.; Schwarzmann, G.; Pohlentz, G.; Sandhoff, K.: Oligosialogangliosides inhibit GM2- and GD3-synthesis in isolated Golgi vesicles from rat liver. Biol. Chem. Hoppe-Seyler, 368, 455-462 (1987) [7] Yohe, H.C.; Yu, R.K.: In vitro biosynthesis of an isomer of brain trisialoganglioside, GT1a. J. Biol. Chem., 255, 608-613 (1980) [8] Busam, K.; Decker, K.: Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases. Eur. J. Biochem., 160, 23-30 (1986) [9] Fishman, P.H.; Bradley, R.M.; Henneberry, R.C.: Butyrate-induced glycolipid biosynthesis in HeLa cells: properties of the induced sialyltransferase. Arch. Biochem. Biophys., 172, 618-626 (1976) [10] Trinchera, M.; Pirovano, B.; Ghidoni, R.: Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b a2-8 sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis. J. Biol. Chem., 265, 18242-18247 (1990) [11] Iber, H.; van Echten, G.; Sandhoff, K.: Substrate specificity of a2,3-sialyltransferases in ganglioside biosynthesis of rat liver golgi. Eur. J. Biochem., 195, 115-120 (1991) [12] Freischutz, B.; Saito, M.; Rahmann, H.; Yu, R.K.: Activities of five different sialyltransferases in fish and rat brains. J. Neurochem., 62, 1965-1973 (1994)

376

2.4.99.8


[13] Sasaki, K.; Kurata, K.; Kojima, N.; Kurosawa, N.; Ohta, S.; Hanai, N.; Tsuji, S.; Nishi, T.: Expression cloning of a GM3-specific a 2,8-sialyltransferase (GD3 synthase). J. Biol. Chem., 269, 15950-15956 (1994) [14] Kojima, N.; Yoshida, Y.; Kurosawa, N.; Lee, Y.-C.; Tsuji, S.: Enzymatic activity of a developmentally regulated member of the sialyltransferase family (STX): evidence for a2,8-sialyltransferase activity toward N-linked oligosaccharides. FEBS Lett., 360, 1-4 (1995) [15] Watanabe, Y.; Nara, K.; Takahashi, H.; Nagai, Y.; Sanai, Y.: The molecular cloning and expression of a2,8-sialyltransferase (GD3 synthase) in a rat brain. J. Biochem., 120, 1020-1027 (1996) [16] Daniotti, J.L.; Martina, J.A.; Giraudo, C.G.; Zurita, A.R.; Maccioni, H.J.F.: GM3 a2,8-sialyltransferase (GD3 synthase): protein characterization and sub-Golgi location in CHO-K1 cells. J. Neurochem., 74, 1711-1720 (2000) [17] Bieberich, E.; Tencomnao, T.; Kapitonov, D.; Yu, R.K.: Effect of N-glycosylation on turnover and subcellular distribution of N-acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells. J. Neurochem., 74, 2359-2364 (2000)

377

Lactosylceramide a-2,3-sialyltransferase

2.4.99.9

1 Nomenclature EC number 2.4.99.9 Systematic name CMP-N-acetylneuraminate:lactosylceramide a-2,3-N-acetylneuraminyltransferase Recommended name lactosylceramide a-2,3-sialyltransferase Synonyms CMP-NeuAc:lactosylceramide a-2,3-sialyltransferase [Swissprot] CMP-acetylneuraminate-lactosylceramide-sialyltransferase CMP-acetylneuraminic acid:lactosylceramide sialytransferase CMP-sialic acid:lactosylceramide-sialyltransferase GM3 synthase GM3 synthetase ganglioside GM3 synthase [Swissprot] SAT1 cytidine monophosphoacetylneuraminate-lactosylceramide a-2,3-sialyltransferase cytidine monophosphoacetylneuraminate-lactosylceramide sialyltransferase ganglioside GM3 synthetase Additional information (cf. EC 2.4.99.2) CAS registry number 125752-90-1

2 Source Organism

378

Homo sapiens [1, 15] Gallus gallus (15 days old embryo [2]; 7-11 days old embryo [4]) [2, 4] Mesocricetus auratus [11] Rattus norvegicus (female Wistar [12]) [3, 5-10, 12-14, 15, 16, 17, 18] Oreochromis mossambicus (cichlid fish [16]) [16] Mus musculus [19, 20]

2.4.99.9


3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + b-d-galactosyl-1,4-b-d-glucosylceramide = CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-b-d-glucosyl-ceramide Reaction type glycosyl group transfer Natural substrates and products S CMP-N-acetylneuraminate + lactosylceramide ( synthesizes the first ganglioside of the gangliotetraose series, GM3 [2]; involved in sequential addition of monosaccharides from sugar nucleotides to nonreducing end of oligosaccharide chains of glycosphingolipids [11]) [2, 11] P CMP + N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-b-d-glucosylceramide Substrates and products S CMP-N-acetylneuraminate + 1-deoxy-1-cholesteryl (N-acetyl)-ethanolaminolactiol ( 115% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + 1-deoxy-1-cholesterylethanolaminolactiol ( 28% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + 1-deoxy-1-cholesterylphospho (N-acetyl)ethanolaminolactiol ( 151% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + 1-deoxy-1-cholesterylphosphoethanolaminolactiol ( 60% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + 2,3-dicholesteryl-1-b-lactosylglycerol ( 113% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + 2-cholesteryl-1-b-lactosylglycerol ( 138% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + 3-cholesteryl-1-b-lactosylglycerol ( 163% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + b-d-galactosyl-1,3-N-acetylgalactoseamin-1, 4-b-d-galactosyl-1,4-b-d-glucosylceramide ( i.e. asialo-ganglioside GM1, sialylated at about 84% the rate of lactosylceramide [6]) (Reversibility: ? [6]) [6]

379


2.4.99.9

P CMP + ? S CMP-N-acetylneuraminate + b-d-galactosyl-1,3-N-acetylgalactoseaminlactosylceramide ( 14% of activity with lactosylceramide [9]) (Reversibility: ? [9]) [9] P CMP + ? S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-b-d-glucosylceramide ( i.e. lactosylceramide, high specificity [9]; preferred acceptors have the general structure b1-O-ceramide, disaccharides are preferred to monosaccharide [6]; no acceptors are fetuin, mucin, a1 -acid glycoprotein, glycophorin or their respective asialo-derivatives, gangliosides GD1b, GM3, GM2, GM1 or GD1a [6]; brain sialyltransferase shows high activity with d18:1-16:0, d18:1-22:1, and d18:0-18:0 lactosylceramide molecular species, specificity changes when the lipid composition of the neuronal microsomal membrane resembles that of liver Golgi membrane lipids [17]) (Reversibility: ? [1-9,11-16,20]) [1-9, 11-20] P CMP + N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-b-d-glucosylceramide ( i.e. ganglioside GM3 [1-9,11-20]) [1-9, 11-20] S CMP-N-acetylneuraminate + b-lactosylcholesterol ( 138% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + cholesteryl-b-lactosylpropane-1,3-diol ( 143% of activity with lactosylceramide [18]) (Reversibility: ? [18]) [18] P ? S CMP-N-acetylneuraminate + galactosylceramide ( sialylated at about 40% the rate of lactosylceramide [6]) (Reversibility: ? [6]) [6] P CMP + ? S CMP-N-acetylneuraminate + glucosylceramide ( sialylated at about 65% the rate of lactosylceramide [6]) (Reversibility: ? [6]) [6] P CMP + ? Inhibitors (4-amidinophenyl)-methanesulfonyl fluoride ( serine protease, 0.02 mM, 48% inhibition of purified sialyltransferase-1 [7]) [7] Ba2+ ( weak [12]) [12] CDP [6, 14] CMP [2, 6, 14] CMPdialdehyde ( kinetics [2]) [2] CTP [6, 14] Ca2+ ( weak [12]) [12] Cd2+ ( strong [12]) [12] Co2+ [12] Cu2+ ( strong [12]) [12] E-64 ( thiol protease inhibitor, 0.0028 mM, 71% inhibition of purified sialyltransferase-1, activation of sialytransferase-1 activity in microsomes [7]) [7]

380

2.4.99.9


EDTA ( 0.1 mM, 71% inhibition of purified sialyltransferase-1 [7]) [7] Ep-475 ( thiol protease inhibitor, 0.0028 mM, 75% inhibition of purified sialyltransferase-1 [7]) [7] Ep-475 ( thiol protease inhibitor, 0.0028 mM, 84% inhibition of purified sialyltransferase-1 [7]) [7] l-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone ( chymotrypsin and thiol protease inhibitor, 0.284 mM, 67% inhibition of purified sialyltransferase-1 [7]) [7] l-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone-HCl ( trypsin and thiol protease inhibitor, 0.135 mM, 73% inhibition of purified sialyltransferase-1 [7]) [7] Mn2+ ( weak [12]) [12] N-3 ( 1 mM, 87% inhibition of purified sialyltransferase-1 [7]) [7] Ni2+ ( strong [12]) [12] Tris buffer ( weak [1]) [1] Triton CF-54 ( at higher concentrations [13]) [13] UDP [2] UDP-N-acetylgalactosamine ( weak [2]) [2] UDPdialdehyde ( kinetics [2]) [2] UDPgalactose ( weak [2]) [2] Zn2+ ( weak [12]; 10 mM, 90% inhibition [9]) [9, 12] a2 -macroglobulin ( 1 unit, 67% inhibition of purified sialyltransferase-1, no inhibition of sialyltransferase-1 activity in microsomes [7]) [7] aprotinin ( serine protease inhibitor, 0.0003 mM, 74% inhibition of purified sialyltransferase-1 [7]) [7] asialofetuin ( weak [11]) [11] dithiothreitol ( 0.1 mM, 89% inhibition of purified sialyltransferase1 [7]) [7] ganglioside GM1a [13] ganglioside GM3 ( 0.5 mM and 1 mM, 43% and 60% inhibition respectively [11]) [11] globotetraosylceramide ( weak [11]) [11] ionic detergents [11] leupeptin ( serine and thiol protease inhibitor, 0.001 mM, 72% inhibition of purified sialyltransferase-1, no inhibition of sialyltransferase-1 activity in microsomes [7]) [7] monoclonal antibody M12GC7 [6] pepstatin A ( 0.001 mM, 72% inhibition of purified sialyltransferase1, activation of sialyltransferase-1 activity in microsomes [7]) [7] Additional information ( inhibition in vivo i.e. stable microsomes not as dramatic as in vitro [7]; not inhibited by Fe2+ , Mg2+ , IAA, 2-mercaptoethanol [12]; glucosylceramide or globotriaosylceramide [11]; not inhibited by EDTA [1,12]; not inhibited by N-ethylmaleimide [4]) [1, 4, 7, 11, 12]

381


2.4.99.9

Activating compounds 2-mercaptoethanol ( 0.18 mM, 392% activation of purified sialyltransferase-1, 1120% activation of sialytransferase-1 activity in microsomes [7]) [7] cardiolipin ( activation [1]) [1] cutscum ( activation, most potent activator [1]; can be replaced by Triton CF-54, Triton X-100 or Triton CF-54/Tween 80, ratio 2:1, activation efficiency in descending order [1]) [1, 11] Myrj 59 ( activation, most potent activator, can be replaced by sodium deoxycholate, Triton CF-54, Tween 20, Triton CF-54/Tween 80, ratio 2:1, Triton X-100 or Tween 80, activation efficiency in descending order [10]) [10] Nonidet P-40 ( slight activation [1]) [1] Triton CF-54 ( activation, most potent activator [11]; inhibits at higher concentrations [13]; 0.15%, required for full activity [9]) [1, 8-11, 13] Triton CF-54/Tween 80 ( ratio 2:1, activation, about 50% as efficient as Cutscum [1]; less than 50% as efficient as Myrj 59 [10]) [1, 10, 11] Triton X-100 ( activation [1,8,10-12]) [1, 8, 10-12] b-octylglucoside [8] lauryldimethylamine oxide ( i.e. Ammonyx LO, nonionic/cationic detergent, 12-15fold higher activation than by Triton CF-54, Triton X-100 or b-octylglucoside [8]) [8] Additional information ( not activated by Tween 20 or 80 [11]) [11] Metals, ions Ca2+ ( activation, slightly less efficient than Mn2+ [11]) [11] Mg2+ ( activation, slightly less efficient than Mn2+ [11]) [11, 15] Mn2+ ( 10 mM, approx. 2fold activation, required for full activity [9]; 15 mM, required for maximal activity [11]) [6, 7, 9, 11] Additional information ( no metal ion requirement [1,12]) [1, 12] Specific activity (U/mg) 0.0000027 ( sialyltransferase-1 activity in membrane fraction from brain [16]) [16] 0.0000045 [1] 0.0000064 ( sialyltransferase-1 activity in membrane fraction from brain [16]) [16] 0.0055 [6] 0.0094 [9] Km-Value (mM) 0.00011 (lactosylceramide) [6] 0.00026 (CMP-N-acetylneuraminate) [6] 0.035 (lactosylceramide) [1] 0.068 (lactosylceramide) [13] 0.075 (lactosylceramide) [15]

382

2.4.99.9


0.08 (lactosylceramide) [9, 12] 0.0815 (lactosylceramide) [2] 0.11 (lactosylceramide) [11] 0.16 (CMP-N-acetylneuraminate) [11] 0.19 (CMP-N-acetylneuraminate) [15] 0.21 (CMP-N-acetylneuraminate) [9] 1.5 (CMP-N-acetylneuraminate) [1, 12] Ki-Value (mM) 0.0853 (CMP-dialdehyde, at 0.1 mM [2]) [2] 0.104 (CMP-dialdehyde, at 0.2 mM [2]) [2] 0.219 (UDP-dialdehyde, at 0.2 mM [2]) [2] pH-Optimum 5.7 [12] 6 [1] 6.2 [15] 6.5 ( broad optimum in cacodylate-HCl buffer [11]) [6, 9, 11] Additional information ( pI: 5.7-6.2 [6]; no effects of buffer on activity [11]) [6, 11] pH-Range 4.5-8 [11] 6.1-7.6 ( detectable activity [6]) [6] Temperature optimum ( C) 37 [15]

4 Enzyme Structure Subunits ? ( x * 60000, SDS-PAGE [6,8]; x * 76000, SDS-PAGE [9]) [6, 8, 9] Posttranslational modification glycoprotein ( N- and O-linked carbohydrate side chains, containing sialic acids in a-2,3 and 2,6-linkage, galactose and galactosamine, branched N-glycans containing mannose [6]) [6]

5 Isolation/Preparation/Mutation/Application Source/tissue HeLa cell ( strain R, elevated enzyme level if cultured in butyrate containing media [1]) [1] P-19 cell ( embryonic carcinoma cell line P19 differentiates into neurons and astrocytes on cell aggregation after treatment with retinoic acid [19]) [19]

383


2.4.99.9

brain [2, 4, 9, 10, 16, 17] cell suspension culture [11] fibroblast ( contact-inhibited cell-line NIL-8 [11]) [11] liver [3, 5-8, 12-14, 18] lymphocyte ( from peripheral blood mononuclear cells, enzyme level is stimulated by growth in the presence of phytohemagglutinin [15]) [15] neuroblastoma cell ( murine neuroblastoma x rat dorsal root ganglion F-11A cells [20]) [20] neuron ( brain neuron [17]) [17, 19] Localization endoplasmic reticulum ( broadley distributed in endoplasmic reticulum [20]) [20] Golgi apparatus ( distribution [3]; luminal side [5]; broadley distributed in Golgi [20]) [3, 5-7, 12-14, 18, 20] membrane [10, 12, 16] microsome [2, 7, 17] Cloning (expression as fusion proteins with red fluorescent protein in murine neuroblastoma F-11A cells [20]) [20]

6 Stability Temperature stability 56 ( t1=2 : 60 s [13]) [13] 100 ( 5 min, inactivation [1]) [1] General stability information , PMSF, leupeptin or pepstatin A stabilize during purification [7] , lauryldimethylamine oxide solubilizes and stabilizes solubilized enzyme during purification and storage [8] Storage stability , -80 C, 25 mM sodium cacodylate, pH 6.5, 15% w/v lauryldimethylamine oxide, 6-12 months, no loss of activity [8]

References [1] Fishman, P.H.; Bradley, R.M.; Henneberry, R.C.: Butyrate-induced glycolipid biosynthesis in HeLa cells: properties of the induced sialyltransferase. Arch. Biochem. Biophys., 172, 618-626 (1976) [2] Cambron, L.D.; Leskawa, K.C.: Inhibition of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase by nucleotides, nucleotide sugars and nucleotide dialdehydes. Biochem. Biophys. Res. Commun., 193, 585590 (1993)

384

2.4.99.9


[3] Trinchera, M.; Pirovano, B.; Ghidoni, R.: Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b a2-8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis. J. Biol. Chem., 265, 18242-18247 (1990) [4] Higashi, H.; Basu, M.; Basu, S.: Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain. J. Biol. Chem., 260, 824-828 (1985) [5] Trinchera, M.; Fabbri, M.; Ghidoni, R.: Topography of glycosyltransferases involved in the initial glycosylations of gangliosides. J. Biol. Chem., 266, 20907-20912 (1991) [6] Melkerson-Watson, L.J.; Sweeley, C.C.: Purification to apparent homogeneity by immunoaffinity chromatography and partial characterization of the GM3 ganglioside-forming enzyme, CMP-sialic acid:lactosylceramide a2,3sialyltransferase (SAT-1), from rat liver Golgi [published erratum appears in J Biol Chem 1991 Oct 15;266(29):19865]. J. Biol. Chem., 266, 4448-4457 (1991) [7] Melkerson-Watson, L.J.; Sweeley, C.C.: Special considerations in the purification of the GM3 ganglioside-forming enzyme, CMP-sialic acid:lactosylceramide a2-3 sialyltransferase (SAT-1): effects of protease inhibitors on rat hepatic SAT-1 activity. Biochem. Biophys. Res. Commun., 175, 325-332 (1991) [8] Melkerson-Watson, L.J.; Sweeley, C.C.: Special considerations in the purification of the GM3 ganglioside forming enzyme, CMP-sialic acid:lactosylceramide a2-3 sialyltransferase (SAT-1): solubilization of SAT-1 with lauryldimethylamine oxide. Biochem. Biophys. Res. Commun., 172, 165-171 (1990) [9] Preuss, U.; Gu, X.; Gu, T.; Yu, R.K.: Purification and characterization of CMP-N-acetylneuraminic acid:lactosylceramide (a2-3) sialyltransferase (GM3-synthase) from rat brain. J. Biol. Chem., 268, 26273-26278 (1993) [10] Gu, X.-B.; Gu, T.-J.; Yu, R.K.: Purification to homogeneity of GD3 synthase and partial purification of GM3 synthase from rat brain. Biochem. Biophys. Res. Commun., 166, 387-393 (1990) [11] Burczak, J.D.; Fairley, J.L.; Sweeley, C.C.: Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells. Biochim. Biophys. Acta, 804, 442-449 (1984) [12] Busam, K.; Decker, K.: Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases. Eur. J. Biochem., 160, 23-30 (1986) [13] Iber, H.; van Echten, G.; Sandhoff, K.: Substrate specificity of a 2-3-sialyltransferases in ganglioside biosynthesis of rat liver golgi. Eur. J. Biochem., 195, 115-120 (1991) [14] Eppler, C.M.; MorrØ, D.J.; Keenan, T.W.: Ganglioside biosynthesis in rat liver: alteration of sialyltransferase activities by nucleotides. Biochim. Biophys. Acta, 619, 332-343 (1980) [15] Basu, S.K.; Whisler, R.L.; Yates, A.J.: Effects of lectin activation on sialyltransferase activities in human lymphocytes. Biochemistry, 25, 2577-2581 (1986) 385


2.4.99.9

[16] Freischutz, B.; Saito, M.; Rahmann, H.; Yu, R.K.: Activities of five different sialyltransferases in fish and rat brains. J. Neurochem., 62, 1965-1973 (1994) [17] Kadowaki, H.; Grant, M.A.: Relationship of membrane phospholipid composition, lactosylceramide molecular species, and the specificity of CMP-Nacetylneuraminate:lactosylceramide a2,3-sialyltransferase to the molecular species composition of GM3 ganglioside. J. Lipid Res., 36, 1274-1282 (1995) [18] Pohlentz, G.; Mokros, A.; Egge, H.: Cholesterol-containing lactose derived neoglycolipids serve as acceptors for sialyltransferases from rat liver Golgi vesicles. Glycoconjugate J., 13, 147-152 (1996) [19] Osanai, T.; Watanabe, Y.; Sanai, Y.: Glycolipid sialyltransferases are enhanced during neural differentiation of mouse embryonic carcinoma cells, P19. Biochem. Biophys. Res. Commun., 241, 327-333 (1997) [20] Bieberich, E.; MacKinnon, S.; Silva, J.; Li, D.D.; Tencomnao, T.; Irwin, L.; Kapitonov, D.; Yu, R.K.: Regulation of Ganglioside Biosynthesis by Enzyme Complex Formation of Glycosyltransferases. Biochemistry, 41, 11479-11487 (2002)

386

Neolactotetraosylceramide a-2,3-sialyltransferase

2.4.99.10

1 Nomenclature EC number 2.4.99.10 Systematic name CMP-N-acetylneuraminate:neolactotetraosylceramide a-2,3-sialyltransferase Recommended name neolactotetraosylceramide a-2,3-sialyltransferase Synonyms SAT-3 cytidine monophosphoacetylneuraminate-neolactotetraosylceramide sialyltransferase sialyltransferase sialyltransferase 3 CAS registry number 83745-06-6

2 Source Organism Gallus gallus (7-12 days old embryo [1]; 10-12 days old embryo [3,6]) [1, 3, 4, 6] Homo sapiens (colon carcinoma [5,6]) [2, 5, 6]

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl1,3-b-d-galactosyl-1,4-d-glucosylceramide = CMP + a-N-acetylneuraminyl2,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-dglucosylceramide Reaction type glycosyl group transfer

387


2.4.99.10

Natural substrates and products S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-d-glucosylceramide ( involved in glycosphingolipid biosynthesis [1]) [1] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-d-glucosylceramide Substrates and products S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-d-glucosylceramide ( transfers sialic acid to the O-3-position of the terminal galactosyl residue of neolactotetraosylceramide [1,3]; acceptor and donor specificity [3]) (Reversibility: ? [1-4]) [1-6] P CMP + a-N-acetylneuraminyl-2,3-b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl-1,4-d-glucosylceramide [1-6] S CMP-N-acetylneuraminate + ganglioside GM1 (Reversibility: ? [3]) [3] P CMP + ganglioside GM3 [3] S CMP-N-acetylneuraminate + gangliotetraosylceramide (Reversibility: ? [1]) [1] P CMP + N-acetylneuraminyl-a-2,3-gangliotetraosylceramide [1] S CMP-N-acetylneuraminate + lacto-N-neohexaosylceramide (Reversibility: ? [3]) [3] P CMP + N-acetylneuraminyl-lacto-N-neohexaosylceramide [3] Inhibitors 5'-CMP [1, 3] AMP ( not inhibitory [1]) [1, 3] Ba2+ [3] CMP-N-acetylneuraminate ( i.e. substrate inhibition by CMP-sialic acid [3]) [3] Co2+ [3] Cu2+ [3] EDTA [3] Fe2+ [3] Fe3+ [3] Hg2+ [3] K+ [3] N-ethylmaleimide [4] Ni2+ [3] Pb2+ [3] Zn2+ [3] neolactotetraosylceramide ( substrate inhibition [3]) [3] sialic acid ( i.e. acetylneuraminate [3]) [3]

388

2.4.99.10


Activating compounds Triton CF-54 [1, 3] Triton CF-54/X-100 ( can replace Triton CF-54 [3]; as efficient as Triton CF-54 or X-100 [1]) [1, 3] Triton DF-16 ( can replace Triton CF-54 [3]) [3] Triton N-57 ( can replace Triton CF-54 [3]) [3] Triton X-100 [1, 3] Tween 20 ( 40% as efficient as Triton CF-54 [3]) [3] Tween 60 ( 27% as efficient as Triton CF-54 [3]) [3] Tween 80 ( 40% as efficient as Triton CF-54 [3]) [3] Additional information ( no activation by Triton GR-7M, TW-30 or sodium taurocholate [3]) [3] Metals, ions Mg2+ [1-3] Mn2+ [3] Km-Value (mM) 0.027 (gangliotetraosylceramide, pH 7.2, 37 C [1]) [1] 0.5 (b-d-galactosyl-1,4-N-acetyl-b-d-glucosaminyl-1,3-b-d-galactosyl1,4-d-glucosylceramide, pH 6.4, 37 C, neolactotetraosylceramide as substrate [1]) [1] 0.67 (CMP-N-acetylneuraminate, 37 C, in presence of lacto-N-hexaosylceramide [3]) [3] 0.9 (lacto-N-neohexaosylceramide, 37 C [3]) [3] pH-Optimum 6 [2] 6.8 ( in the presence of Triton CF-54 and Mg2+ [3]) [3] pH-Range 5.8-7.2 ( about half-maximal activity at pH 5.8 and about 90% of maximal activity at pH 7.2 [3]) [3] Temperature optimum ( C) 37 [4]

5 Isolation/Preparation/Mutation/Application Source/tissue brain [1, 4, 6] colonic cancer cell line [5, 6] lymphocyte ( from peripheral blood mononuclear cells, enzyme level stimulated by phytohemagglutinin [2]) [2] skeletal muscle [3] Localization Golgi membrane [1-6]

389


2.4.99.10

Purification (partial, using centrifugation in a sucrose gradient [1]; purification of a recombinant GST fusion protein using glutathione-agarose affinity chromatography [6]) [1, 6] (partial, using DEAE-Cibacron Blue chromatography after sodium taurocholate solubilization [5]) [5] Cloning (expression of a GST fusion protein in Escherichia coli [6]) [6]

References [1] Basu, M.; Basu, S.; Stoffyn, A.; Stoffyn, P.: Biosynthesis in vitro of sialyl(a 23)neolactotetraosylceramide by a sialyltransferase from embryonic chicken brain. J. Biol. Chem., 257, 12765-12769 (1982) [2] Basu, S.K.; Whisler, R.L.; Yates, A.J.: Effects of lectin activation on sialyltransferase activities in human lymphocytes. Biochemistry, 25, 2577-2581 (1986) [3] Dasgupta, S.; Chien, J.L.; Hogan, E.L.: Sialylation of lacto-N-neohexaosylceramide by sialyltransferase from embryonic chicken muscle. Biochim. Biophys. Acta, 876, 363-370 (1986) [4] Higashi, H.; Basu, M.; Basu, S.: Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain. J. Biol. Chem., 260, 824-828 (1985) [5] Basu, S.S.; Basu, M.; Li, Z.; Basu, S.: Characterization of two glycolipid: a23sialyltransferases, SAT-3 (CMP-NeuAc:nLcOse4Cer a2-3sialyltransferase) and SAT-4 (CMP-NeuAc:GgOse4Cer a2-3sialyltransferase), from human colon carcinoma (Colo 205) cell line. Biochemistry, 35, 5166-5174 (1996) [6] Basu, S.S.; Basu, M.; Dastgheib, S.; Ghosh, S.; Basu, S.: Cloning and expression of SAT-3 involved in SA-Le(x) biosynthesis: inhibition studies with polyclonal antibody against GST-SAT-3 fusion protein. Indian J. Biochem. Biophys., 34, 97-104 (1997)

390

Lactosylceramide a-2,6-N-sialyltransferase

2.4.99.11

1 Nomenclature EC number 2.4.99.11 Systematic name CMP-N-acetylneuraminate:lactosylceramide a-2,6-N-acetylneuraminyltransferase Recommended name lactosylceramide a-2,6-N-sialyltransferase Synonyms CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase CMP-acetylneuraminate-lactosylceramide-sialyltransferase CMP-sialic acid:lactosylceramide sialyltransferase GM3-synthase cytidine monophosphoacetylneuraminate-lactosylceramide sialyltransferase sialyltransferase, cytidine monophosphoacetylneuraminate-lactosylceramide CAS registry number 55071-95-9

2 Source Organism Rattus norvegicus (15 days old albino [1]; Sprague-Dawley [2]) [1, 2] Gallus gallus (20 days old [1]) [1] Oreochromis mossambicus (cichlid fish [2]) [2]

3 Reaction and Specificity Catalyzed reaction CMP-N-acetylneuraminate + b-d-galactosyl-1,4-b-d-glucosylceramide = CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-b-d-glucosylceramide Reaction type glycosyl group transfer

391

Lactosylceramide a-2,6-N-sialyltransferase

2.4.99.11

Substrates and products S CMP-N-acetylneuraminate + b-d-galactosyl-1,4-b-d-glucosylceramide ( i.e. lactosylceramide [1]) (Reversibility: ? [1-2]) [1, 2] P CMP + a-N-acetylneuraminyl-2,6-b-d-galactosyl-1,4-b-d-glucosylceramide ( i.e. ganglioside GM3 [1-2]) [1, 2] Inhibitors endogenous acidic peptide ( heat-stable, amino acid composition [1]) [1] Km-Value (mM) 0.15 (CMP-N-acetylneuraminate) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue brain ( cerebellum excluded [1]) [1, 2] Localization membrane [1, 2] Purification (homogenized, centrifuged, resuspended in 140 mM-cacodylate/HCl buffer pH 6.8 [1]; homogenized, centrifuged, resuspended in 25 mM-cacodylate buffer pH 6.5 containing 0.15% Triton X-100, 75 mM NaCl and 10 mM MnCl2 [2]) [1, 2] (homogenized, centrifuged, resuspended in 25 mM-cacodylate buffer pH 6.5 containing 0.15% Triton X-100, 75 mM NaCl and 10 mM MnCl2 [2]) [2]

References [1] Albarracin, I.; Lassaga, F.E.; Caputto, R.: Purification and characterization of an endogenous inhibitor of the sialyltransferase CMP-N-acetylneuraminate: lactosylceramide a2,6-N-acetylneuraminyltransferase (EC 2.4.99.-). Biochem. J., 254, 559-565 (1988) [2] Freischutz, B.; Saito, M.; Rahmann, H.; Yu, R.K.: Activities of five different sialyltransferases in fish and rat brains. J. Neurochem., 62, 1965-1973 (1994)

392

Dimethylallyltranstransferase

2.5.1.1

1 Nomenclature EC number 2.5.1.1 Systematic name dimethylallyl-diphosphate:isopentenyl-diphosphate dimethylallyltranstransferase Recommended name dimethylallyltranstransferase Synonyms (2E,6E)-farnesyl diphosphate synthetase DMAPP:IPP-dimethylallyltransferase dimethylallyl transferase diprenyltransferase geranyl diphosphate synthase geranyl pyrophosphate synthase geranyl pyrophosphate synthetase geranyl-diphosphate synthase prenyltransferase trans-farnesyl pyrophosphate synthetase Additional information (cf. EC 2.5.1.10, only enzymes for which geranyl diphosphate is not mentioned as substrate or is excluded as substrate are assigned to EC 2.5.1.1, enzymes for which the reaction with geranyl diphosphate or geranyl diphosphate and dimethylallyl diphosphate is reported are assigned to EC 2.5.1.10, cf. EC 2.5.1.29) CAS registry number 9032-79-5

2 Source Organism Tanacetum vulgare [1] Micrococcus lysodeikticus [2] Micrococcus luteus (the activities of EC 2.5.1.1 and EC 2.5.1.29 may be a mixture of 2 enzymes or a single enzyme catalyzing 2 reactions, see EC 2.5.1.29 [3,7]) [3, 7] Lithospermum erythrorhizon [4, 6, 9] Vitis vinifera [5]

393


2.5.1.1

Salvia officinalis [8] Mentha x piperita (cv. Black Mitcham, peppermint [10]) [10] Mentha spicate (spearmint [10]) [10] Arabidopsis thaliana [11] Abies grandis (grand fir [12]) [12]

3 Reaction and Specificity Catalyzed reaction dimethylallyl diphosphate + isopentenyl diphosphate = diphosphate + geranyl diphosphate Reaction type alkenyl group transfer Substrates and products S E-3-methyl-2-pentenyl diphosphate + isopentenyl diphosphate ( 32% of the rate with dimethylallyl diphosphate [3]) (Reversibility: ? [3]) [3] P ? S dimethylallyl diphosphate + isopentenyl diphosphate (Reversibility: ? [1-8,9,11,12]) [1-12] P diphosphate + geranyl diphosphate ( neryl diphosphate is not synthesized [5, 8]; farnesyl diphosphate is not synthesized [5]) [1-12] S farnesyl diphosphate + isopentenyl diphosphate ( the activities of EC 2.5.1.1 and EC 2.5.1.29 may be a mixture of 2 enzymes or a single enzyme with 2 independent catalytic sites, see EC 2.5.1.29 [3,7]) (Reversibility: ? [2,3,7]) [2, 3, 7] P geranylgeranyl diphosphate [2] Inhibitors 5,5'-dithiobis(nitrobenzoic acid) ( 0.004 mM, 50% inhibition [8]) [8] K+ ( 500 mM, 63% inhibition in the presence of Mg2+ [12]) [12] N-ethylmaleimide ( 0.06 mM, 50% inhibition [8]) [8] Triton X-100 ( stimulates farnesyl-transferring activity, inhibits dimethylallyl-transferring activity [3,7]) [3, 7] aminophenylethyl diphosphate ( 15 mM, 90% inhibition [5]) [5] diphosphate ( 10 mM, 85% inhibition [5]; 10 mM, complete inhibition of dimethylallyl- and farnesyl-transfering activity [7]; 0.06 mM, 50% inhibition [12]) [3, 5, 7, 12] geranyl diphosphate ( 0.2 mM, 33% inhibition [4]; 0.2 mM, 50% inhibition [5]; 1.3 mM, 50% inhibition [12]) [4, 5, 12] iodoacetamide ( 20 mM, 60% inhibition of dimethylallyl-transferring activity, no inhibition of farnesyl-transferring activity [3]) [3, 7]

394

2.5.1.1


isopentenyl diphosphate ( above 0.04 mM, 64% inhibition at 0.1 mM [12]) [12] p-mercuribenzenesulfonic acid ( 0.04 mM, 50% inhibition [8]) [8] Activating compounds Triton X-100 ( stimulates farnesyl-transferring activity, inhibits dimethylallyl-transferring activity [3,7]; 0.5%, 3.5fold activation [5]) [3, 5, 7] Tween 80 ( stimulates [7]) [7] Additional information ( not activated by Tween 20, Triton X-100, CHAPS or Nonidet P-40 [12]) [12] Metals, ions Co2+ ( can partially replace Mg2+ in activation [5]; 0.5 mM, 56% of maximal activity obtained with Mg2+ [8]; maximal activity at 0.5 mM, approx. 8% of activity obtained with Mg2+ [12]) [5, 8, 12] Mg2+ ( absolutely required for activity [3-5, 7, 12]; divalent cation required for activity [4, 12]; Mn2+ or Mg2+ are required for activity [5]; maximal activity at 2.5 mM, half-maximal activity at 0.23 and 42 mM respectively [4]; 58% as effective as Mn2+ [5]; Mg2+ most effective [8]; maximal activity at 10 mM [5]; maximal stimulation of farnesyl-transferring activity at 1 mM, maximal stimulation of dimethylallyl-transferring activity at 3 mM [3,7]; maximal activity at 5 mM [12]) [3-5, 7, 8, 12] Mn2+ ( absolutely required [4, 5, 12]; divalent cation required for activity [4, 12]; Mn2+ or Mg2+ are required for activity, more effective than Mg2+ , maximal activity at 10 mM [5]; 0.5 mM, 61% of maximal activity obtained with Mg2+ [8]; activates, slightly less effective than Mg2+ [3, 7]; Mn2+ and Mg2+ most effective [4]; can replace Mg2+ [8]; optimal activity at 10 mM [5]; maximal activity at 5 mM, less effectiv than Mg2+ [12]) [3-5, 7, 8, 12] Ni2+ ( 1 mM, 39% of maximal activity obtained with Mg2+ [8]) [8] Zn2+ ( can partially replace Mg2+ in activation [4]; 0.5 mM, 35% of maximal activity obtained with Mg2+ [8]) [4, 8] Specific activity (U/mg) 0.0176 [5] 0.0182 [3, 7] Km-Value (mM) 0.0056 (dimethylallyl diphosphate) [8] 0.0073 (isopentenyl diphosphate) [8] 0.008 (isopentenyl diphosphate) [3, 7] 0.0085 (isopentenyl diphosphate) [5] 0.014 (dimethylallyl diphosphate) [4] 0.0143 (isopentenyl diphosphate) [12] 0.0167 (dimethylallyl diphosphate) [12] 0.057 (dimethylallyl diphosphate) [5] 0.062 (dimethylallyl diphosphate) [3, 7] 395


2.5.1.1

0.083 (isopentenyl diphosphate) [4] 0.27 (Mg2+ ) [8] 0.455 (mg2+) [12] pH-Optimum 6.8 [4] 7 [8] 7-7.5 [12] 7.4 ( assay at [2]) [2] 7.7 [3, 7] pH-Range 5.5-8 ( approx. 50% of maximal activity at pH 5.5 and 8.0 [4]) [4] 6-8 ( approx. 50% of maximal activity at pH 6.0 and 8.0 [8]; approx. 50% of maximal activity at pH 6.0, 65% of maximal activity at pH 8.0 [12]) [8, 12] Temperature optimum ( C) 30 ( assay at [4,6]) [4, 6] 37 ( assay at [2,3]) [2, 3]

4 Enzyme Structure Molecular weight 53000 ( gel filtration [12]) [12] 68000 ( gel filtration [5, 10]) [5, 10] 70000 ( gel filtration [3,7]) [3, 7] 73000 ( gel filtration [4]) [4] 100000 ( gel filtration [8]) [8] Subunits ? ( x * 36000-38000, immunoblot [11]) [11] dimer ( 1 * 28000 + 1 * 37000, SDS-PAGE [10]; 1 * 28485 + 1 * 36400, mature proteins, deduced from nucleotide sequence [10]) [10] monomer ( 1 * 66000, SDS-PAGE [5]) [5]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture [4-6] leaf ( optimal preparation just before onset of flowering [1]; geranyl diphosphate synthase is probably exclusively localized in the monoterpene-producing oil glands [8]; glandular trichome i.e. oil gland secretory cells [10]) [1, 8, 10] sapling [12]

396

2.5.1.1


Localization chloroplast ( probably 2 isoforms depending on the methionine used to initiate translation: a plastid-targeted isoform and a truncated isoform targeted to the cytosol [11]) [11] cytosol ( very little activity in organelles, no activity in microsomes [9]) [9] soluble [4] Purification (partial [1]) [1] [2] (ammonium sulfate, DEAE-Sephadex, hydroxylapatite, gel filtration [7]) [3, 7] (ammonium sulfate, DEAE-Sephacel, phenyl-Sepharose, Sepadex G-150 [4,6]) [4, 6] [5] (ammonium sulfate, phenyl-Sepharose, Sephadex G-150, partial purification [8]) [8] (dye-ligand interaction chromatography, anion-exchange chromatography [10]) [10] (recombinant enzyme [11]) [11] (DEAE-Sepharose, Mono Q, phenyl-Sepharose, partial purification [12]) [12] Cloning (expression in Escherichia coli [10, 11]) [10, 11]

6 Stability General stability information , stability is greater in phosphate buffer than in MES or Tris buffer [4] , stability is increased by 16-20% sucrose or glycerol, 1 mM EDTA, 0.02 mM DMSF, or 1% bovine serum albumin [4] Storage stability , -20 C, 17 days, 20% loss of activity [2] , -20 C, 50 mM Tris-HCl, pH 7.7, 1 week, no loss of activity [7] , -20 C, 100 mM potassium phosphate, pH 7.75, 16% glycerol, 1 mM EDTA, 0.02 mM PMSF, 15weeks, 65% loss of activity [4] , 4-8 C, 100 mM Tris-HCl, pH 7.5, 24 h, 80% loss of activity [4] , 4-8 C, 100 mM potassium phosphate, pH 7.75, 16% glycerol, 1 mM EDTA, 0.02 mM PMSF, 24 h, 17% loss of activity [4]

397


2.5.1.1

References [1] Banthorpe, D.V.; Bucknall, G.A.; Doonan, H.J.; Doonan, S.; Rowan, M.G.: Biosynthesis of geraniol and nerol in cell-free extracts of Tanacetum vulgare. Phytochemistry, 15, 91-100 (1976) [2] Sagami, H.; Ogura, K.; Seto, S.: A new prenyltransferase from Micrococcus lysodeikticus. Biochem. Biophys. Res. Commun., 85, 572-578 (1978) [3] Sagami, H.; Ogura, K.: Geranylpyrophosphate synthetase-geranylgeranylpyrophosphate synthetase from Micrococcus luteus. Methods Enzymol., 110, 188-192 (1985) [4] Heide, L.; Berger, U.: Partial purification and properties of geranyl pyrophosphate synthase from Lithospermum erythrorhizon cell cultures. Arch. Biochem. Biophys., 273, 331-338 (1989) [5] Clastre, M.; Bantignies, B.; Feron, G.; Soler, E.; Ambid, C.: Purification and characterization of geranyl diphosphate synthase from Vitis vinifera L. cv. muscat de frontignan cell cultures. Plant Physiol., 102, 205-211 (1993) [6] Heide, L.: Geranylpyrophosphate synthase from cell cultures of Lithospermum erythrorhizon. FEBS Lett., 237, 159-162 (1988) [7] Sagami, H.; Ogura, K.: Geranylgeranyl pyrophosphate synthetase lacking geranyl-transferring activity from Micrococcus luteus. J. Biochem., 89, 1573-1580 (1981) [8] Croteau, R.; Purkett, P.T.: Geranyl pyrophosphate synthase: characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis). Arch. Biochem. Biophys., 271, 524-535 (1989) [9] Sommer, S.; Severin, K.; Camara, B.; Heide, L.: Intracellular localization of geranylpyrophosphate synthase from cell cultures of Lithospermum erythrorhizon. Phytochemistry, 38, 623-627 (1995) [10] Burke, C.C.; Wildung, M.R.; Croteau, R.: Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer. Proc. Natl. Acad. Sci. USA, 96, 13062-13067 (1999) [11] Bouvier, F.; Suire, C.; d'Harlingue, A.; Backhaus, R.A.; Camara, B.: Molecular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells. Plant J., 24, 241-252 (2000) [12] Tholl, D.; Croteau, R.; Gershenzon, J.: Partial purification and characterization of the short-chain prenyltransferases, geranyl diphosphate synthase and farnesyl diphosphate synthase, from Abies grandis (grand fir). Arch. Biochem. Biophys., 386, 233-242 (2001)

398

Thiamine pyridinylase

2.5.1.2

1 Nomenclature EC number 2.5.1.2 Systematic name thiamine:base 2-methyl-4-aminopyrimidine-5-methenyltransferase Recommended name thiamine pyridinylase Synonyms pyrimidine transferase thiamin hydrolase thiamin pyridinolase thiamin pyridinylase thiaminase thiaminase I thiamine pyridinolase thiamine pyridinylase CAS registry number 9030-35-7

2 Source Organism no activity in Squilla oratoria muscle [1] no activity in Penaeopsis acclivis muscle [1] no activity in Camboroides japonicus muscle [1] no activity in Camposcia retusa liver or muscle [1] no activity in Plecoglossus altivelis [1] no activity in Astrocanger myriaster [1] Carassius auratus (crucian carp [1]) [1] Cyprius carpio (carp [1,12]) [1, 12, 16] Mugil cephalus (grey mullet [1]) [1] Parasilurus asotus (catfish [1]) [1] Misgurnus anguilli caudatus (loach [1]) [1] Eleotris oxycephala [1] Auxis hira [1] Anguilla japonica (eel [1]) [1] Acanthogobius flavimanus (goby [1]) [1]

399


400

2.5.1.2

Ischikauia steenackeri [1] Odontobutis obscurus [1] Gnathopogon mayedae [1] Rhinogobius similis [1] Hypomessus olidus [1] Eviota abox [1] Leucopsarion petersi [1] Meretrix meretrix (clam [1]) [1] Paphia philippinarum [1] Solen gouldi (razor shell [1]) [1] Umbonium costatum [1] Cristaria plicata (fresh-water mussel [1]) [1] Corbicula leana [1] Circe seripta [1] Viviparus malleatus (mud snail [1]) [1] Clava kochi [1] Mactra veneriformis [1] Ostrea laperousei (oyster [1]) [1] Thiara libertina [1] Mactra sulcataria [1] Anadara inflata [1] Turbo cornutus [1] Haliotis gigantea [1] Haliotis japonica [1] Portunus trituberculatus (crab [1]) [1] Panulirus japonicus (spiny lobster [1]) [1] Charybdis 6-dentata [1] Bacillus thiaminolyticus (BMM strain [1,8]; M strain [2]; strain YUSM I00I [5]; DBM 1068 [13]) [1, 2, 4-6, 8, 13] Bacillus aneurinolyticus (BKA [1]) [1] Clostridium thiaminolyticum (equal to Paenibacillus thiaminolyticum, CKL [1]) [1] Pteridium aquilinum (var. Japonicum, bracken fern [1]) [1] Dicranopteris sp [1] Celosia crista (cockscomb [1]) [1] Polystichum tsumense [1] Polystichum aculeatum (var. Japonicum [1]) [1] Polystichum fortunei [1] Osmunda japonica (osmund royal [1]) [1] Equisetum arvense (horsetail [1,11]) [1, 11] Athyrium nipponicum [1] Polystichum falcatum [1] Lycopodium clavatum [1] Blechnum nipponicum [1] Dryopterus lacera [1] Polypodium ellipticum (var. Typican [1]) [1] Dicranopteris dichotoma [1]

2.5.1.2


Dryopteris erythrosora [1] Marsilea drumondii (nardoo [7]) [7] Marsilea augustifolia [7] Marsilea mutica [7] Pteridium esculentum (bracken fern [7]) [7] Cheilanthes seiberi (rock fern [7]) [7] Velesunio ambiguus (freshwater mussel [7]) [7] Bacillus thiaminolyticus (cloned in Escherichia coli [9-10,14-15]) [9-10, 14-15] Clupea harengus (baltic herring [17]) [17] Salmo salar (baltic salmon [17]) [17] Megasphaera elsdenii [3]

3 Reaction and Specificity Catalyzed reaction thiamine + pyridine = 1-[(4-amino-2-methylpyrimidin-5-yl)methyl]pyridinium + 4-methyl-5-(2-hydroxyethyl)thiazole Reaction type pyrimidyl group transfer Natural substrates and products S thiamine + adenine [7] P ? S thiamine + hydroxyproline [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]4-hydroxypyrrolidine-2-carboxylic acid S thiamine + proline [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]pyrrolidine-2-carboxylic acid Substrates and products S 1'-methylthiamine + aniline ( 22% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 4-amino-5-(anilinomethyl)-1,2dimethylpyrimidin-1-ium S 2'-ethylthiamine + aniline ( 63% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 5-(anilinomethyl)-2-ethylpyrimidin-4-amine S 2,3,4,5-tetrahydroxythiamine + aniline ( 84% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P ? S 2-(1-hydroxyethyl)thiamine + aniline ( 92% of activity with thiamine [16]) (Reversibility: ? [16]) [16]

401


2.5.1.2

P 4-methyl-2-(1-hydroxyethyl)-5-(2-hydroxyethyl)-thiazole + 5-(anilinomethyl)-2-methylpyrimidin-4-amine S 5-chloroethylthiamine + aniline ( 67% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 4-methyl-5-chloroethyl-thiazole + 5-(anilinomethyl)-2-methylpyrimidin4-amine S 5-nor-thiamine + aniline ( 88% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 4-methyl-thiazole + 5-(anilinomethyl)-2-methylpyrimidin-4-amine S O-(b-d-galactosyl)thiamine + aniline ( 63% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 4-methyl-5-(2-hydroxyethyl-O-b-d-galactosyl)-thiazole + 5-(anilinomethyl)-2-methylpyrimidin-4-amine S O-S-diacetylthiamine + aniline ( 50% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 1-acetyl-4-methyl-5-(2-hydroxyethyl-O-acetyl)-thiazole + 5-(anilinomethyl)-2-methylpyrimidin-4-amine S O-benzoylthiamine + aniline ( 91% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 2-(4-methyl-1,3-thiazol-5-yl)ethyl benzoate + 5-(anilinomethyl)-2-methylpyrimidin-4-amine S O-succinylthiamine + aniline ( 83% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 4-methyl-5-(2-hydroxyethyl-O-succinyl)-thiazole + 5-(anilinomethyl)-2methylpyrimidin-4-amine S pyrithiamine + aniline ( 72% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 2-(2-methylpyridin-3-yl)ethanol + 5-(anilinomethyl)-2-methylpyrimidin4-amine S thiamine ( there is degradation of thiamine without a base substrate, possibly the ring-opened form of thiamine functions as the nucleophile [15]; there is activity without base substrate [17]) (Reversibility: ? [15,17]) [15, 17] P ? S thiamine + 2-hydroxyaniline (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(2-hydroxyphenyl)amine S thiamine + 2-mercaptobenzoic acid (Reversibility: ? [7]) [7] P ? S thiamine + 2-mercaptoethanol ( 63% of activity with aniline [16]) (Reversibility: ? [16]) [16] P ? S thiamine + 2-methylbenzeneamine ( i.e. o-toluidine [7]) (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(2-methylphenyl)amine

402

2.5.1.2


S thiamine + 2-pyridinecarboxylic acid ( i.e. a-picolinic acid [7]) (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]-2-carboxypyridinium S thiamine + 3-hydroxyaniline (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(3-hydroxyphenyl)amine S thiamine + 3-methylbenzeneamine ( i.e. m-toluidine [7]) (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(3-methylphenyl)amine S thiamine + 3-pyridinecarboxylic acid ( i.e. b-picolinic acid [7]) (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]-3-carboxypyridinium S thiamine + 4-hydroxyaniline (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(4-hydroxyphenyl)amine S thiamine + 4-methylbenzeneamine ( i.e. p-toluidine [7]) (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(4-methylphenyl)amine S thiamine + 4-pyridinecarboxylic acid ( i.e. g-picolinic acid [7]) (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]-4-carboxypyridinium S thiamine + 6-aminohexanoic acid (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 6-[[(4-amino-2-methylpyrimidin5-yl)methyl]amino]hexanoic acid S thiamine + l-cysteine ( 50% of activity with aniline [16]) (Reversibility: ? [16]) [16] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 2-[[(4-amino-2-ethylpyrimidin-5yl)methyl]amino]-3-mercaptopropanoic acid S thiamine + adenine (Reversibility: ? [7]) [7] P ? S thiamine + a-picoline (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]a-picolinium S thiamine + aniline ( one molecule of thiamine is cleaved releasing one molecule of methylhydroxyethylthiazole for one molecule of enzyme [6]; taken as 100% of activity of purified enzyme [16]; activity found [17]) (Reversibility: ? [1-3, 5-8, 16, 17]) [1-3, 5-8, 16, 17] P 5-(anilinomethyl)-2-methylpyrimidin-4-amine + 4-methyl-5-(2-hydroxyethyl)-thiazole [1, 2, 5-8, 16, 17] S thiamine + b-picoline (Reversibility: ? [7]) [7]

403


2.5.1.2

P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]b-picolinium S thiamine + g-picoline (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]g-picolinium S thiamine + hydroxyproline (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]4-hydroxypyrrolidine-2-carboxylic acid S thiamine + imidazole ( 24% of activity with aniline [16]) (Reversibility: ? [7,16]) [7, 16] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 5-(1H-imidazol-1-yl-methyl)-2methylpyrimidin-4-amine S thiamine + m-aminobenzoic acid (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 3-[[(4-amino-2-methylpyrimidin5-yl)methyl]amino]benzoic acid S thiamine + m-phenylene diamine (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(3-aminophenyl)amine S thiamine + o-aminobenzoic acid (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 2-[[(4-amino-2-methylpyrimidin5-yl)methyl]amino]benzoic acid S thiamine + o-phenylene diamine (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(2-aminophenyl)amine S thiamine + p-aminobenzoic acid (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 4-[[(4-amino-2-methylpyrimidin5-yl)methyl]amino]benzoic acid S thiamine + p-phenylene diamine (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + [(4-amino-2-methylpyrimidin-5yl)methyl]-N-(4-aminophenyl)amine S thiamine + proline (Reversibility: ? [7]) [7] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]pyrrolidine-2-carboxylic acid S thiamine + pyridine ( best substrate with aniline [16]) (Reversibility: ? [4,5,7,16]) [4, 5, 7, 16] P heteropyrithiamine + 4-methyl-5-(2-hydroxyethyl)-thiazole [4, 5, 7, 16] S thiamine + quinoline (Reversibility: ? [7,8]) [7, 8] P 4-methyl-5-(2-hydroxyethyl)-thiazole + 1-[(4-amino-2-methylpyrimidin5-yl)methyl]quinolinium S thiamine + veratrylamine ( the reaction can occur without base substrate [9]) (Reversibility: ? [9]) [9] P bis-(4-amino-2-methyl)pyrimidinyl-3,4-dimethoxybenzylamine + 4methyl-5-(2-hydroxyethyl)-thiazole [9] S thiamine disulfide + aniline ( 26% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P ? 404

2.5.1.2


S thiamine phosphate + aniline ( 64% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 2-(4-methyl-1,3-thiazol-5-yl)ethyl dihydrogenphosphate + 5-(anilinomethyl)-2-methylpyrimidin-4-amine S thiothiamine + aniline ( 12% of activity with thiamine [16]) (Reversibility: ? [16]) [16] P 4-methyl-5-(2-hydroxyethyl)-2-thio-thiazole + 5-(anilinomethyl)-2-methylpyrimidin-4-amine S Additional information ( primary substrates inactivate the enzyme at 0.006 mM and secondary substrates or acceptor bases reactivate the enzyme at 10 mM, each substrate at high concentrations eliminates the inactivation of the other substrate [5]; no activity with 4'deaminothiamine and 4'-oxythiamine suggest that 4'-amino group is essential for enzyme activity [16]) [5, 16] P ? Inhibitors 4-amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine ( complete irreversible inhibition 2 times faster than 4-amino-6-chloro-2-methylpyrimidine [8]) [8] 4-amino-6-chloro-2-methylpyrimidine ( complete irreversible inhibition, activity protected by thiamine and quinolone [8]; inhibitory [9]; this suicide substrate is linked to Cys113 of the enzyme [10]) [8-10] 4-aminopyrimidines ( mostly inhibitory [1]) [1] Ag+ ( 100% inhibition at 1 mM [7]) [7] Cu2+ ( different inhibitory effects at 1 mM [1]; no inhibition [1]; 50% of inhibition at 1 mM [7]) [1, 7] Fe2+ ( different inhibitory effects at 1 mM [1]; no inhibition [1]; 70% of inhibition at 1 mM [7]) [1, 7] HgCl2 ( potent inhibitor at 0.001 mM [7]) [7] Mn2+ ( different inhibitory effects at 1 mM [1]; no inhibition [7]) [1] aniline ( highly inhibitory above 2.4 mM [2]; reactivation at 2 mM [5]) [2] aniline ( inactivation at 0.006 mM [5]) [5] aromatic amines ( inhibited by or without effect [1]) [1] dimethialium ( inactivation at 0.006 mM [5]) [5] heterocyclic amines [1] heteropyrithiamine ( inhibitory at 0.006 mM [4]; inactivation at 0.006 mM that is restored by pyridine at 25 mM and inhibition of restoration of activity by pyridine at 1 mM [5]) [4, 5] mersalyl acid ( potent inhibitor at 0.001 mM [7]) [7] p-chloromercuribenzoate ( 50% of inhibition at 0.2 mM and inhibits reactivation by pyridine [5]; complete inhibition at 0.001 mM [7]) [5, 7] thiamine ( slightly inhibitory above 0.035 mM [2]; reversible inactivation above 0.0003 mM [4]; inactivation at 0.006 mM [5];

405


2.5.1.2

inactivation is proportional to thiamine concentration and the thiamine/enzyme ratio is 1 mol/1 mol [6]) [2, 4-6] thiamine diphosphate ( inactivation at 0.006 mM [5]) [5] Additional information ( chloramphenicol does not affect activity suggesting that the enzyme has an inactive form [4]; primary substrates inactivate the enzyme at 0.006 mM, the lost activity can be restored by incubation at 37 C for 60 min after dialysis or by incubation with secondary substrates or acceptor bases, each substrate at high concentrations abolishes the inactivation of the other substrate [5]; acidic conditions prevent inactivation but low pH does not restore activity after inactivation, quaternary nitrogen linked to a substituted pyrimidine ring through a methylene bridge is shared by compounds that inactivates the enzyme, inactivation alters either the net charge or the structure of the protein or both [6]; not inhibited by EDTA [7]; 4-amino-2-methyl pyrimidine does not inactivate nor competitively inhibits the enzyme [8]) [4-8] Activating compounds 2-mercaptoethanol ( similar effects as pyridine at 10 mM probably due to its action as secondary substrate [5]; reactivates enzyme less effectively than dithiothreitol [6]; complete reactivation at 3.3 mM but inhibition at 33 mM [7]) [5-7] 4,5-dimethylthiazole ( reactivation at 10 mM [5]) [5] 5-(2-chloroethyl)-4-methylthiazole ( reactivation at 10 mM [5]) [5] 5-(2-hydroxyethyl)-4-methylthiazole ( reactivation at 10 mM [5]) [5] aniline ( activated by 1 mM [1]; not activated by 1 mM [1]; reactivation of enzyme at 2 mM [5]) [1, 5] aromatic amines ( increased activity, aniline is the most potent activator, primary amines not directly attached to the benzene ring have no effect [1]) [1] cysteine ( complete reactivation at 3.3 mM but inhibition at 33 mM [7]) [7] dithiothreitol ( similar effects as pyridine at 10 mM probably due to its action as secondary substrate [5]; reactivates 60-100% of the original activity [6]) [5, 6] heterocyclic amines ( pyridine, 2-amino-pyridine, 3-amino-pyridine increase activity [1]; nicotinic acid and quinolone increase activity [1]; most 4-aminopyrimidines increase activity [1]; most 4-aminopyrimidines are inhibitory [1]) [1] pyridine ( at 25 mM restores activity inhibited by heteropyrimidine at 0.006 mM [5]) [5] sulfhydryl compounds ( increase activity, more markedly in bacteria [1]) [1] thioglycollate ( greater reactivation at 33 mM than at 3.3 mM [7]) [7]

406

2.5.1.2


thiosulphate ( greater reactivation at 33 mM than at 3.3 mM [7]) [7] Additional information ( aliphatic amines and taurine have no effect [1]; l-phenylalanine, l-tyrosine, l-tryptophan and g-aceto-g-chloropropyl acetate have no effect [1]) [1] Metals, ions Additional information ( Ca2+ , Mo+, Mn2+ , Zn2+ , Mg2+ have no effect on activity [7]) [7] Km-Value (mM) 0.003 (thiamine, measured in Tris and diphosphate, the same as for thiamine diphosphate [7]) [7] 0.0037 (thiamine) [9] 0.0087 (thiamine, pH 5.8, 25 C, the enzyme does not follow strict Michaelis-Menten kinetics [2]) [2] 0.5 (b-picoline) [7] 0.7 (pyridine) [7] 0.9 (g-picoline) [7] 0.9 (nicotinic acid) [7] 1.4 (aniline, lower maximal activity than pyridine and nicotinic acid [7]) [7] 1.7 (m-phenylene diamine) [7] 1.7 (o-phenylene diamine) [7] 2 (aniline, lower maximal activity than pyridine and nicotinic acid [7]) [7] 2.4 (m-toluidine) [7] 2.5 (pyridine) [7] 2.8 (3-hydroxyaniline) [7] 2.9 (3-aminopyridine) [7] 2.9 (aniline, pH 5.8, 25 C, the enzyme does not follow strict Michaelis-Menten kinetics [2]) [2] 3.3 (a-picoline) [7] 3.3 (p-phenylene diamine) [7] 3.3 (quinoline) [7] 3.4 (g-picolinic acid) [7] 4.2 (2-hydroxyaniline) [7] 5 (o-mercaptobenzoic acid, best cosubstrate [7]) [7] 5 (pyridine) [7] 5.1 (aniline, lower maximal activity than pyridine and nicotinic acid [7]) [7] 6.4 (o-toluidine) [7] 9.5 (adenine) [7] 12.5 (2-aminopyridine) [7] 15.4 (4-aminopyridine) [7] 20 (4-hydroxyaniline) [7] 21.2 (a-picolinic acid) [7]

407


2.5.1.2

25 (b-picolinic acid) [7] 25 (nicotinic acid) [7] 29 (nicotinic acid) [7] 32 (methylamine) [7] 33 (cysteine) [7] 37 (m-aminobenzoic acid) [7] 37 (o-aminobenzoic acid) [7] 38 (6-aminohexanoic acid) [7] 50 (m-nitroaniline) [7] 50 (sulfanilic acid) [7] 55 (nicotinic acid) [7] 77 (sulfanilamide) [7] 111 (proline) [7] 125 (imidazole) [7] 200 (Tris) [7] 200 (p-aminobenzoic acid) [7] 400 (hydroxyproline) [7] Ki-Value (mM) 0.096 (4-amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine) [8] 7.7 (4-amino-6-chloro-2-methylpyrimidine) [9] pH-Optimum 5 [1] 5.5 [1] 6 [1] 6-7 [16] 7.5 [1] 8 [1] 8-9 [7, 11] pH-Range 3-12 [7] 5.8-6.8 ( assay at 25 C [2]) [2] Temperature optimum ( C) 25 ( in 50 mM sodium phosphate at pH 7 [9]) [9] 37 ( in 0.1 M sodium phosphate buffer at pH 5.8 [2]; in 0.1 M disodium hydrogenophosphate buffer [11]) [2, 11] 40 [16] Temperature range ( C) 37 ( after dialysis, 60 min of incubation restore 90% of activity inhibited by thiamine [4,5]; assay at [5]) [4, 5] 37-65 ( enzyme is stable [11]) [11] 50-60 ( maximal activity [1]) [1] 60 ( more than 90% of activity remains after 20 min [2]) [2] 65 ( activity abolished after 20 min [2]) [2]

408

2.5.1.2


70 ( 50% denaturation [11]) [11] 90 ( inactivation after 10 min [1]; inactivation after 15 min [1]) [1] 100 ( inactivation after 10 min [1]; inactivation after 15 min [1]; inactivation after 20 min [1]) [1]

4 Enzyme Structure Molecular weight 42000 ( SDS-PAGE [9]) [9] 44000 ( sedimentation and diffusion data [2]) [2] 55000 ( gel filtration [16]) [16] 93000 ( gel filtration [7]) [7] 107000 ( gel filtration [7]) [7] 110000 ( gel filtration [7]) [7] 115000 ( gel filtration [7]) [7] Subunits monomer ( 1 * 42000by SDS-PAGE [9]; active site between Pro109 and Phe118, includes nucleophilic site Cys113 [10]) [9-10]

5 Isolation/Preparation/Mutation/Application Source/tissue brain [1] cell culture ( addition of yeast extract and Ca2+ in the medium increases production of thiaminase, addition of glutamate or brain heart infusion decreases production [13]) [4-6, 8-9, 13-15] culture filtrate [2] eye ( ball [1]) [1] flower ( less activity than in leaves [1]) [1] frond ( extremely high levels of the enzyme, lush fronds have much more enzyme than old fronds [7]; very low activity in lush fronds [7]; activity found in lush fronds [7]; very low activity in young and old fronds [7]; activity found in lush fronds [7]) [7] gastrointestinal tract ( fluid [17]) [1, 17] gill [1] head [1] heart [1] intestine [1] kidney ( highest activity [1]; high activity [12]) [1, 12] leaf [1]

409


2.5.1.2

liver ( scarcely found [1]; no activity [1]) [1, 16] muscle ( scarcely found [1]; no activity [1]) [1] ovary [1] pancreas ( scarcely found [1]) [1] root ( lush growth [7]) [7] skin [1] spleen ( highest activity found [1]; high activity [12]) [1, 12] stomach [1] testis ( scarcely found [1]) [1] tuber ( very low activity [7]) [7] viscus ( high activity [1]) [1] whole body ( very low activity [1]; activity found [7]; homogenate [17]; vacuum dried and fresh extracts have thiaminase activity, aqueous extract and alcohol extract have no activity [11]) [1, 7, 11, 17] Localization cell associated [4, 16] extracellular [2, 5-6, 8-9, 13-15] lysosome ( enzyme activity is mostly found in lysosomal fraction determined by differential centrifugation, sucrose and percoll gradients [12]) [12] Purification (by ammonium precipitation, CM-cellulose column, DEAE-cellulose column, Cibacron blue Sepharose column, Sephacryl S-300 column, Superdex200 and Superdex-75 column [16]) [16] (by ammonium precipitation, Sephadex G-25 column, Sephadex G-100 column and DEAE-Sephadex column [2]; Tris-glycine polyacrylamide electrophoresis [6]; ammonium precipitation and gel filtration [13]) [2, 6, 13] (by DEAE-cellulose chromatrography, ultrogel AcA 34 column, Sephadex G-100 column and phosphocellulose column [7]) [7] (by ammonium precipitation, DEAE column, Cibacron blue dye affinity column [9]) [9] Crystallization (by vapour-diffusion method [14]) [14] Cloning (cloned in Escherichia coli with Gly6 changed to Ala6 with no apparent effect [9]) [9] Engineering C113S ( inactive mutant at the active nucleophilic site [9]) [9]

410

2.5.1.2


Application medicine ( bacteria can infect organisms and produce thiaminase disease [1]; ferns that have thiaminase produce thiamine deficiency in rat, cattle and horse [1]; plant extracts that have thiaminase activity can be toxic to animals or humans [11]) [1, 11] nutrition ( thiaminase can be used as an effective method for thiamine determination in food and fodder [13]) [13]

6 Stability pH-Stability 3-12 ( stable in this range [7]) [7] 5.8 ( 0.1 M sodium phosphate buffer [2,6]) [2, 6] 6.5 ( 0.15 M citrate-phosphate buffer [5]) [5] 7 ( 50 mM sodium phosphate buffer, 2 mM dithiothreitol, 2 mM EDTA [9,14]) [9, 14] Oxidation stability , addition of dithiothreitol, 2 mM, reduces the loss of activity [9]

References [1] Fujita, A.: Thiaminase. Adv. Enzymol. Relat. Subj. Biochem., 15, 389-421 (1954) [2] Wittliff, J.L.; Airth, R.L.: The extracellular thiaminase I of Bacillus thiaminolyticus. I. Purification and physicochemical properties. Biochemistry, 7, 736-744 (1968) [3] Boyd, J.W.: Studies on thiaminase I activity in ruminant faeces and rumen bacteria. J. Agric. Sci., 104, 637-642 (1985) [4] Suzuki, K.; Ooba, J.I.: Reversible inactivation of cellular thiaminase I in Bacillus thiaminolyticus by thiamine and heteropyrithiamine. J. Biochem., 72, 1053-1055 (1972) [5] Suzuki, K.; Ooba, J.-I.: Reversible inactivation of extracellular thiaminase I in Bacillus thiaminolyticus. I. Inactivation by the primary substrate and reactivation by the secondary substrate. Biochim. Biophys. Acta, 293, 111117 (1973) [6] Agee, C.C.; Airth, R.L.: Reversible inactivation of thiaminase I of Bacillus thiaminolyticus by its primary substrate, thiamine. J. Bacteriol., 115, 957965 (1973) [7] McCleary, B.V.; Chick, B.F.: The purification and properties of a thiaminase I enzyme from nardoo (Marsilea Drumondii). Phytochemistry, 16, 207-213 (1977) [8] Hutter, J.A.; Slama, J.T.: Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate. Biochemistry, 26, 1969-1973 (1987)

411


2.5.1.2

[9] Costello, C.A.; Kelleher, N.L.; Abe, M.; McLafferty, F.W.; Begley, T.P.: Mechanistic studies on thiaminase I. Overexpression and identification of the active site nucleophile. J. Biol. Chem., 271, 3445-3452 (1996) [10] Kelleher, N.L.; Nicewonger, R.B.; Begley, A.P.; McLafferty, F.W.: Identification of modification sites in large biomolecules by stable isotope labeling and tandem high resolution mass spectrometry. The active site nucleophile of thiaminase I. J. Biol. Chem., 272, 32215-32220 (1997) [11] Fabre, B.; Geay, B.; Beaufils, P.: Thiaminase activity in Equisetum arvense and its extracts. Plant. Med. Phytother., 26, 190-197 (1993) [12] Sato, M.; Hayashi, S.; Nishino, K.: Subcellular localization of thiaminase I in the kidney and spleen of carp, Cyprinus carpio. Comp. Biochem. Physiol. A, 108A, 31-38 (1994) [13] Ruml, T.; Silhankova, L.; Brunerova, M.: Purification of thiaminase I for analytical purposes. Potravinarske Vedy, 13, 181-187 (1995) [14] Campobasso, N.; Begun, J.; Costello, C.A.; Begley, T.P.; Ealick, S.E.: Crystallization and preliminary X-ray analysis of thiaminase I from Bacillus thiaminolyticus: space group change upon freezing of crystals. Acta Crystallogr. Sect. D, D54, 448-450 (1998) [15] Wu, M.; Papish, E.T.; Begley, T.P.: Mechanistic studies on thiaminase I. Identification of the product of thiamin degradation in the absence of the nucleophilic cosubstrate. Bioorg. Chem., 28, 45-48 (2000) [16] Bos, M.; Kozik, A.: Some molecular and enzymatic properties of a homogeneous preparation of thiaminase I purified from carp liver. J. Protein Chem., 19, 75-84 (2000) [17] Wistbacka, S.; Heinonen, A.; Bylund, G.: Thiaminase activity of gastrointestinal contents of salmon and herring from the Baltic Sea. J. Fish Biol., 60, 1031-1042 (2002)

412

Thiamine-phosphate diphosphorylase

2.5.1.3

1 Nomenclature EC number 2.5.1.3 Systematic name 2-methyl-4-amino-5-hydroxymethylpyrimidine-diphosphate:4-methyl-5-(2phosphoethyl)-thiazole 2-methyl-4-aminopyrimidine-5-methenyltransferase Recommended name thiamine-phosphate diphosphorylase Synonyms 2-methyl-4-amino-5-hydroxymethylpyrimidinepyrophosphate:4-methyl-5(2'-phosphoethyl)-thiazole 2-methyl-4-aminopyrimidine-5-methenyltransferase TMP pyrophosphorylase TMP-PPase hydroxyethylthiazole kinase/thiamine-phosphate pyrophosphorylase ( bifunctional enzyme with hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase activity [2]) [2] pyrophosphorylase, thiamin phosphate thiamin phosphate pyrophosphorylase thiamin phosphate synthase thiamin-phosphate pyrophosphorylase thiamine monophosphate pyrophosphorylase thiamine phosphate pyrophosphorylase thiamine-phosphate pyrophosphorylase thiamine-phosphate synthase thiaminephosphate pyrophosphorylase CAS registry number 9030-30-2

2 Source Organism Escherichia coli (K12, strain PT-R1 [5]) [4, 5] Saccharomyces cerevisiae (IFO 10483 [2]; mutant resistant to 2-amino-4methyl-5-b-hydroxyethylthiazole, an antimetabolite of 4-methyl-5-b-hydroxyethylthiazole, deficient in activity of both EC 2.5.1.3 and EC 2.7.1.50 [2]; bifunctional enzyme with hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase activity [2]) [1-3, 7] 413


2.5.1.3

Bacillus subtilis [6, 9] Brassica napus (bifunctional enzyme with hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase activity [8]) [8]

3 Reaction and Specificity Catalyzed reaction 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2phosphono-oxyethyl)thiazole = diphosphate + thiamine phosphate ( dissociative SN1 mechanism [6]) Reaction type hydroxymethylpyrimidine group transfer Natural substrates and products S 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + thiazole monophosphate ( involved in biosynthesis of thiamine [1, 7, 8]; the bifunctional enzyme hydroxyethylthiazole kinase/thiaminephosphate pyrophosphorylase catalyzes two sequential steps in the synthesis of thiamin monophosphate from hydroxyethylthiazole [2]; expression is negatively regulated by thiamin [8]) (Reversibility: ? [1, 2, 7, 8]) [1, 2, 7, 8] P ? Substrates and products S 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl5-(2-phosphono-oxyethyl)-thiazole (Reversibility: r [3]; ? [1-9]) [1-9] P thiamine monophosphate + diphosphate [1-9] S 4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole ( poor substrate [6]) (Reversibility: ? [6]) [6] P ? S 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine diphosphate + 4methyl-5-(2-phosphono-oxyethyl)-thiazole ( good substrate [6]) (Reversibility: ? [6]) [6] P ? Inhibitors ADP ( 4 mM, 42.4% inhibition [5]) [4, 5] ATP ( uncompetitive with respect to both substrates [4]; 4 mM, 64.7% inhibition [5]) [4, 5] CTP ( 4 mM, 58.7% inhibition [5]) [4, 5] GTP ( 4 mM, 76.7% inhibition [5]) [4, 5] PCMB ( 0.01 mM, 98.5% inhibition, inhibition prevented by addition of 2-mercaptoethanol in 10fold molar excess [2]) [2] UTP ( 4 mM, 71.6% inhibition [5]) [4, 5]

414

2.5.1.3


acetyl phosphate ( 0.5 mM, 50% inhibition, uncompetitive with respect to both substrates [4]; 4 mM, 82.8% inhibition [5]) [4, 5] diphosphate ( 0.05 mM, 50% inhibition [5]) [3-5] phosphocreatine ( 4 mM, 63.8% inhibition [5]) [4, 5] phosphoenolpyruvate ( 4 mM, 31.1% inhibition [5]) [4, 5] Metals, ions Ca2+ ( stimulates, 20% as effective as Mg2+ [4,5]) [4, 5] Co2+ ( stimulates, 20% as effective as Mg2+ [4,5]) [4, 5] Mg2+ ( required [1, 2, 3, 4, 5]; 1 mM, 3-6fold stimulation [3]; most effective at 6 mM [4, 5]; Km : 0.063 mM [4,5]) [1-5] Mn2+ ( half as effective as Mg2+ [3]; stimulates, at 6 mM equally effective as Mg2+ [4,5]) [3, 4, 5] Zn2+ ( stimulates, 20% as effective as Mg2+ [4,5]) [4, 5] Turnover number (min±1) Additional information [6] Specific activity (U/mg) 0.065 [4, 5] 0.269 [2] Additional information [3] Km-Value (mM) 0.0004 (thiazole monophosphate) [4, 5] 0.00085 (hydroxymethylpyrimidine diphosphate) [4, 5] 0.001 (hydroxymethylpyrimidine diphosphate, pH 9.2, 37 C [3]) [3] 0.007 (thiazole monophosphate, pH 9.2, 37 C [3]) [3] pH-Optimum 8.5 [4] 9.1 [2] 9.2 [3] pH-Range 7-9.1 ( pH 7.0: about 30% of maximal activity, pH 9.1: optimum [2]) [2] 7.7-10 ( about 40% of activity maximum at pH 7.7 and pH 10.0 [3]) [3] Temperature optimum ( C) 40 [4, 5]

4 Enzyme Structure Molecular weight 17000 ( gel filtration [4,5]) [4, 5] 470000 ( hydroxyethylthiazole kinase is a bifunctional enzyme with thiamine-phosphate pyrophosphorylase activity, gel filtration [2]) [2]

415


2.5.1.3

Subunits ? ( x * 58058, calculation from nucleotide sequence [7]) [7] octamer ( 8 * 60000, SDS-PAGE, hydroxyethylthiazole kinase is a bifunctional enzyme with thiamine-phosphate pyrophosphorylase activity [2]) [2]

5 Isolation/Preparation/Mutation/Application Purification [4] (bifunctional enzyme with hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase activity [2]) [2, 3] (from E. coli overexpression strain [6]) [6] Crystallization (hanging-drop vapor diffusion, crystal structure at 1.25 A resolution [9]) [9] Cloning (bifunctional enzyme with hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase activity [7]) [7] (bifunctional enzyme with hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase activity, expression in Escherichia coli [8]) [8]

6 Stability Temperature stability 21 ( room temperature, completely inactivated overnight [3]) [3] 45 ( 5 min, without addition of both substrates, completely inactivated [4,5]) [4, 5] Storage stability , -20 C, stable for several months [4, 5] , -15 C, 6 months, less than 10% loss of activity [3] , -80 C, about 64.4% loss of activity after 1 month [2]

References [1] Camiener, G.W.; Brown, G.M.: The biosynthesis of thiamine. 2. Fractionation of enzyme system and identification of thiazole monophosphate and thiamine monophosphate as intermediates. J. Biol. Chem., 235, 2411-2417 (1960) [2] Kawasaki, Y.: Copurification of hydroxyethylthiazole kinase and thiaminephosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway. J. Bacteriol., 175, 5153-5158 (1993)

416

2.5.1.3


[3] Leder, I.G.: The enzymatic synthesis of thiamine monophosphate. J. Biol. Chem., 236, 3066-3071 (1961) [4] Kayama, Y.; Kawasaki, T.: Purification and properties of thiaminephosphate pyrophosphorylase of Escherichia coli. Arch. Biochem. Biophys., 158, 242248 (1973) [5] Kawasaki, T.: Thiamine phosphate pyrophosphorylase. Methods Enzymol., 62, 69-73 (1979) [6] Reddick, J.J.; Nicewonger, R.; Begley, T.P.: Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism. Biochemistry, 40, 10095-10102 (2001) [7] Nosaka, K.; Nishimura, H.; Kawasaki, Y.; Tsujihara, T.; Iwashima, A.: Isolation and characterization of the THI6 gene encoding a bifunctional thiaminphosphate pyrophosphorylase/hydroxyethylthiazole kinase from Saccharomyces cerevisiae. J. Biol. Chem., 269, 30510-30516 (1994) [8] Kim, Y.S.; Nosaka, K.; Downs, D.M.; Kwak, J.M.; Park, D.; Chung, I.K.; Nam, H.G.: A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis. Plant Mol. Biol., 37, 955-966 (1998) [9] Chiu, H.-J.; Reddick, J.J.; Begley, T.P.; Ealick, S.E.: Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution. Biochemistry, 38, 6460-6470 (1999)

417

Adenosylmethionine cyclotransferase

2.5.1.4

1 Nomenclature EC number 2.5.1.4 Systematic name S-adenosyl-l-methionine alkyltransferase (cyclizing) Recommended name adenosylmethionine cyclotransferase Synonyms adenosylmethioninase cyclotransferase, adenosylmethionine CAS registry number 9030-34-6

2 Source Organism Saccharomyces cerevisiae [1, 2] Escherichia coli (infected with bacteriophage T3 and UV-T3 [3]; infected with bacteriophage T3 [5]) [3, 5] Sus scrofa (pig [4]) [4] Rattus norvegicus (Sprague-Dawley rat [4]) [4] Homo sapiens [4]

3 Reaction and Specificity Catalyzed reaction S-adenosyl-l-methionine = 5'-methylthioadenosine + 2-aminobutan-4-olide ( mechanism [2]) Reaction type nucleophilic substitution [2] Natural substrates and products S S-adenosyl-l-methionine (Reversibility: ir [1]) [1, 2] P 5'-methylthioadenosine + a-amino-g-butyrolactone [1, 2]

418

2.5.1.4


Substrates and products S S-adenosyl-l-methionine (Reversibility: ir [1]) [1-4] P 5'-methylthioadenosine + 2-aminobutan-4-olide [1-4] S S-adenosylethionine (Reversibility: ? [1]) [1] P ? Inhibitors 5'-O-methyladenosine ( weak inhibition [1]) [1] l-methionine ( weak inhibition [1]) [1] Mn2+ [3] S-adenosyl-l-ethionine ( 4 mM inhibits more than 50% [1]; racemic mixture, 0.2 mM, 50% inhibition [3]) [1, 3] S-adenosyl-l-homocysteine ( 4 mM inhibits 25-50% [1]) [1, 3, 4] S-ribosylmethionine ( weak inhibition [1]) [1, 3] SH-binding reagents [3] adenine ( weak inhibition [1]) [1] adenosine ( weak inhibition [1]) [1] allo-S-adenosyl-l-methionine ( 4 mM inhibits more than 50% [1]) [1] dimethyladenosylsulfonium ( weak inhibition [1]) [1] ethylthioadenosine ( 4 mM inhibits more than 50% [1]) [1] methionine methylsulfonium ( weak inhibition [1]) [1] methylthioadenosine ( 4 mM inhibits more than 50% [1]) [1] methylthioinosine ( weak inhibition [1]) [1] Additional information ( no effect: Mg2+ , EDTA [3]) [3] Cofactors/prosthetic groups Additional information ( no cofactor is required [1]) [1] Specific activity (U/mg) 0.061 [4] 0.099 ( porcine liver homogenate supernatant [4]) [4] 0.108 ( pH 5 preparation [4]) [4] 0.117 ( ammonium sulfate-treated, pH 5 enzyme preparation [4]) [4] 3.07 ( E. coli infected with bacteriophage T3 [3]) [3] 28.3 ( E. coli infected with bacteriophage UV-T3 [3]) [3] Additional information ( synthesized enzyme tested according to the method of Gefter et al. [5]) [5] Km-Value (mM) 0.05 (S-adenosyl-l-methionine) [4] 1 (S-adenosyl-l-methionine) [3] Ki-Value (mM) 0.05 (S-adenosyl-l-homocysteine, competitive inhibitor to S-adenosylmethionine [4]) [4] pH-Optimum 8.5 [3]

419


2.5.1.4

pH-Range 6-9 ( about 60% of activity maximum at pH 6 and 9 [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue KB cell ( cells [4]) [4] liver [4] Purification (partial purification by a method that includes precipitation with acetone, differential heat inactivation and precipitation by ammonium sulfate [1]) [1] (method that includes chromatography on DEAE-cellulose [3]) [3]

6 Stability General stability information , not stable to repeated freezing and thawing [3] Storage stability , -10 C, enzyme from E. coli infected with bacteriophage UV-T3 is stable for 2 weeks [3] , 0 C or -10 C, enzyme from E. coli infected with bacteriophage T3 loses all of its activity within 3 days [3]

References [1] Mudd, S.H.: Enzymatic cleavage of S-adenosylmethionine. J. Biol. Chem., 234, 87-92 (1959) [2] Mudd, S.H.: The mechanism of the enzymatic cleavage of S-adenosylmethionine to a-amino-g-butyrolactone. J. Biol. Chem., 234, 1784-1786 (1959) [3] Gefter, M.L.: T3 and UV-T3-induced S-adenosylmethionine cleavage enzyme (Escherichia coli). Methods Enzymol., 17 B, 406-411 (1971) [4] Swiatek, K.R.; Simon, L.N.; Chao, K.-L.: Nicotinamide methyltransferase and S-adenosylmethionine: 5-methylthioadenosine hydrolase. Control of transfer ribonucleic acid methylation. Biochemistry, 12, 4670-4674 (1973) [5] Gazarian, K.G.; Gening, L.V.; Gazarian, T.G.: l-Homoserine: a novel excreted metabolic marker of hepatitis B abnormally produced in liver from methionine. Med. Hypotheses, 58, 279-283 (2002)

420

Galactose-6-sulfurylase

2.5.1.5

1 Nomenclature EC number 2.5.1.5 Systematic name d-galactose-6-sulfate:alkyltransferase (cyclizing) Recommended name galactose-6-sulfurylase Synonyms galactose 6-sulfatase galactose 6-sulfurylase galactose-6-sulfatase porphyran sulfatase sulfatase, porphyran CAS registry number 9030-36-8

2 Source Organism Porphyra sp. [1, 2]

3 Reaction and Specificity Catalyzed reaction eliminates sulfate from the d-galactose 6-sulfate residues of porphyran producing 3,6-anhydrogalactose residues Reaction type sulfate group removal Natural substrates and products S porphyran (, eliminates sulfate from the d-galactose 6-sulfate residues of porphyran producing 3,6-anhydrogalactose residues [1,2]) [1, 2] P sulfate + porphyran containing 3,6-anhydrogalactose residues

421


2.5.1.5

Substrates and products S porphyran (, eliminates sulfate from the d-galactose 6-sulfate residues of porphyran producing 3,6-anhydrogalactose residues [1,2]) (Reversibility: ? [1]) [1, 2] P sulfate + porphyran containing 3,6-anhydrogalactose residues Inhibitors Al3+ [1] Ca2+ [1] Cd2+ [1] Cu2+ [1] EDTA [1, 2] KCN [1, 2] NH4 F [1] NaF [2] Ni2+ [1] Activating compounds borate (, 60% activation at pH 7.6 [1]; , activates [2]) [1, 2] Metals, ions Additional information (, activity is dependent on the presence of a bi- or tervalent cation which is not Mg2+ [1]) [1] pH-Optimum 7.6-7.8 (, sodium tetraborate/HCl buffer [1]) [1] pH-Range 6.8-9.2 (, pH 6.8: about 45% of activity maximum, pH 9.2: about 35% of activity maximum [1]) [1]

5 Isolation/Preparation/Mutation/Application Purification (partial [1]) [1]

6 Stability Storage stability , 4 C, gradual loss of activity in solution [1] , room temperature, stable for at least several months as a freeze-dried powder [1]

422

2.5.1.5


References [1] Rees, D.A.: Enzymatic desulphation of porphyran. Biochem. J., 80, 449-453 (1961) [2] Rees, D.A.: Enzymic synthesis of 3:6-anhydro-l-galactose within porphyran from l-galactose 6-sulphate units. Biochem. J., 81, 347-352 (1961)

423

Methionine adenosyltransferase

2.5.1.6

1 Nomenclature EC number 2.5.1.6 Systematic name ATP:l-methionine S-adenosyltransferase Recommended name methionine adenosyltransferase Synonyms ATP-methionine adenosyltransferase AdoMet synthetase EC 2.4.2.13 (formerly) S-adenosyl-l-methionine synthetase S-adenosylmethionine synthase S-adenosylmethionine synthetase adenosylmethionine synthetase methionine S-adenosyltransferase methionine adenosyltransferase methionine-activating enzyme CAS registry number 9012-52-6

2 Source Organism

424

Sulfolobus solfataricus (2 forms: A and B [11]) [11] Saccharomyces cerevisiae (2 forms: I and II [18]) [13, 14, 18] Escherichia coli (XL1-Blue strain [39,29]) [8, 12, 19, 24, 29, 33, 36, 39] Trypanosoma brucei brucei [9] Oryctolagus cuniculus [1] Rattus norvegicus (3 isoenzymes: MAT-I, MAT-II, MAT-III [6,7]; 2 forms: high-MW form and low-MW form [16]; Sprague-Dawley rats [32]; two MAT III isoforms [31]) [2, 4-7, 16, 17, 21, 25, 27, 31, 32, 34, 36] Pisum sativum [3] Homo sapiens (3 isoenzymes: MAT-I, MAT-II, MAT-III [28]) [4, 15, 22, 23, 28, 30, 33, 36] Homo sapiens (MAT-II [33]) [33] Mus musculus (mouse, 2 isoenzymes: I and II [10]) [10, 35]

2.5.1.6


Bos taurus (bovine [20]) [20, 32] Sus scrofa (pig [32]) [32] Felis catus (cat [32]) [32] Rhesus monkey [32] Catharanthus roseus (Madagascar perwinkle, L. G. Don, line CP3a, three isoenzymes: SAMS 1, 2 and 3 [26] SwissProt-ID: Q96551) [26] Catharanthus roseus (Madagascar perwinkle, L. G. Don, line CP3a, three isoenzymes: SAMS 1, 2 and 3 [26] SwissProt-ID: Q96552) [26] Catharanthus roseus (Madagascar perwinkle, L. G. Don, line CP3a, three isoenzymes: SAMS 1, 2 and 3 [26] SwissProt-ID: Q96553) [26] Leishmania donovani [41] Leishmania infantum [38] Methanococcus jannaschii (hypertermophilic archaeon [37]) [37] Streptomyces coelicolor A3(2) (KO-179 strain. Actinorhodin-overproducer [40]) [40]

3 Reaction and Specificity Catalyzed reaction ATP + l-methionine + H2 O = phosphate + diphosphate + S-adenosyl-lmethionine ( mechanism [1]; steady state ordered bi ter mechanism with ATP adding before l-methionine and S-adenosylmethionine being the first product released, random release of phosphate and diphosphate [15]) Reaction type adenosyl group transfer Natural substrates and products S ATP + l-methionine + H2 O ( the product S-adenosylmethionine is important as a direct metabolic donor of methyl and aamino-n-butyryl groups [7]; used as aminopropyl group donor in synthesis of polyamines and is also the methyl group donor for most cellular methyltransferase reactions [9]; methyl donor in transmethylation reactions and as propylamine donor for polyamine biosynthesis [10]; MAT activity controls cellular glutathione levels, polyamine synthesis and folate cycling [27]; two reaction steps: S-adenosylmethionine synthesis and tripolyphosphate hydrolysis. Tripolyphosphate hydrolysis is the rate determining reaction [31]) (Reversibility: ? [1, 2, 7, 9-11, 27]; ir [37]) [1, 2, 7, 9-11, 27, 37] P S-adenosyl-l-methionine + phosphate + diphosphate [1, 2, 7, 9-11, 37] Substrates and products S 2'-deoxy-ATP + l-methionine + H2 O ( completely specific for ATP [13]) (Reversibility: ? [1-21]; ir [37]) [1-21, 37] P ?

425


2.5.1.6

S 3'-deoxy-ATP + l-methionine + H2 O ( completely specific for ATP [13]) (Reversibility: ? [1-21]; ir [37]) [1-21, 37] P ? S ATP + d-methionine + H2 O (Reversibility: ? [37]) [37] P S-adenosyl-d-methionine + phosphate + diphosphate [37] S ATP + l-ethionine + H2 O (Reversibility: ? [18]) [9, 18, 37] P S-adenosyl-l-ethionine + phosphate + diphosphate [9, 18, 37] S ATP + l-methionine + H2 O ( completely specific for ATP [13]; mechanism [31]) (Reversibility: ? [1-23, 26, 28, 30-33, 37, 39]; ir [37]) [1-23, 26, 28, 30-33, 37, 39] P S-adenosyl-l-methionine + phosphate + diphosphate [1-21] S ATP + l-methionine methyl ester + H2 O (Reversibility: ? [37]) [37] P S-adenosyl-l-methionine methyl ester + phosphate + diphosphate [37] S ATP + methionine + H2 O (Reversibility: ? [3,6,21]) [3, 6, 21] P S-adenosyl-l-methionine + phosphate + diphosphate [3, 6, 21] S CTP + l-methionine + H2 O (Reversibility: ? [37]) [37] P S-cytosyl-l-methionine + phosphate + diphosphate [37] S GTP + l-methionine + H2 O (Reversibility: ? [37]) [37] P S-guanosyl-l-methionine + phosphate + diphosphate [37] S UTP + l-methionine + H2 O (Reversibility: ? [37]) [37] P S-urasyl-l-methionine + phosphate + diphosphate [37] S tripolyphosphate + H2 O ( tripolyphosphatase activity [8, 11, 14, 15, 18, 19]; two isoenzymes with different behavior on exogenous S-adenosylmethionine addition [18]) (Reversibility: ? [8, 11, 14, 15, 18, 19]) [8, 11, 14, 15, 18, 19, 39] P diphosphate + phosphate [8, 11, 14, 15, 18, 19, 39] S Additional information ( ATP can be substituted by 3'deoxy-ATP,8-bromo-ATP, formycin triphosphate, adenyl-5l imidodiphosphate [8, 9, 19]; methionine can be substituted by selenomethionine, a-methyl-dl-methionine [8, 9, 19]; no activity with methional, methioninol, 3-methylthiopropylamine [37]; S-adenosylmethionine synthesis and tripolyphosphatase activity messured for six aspartate-mutants [29]) [8, 9, 19, 29, 37] P ? Inhibitors 1-aminocyclopentanecarboxylic acid [14] 3-morpholinosydnoniimide ( loss of liver MAT activity in vivo [27]) [27] ADP ( 35-50% inhibition with 5 mM [26]) [26] ATP ( ATP and methionine act as a switch between two different MAT III isoforms [31]) [15, 31, 38] CTP ( 60-70% inhibition with 5 mM [26]) [11, 13, 26]

426

2.5.1.6


d-methionine [9] dl-2-amino-trans-4-hexenoic acid [14] dl-ethionine [9] GDP [13] GTP ( not accepted as a substrate but inhibits the reaction in the presence of ATP, 70-80% inhibition with 5 mM [26]; competitive with respect to ATP and noncompetitive with l-methionine [37]) [11, 13, 14, 19, 26, 37] K+ ( above 50 mM [2]) [2-4] l-2-amino-4-hexynoic acid [14] l-2-amino-4-methoxy-cis-but-3-enoic acid [9, 10] l-2-amino-4-methylthio-cis-but-3-enoic acid [10] l-buthionine-(S,R)-sulfoximine ( inhibits glutathione synthesis and this decreases MAT activity in vivo. Prevented by the administration of glutathione-ethyl ester [27]; inactivates hepatic MAT, prevented by the administration of glutathione-ethyl ester [36]) [27, 36] l-ethionine ( competitive with respect to methionine for S-adenosylmethionine formation and noncompetitive with respect to ATP [37]) [37] l-methionine ( ATP and methionine act as a switch between two different MAT III isoforms [31]) [15, 31] l-ornitine ( 10-25% inhibition with 5 mM [26]) [26] Mg2+ ( inhibitory above 8.5 mM [9]) [9] Mn2+ ( inhibition in presence of Mg2+ [9]) [9] N-ethylmaleimide ( time-dependent inactivation of both MAT activities [41]) [41] Na+ ( in presence of Mg2+ [9]) [9] S-adenosyl-l-ethionine [38] S-adenosyl-l-homocysteine ( not inhibitory [11]) [9, 38] S-adenosyl-l-methionine ( inhibits the A form but not the B form [11]; above 0.3 mM inhibits both high-MW and low-MW isoenzymes [16]; inhibition of rat kidney enzyme and rat liver MAT-II, weak inhibition of rat liver MAT-I [6,7]; non competitive with ATP at low methionine concentration [15]; non competitive inhibition [41]; more than 50% inhibition at 1mM concentration [38]; noncompetitive inhibitor with respect to ATP and methionine [37]) [3, 6-9, 11, 15, 16, 19, 37, 38, 41] S-carbamoylcysteine ( competitive with methionine [8,19]) [8, 19] S-nitrosoglutathione ( inactivates MATI/III by 70% [36]; inhibits S-adenosylmethionine sinthetase activity [31]) [36, 31] S-nitrosoglutathione monoethyl ester ( inactivates [36]) [36] S-nitrosylated glutathione ( rapid and dose-dependent loss of enzymatic activity of MAT I/III [27]) [27] S-trifluoromethyl-l-homocysteine [14] SIN-1 ( rapid and dose-dependent loss of enzymatic activity of MAT I/III [27]) [27] TTP [13] UTP [11, 13] 427


2.5.1.6

adenyl-5'-ylimidodiphosphate ( competitive with ATP [8,19]) [8, 19] a,b-methylene-ATP [13] a,b-methylene-adenosine tetraphosphate [13] bacterial lipopolysaccharide ( decreases MAT activity in vivo [27]; results in the accumulation of nitrites and nitrates in serum and in the inactivation of MAT I/III [36]) [27, 36] b,g-methylene-ATP [13] carbon tetrachloride ( depletion of glutathione levels reduces MAT I/ III activities in vivo [36]) [36] cycloleucine ( inhibits only at sub saturating concentrations of methionine [11]) [9, 11] dGTP [13] dimethylsulfoxide ( weak inhibition of liver isoenzyme [6,7]; slight inhibition of g isoenzyme from kidney [17]) [2, 6, 7, 9, 15, 17, 21] diphosphate ( inhibits high-MW isoenzyme, no effect on low-MW enzyme [16]; individually a weak inhibitor, in combination with phosphate there is a marked synergistic effect [2]; inhibition for S-adenosylmethionine and l-methonine [38]) [2, 8, 11, 13, 15, 16, 19, 38] ethionine ( 32-38% inhibition with 5 mM [26]) [26] fumarylacetoacetate ( reduces MAT I/III activity [36]) [36] glycerol ( inhibits kidney isoenzyme g [17]) [17] hydrogen peroxide ( inactives CHO cells-MAT, prevented by desferoxamine. Time- and dose-dependent inactivation of MAT I/III, activity recovered by addition of glutathione [27]; reduces MAT I/III activity [36]) [27, 36] nitric oxide ( inactivates hepatic MAT [36]; two MAT III isoforms, one with low tripolyphosphatase activity that is insensitive to NO and another with high tripolyphosphatase activity that is inhibited by NO [31]) [36, 31] p-chloromercuribenzoate ( a and b isoenzymes completely inhibited, g isoenzyme slightly inhibited [17]) [15, 17] p-chloromercuribenzoate ( reduces MAT I/III activity [36]) [36] phosphate ( no effect [16]; individually is a weak inhibitor, in combination with diphosphate there is a synergistic inhibitory effect [2]; competitive toward both ATP and methionine [37]) [2, 8, 11, 13, 15, 16, 19, 37] putrescine ( 15-25% inhibition with 5 mM [26]) [26] seleno-l-methionine [9] sodium diphosphate [9] spermidine ( 15-34% inhibition with 5 mM [26]) [26] spermine ( 30-40% inhibition with 5 mM [26]) [26] tetrapolyphosphate [11, 13] tripolyphosphate ( competitive with ATP [19, 38]; non competitive with l-methionine [38]; activation or inhibition, depending on isoenzyme, S-adenosylmethionine and tripolyphosphate concentration [17]; competitive with ATP and non competitive

428

2.5.1.6


with l-methionine [15]; strong inhibitor [26]) [8, 13, 15, 17, 19, 20, 26, 38] Additional information ( S-adenosyl(5')-3-methylthiopropylamine does not inhibit [11]; overview of the regulatory properties, effect of l-methionine analogues and influence of l-methionine concentration on activating and inhibiting effects, effect of tripolyphosphate and p-hydroxymercuribenzoate [15]; addition of reducing agents has no effect [26]; MAT is inactivated after 6 h of incubation in hypoxia (3% O2 ) in rat hepatocytes, prevented by Ng -monomethyl-l-arginine methyl ester. Hepatic MAT s a sensible target for free radicals in vivo [27]; reactive oxygen and nitrogen species induce the inactivation of MAT I/III [36]) [11, 15, 26, 27, 36] Additional information ( no inhibition with cycloleucine, lhomocysteine, l-norleucine, l-cis-2-amino-4-methoxy-3-butenoic acid, Sadenosylhomocysteine, 5'-methylthioadenosine, sinefungin [37]) [37] Activating compounds ATP ( tripolyphosphatase activity stimulated by preincubation with ATP and methionine [31]) [31] S-adenosyl-l-methionine ( below 0.3 mM activates low-MW isoenzyme [16]; activation of rat liver MAT-III [6,7]; enhances tripolyphosphatase activity [19]; activates the tripolyphosphatase activity of A isoform [11]; nonessential activator of tripolyphosphatase activity in the range of 0.005-0.100 mM [41]) [6, 7, 11, 16, 19, 41] SH-reagents ( liver enzyme: requirement, tumor enzyme: no requirement [5]) [5] dimethylsulfoxide ( activates isoform from kidney and isoform MAT-III from liver [6,7]; activates a and b isoenzymes from liver [17]; weak activation of the a isozyme from liver [21]; activates the b isozyme from liver 13 to 15-fold [21]) [6, 7, 17, 21] dithiothreitol ( required for a and b isoenzymes activity, not required for g enzyme [17]) [17] glycerol ( activates rat liver isoenzymes a and b [17]) [17] methionine ( stimulation of activity demonstrated in vivo. Tripolyphosphatase activity stimulated by preincubation with ATP and methionine [31]) [31] tripolyphosphates ( activation or inhibition, depending on isoenzyme, S-adenosylmethionine and tripolyphosphate concentration [17]) [17] Additional information ( no requirement for reducing agents [11]; no effect with ADP and methionine or S-adenosylmethionine [31]) [11, 31] Metals, ions Co2+ ( for maximal activation the cation concentration must be at least equal to ATP concentration [8]; can replace Mg2+ with lower relative activity [11]) [8, 11, 19] Cs+ [19]

429


2.5.1.6

K+ ( activation at 25-50 mM [2]; absolute requirement, cannot be replaced by Na+ [17]; both Mg2+ and K+ required for full activity [4]; monovalent cations required for optimal activity, 50 mM K+ sufficient for full activity, can be replaced by NH+4 but not by Na+ [26]; enhances Kcat and decrees Km values for both substrates [37]) [2-4, 17, 19, 26, 37] Li+ [19] Mg2+ ( both Mg2+ and K+ required for full activity [4]; absolute requirement, cannot be replaced by Mn2+ [17]; optimal concentration at 10-20 mM [2]; cation concentration must be at least equal to ATP concentration for maximal activity [8,19]; absolute requirement [9,37]; maximal activation at 40 mM [11]; Mg2+ in excess of that bound to ATP and EDTA is required for optimal activity [15]; strictly dependent on divalent cations with maximum activity at 5mM and Mg2+ fully replaceable by Mn2+ or Co2+ salts [26]; cannot be replaced by Ca2+ [29]) [2-4, 8, 9, 11, 15, 17, 19-21, 26, 29, 37] Mn2+ ( can replace Mg2+ with lower relative activity [11]; optimal concentration at 2 mM [2]; for maximal activation the cation concentration must be at least equal to ATP concentration [8]; can partially replace Mg2+ in activation, inhibition in presence of Mg2+ [9]) [2, 3, 8, 9, 11, 19] NH+4 ( activity is dependent on both divalent Mg2+ or Mn2+ and monovalent cations NH+4 or K+ [3]) [3, 19] Na+ ( slight activation in absence of Mg2+ [9]) [9, 19] Tl+ [19] Additional information ( divalent cations are required for tripolyphosphatase activity [19]) [19] Turnover number (min±1) 0.000013 (tripolyphosphate, G8 mutant, tripolyphosphatase activity [39]) [39] 0.000022 (S-adenosylmethionine, RLL mutant, S-adenosylmethionine synthesis [39]) [39] 0.000027 (S-adenosylmethionine, G6 mutant, S-adenosylmethionine synthesis [39]) [39] 0.000037 (tripolyphosphate, G6 mutant, tripolyphosphatase activity [39]) [39] 0.000038 (tripolyphosphate, G5 mutant, tripolyphosphatase activity [39]) [39] 0.00004 (S-adenosylmethionine, G8 mutant, S-adenosylmethionine synthesis [39]) [39] 0.000073 (tripolyphosphate, RLL and G7 mutants, tripolyphosphatase activity [39]) [39] 0.0001 (tripolyphosphate, wild-type, tripolyphosphatase activity [39]) [39] 0.00013 (S-adenosyl-l-methionine, G5 mutant, S-adenosylmethionine synthesis [39]) [39]

430

2.5.1.6


0.00072 (S-adenosyl-l-methionine, G7 mutant, S-adenosylmethionine synthesis [39]) [39] 0.0053 (ATP) [38] 0.0053 (S-adenosyl-l-methionine) [38] 0.025 (S-adenosyl-l-methionine, wild-type, S-adenosylmethionine synthesis [39]) [39] 0.24 (tripolyphosphate, tripolyphosphase activity [41]) [41] 1.92 (S-adenosyl-l-methionine, S-adenosylmethionine synthesis activity [41]) [41] 7.5 (tripolyphosphate, tripolyphosphatase activity [31]) [31] 35 (S-adenosyl-l-methionine, S-adenosylmethionine synthesis [31]) [31] Specific activity (U/mg) 0.0002 ( activity peak II, adult erythrocytes [22]) [22] 0.0022 [19] 0.00278 [9] 0.039 ( MAT I [34]) [34] 0.058 ( tripolyphosphase activity [41]) [41] 0.096 ( glutathione/glutathione disulfide-refolded MAT III [34]) [34] 0.098 ( MAT III [34]) [34] 0.1 ( glutathione/glutathione disulfide-refolded MAT I [34]) [34] 0.11 ( dithiothreitol-refolded MAT III [34]) [34] 0.2 ( S-adenosylmethionine synthesis activity [41]) [41] 0.28 ( low-MW isozyme [16]) [16] 0.75 [8] 3 ( b isoenzyme from liver [17]) [17] 4.61 ( high-MW isozyme [16]) [16] 7.5 ( a isoenzyme from liver [17]) [17] 11 ( isoenzyme II [18]) [18] 12.2 [15] 12.4 [20] 18.7 ( isoenzyme I [18]) [18] 32.75 ( b form from liver [21]) [21] 39 ( g isoenzyme from liver [17]) [17] 78.24 ( a form from liver [21]) [21] Additional information ( different values in refolding and purification process [34]) [1, 7-9, 11, 15, 34] Km-Value (mM) 0.0013 (tripolyphosphate, wild type, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine [39]) [39] 0.0015 (tripolyphosphate, G7 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine [39]) [39] 0.0016 (tripolyphosphate, G8 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine [39]) [39] 0.002 (ATP, b form from liver [21]) [21] 0.0029 (l-methionine) [32] 431


2.5.1.6

0.003 (l-methionine) [32] 0.003 (tripolyphosphate, wild type, tripolyphosphatase activity, in absence of S-adenosylmethionine [39]) [39] 0.0032 (tripolyphosphate, G6 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine [39]) [39] 0.005 (ATP, a form from liver [21]) [21] 0.0053 (tripolyphosphate, G5 mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine [39]) [39] 0.006 (Mg2+ , a form from liver [21]) [21] 0.007 (Mg2+ , b form from liver [21]) [21] 0.0075 (l-methionine, erythrocyte extract, a and b subunit [22]) [22] 0.008 (tripolyphosphate, G8 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine [39]) [39] 0.0083 (methionine, MAT-II isoenzyme [6]) [6] 0.01 (l-methionine, isoenzyme A [11]) [11, 20] 0.011 (tripolyphosphate, G6 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine [39]) [39] 0.0125 (l-methionine, erythrocyte extract, a subunit [22]) [22] 0.014 (tripolyphosphate, RLL mutant, tripolyphosphatase activity, in absence of S-adenosylmethionine [39]) [39] 0.015 (l-methionine, endogenous MAT II [22]) [22, 28] 0.015 (tripolyphosphate, G7 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine [39]) [39] 0.016 (l-methionine, a2 -transfected MAT II, two kinetic forms [28]) [28] 0.017 (l-methionine, a form from liver [21]) [21] 0.02 (l-methionine, isoenzyme B [11]) [11] 0.02 (l-methionine, recombinant MAT II co-expressing a2 and b subunits [28]) [28] 0.022 (l-methionine, crude extract [22]) [22] 0.022 (l-methionine, one kinetic form of a-two subunit in the presence of b subunit [33]) [33] 0.022-0.024 (l-methionine, ro subunit [22]) [22] 0.024 (l-methionine, wild-type [30]) [30] 0.024 (tripolyphosphate, G5 mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine [39]) [39] 0.026 (ATP, G6 mutant, S-adenosylmethionine synthesis [39]) [39] 0.026 (tripolyphosphate, RLL mutant, tripolyphosphatase activity, in the presence of 0.1 mM of S-adenosylmethionine [39]) [39] 0.03-0.038 (l-methionine, one kinetic form of a subunit in the presence of b subunit [33]) [33] 0.041 (l-methionine, MAT-I isoenzyme [6]) [6] 0.045 (ATP, RLL and G8 mutants, S-adenosylmethionine synthesis [39]) [39] 0.05 (ATP, isoenzyme A [11]) [11, 20]

432

2.5.1.6


0.06 (l-methionine, uninduced E. coli NM522 strain extract a subunit at low l-methionine concentrations [23]) [23] 0.06-0.1 (l-methionine, a-two subunit [33]) [33] 0.065-0.08 (l-methionine, a subunit at low l-methionine concentrations [33]) [33] 0.069 (ATP, G7 mutants, S-adenosylmethionine synthesis [39]) [39] 0.073 (ATP, wild-type S-adenosylmethionine synthesis [39]) [39] 0.075 (l-methionine, a2 -transfected MAT II, two kinetic forms [28]) [28] 0.076 (l-methionine, one kinetic form of a-two subunit in the presence of b subunit [33]) [33] 0.08 (ATP) [2] 0.08 (l-methionine, S283T mutant [30]) [30] 0.08 (l-methionine, induced E. coli NM522 strain extract a subunit at low l-methionine concentrations and uninduced E. coli NM522 strain extract a subunit at high l-methionine concentrations [23]) [23] 0.08-0.09 (l-methionine, a subunit at high l-methionine concentrations. Also one kinetic form of a subunit in the presence of b subunit [33]) [33] 0.087 (ATP, G5 mutant S-adenosylmethionine synthesis [39]) [39] 0.088 (l-methionine, Q113A mutant, S-adenosylmethionine synthesis [30]) [30] 0.092 (l-methionine, wild-type, S-adenosylmethionine synthesis [39]) [39] 0.1 (l-methionine) [26] 0.15 (ATP, isoenzyme B [11]) [11] 0.18 (ATP) [26] 0.2 (l-methionine, induced E. coli NM522 strain extract a subunit at high l-methionine concentrations [23]) [23] 0.215 (l-methionine, MAT-III isoenzyme [6]) [6] 0.22 (ATP, saturated with KCl [37]) [37] 0.22 (l-methionine, wild-type [30]) [30] 0.23 (l-methionine, RLL mutant, S-adenosylmethionine synthesis [39]) [39] 0.24 (l-methionine) [37] 0.25 (ATP) [26] 0.26 (ATP) [37] 0.3 (l-methionine, G6 mutant, S-adenosylmethionine synthesis [39]; saturated with KCl [37]) [39, 37] 0.3 (MgATP2- ) [3] 0.31 (ATP) [26] 0.4 (CTP) [37] 0.4 (methionine) [3] 0.49 (l-methionine, G7 mutant, S-adenosylmethionine synthesis [39]) [39] 0.5 (methionine, b form from liver [21]) [21] 0.62 (GTP) [37] 433


2.5.1.6

0.62 (l-methionine, G8 mutant, S-adenosylmethionine synthesis [39]) [39] 0.74 (l-ethionine) [37] 0.77 (l-methionine, G5 mutant, S-adenosylmethionine synthesis [39]) [39] 1.1 (l-methionine) [37] 1.4 (ATP) [37] 2.2 (UTP) [37] 2.6 (l-methionine methyl esther) [37] 3.3 (l-methionine) [15] 3.5 (d-methionine) [37] 31 (ATP) [15] Additional information ( Km for S-adenosylmethionine synthesis and tripolyphosphatase activity measured for six aspartate-mutants [29]; low-MW isoenzyme shows two different Km values in the range 0.03-10 mM methionine [16]; liver contains two isofunctional enzymes with different Km values [3]; estimation of apparent Km for ATP and lmethionine [18]; Km values for methionine and ATP depend on the concentration of substrates [9]; fifteen adenosine-, ribose- and phosphate modified compounds studied [37]; 2 isofunctional forms with different Km values in liver [5]) [3, 5, 7, 9, 10, 15, 16, 18, 29, 37] Ki-Value (mM) 0.0031 (l-2-amino-4-methoxy-cis-but-3-enoic acid, isoenzyme II [10]) [10] 0.0057 (l-2-amino-4-methylthio-cis-but-3-enoic acid, isoenzyme II [10]) [10] 0.0063 (l-2-amino-4-methoxy-cis-but-3-enoic acid, isoenzyme I [10]) [10] 0.021 (l-2-amino-4-methylthio-cis-but-3-enoic acid, isoenzyme I [10]) [10] 0.035 (tripolyphosphate) [26] 0.05 (S-adenosyl-l-methionine, inhibits the A isoform [11]) [11] 0.05 (tripolyphosphate) [13] 0.18 (tetrapolyphosphate) [13] 0.2 (tripolyphosphate, inhibition with respect to l-methionine [38]) [38] 0.24 (S-adenosyl-l-methionine) [9] 0.25 (tripolyphosphate, inhibition with respect to ATP [38]) [38] 0.3 (diphosphate, inhibition with respect to ATP [38]) [38] 0.35 (diphosphate, inhibition with respect to l-methionine [38]) [38] 0.4 (d-methionine) [9] 0.51 (seleno-l-methionine) [9] 0.71 (dl-ethionione) [9] 0.8 (ATP, noncompetitive inhibition at 0.5-5 mM ATP concentrations [38]) [38]

434

2.5.1.6


0.8 (S-adenosyl-l-methionine, ATP concentrations of 0.5-5 mM [41]) [41] 1.6 (sodium diphosphate) [9] 4 (S-adenosylmethionine, l-methionine concentrations of 0.55 mM [41]; noncompetitive inhibition at 0.5-5 mM l-methionine concentrations [38]) [41, 38] 17 (cycloleucine) [9] 250 (Na+ , in presence of Mg2+ [9]) [9] Additional information ( Ki values for several phosphonate analogues and nucleotides [13,14]) [13, 14] pH-Optimum 7-8 [1] 7-8.3 ( three isoenzymes [26]) [26] 7.5-9.5 ( glutathione/glutathione disulfide-refolded MAT I [34]) [34] 7.7 ( assay at [32]) [32] 7.7-8.8 ( MAT III, MAT I and dithiothreitol-refolded MAT III [34]) [34] 8 ( assay at [8]) [8] 8.1 [11] 8.5-10 ( glutathione/glutathione disulfide-refolded MAT III [34]) [34] 9 ( slight variation in activity between 8 and 10 [9]) [9] pH-Range 7.2-9.3 ( about 50% of activity maximum at pH 7.2 and 9.3 [11]) [11] 7.5-8.5 [15] 7.5-9.5 ( about 70-80% of maximal activity at pH 7.5 and 9.5 [17]) [17] Temperature optimum ( C) 25 ( assay at [8]) [8] 37 ( assay at [7, 9, 14, 15, 25, 26, 31, 32]) [7, 9, 14, 15, 25, 26, 31, 32] 37-45 [26] 70 ( optimal growth temperature for Methanococcus jannaschii: 87 C [37]) [37] 90 [11] Temperature range ( C) 75-110 ( about 50% of activity maximum at 75 C and 110 C [11]) [11]

4 Enzyme Structure Molecular weight 32500 ( b subunit, gel filtration [22]) [22] 435


2.5.1.6

38000-39000 ( MAT II b subunit, gel filtration [33]) [33] 42560 ( SAMS 3, gel filtration [26]) [26] 43000 ( SAMS 2, gel filtration [26]) [26] 43050 ( SAMS 1, gel filtration [26]) [26] 46000 ( 2D-PAGE [40]) [40] 48000 ( monomer, gel filtration and immunoblotting [34]; MAT II, gel filtration [41]) [34, 41] 49000 ( native MAT-II, gel filtration [38]) [38] 51000 ( expression of the a subunit in E. coli, gel filtration [23]) [23] 53000 ( a subunit, gel filtration [22]; expression of the a subunit in E. coli, gel filtration [23]) [22, 23] 60000 ( isoenzyme ro, Peak I, gel filtration [22]) [22] 75000 ( isoenzyme B, gel filtration [11]) [11] 86000 ( recombinant enzyme, gel filtration [37]) [37] 89130 ( glutathione/glutathione disulfide-refolded MAT III, gel filtration [34]) [34] 92700 ( dithiothreitol-refolded MAT III, gel filtration [34]) [34] 97000 ( MAT-III, gel filtration [6]) [6, 7] 100000 ( b isoenzyme from liver, gel filtration [21]) [21] 110000 ( forms I and II, gel filtration [18]; low-MW isoenzyme, gel filtration [16]; MAT III, gel filtration [34]) [16, 18, 34] 120000 ( MAT-II, isolated from kidney and liver, gel filtration [6]) [6, 7] 145000 ( gel filtration [9]) [9] 160000 ( gel filtration [20]; b isoenzyme from liver, gel filtration [17]) [17, 20] 180000 ( isoenzyme A, gel filtration [11]; gel filtration [19]; X-ray crystallography [12]) [8, 11, 12, 19] 185000 ( equilibrium sedimentation studies [15]) [15] 190000 ( g isoenzyme from kidney, gel filtration [17]) [17] 194900 ( glutathione/glutathione disulfide-refolded MAT I, gel filtration [34]) [34] 200000 ( a isoenzyme from liver, gel filtration [21]) [21] 208000 ( MAT-I, gel filtration [6]) [6, 7] 210000 ( a isoenzyme, gel filtration [17]; high-MW isoenzyme, gel filtration [16]; MAT I [34]) [16, 17, 34] 249000 ( recombinant MAT-II, gel filtration [38]) [38] Subunits ? ( x * 43000 [8]; x * 48000 + x * 38000, SDS-PAGE [20]; ro subunit may exist in a monomeric or polymeric form [22]) [8, 15, 20, 22] dimer ( 2 * 47000, MAT-III, SDS-PAGE [6,7]; 2 * 47000, low-MW isoenzyme, SDS-PAGE [16]; 1 * 55000 + 1 * 60000, SDS-PAGE [18]; 2 * 48000, b isoenzyme from liver, SDS-PAGE [21]; substrate and/or reaction products promotes dimer formation [30]; 2*45000 [37]; recombinant enzyme is present in two different oli-

436

2.5.1.6


gomeric forms that can be separated by hydrophobic chromatography and DMSO elution [25]) [6, 7, 16, 18, 21, 30, 41, 38, 37, 25] monomer ( gel filtration [26]) [26] tetramer ( 4 * 48500, high-MW isoenzyme, SDS-PAGE [16]; 4 * 43000, SDS-PAGE [19]; two subunits form a spherical dimer and pairs of these dimers form a tetrameric enzyme [24]; 4 * 48000, a isoenzyme from liver, SDS-PAGE [21]; heterotetramer with subunit composition aa'b2 , a2 b2 or a'2 b2 , where a, a', and b are chains of molecular weight 53000, 51000 and 38000 [15]; gel filtration and native gel electrophoresis [39]; native polyacrylamide gel electrophoresis [29]) [16, 19, 21, 36, 15, 39, 29, 25] Additional information ( a2 and b subunits of MAT II associate spontaneously [28]; a subunit from human kidney and normal or malignant lymphocytes is identical. a subunit expressed in human liver. a subunit is the catalytic subunit whereas b subunit may have a regulatory function [23]; b subunit has a regulatory function. MAT II b subunit associates spontaneously with E. coli MAT a subunit as well as with the recombinant human MAT II a-two subunit [33]) [23, 28, 33]

5 Isolation/Preparation/Mutation/Application Source/tissue Novikoff ascites tumor ( a single form of enzyme [5]) [5] ascites ( L1210 cell line, two isoenzymes [10]) [10] brain [20] erythrocyte [22] erythroleukemia cell [22] kidney ( g isoenzyme in kidney enzyme [17]) [6, 17] lens [2] liver ( two isofunctional forms with different Km values [5, 21]; a and b isozymes [17, 21]; three isofunctional forms [35]; MAT I/III isozymes [28]) [1, 5-7, 16, 17, 21, 25, 27, 28, 31, 34, 35] lymphocyte ( from chronic leukemia cells [15]) [15, 23] shoot ( green of 14-days old of seedlings [3]) [3] spinal cord [32] Additional information ( isoenzyme MAT-II present in all tissues [28]) [28] Localization cytosol [11, 16, 17] Purification (two isoforms: A and B [11]) [11] (partial, two forms: I and II [18]) [18] (partial [8]; method that includes ammonium sulfate fraction, phenylSepharose HR and hydroxylapetite CHT-1 chromatographies and amminohexyl-Sepharose anion exchange [39]; method that includes ammonium sul437


2.5.1.6

fate fraction, phenyl-Sepharose HR chromatographies and amminohexyl-Sepharose anion exchange [29]) [8, 19, 29, 39] (partial [9]) [9] (partial [1]) [1] (partial [2,5]; three isoforms: MAT-I, MAT-II and MAT-III, MAT-III to homogeneity [6]; 2 forms: high-MW form and low-MW form [16]; g isoenzyme from kidney [17]; a isozyme from liver using immunoaffinity chromatography [21]; b isoenzyme from liver using several chromatographic steps [21]; DEAE-Sephacel chromatography and phenyl Sepharose CL-4B chromatography [34]; method that includes Q-Sepharose anion exchange, phenyl-Sepharose CL4B and BioGel gel filtration chromatographies [37]; DEAE-Sepharose, phenyl-Sepharose and blue-Sepharose chromatographies and ultrafiltration [31]) [2, 5-7, 16, 17, 21, 31, 34, 37] (partial [3]) [3] (partial on DEAE-cellulose, two peaks of activity: peak I contains ro isoform and peak II contains subunits a and b [22]) [22] (two methods: the first involved purification of the His-tagged protein under denaturing conditions of 8M urea and the second involved separation of SDS-PAGE [33]) [33] (two isoenzymes: I and II [10]) [10] (partial [20]) [20] (recombinant MAT II purified by a method that includes Ni-agarose bead affinity capture. Alternative method includes Triton X-100 and 8 mM urea buffered extraction of inclusion body phase and dialysis [38]) [38] (recombinant enzyme purified by a method that includes Ni2+ -His binding resin, HiLoad Q-Sepharose and Sephadex-200 chromatography [37]) [37] Renaturation (refolding by direct dialysis or by dilution after treatment with urea under several conditions [34]) [34] (refolding by a fourfold dilution refolding buffer up to a concentration of 2 M urea [41]) [41] Crystallization (method of vapor diffusion, hexagonal bipiramid crystals in the presence of Mg2+ and diphosphate [12]; hexagonal bipyramid crystals of the diphosphate complex grown from a solution containing phosphate, diphosphate, sulfate and Mg2+ . Crystals of the ADP complex grown obtained from a solution containing phosphate, ADP, sulfate and Mg2+ . Crystals of the BrADP complex obtained by a soaking method: pyrophosphate-MAT crystals obtained from a solution containing phosphate, sulfate and Mg2+ , and replaced the mother liquor with a solution containing BrATP, Tris-HCl buffer, Mg2+ and ammonium sulfate [24]) [12, 24]

438

2.5.1.6


Cloning (expressed in Escherichia coli BL21(DE3) [34,25]) [34, 25] (expressed in M15 bacteria [30]; His-tagged recombinant MAT II a2 subunit expressed in COS-1 cells [28]; a subunit expressed in Escherichia coli NM522 strain [23]) [30, 28, 23] (expressed in Escherichia coli M15(Qiagen) [33]) [33] (expressed in Escherichia coli XL-1Blue strain [41]) [41] (His6-tagged fusion protein expressed in Escherichia coli [38]) [38] (expressed in Escherichia coli BL21(DE3)codon plus/pMJ1208-strain with a decahistidine tag on the N-terminus [37]) [37] (three isoenzymes expressed in Escherichia coli [26]) [26]

6 Stability Temperature stability 22 ( stable for several hours, if protein concentration is above 0.1 mg/ml and ionic strength is at least 50 mM [15]) [15] 100 ( 30 min, 90% loss of activity [11]) [11] Additional information ( preincubation with ATP protects against thermal inactivation [11]) [11] General stability information , ATP, preincubation protects against thermal inactivation [11] , glycerol, 10% stabilizes [11] , significant decrease during the interval from 0-8 hr at 23 C after decapitation. From here up to 72 hs post-mortem at 4 C the activity remains at 60% of the initial value [32] Storage stability , -20 C, partially purified isoenzymes, 10 mM Tris/HCl, pH 8, stable for 4 weeks [11] , 4 C, isoenzyme A and B are spontaneously inactivated in 1 month and 1 week, respectively, in presence of glycerol stability increases up to 90% [11] , -70 C, 5 mg/ml enzyme concentration [8] , -70 C, 63% loss of activity after 3 days [9] , 0 C, 50 mM Tris/HCl, pH 7.8, 20% v/v glycerol, 0.2 mM DTT, 0.1 mM EDTA, 10 mM MgCl2 , 0.1 M KCl, partially purified liver enzymes stable for at least 1 month [17] , -70 C, less than 10% loss of activity after 3 months [15] , 2 C, stable for at least 2 months [20]

References [1] Cantoni, G.L.; Durell, J.: Activation of methionine for transmethylation. The methionine-activating enzyme: studies on the mechanism of the reaction. J. Biol. Chem., 225, 1033-1048 (1957) 439


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[2] Geller, A.M.; Kotb, M.Y.S.; Jernigan, H.M.; Kredich, N.M.: Purification and properties of rat lens methionine adenosyltransferase. Exp. Eye Res., 43, 997-1008 (1986) [3] Aarnes, H.: Partial purification and characterization of methionine adenosyltransferase from pea seedlings. Plant Sci. Lett., 10, 381-390 (1977) [4] Tallan, H.H.; Cohen, P.A.: Methionine adenosyltransferase: kinetic properties of human and rat liver enzymes. Biochem. Med., 16, 234-250 (1976) [5] Liau, M.C.; Lin, G.W.; Hurlbert, R.B.: Partial purification and characterization of tumor and liver S-adenosylmethionine synthetases. Cancer Res., 37, 427-435 (1977) [6] Sullivan, D.M.; Hoffman, J.L.: Fractionation and kinetic properties of rat liver and kidney methionine adenosyltransferase isozymes. Biochemistry, 22, 1636-1641 (1983) [7] Hoffman, J.L.: Fractionation of methionine adenosyltransferase isozymes (rat liver). Methods Enzymol., 94, 223-228 (1983) [8] Markham, G.D.; Hafner, E.W.; White Tabor, C.; Tabor, H.: S-adenosylmethionine synthetase (methionine adenosyltransferase) (Escherichia coli). Methods Enzymol., 94, 219-222 (1983) [9] Yarlett, N.; Garofalo, J.; Goldberg, B.; Ciminelli, M.A.; Ruggiero, V.; Sufrin, J.R.; Bacchi, C.J.: S-adenosylmethionine synthetase in bloodstream Trypanosoma brucei. Biochim. Biophys. Acta, 1181, 68-76 (1993) [10] Sufrin, J.R.; Lombardini, J.B.; Alks, V.: Differential kinetic properties of l-2amino-4-methylthio-cis-but-3-enoic acid, a methionine analog inhibitor of S-adenosylmethionine synthetase. Biochim. Biophys. Acta, 1202, 87-91 (1993) [11] Porcelli, M.; Cacciapuoti, G.; Carteni-Farina, M.; Gambacorta, A.: S-adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus. Purification and characterization of two isoforms. Eur. J. Biochem., 177, 273-280 (1988) [12] Gilliland, G.L.; Markham, G.D.; Davies, D.R.: S-adenosylmethionine synthetase from Escherichia coli. Crystallization and preliminary X-ray diffraction studies. J. Biol. Chem., 258, 6963-6964 (1983) [13] Chou, T.-C.; Talalay, P.: Inhibition of ATP: l-methionine S-adenosyltransferase of bakers yeast by structural analogues of ATP. Biochim. Biophys. Acta, 321, 467-474 (1973) [14] Chou, T.-C.; Talalay, P.: The mechanism of S-adenosyl-l-methionine synthesis by purified preparations of bakers yeast. Biochemistry, 11, 10651073 (1972) [15] Kotb, M.; Kredich, N.M.: S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization. J. Biol. Chem., 260, 39233930 (1985) [16] Cabrero, C.; Puerta, J.; Alemany, S.: Purification and comparison of two forms of S-adenosyl-l-methionine synthetase from rat liver. Eur. J. Biochem., 170, 299-304 (1987) [17] Okada, G.; Teraoka, H.; Tsukada, K.: Multiple species of mammalian S-adenosylmethionine synthetase. Partial purification and characterization. Biochemistry, 20, 934-940 (1981) 440

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[18] Chiang, P.K.; Cantoni, G.L.: Activation of methionine for transmethylation. Purification of the S-adenosylmethionine synthetase of bakers yeast and its separation into two forms. J. Biol. Chem., 252, 4506-4513 (1977) [19] Markham, G.D.; Hafner, E.W.; White Tabor, C.; Tabor, H.: S-Adenosylmethionine synthetase from Escherichia coli. J. Biol. Chem., 255, 90829092 (1980) [20] Mitsui, K.-i.; Teraoka, H.; Tsukada, K.: Complete purification and immunochemical analysis of S-adenosylmethionine synthetase from bovine brain. J. Biol. Chem., 263, 11211-11216 (1988) [21] Suman, Y.; Shimizu, K.; Tsukada, K.: Isozymes of S-adenosylmethionine synthetase from rat liver: isolation and characterization. J. Biochem., 100, 67-75 (1986) [22] Langkamp-Henken, B.; Geller, A.M.; LeGros, H.L., Jr.; Price, J.O.; De la Rosa, J.; Kotb, M.: Characterization of distinct forms of methionine adenosyltransferase in nucleated, and mature human erythrocytes and erythroleukemic cells. Biochim. Biophys. Acta, 1201, 397-404 (1994) [23] De La Rosa, J.; Ostrowski, J.; Hryniewicz, M.M.; Kredich, N.M.; Kotb, M.; LeGros, H.L., Jr.; Valentine, M.; Geller, A.M.: Chromosomal localization and catalytic properties of the recombinant a subunit of human lymphocyte methionine adenosyltransferase. J. Biol. Chem., 270, 21860-21868 (1995) [24] Takusagawa, F.; Kamitori, S.; Markham, G.D.: Structure and function of Sadenosylmethionine synthetase: Crystal structures of S-adenosylmethionine synthetase with ADP, BrADP, and PPi at 2.8 ANG. resolution. Biochemistry, 35, 2586-2596 (1996) [25] Mingorance, J.; Alvarez, L.; Pajares, M.A.; Mato, J.M.: Recombinant rat liver S-adenosyl-l-methionine synthetase tetramers and dimers are in equilibrium. Int. J. Biochem. Cell Biol., 29, 485-491 (1997) [26] Schroeder, G.; Eichel, J.; Breinig, S.; Schroeder, J.: Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization. Plant Mol. Biol., 33, 211-222 (1997) [27] Avila, M.A.; Corrales, F.J.; Ruiz, F.; Sanchez-Gongora, E.; Mingorance, J.; Carretero, M.V.; Mato, J.M.: Specific interaction of methionine adenosyltransferase with free radicals. Biofactors, 8, 27-32 (1998) [28] Halim, A.-B.; LeGros, L.; Geller, A.; Kotb, M.: Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells. J. Biol. Chem., 274, 29720-29725 (1999) [29] Taylor, J.C.; Markham, G.D.: The bifunctional active site of S-adenosylmethionine synthetase. Roles of the active site aspartates. J. Biol. Chem., 274, 32909-32914 (1999) [30] Chamberlin, M.E.; Ubagai, T.; Pao, V.Y.; Pearlstein, R.A.; Yang Chou, J.: Structural requirements for catalysis and dimerization of human methionine adenosyltransferase I/III. Arch. Biochem. Biophys., 373, 56-62 (2000) [31] del Pino, M.M.; Corrales, F.J.; Mato, J.M.: Hysteretic behavior of methionine adenosyltransferase III. Methionine switches between two conformations of the enzyme with different specific activity. J. Biol. Chem., 275, 23476-23482 (2000) 441


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[32] Ekegren, T.; Aquilonius, S.M.; Gomes-Trolin, C.: A comparative study of methionine adenosyltransferase activity and regional distribution in mammalian spinal cord. Biochem. Pharmacol., 60, 441-445 (2000) [33] LeGros, H.L., Jr.; Halim, A.-B.; Geller, A.M.; Kotb, M.: Cloning, expression, and functional characterization of the b regulatory subunit of human methionine adenosyltransferase (MAT II). J. Biol. Chem., 275, 2359-2366 (2000) [34] Lopez-Vara, M.C.; Gasset, M.; Pajares, M.A.: Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies. Protein Expr. Purif., 19, 219-226 (2000) [35] Lu, S.C.; Alvarez L.A.; Huang, Z.Z.; Chen, L.; An, W.; Corrales F.J.; Avila, M.A.; Kanel, G.; Mato, J.A.: Methionine adenosyltransferase 1a knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation. Proc. Natl. Acad. Sci. USA, 98, 5560-5565 (2001) [36] Corrales, F.J.; Perez-Mato, I.; Sanchez Del Pino, M.M.; Ruiz, F.; Castro, C.; Garcia-Trevijano, E.R.; Latasa, U.; Martinez-Chantar, M.L.; Martinez-Cruz, A.; Avila, M.A.; Mato, J.M.: Regulation of mammalian liver methionine adenosyltransferase. J. Nutr., 132, 2377S-2381S (2002) [37] Lu, Z.J.; Markham, G.D.: Enzymatic properties of S-adenosylmethionine synthetase from the archaeon Methanococcus jannaschii. J. Biol. Chem., 277, 16624-16631 (2002) [38] Reguera, R.M.; Balana-Fouce, R.; Perez-Pertejo, Y.; Fernandez, F.J.; GarciaEstrada, C.; Cubria, J.C.; Ordonez, C.; Ordonez, D.: Cloning expression and characterization of methionine adenosyltransferase in Leishmania infantum promastigotes. J. Biol. Chem., 277, 3158-3167 (2002) [39] Taylor, J.C.; Takusagawa, F.; Markham, G.D.: The active site loop of S-adenosylmethionine synthetase modulates catalytic efficiency. Biochemistry, 41, 9358-9369 (2002) [40] Okamoto, S.; Lezhava, A.; Hosaka, T.; Okamoto-Hosoya, Y.; Ochi, K.: Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2). J.Bacteriol., 185, 601-609 (2003) [41] Perez-Pertejo, Y.; Reguera, R.M.; Villa, H.; Garcia-Estrada, C.; Balana-Fouce, R.; Pajares, M.A.; Ordonez, D.: Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme. Eur. J. Biochem., 270, 28-35 (2003)

442

UDP-N-Acetylglucosamine 1-carboxyvinyltransferase

2.5.1.7

1 Nomenclature EC number 2.5.1.7 Systematic name phosphoenolpyruvate:UDP-N-acetyl-d-glucosamine 1-carboxyvinyltransferase Recommended name UDP-N-acetylglucosamine 1-carboxyvinyltransferase Synonyms EPT [14] MurA transferase UDP-GlcNAc enolpyruvyl transferase [10, 12] UDP-N-acetylglucosamine 1-carboxyvinyl-transferase UDP-N-acetylglucosamine enolpyruvyltransferase [14, 19] UDP-N-acetylglucosamine enoylpyruvyltransferase enol-pyruvyltransferase [6] enoylpyruvate transferase enoylpyruvatetransferase phosphoenolpyruvate-UDP-acetylglucosamine-3-enolpyruvyltransferase phosphoenolpyruvate:UDP-2-acetamido-2-deoxy-d-glucose 2-enoyl-1-carboxyethyltransferase phosphoenolpyruvate:uridine diphosphate N-acetylglucosamine enolpyruvyltransferase phosphoenolpyruvate:uridine-5'-diphospho-N-acetyl-2-amino-2-deoxyglucose-3-enolpyruvyltransferase phosphopyruvate-uridine diphosphoacetylglucosamine pyruvatetransferase pyruvate-UDP-acetylglucosamine transferase pyruvate-uridine diphospho-N-acetyl-glucosamine transferase pyruvate-uridine diphospho-N-acetylglucosamine transferase pyruvatetransferase, phosphoenolpyruvate-uridine diphosphoacetylglucosamine pyruvic-uridine diphospho-N-acetylglucosaminyltransferase Additional information ( phylogenetic tree [18]) [18] CAS registry number 9023-27-2

443


2.5.1.7

2 Source Organism Enterobacter cloacae (MurA [15-17, 19, 21, 22]; Nr. 30054 [6]; strain NRC 492 [1]; recombinant purified enzyme [15, 19, 21, 22]) [1-3, 6, 8, 12, 14-17, 19, 21, 22] Escherichia coli (gene murA [13]; gene murZ [11]; MurA [12, 20]; MurZ [10,11]; strain K-235 [4]; K-12 [20]; K-12 strain KMBL-146 [4]) [4, 10-13, 20] Bacillus cereus (strain T [4]) [4] Staphylococcus epidermidis (strain Texas 26 [5]) [5] Escherichia coli (murA instead of murZ is recommended to be used to designate the gene at 69.3 min of the chromosome [9]; gene murZ, at 69.3 min of the chromosome [7]) [7, 9] Streptococcus pneumoniae (strain R6 [18]; 2 genes murA1 and murA2, 2 isoforms of enzyme MurA [18]) [18] Chlamydia trachomatis (gene murA [23]; MurA [23]; serovar L2 [23]) [23]

3 Reaction and Specificity Catalyzed reaction phosphoenolpyruvate + UDP-N-acetyl-d-glucosamine = phosphate + UDPN-acetyl-3-O-(1-carboxyvinyl)-d-glucosamine ( exchange of cysteine to aspartate in the active site [23]; N23 is responsible for stabilization of transition states [22]; D305 has a dual role as a general base and an essential binding partner to UDP-GlcNAc [22]; Asn23 and Asp305 are essential in the active site [22]; Lys22 is located near the active site and involved in substrate binding [17]; enyme exhibits an open conformation when substrate-free, and a closed, tightly-packed conformation upon substrate binding [16, 22]; thermodynamical investigation of substrate binding and binding of inhibitory substrate analogue fosfomycin [21]; structure analysis of conformational changes upon substrate binding [15]; the addition step proceeds with protonation of C-3 of phosphoenolpyruvate from the 2-si face [10]; active site SH-group [8]; Cys115 in the active site can act as proton donor, necessary for activity, or as a nucleophile [19]; Cys115 is the active site nucleophile [12-14]; mechanism [11, 16, 19, 22]; mechanism, formation of a covalent intermediate between enzyme and enolpyruvate moiety [3]) Reaction type carboxyvinyl group transfer Natural substrates and products S phosphoenolpyruvate + UDP-N-acetyl-d-glucosamine ( peptidoglycan formation is essential for progression through the developmental cycle as well as for cell division [23]; enzyme activity is essential for the organism, the 2 isoforms can substitute for each other [18];

444

2.5.1.7


pathway for biosynthesis of UDP-N-acetylmuramic acid [1, 4]; enzyme catalyzes the first committed step in the biosynthesis of bacterial cell wall peptidoglycan [2, 6, 8, 9, 11, 13, 14]) [1, 2, 4, 6, 8, 9, 11, 13, 14, 18, 23] P phosphate + UDP-N-acetyl-3-(1-carboxyvinyl)-d-glucosamine Substrates and products S phosphoenol-2-oxobutyrate + UDP-N-acetyl-d-glucosamine (Reversibility: ? [8]) [8] P phosphate + UDP-N-acetylglucosaminyl-enol-2-oxobutyrate [8] S phosphoenolpyruvate + UDP-N-acetyl-d-glucosamine ( thermodynamic parameters of substrate binding, wild-type, C115S and K22 mutants [17]; substrate binding structure [13, 15, 17]; specific for phosphoenolpyruvate and UDP-N-acetyl-d-glucosamine [1]) (Reversibility: r [1, 3, 16, 17, 19, 21, 22]; ? [2, 4-15, 18, 20, 23]) [1-23] P phosphate + UDP-N-acetyl-3-(1-carboxyvinyl)-d-glucosamine ( i.e. UDP-N-acetyl-d-glucosamine-enolpyruvate [1-5, 8, 12]) [1-23] S Additional information ( no activity with (Z)-phosphoenol-2oxobutyrate, phosphoenol-3-bromopyruvate and phosphoenol-3-phenylpyruvate [8]) [8] P ? Inhibitors (E)-3-fluorophosphoenolpyruvate ( kinetics [11]; inactivation [11]; formation of 2 reaction intermediates: a covalent phosphofluorolactyl-enzyme adduct and a free phosphofluorolactyl-UDP-GlcNAc tetrahedral adduct [11]; pseudosubstrate, formation of a tetrahedral intermediate in the reaction pathway, investigation of chirality of the intermediate stereospecifically formed at the active site in D2 O [10]) [10, 11] (Z)-3-fluorophosphoenolpyruvate ( kinetics, mutant C115D [12]; wild-type and mutant C115D, competitive against phosphoenolpyruvate [12]; kinetics [11]; inactivation [11]; formation of 2 reaction intermediates: a covalent phosphofluorolactyl-enzyme adduct and a free phosphofluorolactyl-UDP-GlcNAc tetrahedral adduct [11]; pseudosubstrate, formation of a tetrahedral intermediate in the reaction pathway, investigation of chirality of the intermediate stereospecifically formed at the active site in D2 O [10]) [10, 11] 3-bromopyruvate ( irreversible, inhibitory effect is increased by UDP-GlcNAc [8]) [8] 5,5'-dithiobis(2-nitrobenzoic acid) ( i.e. DTNB [5]; complete inhibition at 0.01 M [5]) [5] Ca2+ ( weak [1]) [1] Hg2+ ( weak [1]) [1] N-ethylmaleimide ( complete inhibition at 0.2 M [5]) [1, 5] PGE-553828 ( competitive against UDP-GlcNAc [20]; inhibition mechanism, kinetics [20]) [20] UDP-N-acetylmuramic acid ( weak [4]) [4, 5] UDP-N-acetylmuramic acid-l-Ala ( weak [4]) [4] 445


2.5.1.7

UDP-N-acetylmuramic acid-l-Ala-d-Glu ( weak [4]; no inhibition [5]) [4] UDP-N-acetylmuramyl-l-Ala-d-Glu-meso-a,e-diaminopimelic acid ( inhibitor binding site is distinct from active site [2]; above 1 mM [4]) [2, 4] fosfomycin ( resistant to fosfomycin inhibition due to exchange of cysteine for aspartate in the active site [23]; binding study by isothermal titration calorimetry [20]; binds covalently to Cys115 [13,19,20]; alkylates Cys115 [12]; t1=2 for inactivation of the wild-type enzyme: 6 s [12]; competitive against phosphoenolpyruvate [12]; inhibits the wild-type, mutant C155D is completely resistant [12]; irreversible, active site SH-group is involved [8]) [8, 12, 13, 16, 17, 19, 20] iodoacetamide ( inhibition by alkylation of the active site Cys155, pH-dependent, no alkylation below pH 7.0, maximum alkylation at pH 9.0 [19]) [19] iodoacetate [1] p-chloromercuribenzoate [1] phosphoenol-2-ketovalerate [8] phosphonomycin ( i.e. l-cis-2-epoxypropylphosphonic acid [4]; irreversible inactivation, requires the presence of UDP-GlcNAc [4,7]; inhibitor binding site is distinct from active site [4]) [4, 7] trypsin ( wild-type and mutant C115S, no protection via individually binding of substrates or inhibitor fosfomycin, but via binding of both, the 2 substrates and inhibitor fosfomycin [16]) [16] uridine diphospho-N-acetylmuramyl-l-Ala-d-g-Glu-meso-a,e-diaminopimelic-acid-d-Ala-d-Ala ( inhibitor binding site is distinct from active site [2]; above 1 mM [4]) [2, 4] Additional information ( no inhibition by pyruvate and fluoropyruvate [8]; no inhibition by UDP-galactose [4]) [4, 8] Activating compounds dithiothreitol ( included in the assay reaction mixture [17,20]) [17, 20] thiol groups ( required for activity [1,3]) [1, 3] Metals, ions Additional information ( not affected by K+ , Na+ , Mg2+ , and Mn2+ [1]) [1] Turnover number (min±1) 24.6 (UDP-GlcNAc, isoform MurA1 [18]) [18] 46.8 (UDP-GlcNAc, isoform MurA2 [18]) [18] 180 (UDP-GlcNAc, wild-type enzyme [19]) [19] 180 (phosphoenolpyruvate, wild-type enzyme [19]) [19] 285 (UDP-N-acetyl-d-glucosamine, recombinant enzyme [7]; cosubstrate phosphoenolpyruvate [7]) [7] 285 (phosphoenolpyruvate, recombinant enzyme [7]; cosubstrate UDP-N-acetyl-d-glucosamine [7]) [7]

446

2.5.1.7


534 (UDP-GlcNAc) [20] Additional information ( kcat values of mutant enzymes for the substrates [19]) [19] Specific activity (U/mg) 0.1-0.12 ( partially purified enzyme [3]) [3] 0.2 ( partially purified enzyme [5]) [5] 0.27 ( purified enzyme [1]) [1] 0.9 [2] 1.8 ( purified native enzyme [6]) [6] 1.9 ( purified enzyme [3]) [3] Additional information ( activity of K22 mutants compared to wildtype in forward and reverse reaction, coupled assay [17]) [3, 17] Km-Value (mM) 0.0004 (phosphoenolpyruvate, wild-type enzyme [12]) [12] 0.0041 (phosphoenolpyruvate) [20] 0.0057 (UDP-GlcNAc) [20] 0.008 (phosphoenolpyruvate, wild-type enzyme [19]) [19] 0.011 (phosphoenolpyruvate, isoform MurA2 [18]) [18] 0.015 (UDP-GlcNAc, wild-type enzyme [12]) [12] 0.016 (UDP-GlcNAc, mutant C115D, pH 8.0 [12]) [12] 0.021 (UDP-GlcNAc, mutant C115D, pH 6.0 [12]) [12] 0.022 (phosphoenolpyruvate, mutant C115D, pH 6.0 [12]) [12] 0.03 (phosphoenolpyruvate) [1] 0.037 (phosphoenolpyruvate, isoform MurA1 [18]; mutant C115D, pH 8.0 [12]) [12, 18] 0.08 (UDP-GlcNAc, wild-type enzyme [19]) [19] 0.12 (UDP-GlcNAc, isoform MurA2 [18]) [18] 0.244 (UDP-GlcNAc, isoform MurA1 [18]) [18] 0.46 (UDP-GlcNAc) [1] 1 (phosphoenolpyruvate) [7] 2.5 (UDP-GlcNAc) [7] Additional information ( kinetics [20]; Km values of mutant enzymes for the substrates [19]) [19, 20] Ki-Value (mM) 0.0086 (fosfomycin, wild-type enzyme [12]) [12] 0.038 (PGE-553828) [20] 0.04 ((Z)-3-fluorophosphoenolpyruvate, at pH 8.0 [12]; wildtype enzyme [12]) [12] 0.08 ((Z)-3-fluorophosphoenolpyruvate, at pH 8.0 [12]; mutant C115D [12]) [12] 0.1 ((Z)-3-fluorophosphoenolpyruvate, at pH 6.0 [12]; mutant C115D [12]) [12] 0.8 (uridine diphospho-N-acetylmuramyl-l-Ala-d-g-Glu-meso-a,e-diaminopimelic-acid-d-Ala-d-Ala) [2] 1 (fosfomycin, at pH 8.0 [12]; mutant C115D [12]) [12]

447


2.5.1.7

1.7 (phosphoenol-2-ketovalerate) [8] 2 (fosfomycin, at pH 6.0 [12]; mutant C115D [12]) [12] 4.9-6.6 (UDP-N-acetylmuramyl-l-Ala-d-Glu-meso-a,e-diaminopimelic acid) [2] Additional information [11] pH-Optimum 6.7-7.4 [1] 7.4 ( assay at [17]) [5, 17] 7.5 ( assay at [18]) [18] 8 ( assay at [20]) [20] Additional information ( determination of pKa value for Cys115: 8.3 [19]) [19] pH-Range 6.5-8.4 [5] Temperature optimum ( C) 25 ( assay at [17]) [17] 37 ( assay at [1,5,20]) [1, 5, 20] 42 [1]

4 Enzyme Structure Molecular weight 34000 ( gel filtration [2]) [2] 37000 ( nondenaturing PAGE [6]) [6] 44780 ( MW deduced from amino acid sequence [6]) [6] 44780 ( electrospray ionization-mass spectrometry [16]) [16] 44800 ( MW deduced from amino acid sequence [7]) [7] Subunits monomer ( 42100, SDS-PAGE [6]; 1 * 41000, SDS-PAGE [2]) [2, 6] Additional information ( structure [15-17,21]; two-domain structure with the active site located between them, substrate binding structure [13]) [13, 15-17, 21]

5 Isolation/Preparation/Mutation/Application Purification (covalent adduct of enzyme and phosphoenolpyruvate [19]; recombinant wild-type and mutants from Escherichia coli [17]; recombinant from Escherichia coli [16]) [1-3, 6, 16, 17] (recombinant from overexpresssing strain [20]) [20] (partial [5]) [5] (native and recombinant from Escherichia coli [7]) [7] (recombinant, 2 isoforms from Escherichia coli [18]) [18] 448

2.5.1.7


Crystallization (X-ray structure analysis of ligated and unligated MurA [15]; crystallization of substrate-free enzyme, two-domain structure with an unusual fold, inside out a/b barrel, which is built up from the 6fold repetititon of one folding unit, structure analysis [14]) [14, 15] (crystallization of the enzyme complexed with UDP-N-acetylglucosamine and fosfomycin, two-domain structure with the active site located between them, structure and substrate binding analysis [13]) [13] Cloning (expression of wild-type and mutants in Escherichia coli [17,22]; DNA and amino acid sequence determination, functional overexpression in Escherichia coli strain JM105 [6,16]) [6, 16, 17, 22] (overexpression in Escherichia coli [20]) [20] (expression from plasmid in Escherichia coli deletion mutant, functional complementation [9]; DNA and amino acid sequence determination and analysis, chromosome mapping at 69.3 min, overexpression in Escherichia coli strain JLM 16 [7]) [7, 9] (genes murA1 and murA2, DNA and amino acid sequence determination, overexpression of both isoforms in Escherichia coli BL21(DE3) [18]) [18] (gene murA, DNA sequence analysis, expression in Escherichia coli under control of the arabinose-inducible, glucose-repressible ara promotor, functional complementation, necessary for viability, of a enzyme-deficient Escherichia coli murA null-mutant, impartment of fosfomycin resistance to the Escherichia coli cell [23]) [23] Engineering C115A ( site-directed mutagenesis, overexpression in Escherichia coli, no activity [12]) [12] C115D ( site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, forms only the phospholactyl-enzyme intermediate adduct, but no UDP-GlcNAc-phosphoenolpyruvate, higher kcat than the wild-type at pH 7.0, enhanced pH-dependency of the reaction [12]) [12] C115E ( site-directed mutagenesis, overexpression in Escherichia coli, gains fosfomycin resistance, enhanced pH-dependency of the reaction, low activity [12]) [12] C115N ( site-directed mutagenesis, overexpression in Escherichia coli, deamination of Asn115 to Asp115 [12]) [12] C115S ( site-directed mutagenesis, overexpression in Escherichia coli, no activity [12,17,19]) [12, 16, 17, 19] C251S ( site-directed mutagenesis, Cys251 is not involved in the catalysis, unaltered biochemical properties [19]) [19] C354S ( site-directed mutagenesis, Cys354 is not involved in the catalysis, unaltered biochemical properties [19]) [19] C381S ( site-directed mutagenesis, Cys381 is not involved in the catalysis, unaltered biochemical properties [19]) [19]

449


2.5.1.7

D305A ( site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity, fosfomycin is not covalently attached to Cys115 [22]) [22] D305C ( site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity [22]) [22] D305E ( site-directed mutagenesis, weaker binding of UDP-GlcNAc, 0.1% activity compared to the wild-type [22]) [22] D305H ( site-directed mutagenesis, weaker binding of UDP-GlcNAc, no activity, fosfomycin is not covalently attached to Cys115 [22]) [22] K22E ( site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wildtype, altered UDP-GlcNAc binding, highly reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin [17]) [17] K22R ( site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wildtype, slightly reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin [17]) [17] K22V ( site-directed mutagenesis, exchange of conserved Lys residue located near the active site and involved in substrate binding leading to conformational changes, shows less than 0.5% activity compared to the wildtype, reduced formation of covalent adduct between active site Cys115 and phosphoenolpyruvate or inhibitor fosfomycin [17]) [17] N23A ( site-directed mutagenesis, reduced activity, 20fold higher apparent dissociation constant for fosfomycin compared to wild-type [22]) [22] N23S ( site-directed mutagenesis, reduced activity, 200fold higher apparent dissociation constant for fosfomycin compared to wild-type [22]) [22] Additional information ( individually inactivation of the 2 isoforms by allelic replacement shows, that the 2 forms can substitute for each other, a double deletion mutant is not viable [18]; construction of a murA(Z) deletion mutant strain without catalytic activity, cells grow only when complemented by the enzyme expressed from a plasmid [9]) [9, 18] Application medicine ( target for development of antibacterial agents [20,23]; enzyme is a target for the antibiotic fosfomycin [13,16,17]) [13, 16, 17, 20, 23]

6 Stability General stability information , EDTA, DTT and dithioerythritol stabilize [5]

450

2.5.1.7


Storage stability , -20 C, loss of activity after 1 week [2] , 0 C or-20 C, 0.2 mg/ml DTT, 40% loss of activity after 2 weeks [1] , 4 C, in 80% ammonium sulfate, loss of activity after 1 week [2] , -15 C, crude enzyme preparation, in presence of 4 mM DTT, loss of 84% activity within 2 weeks [5] , -196 C, 4 mM DTT, stable for up to 6 weeks, partially purified enzyme [5] , -196 C, slightly increased activity after storage of 2 weeks [5] , -70 C, 4 mM DTT, 22% loss of activity in 2 weeks, partially purified enzyme [5]

References [1] Gunetileke, K.G.; Anwar, R.A.: Biosynthesis of uridine diphospho-N-acetylmuramic acid. II. Purification and properties of pyruvate-uridine diphospho-N-acetylglucosamine transferase and characterization of uridine diphospho-N-acetylenopyruvylglucosamine. J. Biol. Chem., 243, 5770-5778 (1968) [2] Zemell, R.I.; Anwar, R.A.: Pyruvate-uridine diphospho-N-acetylglucosamine transferase. Purification to homogeneity and feedback inhibition. J. Biol. Chem., 250, 3185-3192 (1975) [3] Zemell, R.I.; Anwar, R.A.: Mechanism of pyruvate-uridine diphospho-Nacetylglucosamine transferase. Evidence for an enzyme-enolpyruvate intermediate. J. Biol. Chem., 250, 4959-4964 (1975) [4] Venkateswaran, P.S.; Lugtenberg, E.J.J.; Wu, H.C.: Inhibition of phosphoenolpyruvate:uridine diphosphate N-acetylglucosamine enolpyruvyltransferase by uridine diphosphate N-acetylmuramyl peptides. Biochim. Biophys. Acta, 293, 570-574 (1973) [5] Wickus, G.G.; Strominger, J.L.: Partial purification and properties of the pyruvate-uridine diphospho-N-acetylglucosamine transferase from Staphylococcus epidermidis. J. Bacteriol., 113, 287-290 (1973) [6] Wanke, C.; Falchetto, R.; Amrhein, N.: The UDP-N-acetylglucosamine 1carboxyvinyl-transferase of Enterobacter cloacae. Molecular cloning, sequencing of the gene and overexpression of the enzyme. FEBS Lett., 301, 271-276 (1992) [7] Marquardt, J.L.; Siegele, D.A.; Kolter, R.; Walsh, C.T.: Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-Nacetylglucosamine enolpyruvyl transferase. J. Bacteriol., 174, 5748-5752 (1992) [8] Anwar, R.A.; Vlaovic, M.: Effect of phosphoenolpyruvate analogs on the activity of enoylpyruvate transferase and the effect of UDP-N-acetylglucosamine on the reactivity of the active site SH group. Biochim. Biophys. Acta, 616, 389-394 (1980)

451


2.5.1.7

[9] Brown, E.D.; Vivas, E.I.; Walsh, C.T.; Kolter, R.: MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J. Bacteriol., 177, 4194-4197 (1995) [10] Kim, D.H.; Lees, W.J.; Walsh, C.T.: Stereochemical analysis of the tetrahedral adduct formed at the active site of UDP-GlcNAc enolpyruvyl transferase from the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate, in D2 O. J. Am. Chem. Soc., 117, 6380-6381 (1995) [11] Kim, D.H.; Lees, W.J.; Haley, T.M.; Walsh, C.T.: Kinetic characterization of the inactivation of UDP-GlcNAc enolpyruvyl transferase by (Z)-3-fluorophosphoenolpyruvate: evidence for two oxocarbenium ion intermediates in enolpyruvyl transfer catalysis. J. Am. Chem. Soc., 117, 1494-1502 (1995) [12] Kim, D.H.; Lees, W.J.; Kempsell, K.E.; Lane, W.S.; Duncan, K.; Walsh, C.T.: Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin. Biochemistry, 35, 4923-4928 (1996) [13] Skarzynski, T.; Mistry, A.; Wonacott, A.; Hutchinson, S.E.; Kelly, V.A.; Duncan, K.: Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, and enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Structure, 4, 1465-1474 (1996) [14] Schoenbrunn, E.; Sack, S.; Eschenburg, S.; Perrakis, A.; Krekel, F.; Amrhein, N.; Mandelkow, E.: Crystal structure of UDP-N-acetylglucosamine enolpyruvyltransferase, the target of the antibiotic fosfomycin. Structure, 4, 10651075 (1996) [15] Schonbrunn, E.; Svergun, D.I.; Amrhein, N.; Koch, M.H.J.: Studies on the conformational changes in the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyltransferase (MurA). Eur. J. Biochem., 253, 406-412 (1998) [16] Krekel, F.; Oecking, C.; Amrhein, N.; Macheroux, P.: Substrate and inhibitor-induced conformational changes in the structurally related enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS). Biochemistry, 38, 8864-8878 (1999) [17] Samland, A.K.; Amrhein, N.; Macheroux, P.: Lysine 22 in UDP-N-acetylglucosamine enolpyruvyl transferase from Enterobacter cloacae is crucial for enzymatic activity and the formation of covalent adducts with the substrate phosphoenolpyruvate and the antibiotic fosfomycin. Biochemistry, 38, 13162-13169 (1999) [18] Du, W.; Brown, J.R.; Sylvester, D.R.; Huang, J.; Chalker, A.F.; So, C.Y.; Holmes, D.J.; Payne, D.J.; Wallis, N.G.: Two active forms of UDP-N-acetylglucosamine enolpyruvyl transferase in gram-positive bacteria. J. Bacteriol., 182, 4146-4152 (2000) [19] Krekel, F.; Samland, A.K.; Macheroux, P.; Amrhein, N.; Evans, J.N.S.: Determination of the pKa value of C11 5 in MurA (UDP-N-acetylglucosamine enolpyruvyltransferase) from Enterobacter cloacae. Biochemistry, 39, 1267112677 (2000) 452

2.5.1.7


[20] Dai, H.J.; Parker, C.N.; Bao, J.J.: Characterization and inhibition study of MurA enzyme by capillary electrophoresis. J. Chromatogr. B, 766, 123-132 (2002) [21] Samland, A.K.; Jelesarov, I.; Kuhn, R.; Amrhein, N.; Macheroux, P.: Thermodynamic characterization of ligand-induced conformational changes in UDP-N-acetylglucosamine enolpyruvyl transferase. Biochemistry, 40, 9950-9956 (2001) [22] Samland, A.K.; Etezady-Esfarjani, T.; Amrhein, N.; Macheroux, P.: Asparagine 23 and aspartate 305 are essential residues in the active site of UDP-Nacetylglucosamine enolpyruvyl transferase from Enterobacter cloacae. Biochemistry, 40, 1550-1559 (2001) [23] McCoy, A.J.; Sandlin, R.C.; Maurelli, A.T.: In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance. J. Bacteriol., 185, 1218-1228 (2003)

453

tRNA isopentenyltransferase

2.5.1.8

1 Nomenclature EC number 2.5.1.8 Systematic name isopentenyl-diphosphate:tRNA isopentenyltransferase Recommended name tRNA isopentenyltransferase Synonyms D2 -isopentenyl pyrophosphate:tRNA-D2 -isopentenyl transferase D2 -isopentenyl pyrophosphate:transfer ribonucleic acid D2 -isopentenyltransferase isopentenyltransferase, transfer ribonucleate transfer ribonucleate isopentenyltransferase CAS registry number 37277-78-4

2 Source Organism

yeast [1] Escherichia coli (strain K-12 [8]) [2, 3, 8, 11] Lactobacillus acidophilus (ATCC 4963 [4]) [4] Zea mays [5] Arabidopsis thaliana [6] Agrobacterium tumefaciens [7] Caenorhabditis elegans [9] Homo sapiens [10]

3 Reaction and Specificity Catalyzed reaction isopentenyl diphosphate + tRNA = diphosphate + tRNA containing 6-isopentenyladenosine ( mechanism [8,11]) Reaction type alkenyl group transfer

454

2.5.1.8


Natural substrates and products S D2 -isopentenyl diphosphate + tRNA ( biosynthesis of N6 -(D2 isopentenyl)adenosine which several species of tRNA contain adjacent to the 3'-end of the anticodon [1]; regulates global physiology by unknown mechanisms [9]) (Reversibility: ? [1, 9]) [1, 9] P tRNA containing N6 -(D-isopentenyl)adenosine + diphosphate Substrates and products S D2 -isopentenyl diphosphate + tRNA ( specific for D2 -isopentenyl phosphate [1, 2]; no reaction with D3 -isopentenyl diphosphate [1, 2, 5]; no reaction with homologous native tRNA or homologous, permanganate-treated tRNA (permanganate specifically cleaves the D2 -isopentenyl groups of tRNA leaving adenosine residues) [1]; the enzyme from both yeast and rat liver catalyzes a significant incorporation of D2 -isopentenyl groups into untreated E. coli B tRNA [1]; tRNA lacking the isopentenyl modification normally present in vivo [2]; mycoplasma species (kid) tRNA [3]; recognition mode of substrate, structural features of substrate tRNA [11]) (Reversibility: ? [1-11]) [1-11] P tRNA containing N6 -(D-isopentenyl)adenosine + diphosphate [1-4] Inhibitors ADP ( competitive to D2 -isopentenyl diphosphate [8]) [8] ATP ( competitive to D2 -isopentenyl diphosphate [8]) [8] EDTA [1, 2, 5] diphosphate [1, 2, 5] high ionic strength [2] iodoacetamide [1] p-mercuribenzoate [1] Metals, ions Mg2+ ( required [1, 2, 4]; optimal concentration: 5 mM [1]; 3.3 mM [2]; 5-7 mM [4]; 3-5 mM [5]) [1, 2, 4, 5] Mn2+ ( can partially replace Mg2+ in activation [1,2]) [1, 2] Zn2+ ( sequence contains a C2 H2 Zn-finger-like motif [10]) [10] divalent cation ( required for full activity [3]) [3] Specific activity (U/mg) 0.053 [2] Additional information [1, 4, 5] Km-Value (mM) 0.000632 (D2 -isopentenyl diphosphate) [8] 0.003 (D2 -isopentenyl diphosphate) [4] 0.013 (tRNA, from Lactobacillus [4]) [4] Additional information ( KM for tRNA substrates is around 3 nM [8]) [8] Additional information ( KM of substrates and tRNA from various species [5]) [5]

455


2.5.1.8

pH-Optimum 7.5 [2] 7.5-8 [1, 4] 7.8 [5] pH-Range 6-9 ( about 50% of maximum activity at pH 6 and pH 9, below pH 5 activity is totally abolished [2]) [2] 7-9.5 ( pH 7: about 95% of maximum activity, pH 9.5: about 45% of maximum activity [1]) [1] Temperature optimum ( C) 37 [5]

4 Enzyme Structure Molecular weight 55000 [3] 60000 ( gel filtration, SDS-PAGE [5]) [5] Subunits monomer ( 1 * 60000, SDS-PAGE [5]) [5]

5 Isolation/Preparation/Mutation/Application Source/tissue kernel [5] leaf [5] root tip [5] seed [6] Localization mitochondrion [9] Purification (partial [1]) [1] [2, 3] (partial [4]) [4] Cloning [6] [10] Engineering Additional information ( deletion mutant: no enzyme activity, no release of isopentenyladenine into extracellular medium [7]) [7]

456

2.5.1.8


6 Stability General stability information , not stable to ammonium sulfate precipitation [2] Storage stability , 0 C, stable for 1 month [2] , -18 C, 0.59 mg bovine serum albumin/ml, 50% glycerol, purified enzyme loses 80% of activity after 14 days [5] , -18 C, 50% glycerol, partially purified enzyme stable for 3 months [5]

References [1] Kline, L.K.; Fittler, F.; Hall, R.H.: N6 -(D2 -isopentenyl) adenosine. Biosynthesis in transfer ribonucleic acid in vitro. Biochemistry, 8, 4361-4371 (1969) [2] Rosenbaum, N.; Gefter, M.L.: D2 -isopentenylpyrophosphate: transfer ribonucleic acid 2-isopentenyltransferase from Escherichia coli. Purification and properties of the enzyme. J. Biol. Chem., 247, 5675-5680 (1972) [3] Bartz, J.K.; Soell, D.: N6 -(2-isopentenyl) adenosine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli. Biochimie, 54, 31-39 (1972) [4] Holtz, J.; Klämbt, D.: tRNA isopentenyltransferase from Lactobacillus acidophilus ATCC 4963. Hoppe-Seyler's Z. Physiol. Chem., 356, 1459-1464 (1975) [5] Holtz, J.; Klämbt, D.: tRNA isopentenyltransferase from Zea mays L. Characterization of the isopentenylation reaction of tRNA, oligo (A) and other nucleic acids. Hoppe-Seyler's Z. Physiol. Chem., 359, 89-101 (1978) [6] Golovko, A.; Sitbon, F.; Tillberg, E.; Nicander, B.: Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana. Plant Mol. Biol., 49, 161-169 (2002) [7] Gray, J.; Gelvin, S.B.; Meilan, R.; Morris, R.O.: Transfer RNA is the source of extracellular isopentenyladenine in a Ti-plasmidless strain of Agrobacterium tumefaciens. Plant Physiol., 110, 431-438 (1996) [8] Leung, H.C.E.; Chen, Y.; Winkler, M.E.: Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12. J. Biol. Chem., 272, 13073-13083 (1997) [9] Lemieux, J.; Lakowski, B.; Webb, A.; Meng, Y.; Ubach, A.; Bussiere, F.; Barnes, T.; Hekimi, S.: Regulation of physiological rates in Caenorhabditis elegans by a tRNA-modifying enzyme in the mitochondria. Genetics, 159, 147-157 (2001) [10] Golovko, A.; Hjalm, G.; Sitbon, F.; Nicander, B.: Cloning of a human tRNA isopentenyl transferase. Gene, 258, 85-93 (2000) [11] Motorin, Y.; Bec, G.; Tewari, R.; Grosjean, H.: Transfer RNA recognition by the Escherichia coli D2 -isopentenyl-pyrophosphate:tRNA D2 -isopentenyl transferase: dependence on the anticodon arm structure. RNA, 3, 721-733 (1997)

457

Riboflavin synthase

2.5.1.9

1 Nomenclature EC number 2.5.1.9 Systematic name 6,7-dimethyl-8-(1-d-ribityl)lumazine:6,7-dimethyl-8-(1-d-ribityl)lumazine 2,3-butanediyltransferase Recommended name riboflavin synthase Synonyms 6,7-dimethyl-8-ribityllumazine-synthase ( b-subunit of the enzyme complex [19]) [19] heavy riboflavin synthase ( bifunctional enzyme complex that catalyzes formation of riboflavin from 5-amino-6-ribityl-amino-2,4-pyrimidinedione and 3,4-dihydroxy-2-butanone [10]) [10] light riboflavin synthase lumazine synthase ( b subunit of the enzyme complex [27]) [27] riboflavin synthetase riboflavine synthase riboflavine synthetase synthase, riboflavin Additional information ( lumazinesynthase/riboflavin synthase complex, icosahedral capsid of 60 b subunits enclosing a triplet of a subunits [18-20]) [18-20] CAS registry number 9075-82-5

2 Source Organism no activity in Homo sapiens [24] Ashbya gossypii (strain AG33 [1]) [1, 3] Saccharomyces cerevisiae [1-3, 5, 12] Eremothecium ashbyii (purified enzyme [7]) [4, 7, 16] Spinacia oleracea (spinach [5]) [5] Aerobacter aerogenes [3] Pseudomonas sp. [3]

458

2.5.1.9

Riboflavin synthase

Bacillus subtilis (derepressed strain H94 [20]; heavy enzyme form [18]; heavy and light enzyme form [6,12]; pure enzyme from mutant strain H94 [10]; mutant WA 45 [6]; (168M, H94) (flavinogenic), H322 (flavinogenic), (H52, Rib-), (107,Rib-), ATCC 6051, ATCC 6633, B23 [12]) [3, 6, 815, 18-20, 27, 28] Escherichia coli (selenomethionine containing enzyme [24]; gene ribC [22]; strain ATCC 9637 [1]; B [12]) [1, 3, 6, 12, 22-25, 27, 28] Neurospora crassa [3] Lactobacillus plantarum [3, 12] Bacillus globigii (contains heavy and light form of the enzyme [12]) [12] Bacillus licheniformis (contains heavy and light form of the enzyme [12]) [12] Bacillus megaterium (contains heavy and light form of the enzyme [12]) [12] Bacillus polymyxa (contains heavy and light form of the enzyme [12]) [12] Clostridium thermoaceticum (contains heavy and light form of the enzyme [12]) [12] Nocardia rubra [12] Pseudomonas iodinum [12] Streptomyces venezuelae [12] Bacillus stearothermophilus (strain ATCC 8005 [17]) [17] Escherichia coli [18] Photobacterium leiognathi [18] Methanobacterium thermoautotrophicum (gene ribC [21]; Marburg strain [21]) [21] Schizosaccharomyces pombe [26]

3 Reaction and Specificity Catalyzed reaction 2 6,7-dimethyl-8-(1-d-ribityl)lumazine = riboflavin + 4-(1-d-ribitylamino)-5amino-2,6-dihydroxypyrimidine ( Cys-48 plays a nucleophilic role in catalytic mechanism [23]; active site can accomodate 2 ligand molecules [28]; active site [24, 28]; substrate binding site [18, 22, 26]; reaction mechanism [1, 3, 16, 18-20, 22, 26-28]) Reaction type condensation [19] dismutation Natural substrates and products S 6,7-dimethyl-8-ribityllumazine + 6,7-dimethyl-8-ribityllumazine ( biosynthetic pathway [16, 19]; i.e. 6,7-dimethyl-8-(1'-d-ribityl)lumazine [2]) [1-28] P riboflavin + 4-(1'-d-ribitylamino)-5-amino-2,6-dihydroxypyrimidine

459

Riboflavin synthase

2.5.1.9

S Additional information ( salvage cycle for the by-product of the reaction is involved in the de novo synthesis of riboflavin [16]) [16] P ? Substrates and products S 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione + (3R)-3,4-dihydroxy2-butanone 4-phosphate ( strictly regiospecific [19]; 6fold higher reaction rate with the S-enantiomer compared to the R-enantiomer [19]; catalysed by b-subunit [19]) (Reversibility: ? [19]) [19] P ? S 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione + (3S)-3,4-dihydroxy2-butanone ( strictly regiospecific [19]; product is the intermediate of riboflavin synthesis [19]; catalysed by b-subunit [19, 20]) (Reversibility: ? [19, 20, 27, 28]) [19, 20, 27, 28] P 6,7-dimethyl-8-ribityllumazine [19, 27, 28] S 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione + (3S)-3,4-dihydroxy2-butanone 4-phosphate ( strictly regiospecific [19]; 6fold higher reaction rate than with the R-enantiomer [19]; catalysed by bsubunit [19]) (Reversibility: ? [19]) [19] P ? S 6,7-dimethyl-8-[1'-(5'-deoxy-d-ribityl)]lumazine + 6,7-dimethyl-8-[1'-(5'deoxy-d-ribityl)]lumazine (Reversibility: ? [3]) [3] P 5'-deoxyriboflavin + ? [3] S 6,7-dimethyl-8-ribityllumazine + 6,7-dimethyl-8-ribityllumazine (Reversibility: ? [1]) [1] P riboflavin + a compound related to 4-ribitylamino-2,5,6-trihydroxypyrimidine [1] S 6,7-dimethyl-8-ribityllumazine + 6,7-dimethyl-8-ribityllumazine ( 1 riboflavin is bound per monomer in a site at one end of the 6stranded antiparallel b-barrel which is comprised of elements of both monomers [23]; catalysed by the a-subunit [19, 20]; i.e. 6,7-dimethyl-8-(1'-d-ribityl)lumazine [2]) (Reversibility: ir [5]; ? [1-4,6-28]) [1-28] P riboflavin + 4-(1'-d-ribitylamino)-5-amino-2,6-dihydroxypyrimidine [2, 3, 5, 19, 24] S Additional information ( structure-function relationship [26]; substrate channeling, overview [20]; stereospecificity [19]; diacetyl and 3,4-dihydroxy-2-butanone 3-phosphate are no substrates [19]; overview [1]; no activity with lumazine 5'-phosphate [6,21]; b-subunit of heavy riboflavin synthase catalyzes the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6ribitylamino-2,4-pyrimidinedione and a carbohydrate phosphate [9,19]; dismutation of 6,7-dimethyl-8-ribityllumazine yielding riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione is catalyzed by the asubunit [13,19]) [1, 6, 9, 13, 16, 19-21, 26] P ?

460

2.5.1.9

Riboflavin synthase

Inhibitors 2-amino-4,6-dihydroxy-8-d-ribityl-7-pteridinone [3] 5,5'-dithiobis(2-nitrobenzoate) [17] 5,6,7,8-tetrahydro-9-(1'-d-ribityl)isoalloxazine [3] 6,7-dihydroxy-8-ribityllumazine [3] 6,7-dimethyl-8-(1'-d-xylityl)lumazine [3] 6-carboxyalkyl-derivatives of 7-oxo-8-ribityllumazine ( synthesis of diverse derivatives as inhibitors, inhibitor binding site, model, overview [27]) [27] 6-methyl-7-hydroxy-8-ribityllumazine [3, 7, 17] 6-phosphonoalkyl-6-d-ribitylaminopyrimidinedione amides ( inhibit riboflavin synthase [28]; only very weak inhibition of luminazine synthase [28]) [28] 6-phosphonoalkyl-6-d-ribitylaminopyrimidinediones ( inhibit luminazine synthase [28]; no inhibition of riboflavin synthase [28]) [28] 6-phosphonoxyalkyl-derivatives of 7-oxo-8-ribityllumazine ( synthesis of diverse derivatives as inhibitors, inhibitor binding site, model, overview [27]) [27] 7-hydroxy-6-(2-carboxyethyl)-8-(1-d-ribityl)lumazine [7] 7-hydroxy-6-(d-1,2-dihydroxyethyl)-8-(1-d-ribityl)lumazine [7] 7-hydroxy-6-(l-1,2-dihydroxyethyl)-8-(1-d-ribityl)lumazine ( i.e. photolumazine A [7]) [7] 7-hydroxy-6-(p-hydroxyphenyl)-8-(1-d-ribityl)lumazine [7] 7-hydroxy-6-hydroxymethyl-8-(1-d-ribityl)lumazine ( photolumazine B [7]) [7] 7-hydroxy-8-(1-d-ribityl)lumazine ( i.e. photolumazine [7]) [7] Ca2+ [17] Cu2+ [5, 17] Hg2+ [5, 17] Mg2+ [17] Mn2+ [17] Zn2+ [17] avidin ( moderately, no prevention by biotin [5]) [5] iodoacetamide [17] p-chloromercuribenzenesulfonate ( reversible by cysteine or 2-mercaptoethanol [3,5]) [3, 5] p-chloromercuribenzoate [5, 17] riboflavin ( product inhibition, Saccharomyces cerevisiae [3]) [3] Additional information ( molecular modeling of inhibitor binding [27,28]; no effect by Ba2+ , Co2+ , EDTA [17]) [17, 27, 28] Cofactors/prosthetic groups Additional information ( no cofactor requirement [24]) [24] Activating compounds 2,3-dimercapto-1-propanol ( enhances activity [1]) [1] 2-mercaptoethanol ( enhances activity [1]) [1] N-acetylcysteine ( enhances activity [1]) [1] 461

Riboflavin synthase

2.5.1.9

SO23- ( sodium sulfite [12]; enhances activity [1,12]) [1, 12] diacetyl ( activates [16]) [16] Additional information ( assay carried out under reducing condition to avoid side reactions and because of the lability of the enzyme in presence of O2 [5]; no requirement for a cofactor [17]) [5, 17] Metals, ions Mg2+ ( absolute requirement [21]) [21] Additional information ( no effect by Ba2+ , Co2+ [17]) [17] Turnover number (min±1) 0.5 (6,7-dimethyl-8-ribityllumazine, per subunit [22]) [22] 3.36 ((3S)-3,4-dihydroxy-2-butanone, native enzyme complex [20]) [20] 3.36 (5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, native enzyme complex [20]) [20] 4.56 ((3S)-3,4-dihydroxy-2-butanone, hollow b60 capsid [20]) [20] 4.56 (5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, hollow b60 capsid [20]) [20] 11.4 (6,7-dimethyl-8-ribityllumazine, native enzyme complex [20]) [20] 72 (6,7-dimethyl-8-(1'-d-ribityl)lumazine, heavy enzyme, Bacillus subtilis H94 [12]) [12] 120 (6,7-dimethyl-8-(1'-d-ribityl)lumazine, light enzyme, Bacillus subtilis H94 [12]) [12] Specific activity (U/mg) 0.00025 ( partially purified enzyme [5]) [5] 0.0014 ( partially purified enzyme [6]) [6] 0.003 [1] 0.011 ( purified enzyme [17]) [17] 0.027 ( purified enzyme [10]; heavy enzyme [6,10]) [6, 10] 0.032 ( 65 C [21]; purified recombinant enzyme [21]) [21] 0.033 ( purified enzyme [15]; heavy enzyme [12,15]) [12, 15] 0.045 ( 65 C [21]; purified enzyme [21]) [21] 0.052 ( purified enzyme [1]) [1] 0.16 ( purified enzyme [16]) [16] 0.2 ( lumazine synthase activity, reconstituted hollow b6 0 capsids and native enzyme complex [19]) [19] 0.266 ( purified enzyme [1]) [1] 0.5 ( light enzyme [6]) [6] 0.833 ( light enzyme [12]) [12] 1.2-2.8 ( partially purified enzyme [4]) [4] Additional information ( various estimation methods [16]; assay method [5]) [1-5, 7, 13, 16]

462

2.5.1.9

Riboflavin synthase

Km-Value (mM) 0.005 (5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione) [19, 20] 0.01 (6,7-dimethyl-8-(1'-d-ribityl)lumazine, pH 7.0 [5]) [5] 0.013 (6,7-dimethyl-8-(1'-d-ribityl)lumazine, heavy enzyme [12]) [12] 0.014 (6,7-dimethyl-8-(1'-d-ribityl)lumazine, light enzyme [12]) [12] 0.029 (6,7-dimethyl-8-(1'-d-ribityl)lumazine) [1] 0.04 (6,7-dimethyl-8-(1'-d-ribityl)lumazine) [1] 0.045 (6,7-dimethyl-8-(1'-d-ribityl)lumazine, pH 7.5 [5]) [5] 0.13 ((3S)-3,4-dihydroxy-2-butanone) [20] 0.13 ((3S)-3,4-dihydroxy-2-butanone 4-phosphate) [19] 0.13 (6,7-dimethyl-8-(1'-d-ribityl)lumazine) [13, 20] Additional information ( sigmoidal kinetics, kinetic model [20]; Km is temperature-dependent [17]) [17, 20] Ki-Value (mM) 0.0029 (6-methyl-7-hydroxy-8-ribityllumazine) [7] 0.003 (7-hydroxy-6-hydroxymethyl-8-(1-d-ribityl)lumazine) [7] 0.011 (7-hydroxy-8-(1-d-ribityl)lumazine) [7] 0.017 (7-hydroxy-6-(d-1,2-dihydroxyethyl)-8-(1-d-ribityl)lumazine) [7] 0.017 (7-hydroxy-6-(l-1,2-dihydroxyethyl)-8-(1-d-ribityl)lumazine) [7] 0.027 (7-hydroxy-6-(2-carboxyethyl)-8-(1-d-ribityl)lumazine) [7] 0.028 (7-hydroxy-6-(p-hydroxyphenyl)-8-(1-d-ribityl)lumazine) [7] 0.18 (6-methyl-7-hydroxy-8-ribityllumazine) [17] Additional information ( Ki values of several inhibitory 6-phosphonoalkyl-6-d-ribitylaminopyrimidinediones amides [28]; Ki values of several inhibitory 6-phosphonoalkyl-6-d-ribitylaminopyrimidinediones [28]; Ki values of diverse inhibitory 6-carboxyalkyl- and 6-phosphonoxyalkyl-derivatives of 7-oxo-8-ribityllumazine, overview [27]) [27] pH-Optimum 6.5 [4] 6.7-7.2 [17] 6.9 [1] 7 ( assay at [6]) [1, 3, 5, 6] 7.4 ( light enzyme, the pH-optimum of the heavy enzyme is similar, detailed measurement is not possible because of instability at elevated pH [12]) [12] 7.5 ( 6,7-dimethyl-8-ribityllumazine formation [20]; assay at [10]) [5, 10, 20] pH-Range 5.4-8.8 ( about 50% of activity maximum at pH 5.4 and 8.8 [17]) [17] 5.8-8.4 ( about 50% of activity maximum at pH 5.8 and 8.4 [3]) [3]

463

Riboflavin synthase

2.5.1.9

Temperature optimum ( C) 37 ( assay at [1, 3, 5, 6, 10, 15]) [1, 3, 5, 6, 10, 15] 47 [4] 95 [17] Temperature range ( C) 25-45 [5] 50-95 ( 50 C: about 10% of activity maximum, 80 C: about 30% of activity maximum, 95 C: activity maximum [17]) [17]

4 Enzyme Structure Molecular weight 70000 ( light enzyme [12,13]; analytical ultracentrifugation [12]) [12, 13] 70000-80000 ( analytical ultracentrifugation [3]) [3] 75000 [23] 100000 ( heavy riboflavin synthase [10,12-14]; analytical ultracentrifugation [12]) [10, 12-14] 345000 ( gel filtration [17]) [17] Subunits polymer ( 60 * 16000-16200 (b) + 3 * 23500 (a), heavy riboflavin synthase, X-ray studies, analytical ultracentrifugation [10,13,15]; primary structure of b subunit [13]) [10, 13, 15] trimer ( 3 * 23000 [24]; 3 * 22043, light enzyme, calculation from amino acid sequence [11]; 3 * 23500, light enzyme, SDS-PAGE, subunits not covalently linked [12]) [11, 12, 24] Additional information ( structure model, biological implication [26]; each subunit of the homotrimer consists of 2 domains [23]; N-terminal structure, ligand binding site [22,23]; comparison of primary structure [18]; b subunit model, lumazinesynthase/ riboflavin synthase complex, icosahedral capsid of 60 b subunits enclosing a triplet of a subunits [18,19]; subunit structure and organization [15]) [15, 18, 19, 22, 23, 26]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf [5] mycelium [1, 4, 16] Purification [1] [1, 3] (partially [4]) [4, 16]

464

2.5.1.9

Riboflavin synthase

[5] (light enzyme [11,12]; heavy enzyme [12,13,15]) [11-13, 15] (wild-type enzyme and mutants [22]; partially [6]) [1, 6, 22] [17] (1500fold [21]) [21] Renaturation (reconstitution as hollow b60 capsid [19]; dissociation of subunits at pH values above neutrality and renaturation [15]) [15, 19] Crystallization (vapour diffusion method using sitting drops, protein in 1.55 M sodium/ potassium phosphate, pH 8.7, 1 mM 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, X-ray structure analysis, computational structure refinement [18]; large crystals by vapour diffusion method, initial solution: 0.7 M sodium/potassium phosphate, pH 8.7, 0.3 mM 5-nitroso-6-ribitylamino2,4(1H,3H)-pyrimidinedione, protein 2 mg/ml, reservoir solution: 1.3 M sodium/potassium phosphate, pH 8.7, X-ray structure determination and analysis [13]; detailed structure determination and analysis of enzyme complexed with heavy atoms, three-dimensional structure model [10]; from 1.3 M sodium/potassium phosphate, pH 8.7, 0.35 mM 5-nitroso-6-(1'-d-ribitylamino)-2,4(1H,3 H)-pyrimidinedione, X-ray structure determination and analysis [8]) [8, 10, 13, 18] (structure determination by multiwavelength anomalous diffraction method, modeling [24]) [24] (crystallization of enzyme complexed with 6-carboxyethyl-7-oxo-8-ribityllumazine, sitting drop vapour diffusion method against equal amounts of reservoir solution containing 0.1 M bicine, pH 9.0, 65% v/v 2-mehyl-2,4-pentanediol, enzyme solution: 9 mg/ml, 20 mM TrisHCl, pH 7.0, 0.1 M KCl, 10 molar excess of solid 8, X-ray structure determination and analysis, structure model building and refinement, overview [26]) [26] Cloning (cloning of sequence segments of residues 1-97 and 101-213, and expression in Escherichia coli [25]; ribC hyperexpression strain, DNA sequence determination of mutants [22]) [22, 25] (DNA and amino acid sequence determination and analysis, functional expression in riboflavin-deficient Escherichia coli mutant BSV23 [21]) [21] Engineering D143G ( site-directed mutagenesis, soluble protein, too unstable to be purified [22]) [22] D143N ( site-directed mutagenesis, soluble protein, too unstable to be purified [22]) [22] D185L ( site-directed mutagenesis, low remaining activity [22]) [22] E183G ( site-directed mutagenesis, reduced activity [22]) [22] E66G ( site-directed mutagenesis, low remaining activity [22]) [22] E85G ( site-directed mutagenesis, reduced activity [22]) [22] F2A ( site-directed mutagenesis, no remaining activity [22]) [22] 465

Riboflavin synthase

2.5.1.9

F2Y ( site-directed mutagenesis, very low remaining activity [22]) [22] H102Q ( site-directed mutagenesis, very low remaining activity [22]) [22] H97Q ( site-directed mutagenesis, low remaining activity [22]) [22] K137A ( site-directed mutagenesis, low remaining activity [22]) [22] N181G ( site-directed mutagenesis, soluble protein, too unstable to be purified [22]) [22] N45G ( site-directed mutagenesis, slightly reduced activity [22]) [22] N83G ( site-directed mutagenesis, reduced activity [22]) [22] S146G ( site-directed mutagenesis, low remaining activity [22]) [22] S41A ( site-directed mutagenesis, very low remaining activity [22]) [22] T3R ( site-directed mutagenesis, slightly reduced activity, low expression rate [22]) [22] T71A ( site-directed mutagenesis, slightly reduced activity [22]) [22] Y133A ( site-directed mutagenesis, soluble protein, too unstable to be purified [22]) [22] Additional information ( recombinant sequence segment 1-97 forms a homodimer that can bind riboflavin, 6,7-dimethyl-8-ribityllumazine, and trifluoromethyl-substituted 8-ribityllumazine derivatives, and is required for ligand binding, recombinant sequence segment 101-213 is unstable and only partially involved in riboflavin binding [25]; 5 mutants genes cannot be expressed recombinantly in Escherichia coli: C48S, T50R, T67R, T148R, T165R [22]; a F2D deletion mutant construct has no remaining activity [22]) [22, 25] Application medicine ( enzyme is an attractive target for antimicrobial agents, since it is nonexistent in humans [24]) [24]

6 Stability pH-Stability 4-8 ( 0 C, stable for several h in presence of a suitable reducing agent [3]) [3] 5-7.5 ( 0.1 M phosphate, stable [15]) [15] 6-10 ( 26 C, 18 h, stable [17]) [17] 6.3-7.6 ( 55 C, 18 h, stable [17]) [17] 7 ( unstable above, 0.1 M Tris/HCl buffer [15]) [15] Additional information ( heavy enzyme is instable at elevated pH [12]; 5-nitroso-6-ribitylamino-2,4-pyrimidinedione increases stability at elevated pH-values [15]) [12, 15] Temperature stability 0-2 ( cold lability, inclusion of 0.01 M sodium sulfite in the solution used in purification prevents inactivation [1]) [1] 466

2.5.1.9

Riboflavin synthase

60 ( 24 h, no loss of activity, without substrate [17]) [17] 80 ( 1 min, complete loss of activity [5]) [5] 85 ( 10 min, complete loss of activity [17]) [17] 92 ( 1 min, complete loss of activity [1]) [1] Oxidation stability , lability in presence of O2 [5] General stability information , high ionic strength protects yeast enzyme against inactivation [5] , freezing, completely inactivates spinach enzyme [5] , 5-nitroso-6-ribitylamino-2,4-pyrimidinedione highly increases stability at elevated pH-values [15] , phosphate increases stability at neutral pH-values [15] , stable at 26 C in 4 M urea for 18 h, 4 M urea completely inactivates at 55 C [17] , reducing agents, e.g. cysteine, ascorbate or Na2 SO4 stabilize yeast and spinach enzyme [5] Storage stability , -20 C, completely purified enzyme, stable for several months [1] , -90 C or in liquid N2 , stable for several months [3] , frozen, partially purified in dissolved ammonium sulfate precipitation pellet, stable several weeks [1] , frozen, mycelium, several months without loss of activity [16] , 0-4 C, stable for 1 week in presence of saturated ammonium sulfate [5] , -20 C, slow decomposition by formation of large b-subunit aggregates devoid of a-subunits [13] , solution in 0.1 M phosphate, pH 7.0, 10 mM EDTA and 10 mM sodium sulfite, stable for several months [13] , 4 C, 6 months, no loss of activity [17]

References [1] Plaut, G.W.E.: Studies on the nature of the enzymic conversion of 6,7-dimethyl-8-ribityllumazine to riboflavin. J. Biol. Chem., 238, 2225-2243 (1963) [2] Wacker, H.; Harvey, R.A.; Winestock, C.H.; Plaut, G.W.E.: 4-(1'-d-ribitylamino)-5-amino-2,6-dihydroxypyrimidine, the second product of the riboflavin synthetase reaction. J. Biol. Chem., 239, 3493-3497 (1964) [3] Plaut, G.W.E.; Harvey, R.A.: The enzymatic synthesis of riboflavin. Methods Enzymol., 18B, 515-538 (1971) [4] Suzuki, Y.; Nishikawa, Y.; Mitsuda, H.: Catalytic properties of riboflavin synthetase from a high-riboflavinogenic Eremothecium ashbyii. J. Nutr. Sci. Vitaminol., 20, 301-316 (1974)

467

Riboflavin synthase

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[5] Mitsuda, H.; Kawai, F.; Suzuki, Y.: Assay methods, isolation procedures, and catalytic properties of riboflavin synthetase from spinach. Methods Enzymol., 18B, 539-543 (1971) [6] Harzer, G.; Rokos, H.; Otto, M.K.; Bacher, A.; Ghisla, S.: Biosynthesis of riboflavin. 6,7-Dimethyl-8-ribityllumazine 5-phosphate is not a substrate for riboflavin synthase. Biochim. Biophys. Acta, 540, 48-54 (1978) [7] Suzuki, A.; Goto, M.: Photolumazines, new naturally occurring inhibitors of riboflavin synthetase. Biochim. Biophys. Acta, 313, 229-234 (1973) [8] Ladenstein, R.; Ludwig, H.C.; Bacher, A.: Crystallization and preliminary Xray diffraction study of heavy riboflavin synthase from Bacillus subtilis. J. Biol. Chem., 258, 11981-11983 (1983) [9] Neuberger, G.; Bacher, A.: Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine by heavy riboflavin synthase from Bacillus subtilis. Biochem. Biophys. Res. Commun., 139, 1111-1116 (1986) [10] Ladenstein, R.; Schneider, M.; Huber, R.; Bartunik, H.-D.; Wilson, K.; Schott, K.; Bacher, A.: Heavy riboflavin synthase from Bacillus subtilis. Crystal structure analysis of the icosahedral b 60 capsid at 3.3 A resolution. J. Mol. Biol., 203, 1045-1070 (1988) [11] Schott, K.; Kellermann, J.; Lottspeich, F.; Bacher, A.: Riboflavin synthases of Bacillus subtilis. Purification and amino acid sequence of the a subunit. J. Biol. Chem., 265, 4204-4209 (1990) [12] Bacher, A.; Baur, R.; Eggers, U.; Harders, H.-D.; Otto, M.K.; Schnepple, H.: Riboflavin synthases of Bacillus subtilis. Purification and properties. J. Biol. Chem., 255, 632-637 (1980) [13] Bacher, A.: Heavy riboflavin synthase from Bacillus subtilis. Methods Enzymol., 122, 192-199 (1986) [14] Ludwig, H.C.; Lottspeich, F.; Henschen, A.; Ladenstein, R.; Bacher, A.: Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the b subunit. J. Biol. Chem., 262, 1016-1021 (1987) [15] Bacher, A.; Ludwig, H.C.; Schepple, H.: Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation. J. Mol. Biol., 187, 75-86 (1986) [16] Mitsuda, H.; Nakajima, K.; Nadamoto, T.; Yamada, Y.: Riboflavin synthetase from Eremothecium ashbyii and a salvage pathway of the by-product in the enzyme reaction. Methods Enzymol., 66, 307-323 (1980) [17] Suzuki, Y.; Terai, Y.; Abe, S.: Purification and some properties of riboflavin synthetase from Bacillus stearothermophilus ATCC 8005. Appl. Environ. Microbiol., 35, 258-263 (1978) [18] Ladenstein, R.; Ritsert, K.; Huber, R.; Richter, G.; Bacher, A.: The lumazine synthase/riboflavin synthase complex of Bacillus subtilis. X-ray structure analysis of hollow reconstituted b-subunit capsids. Eur. J. Biochem., 223, 1007-1017 (1994) [19] Kis, K.; Volk, R.; Bacher, A.: Biosynthesis of riboflavin. Studies on the reaction mechanism of 6,7-dimethyl-8-ribityllumazine synthase. Biochemistry, 34, 2883-2892 (1995)

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Riboflavin synthase

[20] Kis, K.; Bacher, A.: Substrate channeling in the lumazine synthase/riboflavin synthase complex of Bacillus subtilis. J. Biol. Chem., 270, 16788-16795 (1995) [21] Eberhardt, S.; Korn, S.; Lottspeich, F.; Bacher, A.: Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum. J. Bacteriol., 179, 2938-2943 (1997) [22] Illarionov, B.; Kemter, K.; Eberhardt, S.; Richter, G.; Cushman, M.; Bacher, A.: Riboflavin synthase of Escherichia coli. Effect of single amino acid substitutions on reaction rate and ligand binding properties. J. Biol. Chem., 276, 11524-11530 (2001) [23] Truffault, V.; Coles, M.; Diercks, T.; Abelmann, K.; Eberhardt, S.; Luttgen, H.; Bacher, A.; Kessler, H.: The solution structure of the N-terminal domain of riboflavin synthase. J. Mol. Biol., 309, 949-960 (2001) [24] Liao, D.I.; Wawrzak, Z.; Calabrese, J.C.; Viitanen, P.V.; Jordan, D.B.: Crystal structure of riboflavin synthase. Structure, 9, 399-408 (2001) [25] Eberhardt, S.; Zingler, N.; Kemter, K.; Richter, G.; Cushman, M.; Bacher, A.: Domain structure of riboflavin synthase. Eur. J. Biochem., 268, 4315-4323 (2001) [26] Gerhardt, S.; Schott, A.K.; Kairies, N.; Cushman, M.; Illarionov, B.; Eisenreich, W.; Bacher, A.; Huber, R.; Steinbacher, S.; Fischer, M.: Studies on the reaction mechanism of riboflavin synthase: X-ray crystal structure of a complex with 6-carboxyethyl-7-oxo-8-ribityllumazine. Structure, 10, 13711381 (2002) [27] Cushman, M.; Yang, D.; Gerhardt, S.; Huber, R.; Fischer, M.; Kis, K.; Bacher, A.: Design, synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase. J. Org. Chem., 67, 5807-5816 (2002) [28] Cushman, M.; Yang, D.; Mihalic, J.T.; Chen, J.; Gerhardt, S.; Huber, R.; Fischer, M.; Kis, K.; Bacher, A.: Incorporation of an amide into 5-phosphonoalkyl-6-d-ribitylaminopyrimidinedione lumazine synthase inhibitors results in an unexpected reversal of selectivity for riboflavin synthase vs lumazine synthase. J. Org. Chem., 67, 6871-6877 (2002)

469

Geranyltranstransferase

2.5.1.10

1 Nomenclature EC number 2.5.1.10 Systematic name geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase Recommended name geranyltranstransferase Synonyms farnesyl pyrophosphate synthetase farnesyl-diphosphate synthase farnesylpyrophosphate synthetase geranyl transferase I prenyltransferase Additional information (cf. EC 2.5.1.1 [2-5, 7-10, 12, 13, 15] the enzyme is named EC 2.5.1.1, here only enzymes for which geranyl diphosphate is not mentioned as substrate or is excluded as substrate are assigned to EC 2.5.1.1, enzymes for which reaction with geranyl diphosphate or geranyl diphosphate and dimethylallyl diphosphate is reported are assigned to EC 2.5.1.10) CAS registry number 37277-79-5

2 Source Organism

470

Saccharomyces cerevisiae [10, 12, 15] Ricinus communis (isoenzyme I and II [11]) [11] Pisum sativum [7] Gossypium hirsutum (isopentenyl diphosphate/dimethylallyl diphosphate isomerase and prenyltransferase, i.e. farnesyl diphosphate synthetase, exist as a multiprotein complex [4]) [4] Bacillus subtilis [1] Curcurbita pepo [2] Gallus gallus [3, 12, 15] Homo sapiens [5, 13, 23, 25] Sus scrofa (2 interconvertible enzyme forms which may result from a slow equilibrium between 2 conformational isomers of the protein [8]; interconvertible enzyme forms A and B [9]) [6, 8, 9, 12, 14]

2.5.1.10


Ricinus communis (geranyl transferase I and II [16]) [16] Bacillus stearothermophilus [17, 29] Lupinus albus (white lupin, farnesyl diphosphate synthase 1 [18] SwissProt-ID: P49351) [18] Lupinus albus (white lupin, farnesyl diphosphate synthase 2 [18] SwissProt-ID: P49352) [18] Parthenium argentatum (guayule rubber, farnesyl diphosphate synthase 1 [19] SwissProt-ID: Q24241) [19] Parthenium argentatum (guayule rubber, farnesyl diphosphate synthase 2 [19] SwissProt-ID: Q24242) [19] Hevea brasiliensis (rubber tree [20]) [20] Cinchona robusta [21] Morinda citrifolia [21] Rubia tinctorum [21] Tabernaemontana divaricata [21] Catharanthus roseus (low activity [21]) [21] Agrotis ipsilon (moth [22]) [22] Abies grandis (grand fir [24]) [24] Rattus norvegicus [26, 28] Mus musculus [27]

3 Reaction and Specificity Catalyzed reaction geranyl diphosphate + isopentenyl diphosphate = diphosphate + trans,transfarnesyl diphosphate ( bimolecular nucleophilic substitution, SN2 reaction, proceeds with an inversion of configuration at the diphosphate-bearing carbon of the allylic substrate and removal of the pro-R hydrogen from the C2 of isopentenyl diphosphate in these trans-polyprenol synthetic reactions [13]) Reaction type alkenyl group transfer Natural substrates and products S dimethylallyl diphosphate + isopentenyl diphosphate ( enzyme participates in isoprenoid biosynthesis in eukaryotes [2]) [2] P (E,E)-farnesyl diphosphate + diphosphate S isopentenyl diphosphate + geranyl diphosphate ( enzyme participates in isoprenoid biosynthesis in eukaryotes [2]) [2] P diphosphate + farnesyl diphosphate Substrates and products S dimethylallyl diphosphate + isopentenyl diphosphate ( at 88% the rate of geranyl diphosphate [7]; activity is higher than with geranyl diphosphate [2]; 2 sequential irreversible 1', 4 con-

471


P S P S

P S P

2.5.1.10

densations [13]; no activity with farnesyl diphosphate [29]) (Reversibility: ir [13]; ? [1-3, 7, 9-12, 16, 17, 24]) [1-3, 7, 9-13, 16, 17, 24, 29] (E,E)-farnesyl diphosphate + diphosphate ( farnesyl diphosphate and geranyl diphosphate in molar ratio of 17/4/1 [9]) [1-3, 7, 9-13, 16, 17, 24, 29] farnesyl diphosphate + isopentenyl diphosphate ( less than 5% of the activity with dimethylallyl diphosphate [2]) (Reversibility: ? [2,10,12]) [2, 10, 12] geranyl diphosphate + diphosphate [10] geranyl diphosphate + isopentenyl diphosphate ( all 4 geometrical isomers of farnesyl diphosphate are synthesized from isopentenyl diphosphate alone, isopentenyl diphosphate + geranyl diphosphate or isopentenyl diphosphate + neryl diphosphate, isopentenyl diphosphate/dimethylallyl diphosphate isomerase and prenyl transferase i.e. farnesyl diphosphate synthetase, exist as a multiprotein complex [4]) (Reversibility: ir [13]; ? [1-12,14,16-22]) [1-22] diphosphate + farnesyl diphosphate ( (E,E)-farnesyl diphosphate [1,11]; all 4 geometrical isomers of farnesyl diphosphate [4]) [1-22] neryl diphosphate + isopentenyl diphosphate ( at 0.8% the rate of geranyl diphosphate [7]) (Reversibility: ? [7]) [7] ?

Inhibitors 1-hydroxy-3(methylpentylamino)-propylidene-1,1-bisphosphonate ( 0.00002 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] 2,(3-pyridinyl)-1-hydroxyethylidene-1,1-bisphosphonate ( 0.00001 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] 2-(imidazol-1-yl)-hydroxyetylidene-1,1-bisphosphonate ( 0.000003 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] 3-amino-1-hydroxypropylidene-1,1-bisphosphonate ( 0.0002 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] 4-amino-1-hydroxybutylidene-1,1-bisphosphonate ( trivial name alendronate, 0.00046 mM, 50% inhibition of recombinant enzyme [23]; 0.00005 mM, 505 inhibition of recombinant farnesyl diphosphate synthase [25]) [23, 25] Ca2+ ( 5 mM, 80% inhibition [1]) [1] K+ ( 500 mM, 63% inhibition in the presence of MgCl2 [24]) [24] Mn2+ ( activates at 0.25-0.50 mM, inhibition above [11]; 1 mM, 50% inhibition [10]; strong inhibition above 0.1 mM, activation below [24]) [10, 11, 24] N-ethylmaleimide ( 8 mM, 54% inhbition [11]) [11]

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NE11089 ( 0.0029 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] NE11808 ( 0.00004 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] NE58051 ( 0.00293 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] [(cycloheptylamino)-methylene]bisphosphonate ( 0.00003 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] [1-hydroxy-2-imidazo(1,2-a)pyridin-3-ylethylidene]bisphosphonate ( 0.000003 mM, 50% inhibition of recombinant farnesyl diphosphate synthase [25]) [25] aryl tetrahydropyridine ( different compounds containing an aryl tetrahydropyridine structure are potent inhibitors of farnesyl diphosphate synthase [27]) [27] bacitracin [1] citronellylphosphonate [6] citronellylphosphonylphosphate ( competitive vs. geranyl diphosphate, noncompetitive vs. isopentenyl diphosphate [6]) [6] diphosphate ( 0.66 mM, 50% inhibition [24]) [24] etidronate ( 0.08 mM, 50% inhibition of recombinant enzyme [23]) [23] farnesyl diphosphate ( 0.06 mM, 50% inhibition [24]) [24] geranylphosphonate [6] geranylphosphonylphosphate ( competitive vs. geranyl diphosphate, noncompetitive vs. isopentenyl diphosphate [6]) [6] high ionic strength ( 8-12% activity in 500 mM Tris-HCl compared to 100% activity in 10 mM Tris-HCl [5]) [5] iodoacetamide ( 5 mM, 31% inhibition [2]; 8 mM, 33% inhibition [11]) [2, 11] iodoacetic acid ( 5% inhibition at 1 mM [7]; 10 mM, 80-90% inactivation after 15 min, dithiothreitol protects [5]) [5, 7] isopentenyl diphosphate ( above 0.002 mM, if the concentration of geranyl phosphate is less than 0.002 mM [5]) [5, 13] p-chloromercuribenzoic acid ( 0.15 mM, 882% inhibition of wildtype farnesyl diphosphate synthase, 11% inhibition of C73S/C289S mutant enzyme [17]) [17] p-hydroxymercuribenzoate ( 11% inhibition at 1 mM [7]; 1 mM, 25% inhibition [11]) [7, 11] pamidronate ( 0.0005 mM, 50% inhibition of recombinant enzyme [23]) [23] phenylglyoxal ( 2 mM, inactivation half-life: 20 min, 3,3-dimethylallyl diphosphate and geranyl diphosphate protect [5]; biphasic inactivation with half-lives of 9.6 and 23 min respectively, in absence of Mg2+ or Mn2+ only 1 mM geranyl diphosphate protects, in presence of 1 mM Mg2+ , isopentenyl diphosphate, dimethylallyl diphosphate or geranyl diphosphate give additional protection over that observed with the metal ions [9]) [5, 9, 13] 473


2.5.1.10

phosphate buffer [5] risedronate ( 0.0000039 mM, 50% inhibition of recombinant enzyme [23]) [23] Additional information ( neither iodoacetamide nor N-ethylmaleimide inhibits at concentration less than 5 mM [11]; inhibition by irradiation with the photolabile analog of geranyl diphosphate [12]; not inhibited by 10 mM iodoacetic acid or iodoacetamide [4]; not inhibited by clodronate [23]) [4, 11, 12, 23] Activating compounds 1,4-dithiothreitol ( 2 mM, 1.6fold stimulation [1]; after dialysis against Tris-HCl buffer, pH 7.8 the enzyme becomes completely dependent on dithiols or thiols for activity, half-maximal activation at 0.48 mM [5]) [1, 5, 13] 2-mercaptoethanol ( 10 mM, 1.2fold stimulation [1]) [1] ATP ( 3 mM, required for maximal activity [7]) [7] NH+4 ( 3fold stimulation with geranyl diphosphate as substrate [1]) [1] NH+4 ( 5fold stimulation with dimethylallyl diphosphate as substrate [1]) [1] Triton X-100 ( 0.5%, 3fold activation [1]) [1] Tween 80 ( 0.5%, 2.3fold activation [1]) [1] dithioerythritol ( after dialysis against Tris-HCl buffer, pH 7.8 the enzyme becomes completely dependent on dithiols or thiols for activity, half-maximal activation at 0.63 mM [5]) [5, 13] Additional information ( not activated by Tween 20, Triton X-100, Chaps or Nonidet P-40 [24]) [24] Metals, ions Ca2+ ( 18% of Mg2+ activation at 0.5 mM, inhibition above [24]) [24] Co2+ ( 78% of Mg2+ activation at 1 mM [24]) [24] K+ ( stimulates with geranyl diphosphate as substrate [1]) [1] Mg2+ ( required for activity [1, 2, 4, 5, 7, 10, 11, 13, 24, 28]; Mg2+ or Mn2+ are absolutely required for activity, halfmaximal activation at 0.089 mM [5]; maximum activity at 6 mM [7]; maximum activity at 1 mM [10]; maximum activity at 1-2 mM [11]; maximal activity of farnesol synthesis at 5 mM [4]; maximal activity at 1 mM [24]; maximal activity at 1 mM, inhibition at high concentrations [28]) [1, 2, 4, 5, 7, 10, 11, 13, 24, 28] Mn2+ ( can partially replace Mg2+ , less effective [1,2]; Mn2+ or Mg2+ are absolutely required for activity, half-maximal activation at 0.0037 mM [5]; activation at 0.25-0.50 mM, inhibition above [11]; can replace Mg2+ , maximal activity at 0.1 mM, inhibition above [24]) [1, 2, 5, 11, 13, 24] Ni2+ ( 10% of Mg2+ activation at 0.5 mM, inhibition above [24]) [24]

474

2.5.1.10


Zn2+ ( 91% of Mg2+ activation at 0.5 mM, almost complete inhibition at 5 mM [24]) [24] Additional information ( not activated by Na+ or Li+ [1]) [1] Specific activity (U/mg) 0.0012 [1] 0.04 ( isoenzyme II [11]) [11] 0.06 ( isoenzyme I [11]) [11] 0.35 ( C289F mutant enzyme at 55 C [17]) [17] 0.52 ( recombinant enzyme, substrate geranyl diphosphate [18]) [18] 0.58 ( recombinant enzyme, substrate dimethylallyl diphosphate [18]) [18] 0.914 [12] 0.922 [5, 13] 1.22 [3] 1.3 ( C73F mutant enzyme at 45 C [17]) [17] 1.5 [12] 1.85 [15] 2.33 [15] 2.61 ( wild-type enzyme at 45 C [17]) [17] 3.94 ( C289S mutant enzyme at 55 C [17]) [17] 3.97 ( C73S/C289S mutant enzyme at 55 C [17]) [17] 4.3 ( C73F mutant enzyme at 55 C [17]) [17] 4.69 ( wild-type enzyme at 55 C [17]) [17] 5.22 [10, 12] Additional information ( 12400 units, 1 unit is defined as the amount of enzyme catalysing the incorporation of 100 dmp (disintergrations per minute) of isopentenyl diphosphate into farnesyldiphosphte [7]) [7] Km-Value (mM) 0.00031 (isopentenyl diphosphate) [8] 0.00044 (geranyl diphosphate) [5] 0.0005 (isopentenyl diphosphate, cosubstrate geranyl diphosphate [3]) [3] 0.00058 (geranyl diphosphate) [8] 0.0006 (geranyl diphosphate) [28] 0.00094 (isopentenyl diphosphate) [5] 0.0012 (dimethylallyl diphosphate) [28] 0.0013 (geranyl diphosphate) [2] 0.0018 (geranyl diphosphate) [24] 0.003 (isopentenyl diphosphate) [28] 0.004 (isopentenyl diphosphate) [10] 0.0045 (dimethylallyl diphosphate, 0.042-0.056 mg/ml protein, isoenzyme II, Km values depend on protein concentration [11]) [11] 0.0059 (dimethylallyl diphosphate, 0.042-0.056 mg/ml protein, isoenzyme I, Km values depend on protein concentration [11]) [11] 0.008 (dimethylallyl diphosphate) [10] 475


2.5.1.10

0.009 (dimethylallyl diphosphate) [24] 0.014 (isopentenyl diphosphate, [7]; cosubstrate geranyl diphosphate [10]) [7, 10] 0.0153 (isopentenyl diphosphate) [24] 0.018 (geranyl diphosphate) [1] 0.032 (isopentenyl diphosphate, 0.042-0.056 mg/ml protein, isoenzyme II, Km values depend on protein concentration [11]) [11] 0.036 (geranyl diphosphate, 0.042-0.056 mg/ml protein, isoenzyme I, Km values depend on protein concentration [11]) [11] 0.039 (geranyl diphosphate, 0.042-0.056 mg/ml protein, isoenzyme II, Km values depend on protein concentration [11]) [11] 0.0445 (Mn2+ ) [24] 0.048 (isopentenyl diphosphate, 0.042-0.056 mg/ml protein, isoenzyme I, Km values depend on protein concentration [11]) [11] 0.05 (dimethylallyl diphosphate) [1] 0.0712 (Mg2+ ) [24] 4.7 (isopentenyl diphosphate, wild-type enzyme at 45 C [17]) [17] 4.8 (isopentenyl diphosphate, C73F mutant enzyme at 45 C [17]) [17] 4.9 (geranyl diphosphate, C73F mutant enzyme at 45 C [17]) [17] 5.6 (geranyl diphosphate, C73S/C289S mutant enzyme at 55 C [17]) [17] 5.8 (geranyl diphosphate, wild-type enzyme at 45 C [17]) [17] 6.4 (geranyl diphosphate, C289S mutant enzyme at 55 C [17]) [17] 7.3 (geranyl diphosphate, C73F mutant enzyme at 55 C [17]) [17] 7.5 (geranyl diphosphate, C289F mutant enzyme at 55 C [17]) [17] 8.4 (geranyl diphosphate, wild-type enzyme at 55 C [17]) [17] 11.6 (isopentenyl diphosphate, C73F mutant enzyme at 55 C [17]) [17] 13 (isopentenyl diphosphate, wild-type enzyme at 55 C [17]) [17] 13.3 (isopentenyl diphosphate, C73S/C289S mutant enzyme at 55 C [17]) [17] 14.3 (isopentenyl diphosphate, C289S mutant enzyme at 55 C [17]) [17] 130 (isopentenyl diphosphate, C289F mutant enzyme at 55 C [17]) [17] Ki-Value (mM) 0.0007 (isopentenyl diphosphate) [13] 0.00125 (citronellylphosphonylphosphate, competitive inhibition [6]) [6] 0.0015 (geranylylphosphonylphosphate, competitive inhibition [6]) [6]

476

2.5.1.10


0.0016 (geranylphosphonylphosphate, uncompetitive inhibition [6]) [6] 0.0033 (citronellylphosphonylphosphate, uncompetitive inhibition [6]) [6] pH-Optimum 5.5-6.5 ( mitochondrial enzyme [28]) [28] 5.8-7.6 [10] 6.8 [11] 6.8-7.2 ( partially purified enzyme, probbly part of an multienzyme complex [4]) [4] 7.3-8.8 [5, 13] 7.5 [2] 7.5-8 [24] 7.6 [7] 8.5 [1] pH-Range 5.5-8.5 ( approx. 45% of maximal activity at pH 5.5, approx. 60% of activity maximum at pH 8.5, emzyme I [11]) [11] 6.3-8.2 ( approx. 50% of maximal activity at pH 6.3 and pH 8.2 [7]) [7] Temperature optimum ( C) 37 ( assay at [1, 10, 12, 13]) [1, 10, 12, 13]

4 Enzyme Structure Molecular weight 56000 ( gel filtration, enzyme I, protein concentration 0.02 mg/ml [11]) [11] 60000 ( gel filtration, enzyme II, protein concentration 0.02 mg/ml [11]) [11] 63100 ( enzyme form A, ultracentrifugal analysis [9]) [9] 67000 ( gel filtration [1]) [1] 72500 ( gel filtration, protein concentration 25 mg/ml [11]) [11] 74000 ( gel filtration [5,13]) [5, 13] 75900 ( enzyme form B, ultracentrifugal analysis [9]) [9] 80500 (Saccharomyces cerevisiae, gel filtration) [10] 83000 ( gel filtration [3]) [3] 89000 ( recombinant His-tagged fusion protein, gel filtration [18]) [18] 96000 ( gel filtration [7]) [7] 110000 ( gel filtration [24]) [24]

477


2.5.1.10

Subunits ? ( x * 39000, SDS-PAGE, deduced from nucleotide sequence [19]; x * 47000, immunoblot, deduced from nucleotide sequence [20]) [19, 20] dimer ( 2 * 42900, SDS-PAGE [3]; 2 * 38400, SDSPAGE [5]; 2 * 45000, SDS-PAGE [7]; 2 * 38000, SDS-PAGE of enzyme reduced by mercaptoethanol and alkylated with iodoacetic acid [8]; 2 * 38500, ultracentrifugal analysis in presence of 6 M guanidine, SDS-PAGE [9]; 2 * 43000, SDS-PAGE [10]; 2 * 38750, immunoblot [18]) [3, 5, 7-10, 18] Posttranslational modification Additional information ( homogeneous enzyme is free of lipid and contains no carbohydrate [8]) [8]

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture [21] corpus allatum ( high expression level [22]) [22] epidermis ( latex producing cells [20]) [20] fruit [2] liver [3, 5, 6, 8, 9, 15, 28] phloem ( latex producing cells [20]) [20] prostate ( epithelial cells, expression is up-regulated in Dunning rat cancer cell lines [26]) [26] sapling [24] seed ( germinating seed [16]) [16, 18] seedling [7] stembark [19] Localization cytosol ( high activity [28]) [28] mitochondrial inner membrane [28] mitochondrial matrix [28] mitochondrion ( 1/6 of the cytosolic activity [28]) [28] Purification (ammonium sulfate, affinity chromatography [15]; ammonium sulfate, DEAE-cellulose, hydroxylapatite, isoelectric focusing [10]) [10, 15] [11] (ammonium sulfate, CM-52, Sephadex G-200, DEAE-52 [7]) [7] (ammonium sulfate, DEAE-cellulose, DEAE-Sephadex, Sepharose 6 B, 3 proteins after electrophoresis [4]) [4] (ammonium sulfate, Sephadex G-100, hydroxylapatite, DEAE-Sephadex A-50 [1]) [1] (ammonium sulfate, Sephadex G 200, hydroxylapatite [2]) [2]

478

2.5.1.10


(ammonium sulfate, affinity chromatography [15]; ammonium sulfate, DEAE-cellulose, hydroxylapatite [3]) [3, 15] (ammonium sulfate, CM-52, DE-52, butyl-agarose, calcium phosphate gel [5]) [5] (acid treatment, DE-52 cellulose, hydroxylapatite [8]) [8] (recombinant enzyme, Ni2+ -affinity column, Mono Q [18]) [18] (DEAE-Sepharose, Mono Q, phenyl-Sepharose [24]) [24] Crystallization [3, 12] Cloning (expression in Escherichia coli [23]) [23, 25] (overexpression of C73F, C289F, C73S, C289S and C73/C289S mutant enzyme in Escherichia coli [17]; expression of wild-type, S82F, S82Y, S82W, L83F, L83Y, I84F and I84Y mutant enzymes in Escherichia coli [29]) [17, 29] (expression in Escherichia coli [18]) [18] (expression in Escherichia coli [19]) [19] (expression in Escherichia coli [19]) [19] (expression in yeast [20]) [20] (cloning of cDNA [22]) [22] (cloning of cDNA [18]) [18] Engineering C289F ( approx. 1/13 of wild-type activity at 55 C [17]) [17] C289S ( similar activity like wild-type [17]) [17] C73F ( heat sensitive mutant [17]) [17] C73S ( similar activity like wild-type [17]) [17] C73S/C289S ( similar activity like wild-type [17]) [17] I84F ( 7.2% of wild-type activity with dimethylallyl diphosphate [29]) [29] I84Y ( similiar activity as wild-type [29]) [29] L83F ( similiar activity as wild-type [29]) [29] L83Y ( similiar activity as wild-type [29]) [29] S82F ( produces exclusively geranyl diphosphate, i.e. the mutant enzyme has changed from a farnesyl diphosphate synthase to a geranyl diphosphate synthase [29]) [29] S82W ( no prenyl transferase activity [29]) [29] S82Y ( similiar activity as wild-type [29]) [29]

6 Stability Temperature stability 55 ( wild-type: stable for 30 min, C73F mutant: complete loss of activity after 20 min [17]) [17]

479


2.5.1.10

Oxidation stability , enzyme seems to be susceptible to oxidation when treated with Cu2+ , forming an intrasubunit disulfide bond rather than intersubunit bonds [17] General stability information , thiol reducing agents are detrimental for stability [13] Storage stability , -18 C, 10 mM phosphate buffer, pH 8.0, 20 mM 2-mercaptoethanol, 105 glycerol, 4-5 days, 50% loss of activity [4] , -20 C, at least 1 week, no loss of activity [1] , 4 C, 5 days, 36% loss of activity [1] , crystalline suspension, 1 year, no loss of activity [12] , crystalline suspension, at least 2 months, no loss of activity [3] , 4 C, 10 mM Tris-HCl, pH 7.2-7.8, without any additives, less than 5% loss in 24 h [5] , 4 C, precipitate in neutral (NH4 )2 SO4 solution at 60% saturation, 1 year, 50-70% loss of activity [5, 13] , -80 C, 50% glycerol, 14 mM 2-mercaptoethanol, at least 2 weeks, no loss of activity [18] , -80 C, 3 months, no loss of activity [24]

References [1] Takahashi, I.; Ogura, K.: Farnesyl pyrophosphate synthetase from Bacillus subtilis. J. Biochem., 89, 1581-1587 (1981) [2] Ogura, K.; Nishino, T.; Seto, S.: The purification of prenyltransferase and isopentenyl pyrophosphate isomerase of pumpkin fruit and their some properties. J. Biochem., 64, 197-203 (1968) [3] Reed, B.C.; Rilling, H.C.: Crystallization and partial characterization of prenyltransferase from avian liver. Biochemistry, 14, 50-54 (1975) [4] Widmaier, R.; Howe, J.; Heinstein, P.: Prenyltransferase from Gossypium hirsutum. Arch. Biochem. Biophys., 200, 609-616 (1980) [5] Barnard, G.F.; Popjak, G.: Human liver prenyltransferase and its characterization. Biochim. Biophys. Acta, 661, 87-99 (1981) [6] Popjak, G.; Hadley, C.: Inhibition of liver prenyltransferase by citronellyl and geranyl phosphonate and phosphonylphosphate. J. Lipid Res., 26, 1151-1159 (1985) [7] Allen, B.E.; Banthorpe, D.V.: Partial purification and properties of prenyltransferase from Pisum sativum. Phytochemistry, 20, 35-40 (1981) [8] Yeh, L.-S.; Rilling, H.C.: Purification and properties of pig liver prenyltransferase: interconvertible forms of the enzyme. Arch. Biochem. Biophys., 183, 718-725 (1977) [9] Barnard, G.F.; Popjak, G.: Characterization of liver prenyl transferase and its inactivation by phenylglyoxal. Biochim. Biophys. Acta, 617, 169-182 (1980)

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[10] Eberhardt, N.L.; Rilling, H.C.: Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties. J. Biol. Chem., 250, 863-866 (1975) [11] Green, T.R.; West, C.A.: Purification and characterization of two forms of geranyl transferase from Ricinus communis. Biochemistry, 13, 4720-4729 (1974) [12] Rilling, H.C.: Eukaryotic prenyltransferases. Methods Enzymol., 110, 145152 (1985) [13] Barnard, G.F.: Prenyltransferase from human liver. Methods Enzymol., 110, 155-171 (1985) [14] Cornforth, J.W.; Cornforth, R.H.; Popjak, G.; Yengoyan, L.: Studies on the biosynthesis of cholesterol. XX. Steric course of decarboxylation of 5-pyrophosphomevalonate and of the carbon to carbon bond formation in the biosynthesis of farnesyl pyrophosphate. J. Biol. Chem., 241, 3970-3987 (1966) [15] Bartlett, D.L.; King, C.-H. R.; Poulter, C.D.: Purification of farnesylpyrophosphate synthetase by affinity chromatography. Methods Enzymol., 110, 171-184 (1985) [16] Green, T.R.; Dennis, D.T.; West, C.A.: Compartemamtation of isopentenyl pyrophosphate isomerase and prenyl transferase in developing castor bean endosperm. Biochem. Biophys. Res. Commun., 64, 976-982 (1975) [17] Koyama, T.; Obata, S.; Saito, K.; Takeshita-Koike, A.; Ogura, K.: Structural and functional roles of the cysteine residues of Bacillus stearothermophilus farnesyl diphosphate synthase. Biochemistry, 33, 12644-12648 (1994) [18] Attucci, S.; Aitken, S.M.; Gulick, P.J.; Ibrahim, R.K.: Farnesyl pyrophosphate synthase from white lupine: molecular cloning, expression, and purification of the expressed protein. Arch. Biochem. Biophys., 321, 493-500 (1995) [19] Pan, Z.; Herickhoff, L.; Backhaus, R.A.: Cloning, characterization, and heterologous expression of cDNAs for farnesyl diphosphate synthase from the guayule rubber plant reveals that this prenyltransferase occurs in rubber particles. Arch. Biochem. Biophys., 332, 196-204 (1996) [20] Adiwilaga, K.; Kush, A.: Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis). Plant Mol. Biol., 30, 935-946 (1996) [21] Ramos-Valdivia, A.C.; Van Der Heijden, R.; Verpoorte, R.: Isopentenyl diphosphate isomerase and prenyltransferase activities in Rubiaceous and Apocynaceous cultures. Phytochemistry, 48, 961-969 (1998) [22] Castillo-Gracia, M.; Couillaud, F.: Molecular cloning and tissue expression of an insect farnesyl diphosphate synthase. Eur. J. Biochem., 262, 365-370 (1999) [23] Bergstrom, J.D.; Bostedor, R.G.; Masarachia, P.J.; Reszka, A.A.; Rodan, G.: Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch. Biochem. Biophys., 373, 231-241 (2000) [24] Tholl, D.; Croteau, R.; Gershenzon, J.: Partial purification and characterization of the short-chain prenyltransferases, gernayl diphospate synthase and farnesyl diphosphate synthase, from Abies grandis (grand fir). Arch. Biochem. Biophys., 386, 233-242 (2001) 481


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[25] Dunford, J.E.; Thompson, K.; Coxon, F.P.; Luckman, S.P.; Hahn, F.M.; Poulter, C.D.; Ebetino, F.H.; Rogers, M.J.: Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates. J. Pharmacol. Exp. Ther., 296, 235-242 (2001) [26] Jiang, F.; Yang, L.; Cai, X.; Cyriac, J.; Shechter, I.; Wang, Z.: Farnesyl diphosphate synthase is abundantly expressed and regulated by androgen in rat prostatic epithelial cells. J. Steroid Biochem. Mol. Biol., 78, 123-130 (2001) [27] Gwaltney, S.L.; O'Connor, S.J.; Nelson, L.T.J.; Sullivan, G.M.; Imade, H.; Wang, W.; Hasvold, L.; Li, Q.; Cohen, J.; Gu, W.-Z.; Tahir, S.K.; Bauch, J.; Marsh, K.; Ng, S.-C.; Frost, D.J.; Zhang, H.; Muchmore, S.; Jakob, C.G.; Stoll, V.; Hutchins, C.; Rosenberg, S.H.; Sham, H.L.: Aryl tetrahydropyridine inhibitors of farnesyltransferase: bioavailable analogues with improved cellular potency. Bioorg. Med. Chem. Lett., 13, 1363-1366 (2003) [28] Runquist, M.; Ericsson, J.; Thelin, A.; Chojnacki, T.; Dallner, G.: Isoprenoid biosynthesis in rat liver mitochondria. Studies on farnesyl pyrophosphate synthase and trans-prenyltransferase. J. Biol. Chem., 269, 5804-5809 (1994) [29] Narita, K.; Ohnuma, S.-I.; Nishino, T.: Protein design of geranyl diphosphate synthase. Structural features that define the product specificities of prenyltransferases. J. Biochem., 126, 566-571 (1999)

482

trans-Octaprenyltranstransferase

2.5.1.11

1 Nomenclature EC number 2.5.1.11 Systematic name (E)-octaprenyl-diphosphate:isopentenyl-diphosphate octaprenyltranstransferase Recommended name trans-octaprenyltranstransferase Synonyms SPP synthase all-trans-nonaprenyl-diphosphate synthase nonaprenyl pyrophosphate synthetase polyprenylpyrophosphate synthetase solanesyl pyrophosphate synthetase solanesyl-diphosphate synthase synthetase, solanesyl pyrophosphate terpenoidallyltransferase terpenyl pyrophosphate synthetase trans-octaprenyltranstransferase trans-prenyltransferase CAS registry number 64763-52-6

2 Source Organism

Morus bombysis (mulberry [6]) [6] Micrococcus luteus [1, 3, 4, 5, 7, 8] Micrococcus lysodeikticus [2] Rattus norvegicus [9, 14] Escherichia coli (wild-type and mutants [11]) [4, 11, 13] Arabidopsis thaliana [10] Rhodobacter capsulatus [12] Spinacia oleracea [15]

483


2.5.1.11

3 Reaction and Specificity Catalyzed reaction all-trans-octaprenyl diphosphate + isopentenyl diphosphate = diphosphate + all-trans-nonaprenyl diphosphate ( mechanism [8]) Reaction type alkenyl group transfer Natural substrates and products S all-trans-octaprenyl diphosphate + isopentenyl diphosphate ( supplies the prenyl side chain of menaquinone-9 [2]; provides isoprenoid precursors for menaquinones [3]; production of prenylquinone side chains of plastoquinone and ubiquinone [10]; determines the side chain of ubiquinone [11,12,13]) [2, 3, 10, 11, 12, 13] P polyprenyldiphosphate + diphosphate Substrates and products S (E,E,E)-geranylgeranyl diphosphate + isopentenyl diphosphate ( about 25% of the activity with geranyl diphosphate [9]) (Reversibility: ? [2, 3, 6, 9, 10, 15]) [2, 3, 6, 9, 10, 15] P ? S farnesyl diphosphate + isopentenyl diphosphate ( about 60% the rate with geranyl diphosphate [9]) (Reversibility: ? [1-11, 13, 14, 15]) [1-11, 13, 14, 15] P polyprenyl diphospate + diphosphate ( distribution of polyprenyl diphosphates changes depending on Mg2+ concentration [1,3]; 0.5 mM Mg2+ : prenyl products ranging in carbon chain from C-20 to C-40, 20 mM Mg2+ : octaprenyl diphosphate and solanesyl diphosphate as main products [1]; chain-length distribution of reaction products changes according to the concentration of the complex formed between isopentenyl diphosphate and Mg2+ [5]; a series of ficaprenyl-type Z,E-mixed polyprenyl diphosphates with carbon chain length ranging from C-40 to C-60 [6]; weak production of (all-E)-nonaprenyl diphosphate + (all-E)-octaprenyl diphosphate [7]; with MgCl2 : C-45 diphosphate as the longest product, with MnCl2 or CoCl2 : C-50 diphosphate and C-55 diphosphate as the longest product [4]; main product is solanesyl diphosphate [10]; product after expression in yeast: octaprenyl diphosphate [13]; products: nona- and decaprenyl diphosphate [14]) [1, 4-6, 10, 13, 14] S farnesyl imidodiphosphate + isopentenyl diphosphate (Reversibility: ? [8]) [8] P solanesyl diphosphate + ? [8] S farnesyl methylenediphosphonate + isopentenyl diphosphate (Reversibility: ? [8]) [8] P solanesyl diphosphate + ? [8] S farnesyl phosphosulfate + isopentenyl diphosphate (Reversibility: ? [8]) [8]

484

2.5.1.11


P solanesyl diphosphate + ? [8] S geranyl diphosphate + isopentenyl diphosphate ( (E)geranyl diphosphate, no activity with (Z)-geranyl diphosphate i.e. nerol diphosphate [9]; stereospecificity: 2-pro-S hydrogen of isopentenyl diphosphate is lost during the formation of the product [6]) (Reversibility: ? [1-3, 6-9, 10, 12, 15]) [1-3, 6-9, 10, 12, 15] P polyprenyl diphosphates + diphosphate ( distribution of polyprenyl diphosphates changes depending on Mg2+ concentration [1, 3, 5]; 0.1 mM Mg2+ : heptaprenyl diphosphate and its shorter prenyl homologues [1]; 0.2 mM Mg2+ : major product is heptaprenyl diphosphate, octaprenyl diphosphate appears as the longest product [1]; 2 mM Mg2+ : solanesyl diphosphate appears as the longest product [1]; 20 mM Mg2+ : octaprenyl diphosphate and solanesyl diphosphate are the main products [1]; all-trans octaprenyl diphosphate and all-trans nonaprenyl diphosphate, no accumulation of prenyl diphosphate with chain length shorter than C-40 [2]; all-trans-prenyl diphosphates ranging in carbon number up to C-45 [3]; a series of ficaprenyl-type Z,E-mixed polyprenyl diphosphates with carbon chain length ranging from C-40 to C-60 [6]; solanesyl diphosphate + unidentified polyprenyl metabolite [9]; main product is solanesyl diphosphate [10]; main product is solanesyl diphosphate, which results in the synthesis of Q-9, minor products are octaprenol, hexaprenol and decaprenol [12]; chloroplast enzyme produces C50-C65 chains, microsomal enzyme produces C70-C85 chains [15]) [1-3, 5, 6, 9, 10, 12, 15] S geranyl imidodiphosphate + isopentenyl diphosphate (Reversibility: ? [8]) [8] P solanesyl diphosphate + ? [8] S geranyl methylenediphosphonate + isopentenyl diphosphate (Reversibility: ? [8]) [8] P solanesyl diphosphate + ? [8] S geranyl phosphosulfate + isopentenyl diphosphate (Reversibility: ? [8]) [8] P solanesyl diphosphate + ? [8] S Additional information ( no substrate: (Z,E,E)-geranylgeranyl diphosphate [2,3]; no substrate: dimethylallyl phosphate [8]; no substrate: geranyl phosphate [8]; enzyme does not accept C5 or C6 allylic diphosphates as primer substrates [10]) [2, 3, 8, 10] P ? Inhibitors (E,E,E)-geranylgeranyl diphosphate [9] Ca2+ ( complete inactivation at 1 mM in the absence of Mg2+ [9]) [9] SDS ( complete inactivation at 0.1% [9]) [9] Triton X-100 ( 73% inactivation at 0.1% [9]) [9] Tween 80 ( activates at 0.005%, inhibits at 0.1% [9]) [9] Zn2+ ( complete inactivation at 1 mM in the absence of Mg2+ [9]) [9] deoxycholate ( 80% inactivation at 0.1% [9]) [9]

485


2.5.1.11

imidodiphosphate [8] methylenediphosphonate [8] solanesyl diphosphate [9] taurodeoxycholate ( 67% inactivation at 0.1% [9]) [9] Activating compounds Triton X-100 ( stimulates [6]) [6] Tween 80 ( 0.05-0.1%, 2-fold stimulation [3]; activates at 0.005%, inhibits at 0.1% [9]) [3, 9] bacitracin ( 0.03-0.06% stimulates 2fold [3]; dependent on concentration [7]) [3, 7] bovine serum albumin ( stimulates, dependent on concentration [7]) [7] digitonin ( activates [9]) [9] taurodeoxycholate ( activates [9]) [9] Additional information ( not dependent on cytosolic protein factors [9]; enzyme is stimulated by a high molecular mass fraction HMF which is separated from cell-free extracts of the same bacterium, probably containing a factor which binds to polyprenyl products and removes them from the active site of the enzyme to facilitate and maintain the turnover of catalysis [7]) [7, 9] Metals, ions Mg2+ ( activates [9]; required [3]; changes distribution of polyprenyl diphosphates as products depending on concentration [1,3]; geranyl diphosphate + isopentenyl diphosphate: optimum concentration 2 mM [1]; farnesyl diphosphate + isopentenyl diphosphate: optimum concentration 0.5 mM [1]; concentration dependent stimulation between 1 and 4 mM [9]; optimal activity at 5 mM [14]) [1, 3, 5, 8, 9, 14] Mn2+ ( less effective than Mg2+ in activation [3]; 0.1 and 0.2 mM [9]) [3, 8, 9] Additional information ( alteration of polyprenyl diphosphate products depending on divalent metal ion, with MgCl2 : C-45 diphosphate as the longest product, with MnCl2 or CoCl2 : C-50 diphosphate and C-55 diphosphate as the longest products [4]) [4] Turnover number (min±1) 1260 (geranylgeranyl diphosphate) [10] 1782 (farnesyl diphosphate) [10] Specific activity (U/mg) 0.000787 [3] Additional information [7] Km-Value (mM) 0.00068 (isopentenyl diphosphate) [14] 0.00075 (farnesyl diphosphate, stimulated by a high molecular mass fraction [7]) [3, 7]

486

2.5.1.11


0.00076 (farnesyl diphosphate, without stimulation by a high molecular mass fraction [7]) [7] 0.001 (farnesyl methylenediphosphonate) [8] 0.00161 (geranyl diphosphate) [10] 0.0043 (geranyl diphosphate) [8] 0.0052 (farnesyl diphosphate) [8] 0.0054 (geranyl methylenediphosphonate) [8] 0.0057 (farnesyl imidodiphosphate) [8] 0.00573 (farnesyl diphosphate) [10] 0.0077 (geranyl imidodiphosphate) [8] 0.0133 (farnesyl phosphosulfate) [8] 0.017 (isopentenyl diphosphate, stimulated by a high molecular mass fraction [7]) [7] 0.023 (isopentenyl diphosphate, without stimulation by a high molecular mass fraction [7]) [7] 0.025 (farnesyl diphosphate) [3] 0.0909 (geranyl phosphosulfate) [8] pH-Optimum 7-8 [3] 7.4 ( assay at [1-3]) [1-3] 8 [9, 10] Temperature optimum ( C) 30 ( assay at [13]) [13] 37 ( assay at [1-4,7]) [1-4, 7]

4 Enzyme Structure Molecular weight 74000-78000 ( gel filtration [7]) [7] 78000 ( gel filtration [2,3]) [2, 3] 79000 ( SDS-PAGE after treatment with protein crosslinker disuccinimidyl suberate [11]) [11] 108000 ( gel filtration [10]) [10] Subunits dimer ( 2 * 34000, SDS-PAGE [7]; 2 * 46600 [10]; heterodimer [11]) [7, 10, 11]

5 Isolation/Preparation/Mutation/Application Source/tissue leaf [6, 15] liver [9, 14]

487


2.5.1.11

Localization chloroplast [15] cytosol [14] microsome ( cytoplasmic surface of rough and smooth microsomes [9]) [9, 15] mitochondrial inner membrane [14, 15] Purification (partial [6]) [6] (18.7fold [3]; partial [4]; homogeneity [5,7]) [3, 4, 5, 7] [2] (partial [4]; wild type and A79Y mutant [11]) [4, 11] [10] Cloning [11, 13] [10] Engineering A79Y ( no functional activity when expressed in E. coli [11]) [11] A79Y ( produces mainly Q-6 when expressed in E. coli [11]) [11] K170A ( no functional activity when expressed in E. coli [11]) [11] K170G ( no functional activity when expressed in E. coli [11]) [11] K235L ( produces mainly Q-8 and lesser amounts of Q-5 and Q-7 when expressed in Escherichia coli [11]) [11] R321A ( produces mainly Q-8 and lesser amounts of Q-5 and Q-7 when expressed in Escherichia coli [11]) [11] R321D ( temperature sensitive [11]) [11] R321V ( produces mainly Q-6 and lesser amounts of Q-5 and Q-7 when expressed in Escherichia coli, produces mainly Q-6, Q-5 and Q-7 and lesser amounts of Q-8 when expressed in yeast [11]) [11] Y37A ( no functional activity when expressed in E. coli [11]) [11] Y37A/Y38A ( produces mainly Q-6 and Q-7 and lesser amounts of Q-8 when expressed in Escherichia coli, produces mainly Q-8 and Q-7 and lesser amounts of Q-5 and Q-6 when expressed in yeast [11]) [11] Y38A ( produces mainly Q-7 and lesser amounts of Q-6 and Q-8 when expressed in E. coli, produces mainly Q-8 and lesser amounts of Q-7 when expressed in yeast [11]) [11] Y38A/R321V ( produces mainly Q-6 and lesser amounts of Q-5 and Q-7 when expressed in Escherichia coli, produces mainly Q-5 and lesser amounts of Q-5 and Q-8 when expressed in yeast [11]) [11] Y38A/R321V ( produces mainly Q-8 and lesser amounts of Q-7 when expressed in Escherichia coli, enzyme is temperature sensitive [11]) [11]

488

2.5.1.11


6 Stability Temperature stability 40 ( stable below for 10 min at pH 8 [10]) [10] 43 ( no growth of R321A and R321D mutants [11]) [11] 55 ( 6 min, about 80% loss of activity [7]) [7] Storage stability , -20 C, partially purified enzyme is stable for at least 2 weeks [3]

References [1] Fujii, H.; Sagami, H.; Koyama, T.; Ogura, K.; Seto, S.: Variable product specificity of solanesyl pyrophosphate synthetase. Biochem. Biophys. Res. Commun., 96, 1648-1653 (1980) [2] Sagami, H.; Ogura, K.; Seto, S.: Solanesyl pyrophosphate synthetase from Micrococcus lysodeikticus. Biochemistry, 16, 4616-4622 (1977) [3] Sagami, H.; Ogura, K.: Nonaprenylpyrophosphate synthetase from Micrococcus luteus. Methods Enzymol., 110, 206-209 (1985) [4] Ohnuma, S.-i.; Koyama, T.; Ogura, K.: Alteration of the product specificities of prenyltransferases by metal ions. Biochem. Biophys. Res. Commun., 192, 407-412 (1993) [5] Ohnuma, S.-i.; Koyama, T.; Ogura, K.: Chain length distribution of the products formed in solanesyl diphosphate synthase reaction. J. Biochem., 112, 743-749 (1992) [6] Koyama, T.; Kokubun, Y.; Ogura, K.: Polyprenyl diphosphate synthase from mulberry leaves: stereochemistry of hydrogen elimination in the prenyltransferase reaction. Phytochemistry, 27, 2005-2009 (1988) [7] Ohnuma, S.-i.; Koyama, T.; Ogura, K.: Purification of solanesyl-diphosphate synthase from Micrococcus luteus. A new class of prenyltransferase. J. Biol. Chem., 266, 23706-23713 (1991) [8] Gotoh, T.; Koyama, T.; Ogura, K.: Farnesyl diphosphate synthase and solanesyl diphosphate synthase reactions of diphosphate-modified allylic analogs: the significance of the diphosphate linkage involved in the allylic substrates for prenyltransferase. J. Biochem., 112, 20-27 (1992) [9] Teclebrhan, H.; Olsson, J.; Swiezewska, E.; Dallner, G.: Biosynthesis of the side chain of ubiquinone:trans-prenyltransferase in rat liver microsomes. J. Biol. Chem., 268, 23081-23086 (1993) [10] Hirooka, K.; Bamba, T.; Fukusaki, E.-I.; Kobayashi, A.: Cloning and kinetic characterization of Arabidopsis thaliana solanesyl diphosphate synthase. Biochem. J., 370, 679-686 (2003) [11] Kainou, T.; Okada, K.; Suzuki, K.; Nakagawa, T.; Matsuda, H.; Kawamukai, M.: Dimer formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length determination of ubiquinone. J. Biol. Chem., 276, 78767883 (2001)

489


2.5.1.11

[12] Okada, K.; Kamiya, Y.; Zhu, X.; Suzuki, K.; Tanaka, K.; Nakagawa, T.; Matsuda, H.; Kawamukai, M.: Cloning of the sdsA gene encoding solanesyl diphosphate synthase from Rhodobacter capsulatus and its functional expression in Escherichia coli and Saccharomyces cerevisiae. J. Bacteriol., 179, 5992-5998 (1997) [13] Okada, K.; Suzuki, K.; Kamiya, Y.; Zhu, X.; Fujisaki, S.; Nishimura, Y.; Nishino, T.; Nakagawa, T.; Kawamukai, M.; et al.: Polyprenyl diphosphate synthase essentially defines the length of the side chain of ubiquinone. Biochim. Biophys. Acta, 1302, 217-223 (1996) [14] Runquist, M.; Ericsson, J.; Thelin, A.; Chojnacki, T.; Dallner, G.: Isoprenoid biosynthesis in rat liver mitochondria. Studies on farnesyl pyrophosphate synthase and trans-prenyltransferase. J. Biol. Chem., 269, 5804-5809 (1994) [15] Sakaihara, T.; Honda, A.; Tateyama, S.; Sagami, H.: Subcellular fractionation of polyprenyl diphosphate synthase activities responsible for the syntheses of polyprenols and dolichols in spinach leaves. J. Biochem., 128, 1073-1078 (2000)

490

Glutathione S-alkyltransferase

2.5.1.12

1 Nomenclature EC number 2.5.1.12 (deleted, included in EC 2.5.1.18) Recommended name glutathione S-alkyltransferase

491

Glutathione S-aryltransferase

1 Nomenclature EC number 2.5.1.13 (deleted, included in EC 2.5.1.18) Recommended name glutathione S-aryltransferase

492

2.5.1.13

Glutathione S-aralkyltransferase

2.5.1.14

1 Nomenclature EC number 2.5.1.14 (deleted, included in EC 2.5.1.18) Recommended name glutathione S-aralkyltransferase

493

Dihydropteroate synthase

2.5.1.15

1 Nomenclature EC number 2.5.1.15 Systematic name (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyldiphosphate:4-aminobenzoate 2-amino-4-hydroxydihydropteridine-6-methenyltransferase Recommended name dihydropteroate synthase Synonyms 7,8-dihydropteroate synthase 7,8-dihydropteroate synthetase 7,8-dihydropteroic acid synthetase DHPS dihydropteroate diphosphorylase dihydropteroate synthetase dihydropteroic synthetase synthase, dihydropteroate CAS registry number 9055-61-2

2 Source Organism

494

Lactobacillus plantarum [1] Escherichia coli [2, 3, 6, 13] Plasmodium berghei [4] Plasmodium chabaudi [5, 7] Escherichia coli MC4100 [8] Pneumocystis carinii (QSAR studies of a series of sulfa drugs [14]) [9, 14] Plasmodium falciparum [10, 20] Pisum sativum [11, 21] Staphylococcus aureus [12] Camphylobacter jejuni [15] Streptococcus pneumoniae [16, 19] Mycobacterium tuberculosis H37Rv [17] Mycobacterium leprae (SwissProt-ID: O69530) [17, 18] Mycobacterium leprae (SwissProt-ID: U15180) [18]

2.5.1.15


3 Reaction and Specificity Catalyzed reaction (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyldiphosphate + 4-aminobenzoate = diphosphate + 7,8-dihydropteroate Reaction type aryl group transfer Natural substrates and products S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoate (Reversibility: ? [1, 3, 6, 7, 9-12, 16, 18, 21]) [1, 3, 6, 7, 9-12, 16, 18, 21] P diphosphate + 7,8-dihydropteroate [1, 3, 7, 9] S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoylglutamate (Reversibility: ? [1]) [1] P diphosphate + dihydrofolate [1] S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfamethoxazole (Reversibility: ? [6]) [6] P diphosphate + dihydropterin-sulfamethoxazole [6] Substrates and products S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoate (Reversibility: ? [1-13, 16-18, 21]) [1-13, 16-18, 21] P diphosphate + 7,8-dihydropteroate [1-13, 16-18, 21] S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + 4-aminobenzoylglutamate (Reversibility: ? [1]) [1] P diphosphate + dihydrofolate [1] S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfamethoxazole (Reversibility: ? [6]) [6] P diphosphate + dihydropterin-sulfamethoxazole [6] S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfanilamide (Reversibility: ? [6]) [6] P diphosphate + dihydropterin-sulfamilamide [6] S 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate + sulfathiazole (Reversibility: ? [6]) [6] P diphosphate + dihydropterin-sulfathiazole [6] Inhibitors 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine [5] 4,4-diaminodiphenyl sulfone [4] 7,8-dihydropteroic acid [2, 3, 11, 21] dapsone [10, 17, 18] dihydrofolate [16] dihydrofolate monoglutamate [21] folate [16] p-aminobenzoate [5]

495


2.5.1.15

p-aminobenzoylglutamate ( weak [1]) [1] p-aminosalicylate [5, 17] pyrimethamine [20] sulfadiazine [4] sulfadoxine [10, 20] sulfaguanidine [5, 7] sulfamethoxazole ( competitive to 4-aminobenzoate [6]) [6, 10, 16, 17] sulfamethoxypyridazine [17] sulfanilamide [5, 6, 7, 16] sulfanilic acid [5] sulfaquinoxazoline [16] sulfathiazole ( competitive to 4-aminobenzoate [3, 5, 6, 7, 10, 13, 15, 19]) [3, 5, 6, 7, 10, 13, 15, 19] sulfonamide ( competitive to 4-aminobenzoate [3, 5, 6, 7]) [3, 5, 6, 7] tetrahydrofolate monoglutamate [21] Metals, ions Mg2+ ( required to synthesize product [1, 2, 3, 5, 11]) [1, 2, 3, 5, 11] Specific activity (U/mg) 0.0000483 ( crude extract of Escherichia coli MG1655 [13]) [13] 0.174 ( recombinant enzyme [17]) [17] 0.29 [16] 0.425 [11] 0.596 ( recombinant enzyme [17]) [17] 1-2 [6] 3.7 [8] 15.78 [5, 7] 30 [2, 3] Additional information ( specific activity in different strains [13]; specific activity in plant enzyme and recombinant enzyme [21]) [13, 21] Km-Value (mM) 0.00012 (sulfamethoxazole) [6] 0.00037 (p-aminobenzoic acid) [17] 0.0005 (4-aminobenzoate) [8] 0.00057 (4-aminobenzoate) [6] 0.0006 (p-aminobenzoic acid) [11, 17] 0.00085 (p-aminobenzoic acid, sulfonamide resistant enzyme [15]) [15] 0.001 (sulfathiazole) [6] 0.0014 (2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate) [4]

496

2.5.1.15


0.0015 (4-aminobenzoate) [5, 7] 0.0019 (2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine diphosphate) [8] 0.0025 (4-aminobenzoic acid) [2, 3] 0.0028 (4-aminobenzoic acid) [4] 0.0048 (sulfanilamide) [6] 0.03 (6-hydroxymethyldihydropteridine diphosphate) [11] Additional information ( activity in Escherichia coli C600 and recombinant strains [18]; activity in Streptococcus pneumoniae strains with different repeats of amino acids [19]; values for recombinant enzymes [13]; activity in plant enzyme and recombinant enzyme [21]; values for recombinant enzymes [10]) [10, 13, 18, 19, 21] Ki-Value (mM) 0.000011 (dapsone) [17] 0.000013 (dapsone) [17] 0.000025 (sulfamethoxypyridazine) [17] 0.000028 (sulfamethoxazole) [17] 0.00003 (sulfamethoxazole) [17] 0.000031 (sulfamethoxypyridazine) [17] 0.00013 (sulfamethoxazole) [6] 0.00053 (sulfonamide) [6] 0.00059 (sulfamethoxazole) [16] 0.00085 (sulfanilamide) [16] 0.005-0.008 (7,8-dihydropteroate, inhibitor of p-aminobenzoic acid [11]) [11] 0.0057 (sulfanilamide) [6] 0.007-0.011 (7,8-dihydropteroate, inhibitor of 6-hydroxymethyl-7,8dihydropteridine diphosphate [11]) [11] 0.014 (sulfathiazole) [7] 0.015 (folate) [16] 0.023 (p-aminosalicylic acid) [5] 0.025 (sulfaquinoxazoline) [16] 0.043 (2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine) [5] 0.069 (sulfaguanidine) [5, 7] 0.13 (sulfanilamide) [5, 7] 0.14 (sulfanilic acid) [5] 0.5 (sulfathiazole, resistant enzyme [15]) [15] 1 (p-aminobenzoylglutamate) [1] Additional information ( Ki values of sulfadoxine for recombinant enzymes [10]; Ki values of sulfathiazole for wild-type and mutant enzyme [13]; Ki values of folate derivatives [21]; Ki values of sulfathiazole inhibition on Streptococcus pneumoniae strains with different repeats of amino acids [19]) [10, 13, 19, 21] pH-Optimum 8 [17] 8.5 [2, 3, 4] 497


2.5.1.15

8.5-9 [7] 8.7 [5] 9 [11] 9 [17] pH-Range 7-10.5 ( 70% of maximal activity at pH 7.0 and 10.5 [3]) [3] Temperature optimum ( C) 30 ( assay at [11,21]) [11, 21] 37 ( assay at [2,3,7,8,10]) [2, 3, 7, 8, 10]

4 Enzyme Structure Molecular weight 50000 ( gel filtration [2]) [2] 52000 ( polyacrylamide gel [3]) [3] 52000-54000 ( gel filtration [8]) [8] 53000 ( SDS-PAGE analysis of soluble protein [21]) [21] 60000 ( gel filtration of Mycobacterium tuberculosis and Mycobacterium leprae dihydropteroate synthase [17]) [17] 61300 [12] 71500 ( polyacrylamide gel [9]) [9] 190000 ( gel filtration [5,7]) [5, 7] 222000 ( gel filtration [10]) [10] 280000-300000 ( gel filtration [11]) [11] Subunits dimer ( 2 * 30000, SDS-PAGE [8]) [8, 12, 17]

5 Isolation/Preparation/Mutation/Application Source/tissue erythrocyte [5, 7] leaf [11, 21] Localization cytosol [8] mitochondrion [11, 21] Purification (Q-Sepharose column [13]) [13] (gel filtration on Sephadex G-100 [2,6]) [2, 6] (enzyme from recombinant clones in Sephacryl S-300 HR, enzyme isolated form Escherichia coli, several chromatographic columns [10]) [10] (anion exchange and affinity column [11]) [11] (recombinant enzyme, affinity column [21]) [21]

498

2.5.1.15


(Superdex-200 HR and other chromatographic columns [16]) [16] (gel filtration and ion-exchange chromatography [19]) [19] (precipitation with ammonium sulfate followed by column chromatography [1,3,5,7,8]) [1, 3, 5, 7, 8] (mycobacterial enzymes, from DEAE-Sepharose and other chromatography steps [17]) [17] Cloning (a single polypeptide, Fas protein, expressed in cultured Spodoptera frugiperda SF9 insect cells [9]) [9] (expression in Escherichia coli [10]) [10] (expression of recombinant enzyme in Escherichia coli [21]) [21] (expression in Escherichia coli [12]) [12] (cloning of sulfonamide resistant strain [15]) [15] [16] (expression in different Escherichia coli strains [19]) [19] (expression in dihydroxypteroate synthase deficient strain of Escherichia coli [17]) [17] (expression in Escherichia coli C600 and in Escherichia coli folP knockout mutant [18]) [18]

6 Stability Temperature stability 60 ( 10 min, loss of activity [1]) [1] 100 ( 60 min, 75% loss of activity [3]) [2, 3] Storage stability , -20 C, sucrose, indefinitely stable [3] , -20 C or -70 C, 20 mM Tris/HCl, pH 8.0, 5 weeks [8] , -80 C, pH 7.0, 20% glycerol, 10-30% loss of activity in 1 month [17]

References [1] Shiota, T.; Baugh, C.M.; Jackson, R.; Dillard, R.: The enzymatic synthesis of hydroxymethyldihydropteridine pyrophosphate and dihydrofolate. Biochemistry, 8, 5022-5028 (1969) [2] Richey, D.P.; Brown, G.M.: The biosynthesis of folic acid. IX. Purification and properties of the enzymes required for the formation of dihydropteroic acid. J. Biol. Chem., 244, 1582-1592 (1969) [3] Richey, D.P.; Brown, G.M.: Hydroxymethyldihydropteridine pyrophosphokinase and dihydropteroate synthetase from Escherichia coli. Methods Enzymol., 18B, 765-771 (1971) [4] McCullough, J.L.; Maren, T.H.: Dihydropteroate synthetase from Plasmodium berghei: isolation, properties, and inhibition by dapsone and sulfadiazine. Mol. Pharmacol., 10, 140-145 (1974) 499


2.5.1.15

[5] Walter, R.D.; Königk, E.: Biosynthesis of folic acid compounds in plasmodia. Purification and properties of the 7,8-dihydropteroate-synthesizing enzyme from Plasmodium chabaudi. Hoppe-Seyler's Z. Physiol. Chem., 355, 431-437 (1974) [6] Roland, S.; Ferone, R.; Harvey, R.J.; Styles, V.L.; Morrison, R.W.: The characteristics and significance of sulfonamides as substrates for Escherichia coli dihydropteroate synthase. J. Biol. Chem., 254, 10337-10345 (1979) [7] Walter, R.D.; Königk, E.: 7,8-Dihydropteroate-synthesizing enzyme from Plasmodium chabaudi. Methods Enzymol., 66, 564-570 (1980) [8] Talarico, T.L.; Dev, I.K.; Dallas, W.S.; Ferone, R.; Ray, P.H.: Purification and partial characterization of 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase and 7,8-dihydropteroate synthase from Escherichia coli MC4100. J. Bacteriol., 173, 7029-7032 (1991) [9] Volpe, F.; Ballantine, S.P.; Delves, C.J.: The multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase. Eur. J. Biochem., 216, 449-458 (1993) [10] Triglia, T.; Menting, J.G.T.; Wilson, C.; Cowman, A.F.: Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum. Proc. Natl. Acad. Sci. USA, 94, 13944-13949 (1997) [11] Rebeille, F.; Macherel, D.; Mouillon, J.M.; Garin, J.; Douce, R.: Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria. EMBO J., 16, 947-957 (1997) [12] Hampele, I.C.; Drcy, A.; Dale, G.E.; Kostrewa, D.; Nielsen, J.; Oefner, C.; Page, M.G.P.; Schonfeld, H.J.; Stuber, D.; Then, R.L.: Structure and function of the dihydropteroate synthase from Staphylococcus aureus. J. Mol. Biol., 268, 21-30 (1997) [13] Vedantam, G.; Nichols, B.P.: Characterization of a mutationally altered dihydropteroate synthase contributing to sulfathiazole resistance in Escherichia coli. Microbiol.Drug Resist., 4, 91-97 (1998) [14] Johnson, T.; Khan, I.A.; Avery, M.A.; Grant, J.; Meshnick, S.R.: Quantitative structure-activity relationship studies of a series of sulfa drugs as inhibitors of Pneumocystis carinii dihydropteroate synthetase. Antimicrob. Agents Chemother., 42, 1454-1458 (1998) [15] Gibreel, A.; Skold, O.: Sulfonamide resistance in clinical isolates of Campylobacter jejuni: mutational changes in the chromosomal dihydropteroate synthase. Antimicrob. Agents Chemother., 43, 2156-2160 (1999) [16] Vinnicombe, H.G.; Derrick, J.P.: Dihydropteroate synthase from Streptococcus pneumoniae: characterization of substrate binding order and sulfonamide inhibition. Biochem. Biophys. Res. Commun., 258, 752-757 (1999) [17] Nopponpunth, V.; Sirawaraporn, W.; Greene, P.J.; Santi, D.V.: Cloning and expression of Mycobacterium tuberculosis and Mycobacterium leprae dihydropteroate synthase in Escherichia coli. J. Bacteriol., 181, 6814-6821 (1999)

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[18] Williams, D.L.; Spring, L.; Harris, E.; Roche, P.; Gillis, T.P.: Dihydropteroate synthase of Mycobacterium leprae and dapsone resistance. Antimicrob. Agents Chemother., 44, 1530-1537 (2000) [19] Haasum, Y.; Strom, K.; Wehelie, R.; Luna, V.; Roberts, M.C.; Maskell, J.P.; Hall, L.M.C.; Swedberg, G.: Amino acid repetitions in the dihydropteroate synthase of Streptococcus pneumoniae lead to sulfonamide resistance with limited effects on substrate Km . Antimicrob. Agents Chemother., 45, 805809 (2001) [20] Chaparro, J.; Rojas, M.O.; Wasserman, M.: Plasmodium falciparum: underestimation of dihydrofolate reductase and dihydropteroate synthase polymorphism in field samples: a technical shortcoming of nested PCR assays with mutation-specific primers. Exp. Parasitol., 99, 115-122 (2001) [21] Mouillon, J.M.; Ravanel, S.; Douce, R.; Rebeille, F.: Folate synthesis in higher-plant mitochondria: coupling between the dihydropterin pyrophosphokinase and the dihydropteroate synthase activities. Biochem. J., 363, 313319 (2002)

501

Spermidine synthase

2.5.1.16

1 Nomenclature EC number 2.5.1.16 Systematic name S-adenosylmethioninamine:putrescine 3-aminopropyltransferase Recommended name spermidine synthase Synonyms PAPT [34] SPDS [27, 28] aminopropyltransferase aminopropyltransferase spermidine synthase putrescine aminopropyltransferase spermidine synthetase synthase, spermidine Additional information (not identical with EC 2.5.1.22 or EC 2.5.1.23) CAS registry number 37277-82-0

2 Source Organism Rattus norvegicus (male Sprague Dawley strain [10, 16]; Wistar strain [12, 17, 25]) [1-3, 6, 7, 8, 9, 10, 12, 15-18, 21, 25, 29, 31] Sus scrofa (pig [4]) [4] Homo sapiens [8] Bos taurus [8, 9, 13, 14, 21] Escherichia coli (W strain [3,22]) [3, 18, 20, 22-25] Neurospora crassa [3] Saccharomyces cerevisiae (SPE3 gene [32]) [3, 32] Trypanosoma brucei brucei [5] Brassica pekinensis (Chinese cabbage, var Pak Choy [11,19,20]) [11, 19, 20] Spinacia oleraceae (spinach [11]) [11] Serratia marcescens [18] Pseudomonas aeruginosa [18] Senecio vulgaris [26]

502

2.5.1.16

Spermidine synthase

Arabidopsis thaliana (SPDS1 and SPDS2, multiprotein complex with SPMS [27]) [27, 28] Nicotiana sylvestris [28] Hyoscyamus niger [28] Glycine max (soybean [30,31]) [30, 31] Gallus gallus (chicken [33]) [33] Thermotoga maritima [34]

3 Reaction and Specificity Catalyzed reaction S-adenosylmethioninamine + putrescine = 5'±S-methyl-5'-thioadenosine + spermidine ( first discovered [20]; Michaelis-Menten kinetics [8]; ping-pong reaction mechanism [13, 24]; single displacement reaction [18]; ping-pong mechanism with a propylaminated form of the enzyme as an obligatory intermediate [18]; uni uni uni uni ping pong mechanism [30]) Reaction type aminopropyl group transfer Natural substrates and products S S-adenosyl-3-methylthio-1-propylamine + 1,4-diaminobutane ( i.e. S-adenosylmethioninamine or decarboxylated S-adenosylmethionine + putrescine, involved in polyamine biosynthetic pathway [1]) (Reversibility: ? [1, 9]) [1-7, 9, 10, 12, 13, 16, 18, 2023, 25, 28-31, 33, 34] P 5'-methylthioadenosine + spermidine [1-3, 910, 18, 20, 22, 23, 25, 28-31, 33, 34] Substrates and products S S-5'-deoxyadenosyl-5'-3-butylthiopropylamine + 1,4-diaminobutane (Reversibility: ? [16]) [16] P ? S S-5'-deoxyadenosyl-5'-3-ethylthiopropylamine + 1,4-diaminobutane (Reversibility: ? [16]) [16] P ? S S-5'-deoxyadenosyl-5'-3-propylthiopropylamine + 1,4-diaminobutane (Reversibility: ? [16]) [16] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 1,3-diaminopropane ( weak substrate [34]) (Reversibility: ? [34]) [34] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 1,4-butene-2-diamine (Reversibility: ? [9]) [9] P ?

503

Spermidine synthase

2.5.1.16

S S-adenosyl-(5')-3-methylthio-1-propylamine + 1,4-diamino-2-butene (Reversibility: ? [16]) [16] P 5'-methylthioadenosine + N-3-aminopropyl-1,4-diamino-2-butene ( in a lower yield than with the natural combination [16]) [16] S S-adenosyl-(5')-3-methylthio-1-propylamine + 1,4-diamino-2-butene (Reversibility: ? [16]) [16] P N-3-aminopropyl-1,4-diamino-2-butene ( forms with a low yield [16]) [16] S S-adenosyl-(5')-3-methylthio-1-propylamine + 1,4-diaminobutane (Reversibility: ? [9]) [9] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 1,4-diaminobutane ( synthetic diamines are substrates too [6]; derivatives of 1,4-diaminobutane are substrates too. Substitution of 1,4-diaminobutane in the immediate vicinity of an amino group produces weak substrates, whereas substituents in the 2-position are tolerated [9]; the reaction is linear from 1 to 10 nmol of spermidine [11]; analogues of decarboxylated S-adenosylmethionine act as propylamine donors [13, 16]; the enzyme transfers only the aminopropyl group, not an aminoethyl or aminobutyl groups [16]; sulfonium analogs must carry the aminopropyl group to qualify as a substrate [16]; linear at least up to 6 nmol of spermidine formed, linearity not maintained in the absence of DTT [25]) (Reversibility: ir [22]; ? [9, 18, 20, 22, 23]) [1-7, 9, 12, 13, 16, 18, 20-23, 25, 28-31, 33, 34] P 5'-methylthioadenosine + spermidine ( + H+ [30]) [1-7, 9, 12, 13, 16, 18, 20-23, 25, 28-31, 33, 34] S S-adenosyl-(5')-3-methylthio-1-propylamine + 1,6-diaminohexane ( 1% the reaction rate than with 1,4-diaminobutane [13]) (Reversibility: ? [13, 21]) [13, 21] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 1-methyl-1,4-butanediamine (Reversibility: ? [9]) [9] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 2,2-difluoro-1,4-diaminobutane (Reversibility: ? [9]) [9] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 2-chloro-1,4-diaminobutane (Reversibility: ? [9]) [9] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 2-hydroxy-1,4 butanediamine (Reversibility: ? [9]) [9] P ? S S-adenosyl-(5')-3-methylthio-1-propylamine + 2-methyl-1,4-diaminobutane (Reversibility: ? [9]) [9] P ?

504

2.5.1.16

Spermidine synthase

S S-adenosyl-(5')-3-methylthio-1-propylamine + spermidine ( at a reduced rate [3, 22, 23]; weak substrate [34]) (Reversibility: ? [3, 22, 23, 34]) [3, 22, 23, 34] P 5'-methylthioadenosine + spermine ( the reaction rate is slower than with 1,4-diaminobutane [22]) [22, 25] S S-adenosyl-3-butylthio-1-propylamine + 1,4-diaminobutane ( prostate enzyme [9]) (Reversibility: ? [9]) [9, 13] P ? S S-adenosyl-3-carboxymethylthio-1-propylamine + 1,4-diaminobutane (Reversibility: ? [13]) [13] P ? S S-adenosyl-3-ethylthio-1-propylamine + 1,4-diaminobutane ( prostate enzyme [9]) (Reversibility: ? [9, 13]) [9, 13] P ? S S-adenosyl-3-methyl-1-propylamine + 1,4-diaminobutane (Reversibility: ? [9]) [9] P ? S S-adenosyl-3-methylthio-1-propylamine + 1,3-diaminopentane ( poor substrate [1]) (Reversibility: ? [1]) [1] P ? S S-adenosyl-3-methylthio-1-propylamine + 1,5-diaminopentane ( cadaverine [3]; 6% the reaction rate than with 1,4-diaminobutane [13]; 20 times less active than 1,4-diaminobutane at the same concentration [25]; weak substrate [34]) (Reversibility: ? [1, 3, 7, 13, 21-23, 25, 34]) [1, 3, 7, 13, 21-23, 25, 28, 34] P 5'-methylthioadenosine + N-(3-aminopropyl)-1,5-diaminopentane ( aminopropylcadaverine [25]) [22, 25] S S-adenosyl-3-propylthio-1-propylamine + 1,4-diaminobutane ( prostate enzyme [9]) (Reversibility: ? [9, 13]) [9, 13] P ? S S-adenosyl-3-thiopropylamine sulfone + 1,4-diaminobutane (Reversibility: ? [10]) [10] P ? S S-adenosyl-3-thiopropylamine sulfoxide + 1,4-diaminobutane (Reversibility: ? [10]) [10] P ? S S-adenosyl-5',1-methyl-3-methylthiopropylamine + 1,4-diaminobutane ( prostate enzyme [9]) (Reversibility: ? [9]) [9] P ? S S-adenosyl-5',1-methyl-3-methylthiopropylamine + 1,4-diaminobutane ( brain enzyme [9]) (Reversibility: ? [9]) [9] P ? S S-adenosyl-l-ethionine + 1,4-diaminobutane (Reversibility: ? [10]) [10] P ? S S-adenosyl-l-homocysteine sulfone + 1,4-diaminobutane (Reversibility: ? [10]) [10] 505

Spermidine synthase

2.5.1.16

P ? S S-adenosyl-l-methionine + 1,4-diaminobutane (Reversibility: ? [10]) [10] P ? Inhibitors 1,3-diaminopropane ( 34% inhibition [13]; 96% inhibition at 0.01 mM and 100% inhibition at 0.1 mM [26]) [13, 26] 1,5-diaminopentane ( weak, competitive [25]) [25] 1,6-diaminohexane ( 23% inhibition [13]) [13] 1-aminooxy-3-aminopropane ( brain enzyme, specific, no inhibition of spermine synthase, reversible, competitive with 1,4-diaminobutane [14]) [14] 2-mercaptoethylamine ( competitive with 1,4-diaminobutane, reversible, its removal restores the enzyme activity, the amino group is necessary to inhibit the enzyme [17]) [17] 5'-deoxy-5'-methylthioadenosine ( weak [11]; above 0.05 mM [25]) [11, 25] 5'-deoxy-5'-methylthiotubercidine ( weak [11]) [11, 25] 5'-ethylthioadenosine ( strong [7]) [7, 10, 13, 17, 22, 23] 5'-methylthioadenosine ( strong [7]; W strain [3]; non-competitive, mixed type inhibition [13]; weak inhibition [21]; 83% inhibition at 0.01 mM and 22% inhibition at 1 mM [26]) [3, 7, 8, 10, 13, 21, 26] 5'-methylthiotubercidin ( in vivo and in vitro, strong [7]) [7, 10] DTNB ( potent inhibition, activity is restored to more than 95% with 0.1 mM DTT [30]) [30] N,N'-bis-(2-aminoethyl)propane-1,3-diamine ( strong, 52% inhibition [13]) [13] N-ethylmaleimide ( potent inhibition [30]) [22, 23, 25, 30] PCMB [25] S-5'-deoxyadenosyl(5')-2-ethylthioethylamine ( uncompetitive with S-adenosyl-3-methylthio-1-propylamine at high concentrations but competitive with S-5'-deoxyadenosyl-5'-3-ethylthiopropylamine [16]) [16] S-5'-deoxyadenosyl(5')-2-methylthioethylamine ( prostate [7]; the most potent inhibitor, competitive with decarboxylated S-adenosylmethionine and S-5'-deoxyadenosyl-(5')-3-ethylthiopropylamine [16]) [7, 16] S-5'-deoxyadenosyl(5')-2-thioethylamine ( weak [16]) [16] S-5'-deoxyadenosyl(5')-3-butylthiopropylamine ( weak [16]) [16] S-5'-deoxyadenosyl(5')-3-thiopropylamine ( weak [16]) [16] S-5'-deoxyadenosyl(5')-4-methylthiobutylamine ( weak [16]) [16] S-5'-deoxyadenosyl(5')-4-thiobutylamine ( uncompetitive with Sadenosyl-3-methylthio-1-propylamine [16]) [16] S-adenosyl(5')-2-methylthioethylamine [13] S-adenosyl(5')-3-methylthiopropanol ( kinetics [24]) [24]

506

2.5.1.16

Spermidine synthase

S-adenosyl-(5')-3-methylthio-1-propylamine ( at concentrations above 0.05 mM [11,26]; competitive substrate inhibition [24,25]; complete inhibition at 0.080 mM [26]) [11, 24, 25, 26] S-adenosyl-1,8-diamino-3-methylthiooctane ( less efficient than the thioether [7]) [7] S-adenosyl-1,8-diamino-3-thiooctane ( 50% inhibition at 0.0025 mM [4]; potent, 50% inhibition at 0.025 mM [5]; transition-state analogue, potent inhibition [11, 15]; specific and more potent inhibitor than the corresponding fully-charged methyl sulfonium salt [15]; prostate [7]; competitive with 1,4-diaminobutane, potent inhibition, stronger than dicyclohexylamine in vitro, but not in vivo, at concentrations of S-adenosyl-3-methylthio-1-propylamine and 1,4-diaminobutane higher than those normally present in vivo. At concentration of substrates that approximate in vivo conditions, more than 20fold stronger inhibition [18]; IC50 of 0.0002 mM [34]) [4, 5, 7, 11, 14, 15, 18, 34] S-adenosyl-3-thiopropylamine sulfone [7] S-adenosyl-4-methylthiobutyric acid [7, 10] S-adenosyl-l-ethionine [10] S-adenosyl-l-homocysteine sulfone [7, 10] S-adenosyl-l-methionine [10] S-inosyl(5')-3-methylthiopropylamine ( not S-inosyl(5')-3-methylthiopropanol [24]) [24] S-tubercidinylmethionine [10] agmatine ( 89% inhibition at 0.1 mM and 61% inhibition at 1 mM [26]) [26] a,w-diamines ( with 3 carbon atoms, at 0.25 mM, not at 2.5 mM substrate concentration [7]) [7] arginine ( 98% inhibition at 0.01 mM, 95% inhibition at 0.1 mM and 84% inhibition at 1 mM [26]) [26] bis-cyclohexylammonium sulfate ( in vitro, 0.2 mM provokes strong decrease in cellular spermidine content, inhibition in cell growth, decrease in DNA synthesis, alterations in cell morphology, disorganization of microfilaments and changes in microtubule network structure, impairs the rate of microtubule reappearance after disruption with colchicine, alterations are prevented with spermidine addition [33]) [33] cadaverine ( 26-39% inhibition [13]; weak [16]; 77% inhibition at 0.01 mM and 22% inhibition at 1 mM [26]) [7, 13, 16, 26] cyclohexylamine ( in vitro, 50% inhibition at 0.015 mM, competitive with 1,4-diaminobutane [5]; 50% inhibition at 0.0081 mM, competitive with 1,4-diaminobutane [4]; strong [11]) [4, 5, 7, 11, 26] deaminated analogs of decarboxy-S-adenosyl-(5')-3-methylthiopropylamine ( competitive inhibition with 1,4-diaminobutane and non-competitive with decarboxy-S-adenosyl-(5')-3-methylthiopropylamine [24]) [24] dicyclohexylamine ( in vitro, 50% inhibition at 0.003 mM, competitive with 1,4-diaminobutane [5]; strong [7, 11]; prostate [7]; potent inhibition, reduces the rate of spermidine synthesis by more than 90% at 0.05 mM [18]) [5, 7, 11, 14, 18] 507

Spermidine synthase

2.5.1.16

exo-2-aminonorbornane ( potent and selective, 98% inhibition at 0.1 mM in the presence of 1 mM of putrescine [29]) [29] homospermidine ( 95% inhibition at 0.01 mM, 92% inhibition at 0.1 mM and 30% inhibition at 1 mM [26]) [26] p-hydroxymercuribenzoate ( 2-mercaptoethanol restores activity [22]) [22, 23] spermidine ( not competitive, mixed type inhibition [13]; W strain [3]; product inhibition at physiological concentrations [26]) [3, 13, 22, 23, 26] spermine ( 99% inhibition at 0.1 mM and 90% inhibition at 1 mM [26]) [26] substrate and product analogues ( overview [10]) [10] sulfhydryl reagents ( W strain [3]) [3] sulfoxide and sulfone derivatives of decarboxylated S-adenosyl-l-homocysteine [10] trans-4-methylcyclohexylamine ( potent, 50% inhibition at 0.0017 mM, competitive with 1,4-diaminobutane [4]; potent and selective, 98% inhibition at 0.1 mM in the presence of 1 mM of putrescine, in vitro. In vivo, intraperitoneal administration causes effective decrease in spermidine content in prostate, and oral administration causes 28%, 21% and 33% decrease in spermidine content in prostate, liver and kidney, respectively [29]) [4, 29] triethylenetetramine ( 39% inhibition [13]) [13] Additional information ( no inhibition by putrescine up to 10 mM [25]; polyamines [7]; S-adenosyl-l-homocysteine [10]; no inhibition by 2-mercaptoethanol at 0.1 to 1 mM [17]; no inhibition by phenylhydrazine, semicarbazide, sodium borohydride, NaCN, KCl, NH4 Cl, MgCl2 , CaCl2 , NaNO3, Na2 SO4, Na2 HPO4 [22]; inhibition by end products of reaction [22,23]; no inhibition by carbonyl binding reagents [23]; no inhibition by 1,4-diaminobutane [24,25]) [7, 10, 17, 22-25] Cofactors/prosthetic groups Additional information ( no cofactor requirement [3]) [3] Metals, ions Ca2+ ( 0.01 mM, activity increases by 200% [30]) [30] Co2+ ( 0.01 mM, activity increases by 352% [30]) [30] Cu2+ ( 0.01 mM, activity increases by 230% [30]) [30] K+ ( 10 mM, activity increases by 20% [25]; not effective [30]) [25] Mg2+ ( 10 mM, activity increases 20% [25]) [25] Na+ ( 10 mM, activity increases by 20% [25]; not effective [30]) [25] Additional information ( no metal ion requirement [3]; Li+ , Mn2+ and Zn2+ , not effective [30]) [3, 30]

508

2.5.1.16

Spermidine synthase

Specific activity (U/mg) 0.008 [16] 0.055 [10] 0.076 [30] 0.66 [8] 0.712 [21] 1.07 [19] 1.12 [13] 1.28 [20] 1.3 [21, 25] 1.83 [22, 23] Km-Value (mM) 0.0003 (S-adenosyl-3-methylthio-1-propylamine, pH 7.4, 37 C [13]) [13] 0.0004 (S-adenosyl-(5')-3-methylthio-1-propylamine, pH 8.0, 37 C [30]) [30] 0.0022 (S-adenosyl-3-methylthio-1-propylamine, pH 8.0, 37 C, fluorometric assay [22]; pH 8.2, 37 C, spectrophotometric assay [23]) [22, 23] 0.004 (S-adenosyl-(5')-3-methylthio-1-propylamine, pH 7.5, 34 C [26]) [26] 0.0067 (S-adenosyl-3-methylthio-1-propylamine, pH 8.8, 37 C, crude extracts [11]; pH 7.4, 37 C, partially purified enzyme [11]) [11] 0.007 (S-adenosyl-3-methylthio-1-propylamine, pH 7.4, 37 C, spleen [8]) [8] 0.01 (S-adenosyl-(5')-3-methylthio-1-propylamine, pH 7.2, 37 C [21]) [21] 0.012 (1,4-diaminobutane, pH 8.8, 37 C, spectrophotometric assay [22]; pH 8.2, 37 C, spectrophotometric assay [23]) [22, 23] 0.02 (1,4-diaminobutane, pH 7.5, 37 C [34]) [34] 0.021 (1,4-diaminobutane, pH 7.5, 34 C [26]) [26] 0.03 (1,4-diaminobutane, pH 7.2, 37 C [21]) [21] 0.032 (1,4-diaminobutane, pH 8.8, 37 C, crude extracts [11]; pH 7.4, 37 C, partially purified enzyme [11]) [11] 0.0325 (1,4-diaminobutane, pH 8.0, 37 C [30]) [30] 0.036 (1,4-diaminobutane, pH 7.5, 37 C, in the presence of 2-mercaptoethylamine [17]) [17] 0.04 (1,4-diaminobutane, pH 7.4, 37 C [13]) [13] 0.08 (1,4-diaminobutane, pH 7.4, 37 C, spleen [8]) [8] 0.09 (1,4-diaminobutane, pH 7.5, 37 C [18]) [18] 0.1 (1,4-diaminobutane, pH 7.2, 37 C [21,25]) [21, 25] 0.1 (1,4-diaminobutane, pH 7.2, 37 C [21]) [21] 0.1 (1,4-diaminobutane, pH 7.2, 37 C [25]) [25] 0.2 (1,4-diaminobutane, pH 7.5, 37 C [18]) [18] 1.1 (S-adenosyl-(5')-3-methylthio-1-propylamine, pH 7.2, 37 C [25]) [25] Additional information ( kinetic study [13]) [13]

509

Spermidine synthase

2.5.1.16

Ki-Value (mM) 0.000008 (S-adenosyl-1,8-diamino-3-thiooctane, prostate [18]) [18] 0.00004 (trans-4-methylcyclohexylamine) [4] 0.00005 (S-adenosyl-1,8-diamino-3-thiooctane) [18] 0.00015 (dicyclohexylamine, prostate [18]) [18] 0.00026 (S-5'-deoxyadenosyl-(5')-2-methylthioethylamine, 0.00350.007 mM [16]) [16] 0.0003 (dicyclohexylamine) [18] 0.0011 (S-5'-deoxyadenosyl-(5')-2-methylthioethylamine, 0.4 mM [16]) [16] 0.0015 (S-5'-deoxyadenosyl-(5')-2-methylthioethylamine, 0.3 mM [16]) [16] 0.0019 (S-5'-deoxyadenosyl-(5')-2-methylthioethylamine, 0.2 mM [16]) [16] 0.0023 (1-aminooxy-3-aminopropane) [14] 0.005 (2-mercaptoethylamine) [17] pH-Optimum 7.5 [34] 7.7 ( in Gly-NaOH buffer [26]) [26] 8.5 ( activity is 20% higher in Tris-HCl buffer than in phosphate buffer at the same pH [30]) [30] 8.8 ( in Gly-NaOH buffer [11,20]) [11, 20] 9.6 ( 190% of the activity, taking pH 7 as 100% [13]) [13] 10 [25] 10.3 ( W strain [3]) [3] 10.4 ( 65% rate of reaction at pH 8.6 and 93% rate of reaction at pH 10.6 [22]) [22, 23] Additional information ( pI 5.1, in spleen [8]; pI 5.22 [13]; at a given pH, the activity of the enzyme in Tris buffer is lower than in phosphate or Gly-NaOH buffers [20]; pI 5.3 [25]; pI 4.61 for SPDS1 and pI 4.32 for SPDS2 [27]) [8, 13, 20, 25, 27] pH-Range 6.5-9.8 ( about 60% of maximal activity at pH 6.5 and about 85% of maximal activity at pH 9.8 [13]) [13] 8.6-10.6 ( about 65% of maximal activity at pH 8.6 and about 90% of maximal activity at pH 10.6 [22]) [22] Additional information ( rapidly inactivated at pH more than 9.0 in Gly-NaOH buffer. The activity of the enzyme is generally lower in Tris buffer than in Gly-NaOH buffer [11]; half-maximal activities at pH 7.2 and 8.4 [26]) [11, 26] Temperature optimum ( C) 28 ( assay at [19]) [19] 34 ( assay at [26]) [26]

510

2.5.1.16

Spermidine synthase

37 ( assay at [1, 8, 10-13, 16-18, 20-25]) [1, 8, 1013, 16-18, 20-25] 90 ( more active than at 37 C [34]) [34]

4 Enzyme Structure Molecular weight 34000-35000 ( prediction from cDNA sequences [28]) [28] 37000 ( gel filtration [26]) [26] 62000 ( spleen, pore gradient gel electrophoresis [8]; brain, pore gradient gel electrophoresis [8]) [8] 70000 ( SDS-PAGE [13]) [13] 70600 ( W strain, sedimentation equilibrium centrifugation [22]) [22] 72000 ( W strain [23]) [23] 73000 ( W strain [3,22]; gel filtration [21,25]; gel filtration [21]) [3, 21, 22, 25] 74000 ( gel filtration and sucrose density gradient centrifugation [5]; gel filtration and SDS-PAGE [30]) [5, 30] 81000 ( gel filtration [11,19,20]) [11, 19, 20] 131000 ( gel filtration [34]) [34] 650000-750000 ( gel filtration [27]) [27] Subunits dimer ( 2 * 37000, SDS-PAGE [21,25]; 2 * 30000-35000, SDS-PAGE [22]; 2 * 35000, SDS-PAGE [8]; 2 * 35800, SDS-PAGE [13,21]; 2 * 37000-38000, high speed sedimentation equilibrium centrifugation in 6 M guanidine-HCl and 0.1 M 2-mercaptoethanol [22]; 2 * 35000, sedimentation equilibrium centrifugation and SDS-PAGE [23]) [8, 13, 21, 22, 23, 25] monomer ( 1 * 74000, gel filtration and SDS-PAGE [30]) [30] tetramer ( HPLC gel filtration [34]) [34] Additional information ( protein complexes [27]) [27]

5 Isolation/Preparation/Mutation/Application Source/tissue brain [2, 3, 8, 9, 13, 14, 21, 29, 31] fibroblast ( embryo, 7 days old [33]) [33, 33] kidney [8, 29, 31] leaf ( protoplasts from healthy or turnip yellow mosaic virus infected leaves [19]) [11, 19, 20, 28] liver [3, 29, 31] lung [8] pancreas [8, 12]

511

Spermidine synthase

2.5.1.16

placenta [8] prostate gland ( ventral [17,21]) [1, 10, 12, 15-18, 21, 25, 29] root ( 10 days old [26]) [26, 28] seed ( 1 day old, maximum activity [31]) [31] small intestine [12] spleen [8] stem ( 3 days old [30]) [28, 30] uterus [12] Additional information ( tissue distribution [8,12]) [8, 12] Localization cytosol [8, 30] Purification (partial [3,16]; chromatography on Sephadex G-25 and DEAE-cellulose chromatography [16]; ammonium sulfate fractionation, chromatography on DEAE-cellulose, affinity chromatography, 265fold purified [17]; ventral prostate [18]; extraction, ammonium sulfate fractionation, chromatography on DEAE-cellulose, affinity chromatography, gel filtration, 4480fold purified [21]; affinity chromatography on S-adenosyl(5')-3-thiopropylamine-Sepharose [25]) [3, 8, 16, 17, 21, 25] (ammonium sulfate fractionation, chromatography on DEAE-Cellulose, affinity chromatography, 8700fold purified [8]) [8] (affinity chromatography [8]; affinity chromatography with S-adenosyl5'-3-thiopropylamine linked to Sepharose, 11000fold purified [13]; extraction, ammonium sulfate fractionation, chromatography on DEAE-Cellulose, chromatography on hydroxyapatite, chromatography on ATPA-Sepharose, gel filtration [21]) [8, 13, 21] (2000fold purified [22]; extraction, ammonium sulfate fractionation, chromatography on calcium phosphate cellulose, DEAE-Sephadex, chromatography on hydroxyapatite, disc gel electrophoresis [23]) [3, 22, 23] (ammonium sulfate fractionation, acetone precipitation and chromatography on Sephadex G-100, 160fold purified [11]; ammonium sulfate fractionation, affinity chromatography and gel filtration, 1900fold purified [19]; streptomycin and ammonium sulfate fractionation, acetone precipitation and chromatography on Sephadex G-100, 160fold purified [20]) [11, 19, 20] (partial, streptomycin sulfate precipitation, ammonium sulfate fractionation and gel filtration, 18fold purified [26]) [26] (ammonium sulfate fractionation, chromatography on DEAE-Sephacel, chromatography on Sephacryl S-300, chromatography on aminooctyl-Sepharose and ATPA-Sepharose, 319fold purified [30]) [30] (metal affinity chromatography [28]) [28] Crystallization (hanging drop vapour diffusion, multiwavelength anomalous diffraction from selenomethionine containing crystals [34]) [34]

512

2.5.1.16

Spermidine synthase

Cloning (expression in E.coli [28]) [28]

6 Stability pH-Stability 6 ( no activity detected in phosphate buffer [20]) [20] 10 ( 95% loss of activity after 30 min [25]) [25] 10 ( no activity detected in Gly-NaOH buffer [20]) [20] 10.4 ( above, rapid loss of activity [25]) [25] Temperature stability 25 (stable over 30 min [30]) [30] 37 (above, progressive inactivation [26]) [26] 37 (stable over 30 min [30]) [30] 50 ( at least 30 min stable in 0.01 M Tris-HCl buffer, pH 8, 1 mM EDTA [22]; 95% loss of activity over 30 min [30]) [22, 23, 30] General stability information , DTT stabilizes [25] , DTT and/or EDTA do not stabilize [11, 20] , dialysis against glycine-NaOH buffer, pH 8.8 inactivates [11, 20] , heat-inactivated enzyme preparation does not restore the activity [20] , very labile in crude extracts [11] , S-adenosyl-(5')-3-methylthio-1-propylamine protects from inhibitors when preincubated [30] , tetramerization stabilizes [34] , freeze-thawing inactivates [22, 23, 25, 30] Storage stability , 0 C, 30% loss of activity after 4 months [25] , 0 C, 50% loss of activity in 2 weeks [16] , -80 C, 20% glycerol, rapid loss of activity in pancreas preparations [8] , -80 C, 20% glycerol, stable for spleen tissue enzyme during storage and freeze-thawings [8] , -20 C, partially purified enzyme, several weeks with less than 20% loss of activity [22] , 0-4 C, purified enzyme, 10-20% loss of activity within 6 months [22, 23] , -15 C, disrupted protoplasts lose more than 90% of activity within 1 week [20] , -20 C, about 30% loss of activity in the final enzyme preparation within 1 month [20] , -20 C, 30% loss of activity in 1 month, after final filtration through Sephadex [11] , -20 C, stable after the acetone precipitation [20] , -20 C, stable for at least 3 months after acetone precipitation [11] , 4 C, unstable [19]

513

Spermidine synthase

2.5.1.16

, -20 C, partially purified enzyme, 0.1 M NaCl and 1% bovine serum albumin, loss of 50% of activity after 15 days [26] , 4 C, crude extracts, loss of activity within 24 hours [26] , 0-4 C, 10% loss of activity after several months [30] , 0 C, in buffer containing 3 M NaCl, 1 mM DTT and 0.05-0.25 mM decarboxylated S-adenosylmethionine, several months [21]

References [1] Pegg, A.E.; Shuttleworth, K.; Hibasami, H.: Specificity of mammalian spermidine synthase and spermine synthase. Biochem. J., 197, 315-320 (1981) [2] Hannonen, P.; Jänne, J.; Raina, A.: Partial purification and characterization of spermine synthase from rat brain. Biochim. Biophys. Acta, 289, 225-231 (1972) [3] Tabor, H.; White Tabor, C.: Biosynthesis and metabolism of 1,4-diaminobutane, spermidine, spermine, and related amines. Adv. Enzymol. Relat. Areas Mol. Biol., 36, 203-268 (1972) [4] Shirahata, A.; Morohoshi, T.; Samejima, K.: trans-4-Methylcyclohexylamine, a potent new inhibitor of spermidine synthase. Chem. Pharm. Bull., 36, 3220-3222 (1988) [5] Bitonti, A.J.; Kelly, S.E.; McCann, P.P.: Characterization of spermidine synthase from Trypanosoma brucei brucei. Mol. Biochem. Parasitol., 13, 21-28 (1984) [6] Coward, J.K.; Anderson, G.L.; Tang, K.C.: Aminopropyltransferase subunits and inhibitors. Methods Enzymol., 94, 286-294 (1983) [7] Pegg, A.E.: Inhibition of aminopropyltransferases. Methods Enzymol., 94, 294-297 (1983) [8] Kajander, E.O.; Kauppinen, L.I.; Pajula, R.L.; Karkola, K.; Eloranta, T.O.: Purification and partial characterization of human polyamine synthases. Biochem. J., 259, 879-886 (1989) [9] Sarhan, S.; Dezeure, F.; Seiler, N.: Putrescine derivatives as substrates of spermidine synthase. Int. J. Biochem., 19, 1037-1047 (1987) [10] Hibasami, H.; Borchardt, R.T.; Chen, S.Y.; Coward, J.K.; Pegg, A.E.: Studies of inhibition of rat spermidine synthase and spermine synthase. Biochem. J., 187, 419-428 (1980) [11] Sindhu, R.K.; Cohen, S.S.: Propylamine transferases in Chinese Cabbage leaves. Plant Physiol., 74, 645-649 (1984) [12] Raina, A.; Pajula, R.L.; Eloranta, T.: A rapid assay method for spermidine and spermine synthases. Distribution of polyamine-synthesizing enzymes and methionine adenosyltransferase in rat tissues. FEBS Lett., 67, 252-255 (1976) [13] Raina, A.; Hyvönen, T.; Eloranta, T.; Voutilainen, M.; Samejima, K.; Yamanoha, B.: Polyamine synthesis in mammalian tissues. Isolation and characterization of spermidine synthase from bovine brain. Biochem. J., 219, 9911000 (1984)

514

2.5.1.16

Spermidine synthase

[14] Khomutov, R.M.; Hyvönen, T.; Karvonen, E.; Kauppinen, L.; Paalanen, T.; Paulin, L.; Eloranta, T.; Pajula, R.L.; Andersson, L.C.; Pösö, H.: 1-Aminooxy-3-aminopropane, a new and potent inhibitor of polyamine biosynthesis that inhibits ornithine decarboxylase, adenosylmethionine decarboxylase and spermidine synthase. Biochem. Biophys. Res. Commun., 130, 596602 (1985) [15] Tang, K.C.; Pegg, A.E.; Coward, J.K.: Specific and potent inhibition of spermidine synthase by the transition-state analog, S-adenosyl-3-thio-1,8-diaminooctane. Biochem. Biophys. Res. Commun., 96, 1371-1377 (1980) [16] Samejima, K.; Nakazawa, Y.: Action of decarboxylated S-adenosylmethionine analogs in the spermidine-synthesizing system from rat prostate. Arch. Biochem. Biophys., 201, 241-246 (1980) [17] Hibasami, H.; Kawase, M.; Tsukada, T.; Maekawa, S.; Sakurai, M.; Nakashima, K.: 2-Mercaptoethylamine, a competitive inhibitor of spermidine synthase in mammalian cells. FEBS Lett., 229, 243-246 (1988) [18] Pegg, A.E.; Bitonti, A.J.; McCann, P.P.; Coward, J.K.: Inhibition of bacterial aminopropyltransferases by S-adenosyl-1,8-diamino-3-thiooctane and by dicyclohexylamine. FEBS Lett., 155, 192-196 (1983) [19] Yamanoha, B.; Cohen, S.S.: S-adenosylmethionine decarboxylase and spermidine synthase from Chinese Cabbage. Plant Physiol., 78, 784-790 (1985) [20] Sindhu, R.K.; Cohen, S.S.: Putrescine aminopropyltransferase (spermidine synthase) of Chinese Cabbage. Methods Enzymol., 94, 279-285 (1983) [21] Samejima, K.; Raina, A.; Yamanoha, B.; Eloranta, T.: Purification of putrescine aminopropyltransferase (spermidine synthase) from eukaryotic tissues. Methods Enzymol., 94, 270-276 (1983) [22] Bowman, W.H.; White Tabor, C.; Tabor, H.: Spermidine biosynthesis. Purification and properties of propylamine transferase from Escherichia coli. J. Biol. Chem., 248, 2480-2486 (1973) [23] White Tabor, C.; Tabor, H.: Putrescine aminopropyltransferase (Escherichia coli). Methods Enzymol., 94, 265-270 (1983) [24] Zappia, V.; Cacciapuoti, G.; Pontoni, G.; Oliva, A.: Mechanism of propylamine-transfer reactions. Kinetic and inhibition studies on spermidine synthase from Escherichia coli. J. Biol. Chem., 255, 7276-7280 (1980) [25] Samejima, K.; Yamanoha, B.: Purification of spermidine synthase from rat ventral prostate by affinity chromatography on immobilized S-adenosyl(5)3-thiopropylamine. Arch. Biochem. Biophys., 216, 213-222 (1982) [26] Graser, G.; Hartmann T.: Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.. Planta, 211, 239-245 (2000) [27] Panicot, M.; Minguet, E.G.; Ferrando, A.; Alcµzar, R.; Blµzquez, M.A.; Carbonell, J.; Altabella, T.; Koncz, C.; Tiburcio, A.F.: A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant Cell, 14, 2539-2551 (2002) [28] Hashimoto T.; Tamaki, K.; Suzuki, K.; Yamada, Y.: Molecular cloning of plant spermidine synthases. Plant Cell Physiol., 39, 73-79 (1998)

515

Spermidine synthase

2.5.1.16

[29] Shirahata, A.; Takahashi, N.; Beppu, T.; Hosoda, H.; Samejima, K.: Effects of inhibitors of spermidine synthase and spermine synthase on polyamine synthesis in rat tissue. Biochem. Pharmacol., 45, 1897-1903 (1993) [30] Yoon, S.O.; Lee, Y.S.; Lee, S.H.; Cho, Y.D.: Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes. Biochim. Biophys. Acta, 1475, 17-26 (2000) [31] Lee. S.; Cho, Y.: A new assay method for spermidine and spermine synthases using antibody against MTA. J. Biochem. Mol. Biol., 30, 443-447 (1997) [32] Friesen, H.; Tanny, J.C.; Segall, J.: SPE3, which encodes spermidine synthase, is required for full repression through NREDIT in Saccharomyces cerevisiae. Genetics, 150, 59-73 (1998) [33] Caruso, A.; Pellati, A.; Bosi, G.; Arena, N.; Stabellini, G.: Effects of spermidine synthase inhibition on cytoskeletal organization in cultured chick embryo fibroblasts. Eur. J. Histochem., 38, 245-252 (1994) [34] Korolev, S.; Ikeguchi, Y.;Skarina T.; Beasley, S.; Arrowsmith, C.; Edwards, A.; Joachimiak, A.; Pegg, A.E.; Savchenko, A.: The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor. Nat. Struct. Biol., 9, 27-31 (2002)

516

Cob(I)yrinic acid a,c-diamide adenosyltransferase

2.5.1.17

1 Nomenclature EC number 2.5.1.17 Systematic name ATP:cob(I)yrinic acid-a,c-diamide Cob-adenosyltransferase Recommended name cob(I)yrinic acid a,c-diamide adenosyltransferase Synonyms ATP:cob(I)alamin transferase (ATR) ATP:corrinoid adenosyltransferase CobA adenosyltransferase, vitamin B12s aquacob(I)alamin adenosyltransferase aquocob(I)alamin adenosyltransferase vitamin B12s adenosyltransferase CAS registry number 37277-84-2

2 Source Organism

Clostridium tetanomorphum [1-4] Bos taurus [12] Homo sapiens [12] Lactobacillus leichmannii [3] Lactobacillus delbrueckii [3] Propionibacterium shermanii [2, 3] Protaminobacter ruber [7] Pseudomonas denitrificans (recombinant strain SC510 Rifr containing plasmid pXL227, this DNA seuqence encodes for several enzymes [5,6]) [5, 6] Salmonella typhimurium (structural gene cob A [8]) [8] Salmonella enterica (serovar typhimurium [9,10,11]) [9, 10, 11] Salmonella enterica (serovar typhimurium, structural gene pduO, this DNA sequence encodes for several enzymes [10]) [10]

517


2.5.1.17

3 Reaction and Specificity Catalyzed reaction ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide ATP + cobinamide = triphosphate + adenosylcobinamide Reaction type adenosyl group transfer Natural substrates and products S ATP + cob(I)alamin ( involved in vitamin B12 metabolism [1]; the main role of this enzyme is apparently the conversion of inactive cobalamins to adenosyl cobalamin for 1,2 propanediol degradation [10]) [1-5, 10] P tripolyphosphate + a-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide Substrates and products S ATP + cob(I)alamin ( transfers the adenosyl-group of ATP to the reduced cobalt atom of the cobalamin molecule [1]; stereospecific process which proceeds with overall inversion of configuration at C-5' of the adenosyl moiety [4]) (Reversibility: r [2]; ? [3, 5, 8, 9, 10, 11]) [1-12] P tripolyphosphate + a-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide (adenosylcobalamin, B12 -coenzyme or deoxyadenosyl-B12 ) [1-5, 8-11] S cob(I)inamide + ATP [5] P 5'-deoxy-5'-adenosyl-cob(I)inamide + polyphosphate [5] S cob(I)yric acid + ATP [5] P 5'-deoxy-5'-adenosyl-cob(I)yric acid + polyphosphate [5] S cob(I)yrinic acid a,c-diamide + ATP [5] P 5'-deoxy-5'-adenosyl-cob(I)yrinic acid a,c-diamide + polyphosphate [5] S cyanocob(I)alamin + ATP [1] P tripolyphosphate + a-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide [1] S Additional information ( S-adenosylmethionine, vitamin B12r or B12a , AMP, ADP are no substrates [1,2]; hydroxocobamides or cyanocobamides in which benzimidazole replaces 5,6-dimethylbenzimidazole, or cyanocobamide in which an adenine group replaces 5,6-dimethylbenzimidazole, can also act as substrates [2]; cobyrinic acid is not a substrate [5]; UTP, GTP, ITP are poor substrates [2]; GTP, CTP show only small activity as substrates [7]; crucial role of the 2'OH group of the ribosyl moiety of ATP, the g-phosphate of ATP is critical for positioning the target for nucleophilic attack, differences in the base of the nucleotide have no effect on enzyme activity, 2'-deoxynucleotides fails to serve as substrates [11]) [1-7, 11] P ?

518

2.5.1.17


Inhibitors 5-mercapto-5'-deoxyadenosine-5'-triphosphate ( complete inhibition at a 3 mM concentration [11]) [11] Ca2+ [3] Cd2+ [3] EDTA [1, 2] Mn2+ ( above 10 mM [3]) [3] Zn2+ [3] diphosphate [1-3] hydrogenobyrinic acid a,c-diamide ( cobalt-free analogue of cobyrinic acid a,c-diamide [5]) [5] trimetaphosphate [1, 2] triphosphate [11] tripolyphosphate [1-3, 5] Additional information ( no inhibition by phosphate [1]; no inhibition by cobyrinic acid, hydrogenobyrinic acid [5]; a,b-methylene-substituted derivatives of ATP are poor inhibitors [11]) [1, 5, 11] Metals, ions Co2+ ( activation, less effective than Mn2+ [1,2]) [1, 2, 8] Cs+ ( activation [3]) [3] K+ ( activation [3]; may be acting upon a step in the reduction of hydroxocobamide [3]) [2, 3, 7-10, 12] Mg2+ ( activation, less effective than Mn2+ [1-3,7]) [1-3, 7-12] Mn2+ [1-3, 5, 7-9] Na+ ( activation [3]) [3] Zn2+ ( activation, less effective than Mn2+ [1,2]) [1, 2] Additional information ( no activation by Ca2+ or Cd2+ [1,2]) [1, 2] Specific activity (U/mg) 0.000125 ( pH 8.0, 37 C [3]; activity measured using washed ribosomes [3]) [3] 0.047 ( pH 8.0, 37 C [12]; bovine ATR containing mitochondrial targeting sequence, expressed in E. coli, soluble fraction [12]) [12] 0.053 ( pH 8.0, 37 C [8]; activity at final purification step [8]) [8] 0.061 ( pH 8.0, 37 C [12]; human ATR containing mitochondrial targeting sequence, expressed in E. coli and forming inclusion bodies [12]) [12] 0.089 ( pH 8.0, 30 C [5]; in the presence of cobalamin as substrate [5]) [5] 0.098 ( pH 8.0, 37 C [12]; human ATR with no mitochondrial targeting sequence, expressed in E. coli and forming inclusion bodies [12]) [12] 0.13 ( pH 8.0, 30 C [5]; in the presence of cobyrinic acid a,cdiamide as substrate [5]) [5] 519


2.5.1.17

0.14 ( pH 8.0, 30 C [5]; in the presence of cobyric acid as substrate [5]) [5] 0.18 ( pH 8.0, 30 C [5]; in the presence of cobinamide as substrate [5]) [5] 0.312 ( pH 8.0, 37 C [10]; activity of inclusion bodies formed with enzyme expressed in E. coli [10]) [10] Additional information ( data for ATR inclusion bodies from the enzyme expressed in E. coli with no mitochondrial targeting sequence also available [12]) [12] Km-Value (mM) 0.0028 (ATP, pH 8.0, 37 C [8]) [8] 0.0052 (cob(I)alamin, pH 8.0, 37 C [8]) [8] 0.01 (cyanocob(I)alamin, pH 8.0, 37 C, i.e. vitamin B12 , reduced form [1]) [1, 2] 0.016 (ATP, pH 8.0, 37 C [1]) [1, 2] 0.25 (ATP) [7] 1.3 (ATP, pH 8.0, 37 C [3]) [3] pH-Optimum 8 ( phosphate or Tris buffer [1]; assay at [5, 8, 9, 12]) [1, 5, 7-9, 12] Temperature optimum ( C) 30 ( assay at [5]) [5] 37 ( assay at [1, 3, 8-10, 12]) [1, 3, 8-10, 12] 50 [7]

4 Enzyme Structure Molecular weight 36600 ( calculated from amino acid sequence [10]) [10] 37000 ( SDS-PAGE of recombinant enzyme expressed in E. coli [10]) [10] 42000 ( non-denaturing PAGE [8]) [8] 44000 ( gel filtration [7]) [7] 52000 ( SDS-PAGE, recombinant enzyme without mitochondrial targeting sequence [12]) [12] 54000 ( calculated from amino acid sequence of recombinant enzyme without mitochondrial targeting sequence [12]) [12] 55000 ( calculated from amino acid sequence of recombinant enzyme without mitochondrial targeting sequence [12]) [12] 56000 ( SDS-PAGE, recombinant enzyme with mitochondrial targeting sequence [12]) [12] 56000 ( HPLC gel filtration [5]) [5] 58000 ( calculated from amino acid sequence of recombinant enzyme with mitochondrial targeting sequence [12]) [12]

520

2.5.1.17


58000 ( calculated from amino acid sequence of recombinant enzyme with mitochondrial targeting sequence [12]) [12] 70000 ( PAGE [5]) [5] Subunits dimer ( 2 * 28000,SDS-PAGE [5]; 2 * 25000, SDS-PAGE [8]) [5, 8]

5 Isolation/Preparation/Mutation/Application Source/tissue liver ( mRNA expression detected [12]) [12] skin ( skin fibroblasts show low abundance [12]) [12] Localization ribosome [3] soluble ( from skin cells [12]) [1, 3, 5, 8, 12] Additional information ( subcellular distribution [3]) [3] Purification (protamine sulfate, heat treatment and chromatography on Sephadex G25 and DEAE-cellulose [1,2]) [1, 2] (centrifugation and SDS-PAGE [12]) [12] (partial purification of the enzyme is achieved from ribosomal fraction by salt extraction and ammonium sulfate fractionation, followed by two chromatographic steps [3]) [3] [7] (centrifugation, MonoQ chromatography and gel filtration [5]) [5] (ammonium sulfate precipitation followed by two chromatographic steps [8]) [8] Cloning (expressed in Escherichia coli [12]) [12] (expressed in Escherichia coli [12]) [12] (structural gene cobO [6]) [6] (structural gene cobA [10]) [10] (structural gene pduO [10]) [10]

6 Stability Temperature stability 4 ( stable for several weeks with dithiothreitol [8]) [8] 55 ( sharp decrease in activity with preincubation at 55 C or higher [8]) [8] 60 ( no appreciable loss of activity after 10 min [1]) [1]

521


2.5.1.17

General stability information , DTT stabilizes during purification and storage, removal leads to complete loss of activity [5] , DTT required to maintain activity [8] , 2-mercaptoethanol stabilizes during purification [1, 3] , repeated freeze-thawing inactivates [1, 3] Storage stability , -20 C, lyophilized, at least 1 week [1] , 4 C, about 1 month [1] , -20 C, more than 50% loss of activity within 1 week, with 50% glycerol only 20% loss of activity [3] , DTT stabilizes during storage [3] , 2-mercaptoethanol stabilizes during storage [1, 3]

References [1] Vitols, E.; Walker, G.A.; Huennekens, F.M.: Enzymatic conversion of vitamin B-12s to a cobamide coenzyme, a-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide (adenosyl-B-12). J. Biol. Chem., 241, 1455-1461 (1966) [2] Mudd, S.H.: The adenosyltransferases. The Enzymes, 3rd. Ed. (Boyer P.D. ed.), 8, 121-154 (1973) [3] Beck, W.S.: Ribosome-associated vitamin B12s adenosylating enzyme of Lactobacillus leichmannii. Methods Enzymol., 67, 41-56 (1980) [4] Parry, R.J.; Ostrander, J.M.; Arzu, I.Y.: Studies of enzyme stereochemistry. Elucidation of the stereochemistry of the reaction catalyzed by cob(I)alamin adenosyltransferase. J. Am. Chem. Soc., 107, 2190-2191 (1985) [5] Debussche, L.; Couder, M.; Thibaut, D.; Cameron, B.; Crouzet, J.; Blanche, F.: Purification and partial characterization of Cob(I)alamin adenosyltransferase from Pseudomonas denitrificans. J. Bacteriol., 173, 6300-6302 (1991) [6] Crouzet, J.; Levy-Schil, S.; Cameron, B.; Cauchois, L.; Rigault, S.; Rouyez, M.-C.; Blanche, F.; Debussche, L.; Thibaut, D.: Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase. J. Bacteriol., 173, 6074-6087 (1991) [7] Sato, K.; Nakashima, T.; Shimizu, S.: Assay, purification and characterization of cob(I)alamin adenosyltransferase of Protaminobacter ruber. J. Nutr. Sci. Vitaminol., 30, 405-413 (1984) [8] Suh, S.-J.; Escalante-Semerena, J.C.: Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium. J. Bacteriol., 177, 921-925 (1995) [9] Fonseca, M.V.; Escalante-Semerena, J.C.: An in vitro reducing system for the enzymic conversion of cobalamin to adenosylcobalamin. J. Biol. Chem., 276, 32101-32108 (2001)

522

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[10] Johnson, C.L.; Pechonick, E.; Park, S.D.; Havemann, G.D.; Leal, N.A.; Bobik, T.A.: Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene. J. Bacteriol., 183, 1577-1584 (2001) [11] Fonseca, M.V.; Buan, N.R.; Horswill, A.R.; Rayment, I.; Escalante-Semerena, J.C.: The ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica requires the 2'-OH group of ATP for function and yields inorganic triphosphate as its reaction byproduct. J. Biol. Chem., 277, 3312733131 (2002) [12] Leal, N.A.; Park, S.D.; Kima, P.E.; Bobik, T.A.: Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant. J. Biol. Chem., 278, 9227-9234 (2003)

523

Glutathione transferase

2.5.1.18

1 Nomenclature EC number 2.5.1.18 Systematic name RX:glutathione R-transferase Recommended name glutathione transferase Synonyms GSH S-transferase GSHTase-P S-(hydroxyalkyl)glutathione lyase glutathione S-alkyl transferase glutathione S-aralkyltransferase glutathione S-aryltransferase glutathione S-transferase glutathione S-transferase X Additional information ( overview of nomenclature of rat glutathione transferase isoenzymes [8]) [8] CAS registry number 50812-37-8

2 Source Organism Mus musculus (3 forms, products of different genes: MI, MII, MIII [1]; multiple forms [33]; wild-type and pi null mutant [59]) [1, 8, 33, 59] Bos taurus (liver: 2 major forms: I and II [10]; retina: 2 isoenzymes [11]) [10, 11] Rattus norvegicus (various isoenzymes [17]; male specific-pathogen-free Sprague Dawley [23]; 3 isoenzymes: 1, 2.1, 2.2 [47]; at least 7 forms [38]) [2-5, 8, 12, 14, 17, 18, 23, 24, 26, 27, 30, 31, 37-39, 43, 45, 47] Gallus gallus [6] Homo sapiens (basic and acid isoenzyme [16]; 13 forms [22]; polycythaemia patients [28]; multiple forms [35]; transferase P1-1 [63]) [7, 8, 12-16, 20, 22, 25, 28, 29, 31, 34-36, 50, 51, 63, 67] Hevea brasiliensis (rubber tree, 5 forms [9]) [9] Salmo gaidnerii (rainbow trout [19]) [19]

524

2.5.1.18


Sus scrofa (5 major and 3 minor forms [21]) [21, 41, 64] Wiseana cervinata (porina moth [32]) [32] Cavia porcellus [40] Ovis aries (sheep, 7 cationic isoenzymes, 5 anionic isoenzymes [46]) [8, 46] Macaca fuscata (Japanese monkey [42]) [42] Macaca fascicularis (crab-eating monkey [42]) [42] Macaca mulatta (rhesus monkey [42,48]) [42, 48] Zea mays (2 forms: GST I, GST II [44]) [14, 44, 54] Pisum sativum (pea [14,54]) [14, 54] Lucilia cuprina (sheep blowfly [49]) [49] Oryza sativa (rice, v. Tequing, indica [52]) [52] mammalia (cytosolic enzymes, referred to as a, m, p and , and microsomal enzyme [53]) [53] Schistosoma mansoni [53, 57] Triticum aestivum [54, 62] Medicago sativa (alfalfa [54]) [54] Trifolium repens (white clover [54]) [54] Phaseolus vulgaris (French bean [54]) [54] Phaseolus coccineus (runner bean [54]) [54] Glycine max [55] Curcubita maxima (pumpkin [56]) [56] Setaria faberi (giant foxtail, grass weed [58]) [58] Ascaris suum [60] Schistosoma japonicum [61] Hordeum vulgare (barley [65]) [65] Ilex sp. (squid [66]) [66]

3 Reaction and Specificity Catalyzed reaction RX + glutathione = HX + R-S-G ( mechanism [14, 49, 61]; random steady state mechanism [32]; kinetic mechanism [50, 53]) Reaction type aryl group transfer Natural substrates and products S RX + glutathione ( may be involved in preventing lipid peroxidation [2]; microsomal enzyme is likely to be involved in drug metabolism [4]; participates in detoxification [3, 8, 13, 14, 57, 58, 59]; phase II detoxification mechanism, isomerization of 3-ketosteroids and biosynthesis of peptide leukotrienes [53]; first step in mercapturic acid synthesis [8, 12, 14]; detoxification of products of oxidative metabolism, e.g. 4-hydroxyalkenals, epoxides, organic hydroperoxides [23]; biotransformation and detoxification of electrophilic xenobiotics [24]; involved both with biliru525


2.5.1.18

bin transport and detoxification of electrophils [35]; detoxification of electrophilic products of cytochrome P-450-dependent reactions, toxic catabolite of heme, bilirubin is bound and thereby neutralized [38]) [2, 3, 4, 8, 12, 14, 23, 24, 35, 38, 53, 57, 58, 59] P HX + R-S-G Substrates and products S RSSR + glutathione (Reversibility: ? [14]) [14] P glutathione-SSR + R-SH [14] S RX + glutathione ( RX: R: aliphatic, aromatic or heterocyclic, X: sulfate, nitrite or halide, enzyme also catalyzes: the addition of aliphatic epoxides and arene oxides to glutathione, the reduction of polyol nitrate by glutathione to polyol and nitrite, certain isomerization reactions and disulfide interchange [1-4, 6, 9, 10, 15, 16, 18, 20-24, 27, 29, 32, 33, 37, 40, 41, 44, 45]) (Reversibility: ? [1-4, 6, 9, 10, 15, 16, 18, 20-24, 27, 29, 32, 33, 37, 40, 41, 44, 45]) [1-4, 6, 9, 10, 15, 16, 18, 20-24, 27, 29, 32, 33, 37, 40, 41, 44, 45] P HX + R-S-G S glutathione + 1,2-dichloro-4-nitrobenzene ( enzyme MIII [1]; at a low rate [4]) (Reversibility: ? [1-4, 6, 9, 12, 14, 17, 20, 23, 24, 27, 30, 32, 33, 37, 42, 43, 45, 47, 48]) [1-4, 6, 9, 12, 14, 17, 20, 23, 24, 27, 30, 32, 33, 37, 42, 43, 45, 47, 48] P ? S glutathione + 1,2-dinitrobenzene (Reversibility: ? [10, 41]) [10, 41] P ? S glutathione + 1,2-epoxy-3-(4-nitrophenoxy)propane ( isoenzyme E [12]; anionic isozyme [46]) (Reversibility: ? [1, 6, 10, 12, 14, 15, 23, 24, 27, 29, 33, 43, 45-48, 52]) [1, 6, 10, 12, 14, 15, 23, 24, 27, 29, 33, 43, 45-48, 52] P ? S glutathione + 1,5-dinitrophenol (Reversibility: ? [41]) [41] P ? S glutathione + 1-chloro-2,4-dinitrobenzene ( enzyme has only weak activity towards the substrate [29]; best substrate [42, 45]; low activity towards the substrate [57]) (Reversibility: ? [1-6, 9, 10-12, 14-16, 18, 2027, 29, 30, 32, 33, 35-39, 41-43, 45-47, 50, 52, 54-58, 60-65, 67]) [1-6, 9, 1012, 14-16, 18, 20-27, 29, 30, 32, 33, 35-39, 41-43, 45-47, 50, 52, 54-58, 60-65, 67] P S-2,4-dinitrophenylglutathione + HCl [25, 26, 50] S glutathione + 1-fluoro-2,4-dinitrobenzene (Reversibility: ? [63]) [63] P ? S glutathione + 3,4-dichloro-1-nitrobenzene (Reversibility: ? [15, 39]) [15, 39] P ?

526

2.5.1.18


S glutathione + 3,4-dinitrobenzoic acid (Reversibility: ? [10,41]) [10, 41] P ? S glutathione + 4-hydroxynonenal (Reversibility: ? [1]) [1] P ? S glutathione + 4-nitrobenzyl chloride (Reversibility: ? [6, 11, 24, 27, 29, 32, 33, 37, 40-43, 45, 48]) [6, 11, 24, 27, 29, 32, 33, 37, 40-43, 45, 48] P ? S glutathione + 4-nitrophenyl acetate (Reversibility: ? [23, 27, 43]) [23, 27, 43] P ? S glutathione + 4-nitrophenyl bromide (Reversibility: ? [48]) [48] P ? S glutathione + 4-nitropyridine (Reversibility: ? [27]) [27] P ? S glutathione + 5-nitrofurfural (Reversibility: ? [10, 41]) [10, 41] P ? S glutathione + 5-nitrofurfural diacetal (Reversibility: ? [10, 41]) [10, 41] P ? S glutathione + 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole ( poor substrate [50]) (Reversibility: ? [50]) [50] P ? S glutathione + acrolein (Reversibility: ? [51]) [51] P ? S glutathione + adenine propenal (Reversibility: ? [51]) [51] P ? S glutathione + alachlor ( i.e. 2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide [44]; herbicide [58]) (Reversibility: ? [44, 58]) [44, 58] P ? S glutathione + androst-5-ene-3,17-dione (Reversibility: ? [23, 27]) [23, 27] P ? S glutathione + anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (Reversibility: ? [17, 18]) [17, 18] P ? S glutathione + atrazine ( herbicide [58]) (Reversibility: ? [58]) [58] P ? S glutathione + benzo(a)pyrene 4,5-oxide (Reversibility: ? [5, 25]) [5, 25] P ?

527


2.5.1.18

S glutathione + bromosulfophthalein (Reversibility: ? [24, 27, 29]) [24, 27, 29] P ? S glutathione + cumene hydroperoxide ( isozyme MI [1]) (Reversibility: ? [1, 2, 15-17, 20-23, 27, 29, 43, 46, 52, 54, 60, 65]) [1, 2, 15-17, 20-23, 27, 29, 43, 46, 52, 54, 60, 65] P ? S glutathione + ethacrynic acid ( low activity towards substrate [57]) (Reversibility: ? [6, 14, 16, 23, 24, 27, 29, 32, 43, 52, 57, 60, 67]) [6, 14, 16, 23, 24, 27, 29, 32, 43, 52, 57, 60, 67] P ? S glutathione + fenchlorim (Reversibility: ? [52]) [52] P ? S glutathione + fenoxaprop ( herbicide [65]) (Reversibility: ? [65]) [65] P 4-hydroxyphenoxypropanoic acid + glutathione conjugate [65] S glutathione + fluorodifen (Reversibility: ? [54, 58, 62, 65]) [54, 58, 62, 65] P 4-nitrophenol + 2-nitro-4-trifluoromethylphenol-glutathione [54, 65] S glutathione + hydroxyethyldisulfide (Reversibility: ? [57]) [57] P ? S glutathione + indomethane (Reversibility: ? [32, 40]) [32, 40] P ? S glutathione + iodomethane (Reversibility: ? [14, 32]) [14, 32] P ? S glutathione + linoleic acid hydroperoxide ( cationic isozyme [46]) (Reversibility: ? [27, 46, 65]) [27, 46, 65] P ? S glutathione + metolachlor ( herbicide [55, 58]) (Reversibility: ? [52, 55, 58]) [52, 55, 58] P ? S glutathione + naphthalene oxide (Reversibility: ? [40]) [40] P ? S glutathione + petrilachlor (Reversibility: ? [52]) [52] P ? S glutathione + styrene 7,8-oxide (Reversibility: ? [25]) [25] P ? S glutathione + styrene oxide (Reversibility: ? [15, 16, 40]) [15, 16, 40] P ? S glutathione + tert-butyl hydroperoxide ( anionic isozyme [46]) (Reversibility: ? [27, 46]) [27, 46] P ? 528

2.5.1.18


S glutathione + trans-4-phenyl-3-buten-2-one ( low activity [37]) (Reversibility: ? [6, 15, 20, 23-25, 37, 39, 42, 45, 47, 48]) [6, 15, 20, 23-25, 37, 39, 42, 45, 47, 48] P ? S Additional information ( obligate requirement for reduced glutathione as thiol donor [6]; specificity [1, 8, 10, 16, 20, 23, 27, 29, 32, 41-43, 45, 46]; assays for differentiation of glutathione S-transferase isoenzymes from rat and human by different specific acitivities with selected substrates [31]; glutathione transferase X: no reaction of menaphthyl sulfate with glutathione, potent in inactivation of tert-10,11-epoxy-r-8,tert-9-dihydroxy8,9,10,11-tetrahydrobenz/a/anthracene [45]; no substrate: 4-nitrobenzyl chloride [3, 9, 32]; no substrate: 1,2-epoxy-3-(4-nitrophenoxy)propane [3, 9, 32]; no substrate: ethacrynic acid [3]; no substrate: bromosulfophthalein [3, 9, 32]; no substrate: methyl iodide [9]; no substrate: 4-nitropyridine N-oxide [32]; enzyme has also selenium-independent peroxidase activity [11, 20]; any compound bearing a sufficiently electrophilic atom may be attacked, reactions with C, S, N and O atoms are possible [12]; catalyzes the reaction of glutathione with a large number of compounds bearing an electrophilic carbon to form the corresponding thioether [14]; isomerization of D5 - to D4 - unsaturated 3-keto steroids [14]; no substrates: sulfobromophthalein and hydrogen peroxide [15]; no substrate: trans-4-phenyl-3-buten-2-one [3, 18]; conjugation of glutathione with base propenals and related alkenes, A1-1 and M1-1 are more active with 4-hydroxyalkenals than with base propenals [51]; catalyzes S-conjugation between the thiol group of glutathione and an electrophilic moiety in the hydrophobic and toxic sustrate [53]; GSTC: activity towards 1-chloro-2,4-dinitrobenzene, GSTF: activity towards the herbicide fluorodifen, GPOX: peroxidase activity, overview: xenobiotic substrates [54]; enzyme has glutathione-dependent dehydroascorbate reductase and thiol transferase activity [57]; enzyme has also activity towards reactive carbonyls [60]) [1-3, 6, 8-10, 12, 14, -18, 20, 21, 23, 27, 29, 31, 32, 41-43, 45, 46, 51, 53, 54, 57, 60] P ? Inhibitors 1-naphthol [9] 2,4,6-trinitrobenzenesulfonate [48] 3,3',5,5'-tetrabromophenolphthalein [9] Cd2+ ( 3.3 mM [48]) [10, 48] Cibacron Blue ( 50% inhibition at 0.00009 mM [60]) [1, 23, 52, 60] Co2+ ( weak [10]; 3.3 mM [48]) [10, 48] Cu2+ ( 3.3 mM [48]) [10, 48] Evan's blue [57]

529


2.5.1.18

Hg2+ ( 3.3 mM [48]) [10, 48] N-ethylmaleimide ( completely inhibits form IX and X at 20 mM after 30 min, but not forms I- VIII [22]; complete loss of activity after 30 min at 5 mM [28]; weak [48]) [22, 28, 48] NaCl ( 0.2 M, 50% inhibition [32]) [32] Ni2+ ( weak [10]; 3.3 mM [48]) [10, 48] Rose Bengal [9, 23] S-(2,4-dinitrophenyl)glutathione ( product inhibition [50]) [32, 33, 50] S-(4-azidophenacryl)-glutathione ( competitive inhibitor, 27% loss of activity after 5 min [67]) [67] S-(4-bromobenzyl)glutathione [1, 23] S-hexylglutathione [1, 51, 52] S-methylglutathione ( competitive [36]) [36] Zn2+ ( 3.3 mM [48]) [10, 48] albendazole ( 50% inhibition at 0.52 mM [60]) [60] alizarin ( 50% inhibition at 0.0042 mM [60]) [60] benzene hexachloride [48] bilirubin ( 50% inhibition at 0.01-0.02 mM [22]; no inhibitor, bilirubin binding site is different from catalytic site [36]; 50% inhibition at 0.009 mM [60]) [22, 33, 36, 60] bithionol ( 50% inhibition at 0.0077 mM [60]) [60] brassylic acid [9] bromocresol green [9] bromosulfophthalein ( inhibits only minor form, but not major form [29]; 50% inhibition at 0.001 mM [60]) [1, 16, 17, 20, 23, 29, 52, 57, 60] bromothymol blue [9] butanol ( 50% inactivation at 5% [10]) [10] chalcone ( 50% inhibition at 0.045 mM [60]) [60] chlorophenol red [9] chlorotriphenyltin [52] ellagic acid ( 50% inhibition at 0.0006 mM [60]) [60] eosin [9] epichlorohydrin ( 3 mM, competitive [40]) [40] fluorescein [9] guanidine hydrochloride ( denaturation at 6 M [40]; reversible denaturation at 3.2 M [48]) [40, 48] hematin ( non competitive with transferase activity [22]) [1, 16, 20, 22, 23, 29, 57] hemin ( competitive with 1-chloro-2,4-dinitrobenzene, non-competitive with glutathione [6]) [6] hexachlorophene ( 50% inhibition at 0.00034 mM [60]) [60] indocyanine green ( inhibition of transferase B is greater than inhibition of transferase AA [31]) [31] indomethacin [23] iodoacetate ( weak [48]) [48, 52] 530

2.5.1.18


lithocholate ( 50% inhibition at 0.1 mM [60]) [60] naphthalene oxide ( 0.09 mM, competitive [40]) [40] phenol red [9] phenolphthalein [9] phenylglyoxal [48] phthalein [9] protoporphyrin IX ( 50% inhibition at 0.03 mM [60]) [60] purpurgallin ( 50% inhibition at 0.0052 mM [60]) [60] quercetin ( 50% inhibition at 0.0006 mM [60]) [60] sodium dodecyl sulfate ( denaturates irreversibly at 0.03 M [48]) [48] sulfophenolphthalein [9] sulfophthalein [1, 9, 23] tributyltin acetate [1, 16, 20, 23] tridiphane [52] triethyltin bromide [1, 17, 23] triphenyltin chloride ( 50% inhibition at 0.0003 mM [60]) [23, 60] undecanedioic acid [9] urea ( denaturates at 7.2 M, reversible [48]; 25% inactivation at 0.7 M [66]) [48, 66] vinylpyridine ( weak [48]) [48] vitamin K [9] Additional information ( difference in sensitivity to inhibition of enzyme MI, MII and MIII [1]; of seven major rat isoenzymes [2]) [1, 2] Activating compounds N-ethylmaleimide ( 8fold [4]) [1, 4] Metals, ions CuCl2 ( 0.05 mM and 0.5 mM increases activity of GPOX [54]) [54] K+ ( no enzyme activity with potassium phosphate concentrations below 20 mM, optimal concentration 50 mM [44]) [44] Turnover number (min±1) 16.8 (glutathione, S9A mutant, cosubstrate: 1-chloro-2,4-dinitrobenzene [49]) [49] 270 (1-chloro-2,4-dinitrobenzene, minor enzyme form [29]) [29] 440 (glutathione) [43] 460 (glutathione, minor enzyme form [29]) [29] 733 (glutathione, major enzyme form [29]) [29] 1100 (1-chloro-2,4-dinitrobenzene, major enzyme form [29]) [29] 1620 (1-chloro-2,4-dinitrobenzene, isoenzyme 4-4, cofactor: glutathione [23]) [23] 3180 (glutathione, wild-type, cosubstrate: 1-chloro-2,4-dinitrobenzene [49]) [49] 3240 (1-chloro-2,4-dinitrobenzene, isoenzyme 3-3, cofactor: glutathione [23]) [23]

531


2.5.1.18

3780 (1-chloro-2,4-dinitrobenzene, isoenzyme 1-2, cofactor: glutathione [23]) [23] 3900 (1-chloro-2,4-dinitrobenzene, isoenzyme 3-4, cofactor: glutathione [23]) [23] 4560 (1-chloro-2,4-dinitrobenzene) [63] 4680 (1-chloro-2,4-dinitrobenzene, isoenzyme 1-1, cofactor: glutathione [23]) [23] 4920 (1-chloro-2,4-dinitrobenzene, isoenzyme 3-?, cofactor: glutathione [23]) [23] 6360 (1-fluoro-2,4-dinitrobenzene) [63] 8640 (glutathione, Y113F mutant, cosubstrate: 1-chloro-2,4-dinitrobenzene [49]) [49] 45680 (1-chloro-2,4-dinitrobenzene, transferase P1-1, cofactor: glutathione [50]) [50] Specific activity (U/mg) 0.00242 [2] 0.0029-0.0036 ( with 1-chloro-2,4-dinitrobenzene as substrate [67]) [67] 0.01 ( with trans-4-phenyl-3-buten-2-one as substrate [15]) [15] 0.024 ( with metolachlor as substrate [55]) [55] 0.03 ( with 1-chloro-2,4-dinitrobenzene + glutathione or trans-4phenyl-3-buten-2-one as substrate [6]) [6] 0.03 ( with cumene hydroperoxide as substrate [15]) [15] 0.07 ( with styrene oxide as substrate [15]) [15] 0.11 ( with 3,4-dichloro-1-nitrobenzene as substrate [15]) [15] 0.12 ( with cumene hydroperoxide as substrate [27]) [27] 0.13 [5] 0.17 [64] 0.18 ( with 4-nitrophenyl acetate as substrate [27]) [27] 0.26 ( with ethacrynic acid as substrate [60]) [60] 0.312 ( with cumene hydroperoxide as substrate [60]) [60] 0.323 [56] 0.34 ( with ethacrynic acid as substrate [27]) [27] 0.37 ( with 1,2-epoxy-3-(4-nitrophenoxy)propane as substrate [15]) [15] 0.4 ( with 1,2-epoxy-3-(4-nitrophenoxy)propane as substrate [27]) [27] 0.41 [42] 0.49 ( with 1,2-epoxy-3-(4-nitrophenoxy)propane as substrate [6]) [6] 0.7 ( with adenine propenal, isoenzyme A1-1 [51]) [51] 0.75 ( with 4-nitrobenzyl chloride as substrate [6]) [6] 0.81 ( isoenzyme B [12]) [12] 0.83 ( with ethacrynic acid as substrate [6]) [6] 0.84-0.92 ( with glutathione as substrate [67]) [67]

532

2.5.1.18


0.86 ( with acrolein, isoenzyme A1-1 [51]) [51] 0.922 ( isoenzyme C [37]) [37] 1.5-8.3 ( with cumene hydroperoxide as substrate [22]) [22] 1.769 ( isoenzyme A [37]) [37] 1.8 ( with 1-chloro-2,4-dinitrobenzene as substrate [27]) [27] 2 ( isoenzyme C [12]) [12] 2.3 ( with 1,2-dinitrobenzene [10]) [10] 2.7 ( with 1,2-dichloro-4-nitrobenzene as substrate [45]) [45] 3.44 [6] 3.6-11.8 ( with cumene hydroperoxide as substrate [21]) [21] 3.7 ( with adenine propenal, isoenzyme M1-1 [51]) [51] 4.1 ( isoenzyme A [12]) [12] 4.9 ( with 1,2-dichloro-4-nitrobenzene as substrate [55]) [55] 6 ( with 1-chloro-2,4-dinitrobenzene as substrate [65]) [65] 7.05 ( with acrolein, isoenzyme M1-1 [51]) [51] 7.9-41.1 ( with 1-chloro-2,4-dinitrobenzene as substrate [21]) [21] 8-12 [61] 10.83 [43] 12.72 ( more anionic isoenzyme [11]) [11] 13.8 ( lung enzyme [39]) [39] 14 ( isoenzyme AA [12]) [12] 14.5 ( cationic isozymes [46]) [46] 15 ( with 1-chloro-2,4-dinitrobenzene [10]) [10] 15.47 ( less anionic isoenzyme [11]) [11] 16 ( isoenzyme b [12]) [12] 16.4 [16] 16.7-64.1 ( with 1-chloro-2,4-dinitrobenzene as substrate [23]) [23] 17 ( isoenzyme g [12]) [12] 17-64 [26] 20.4 ( anionic isozymes [46]) [46] 21 ( GST II [44]) [44] 21.5 ( with 1-chloro-2,4-dinitrobenzene as substrate [45]) [45] 26.3 ( with acrolein, isoenzyme P1-1 [51]) [51] 26.7 ( liver enzyme [39]) [39] 29.2 ( isoenzyme E [12]) [12] 29.4 [58] 30.9 [3] 33-94 ( with 1-chloro-2,4-dinitrobenzene as substrate [22]) [22] 34 ( isoenzyme e [12]) [12] 37 ( isoenzyme d [12]) [12] 38.2 ( with 1,2-dichloro-4-nitrobenzene as substrate [60]) [60] 52 [35] 56.5-79.4 [25] 66 ( species with very low isoelectric point from erythrocytes [12]; GST I [44]) [12, 44]

533


2.5.1.18

77 ( with adenine propenal, isoenzyme P1-1 [51]) [51] 80-272 ( with 1-chloro-2,4-dinitrobenzene as substrate [20]) [20] 82.27 ( GST I-I [52]) [52] 83 [34] 87 [16] 93.21 ( GST II-III [52]) [52] 104 [22] 105 ( with 1-chloro-2,4-dinitrobenzene as substrate [15]) [15] 108 [32] 124 [41] 130 [24] 148 [1] 254.9 [7] Additional information [9, 40] Km-Value (mM) 0.0006 (glutathione, not activated [4]) [4] 0.001-0.002 (glutathione, activated with N-ethylmaleimide [4]) [4] 0.005 (fluorodifen) [58] 0.006 (1-chloro-2,4-dinitrobenzene, not activated [4]) [4] 0.022 (fluorodifen) [62] 0.025 (fluorodifen) [54] 0.03 (1-chloro-2,4-dinitrobenzene, activated with N-ethylmaleimide [4]) [4] 0.03 (metolachlor) [58] 0.033-0.045 (glutathione) [9] 0.09 (glutathione, recombinant enzyme [67]) [67] 0.1 (glutathione, isozyme I [10]) [10] 0.11 (metolachlor) [55] 0.12 (alachlor) [58] 0.15-0.2 (1-chloro-2,4-dinitrobenzene) [9] 0.16 (glutathione, isozyme II [10]) [10] 0.22 (glutathione, placental enzyme [67]) [67] 0.228 (glutathione) [62] 0.23 (glutathione) [43] 0.23 (hydroxyethyldisulfide) [57] 0.232 (glutathione) [55] 0.25 (glutathione) [48] 0.3 (glutathione, transferase 1 and 2.2 [47]) [47] 0.32 (glutathione) [57] 0.35 (reduced glutathione) [6] 0.424 (1-chloro-2,4-dinitrobenzene) [62] 0.44 (1-chloro-2,4-dinitrobenzene, transferase 2.2 [47]) [47] 0.5 (glutathione, major form [29]) [15, 29] 0.503 (1-chloro-2,4-dinitrobenzene) [55]

534

2.5.1.18


0.53 (1-chloro-2,4-dinitrobenzene) [58] 0.55 (1-chloro-2,4-dinitrobenzene, isozyme I [10]) [10] 0.55 (atrazine) [58] 0.55 (glutathione) [54] 0.6 (1-chloro-2,4-dinitrobenzene, major form [29]) [29] 0.6 (alachlor, i.e. 2-chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide [44]) [44] 0.7 (1-chloro-2,4-dinitrobenzene, transferase 1 [47]) [47] 0.94 (1-chloro-2,4-dinitrobenzene) [43] 1.3 (1-chloro-2,4-dinitrobenzene, transferase 2.1 [47]) [47] 1.3 (3,4-dinitrobenzoic acid, isozymes I and II [10]) [10] 1.4 (1,2-dichloro-4-nitrobenzene) [48] 1.41 (1-chloro-2,4-dinitrobenzene, less anionic transferase [11]) [11] 1.8 (1-chloro-2,4-dinitrobenzene, more ionic transferase [11]) [11] 1.9 (1-chloro-2,4-dinitrobenzene, minor form [29]) [29] 1.92 (1-chloro-2,4-dinitrobenzene) [6] 2 (1,2-dinitrobenzene, isozymes I and II [10]) [10] 2 (glutathione, minor form [29]) [29] 2.1 (1-chloro-2,4-dinitrobenzene) [15] 2.57 (1-chloro-2,4-dinitrobenzene, placental enzyme [67]) [67] 2.8 (1-chloro-2,4-dinitrobenzene, GST II [44]) [44] 3 (1-chloro-2,4-dinitrobenzene, GST I [44]) [44] 3.3 (glutathione) [58] 5.6 (glutathione, transferase 2.1 [47]) [47] 6.6 (1-chloro-2,4-dinitrobenzene, recombinant enzyme [67]) [67] Additional information [11, 15, 23, 29, 33, 40] Ki-Value (mM) 0.001 (hemin, with reduced glutathione [6]) [6] 0.002 (hematin, major form [29]) [29] 0.003 (indocyanine green, transferase B [31]) [31] 0.0056-0.011 (bilirubin) [33] 0.017-0.036 (S-(2,4-dinitrohenyl)glutathione) [33] 0.032 (hematin, minor form [29]) [29] 0.037 (naphthalene oxide) [40] 0.068 (bromosulfophthalein, minor form [29]) [29] 0.1 (S-methylglutathione) [36] 0.1 (indocyanine green, transferase AA [31]) [31] 1.49 (epichlorohydrin) [40] pH-Optimum 6.5 ( more anionic transferase [11]) [11] 6.5 ( more anionic transferase [11]; GST I-I [52]) [11, 52] 6.5-7.5 ( less anionic transferase [11]) [11, 41] 6.5-8 [2]

535


2.5.1.18

7 ( GST II-III [52]) [52] 7.5 [10, 47] 7.5-8.5 [32] 7.5-9 ( 1,2-dichloro-4-nitrobenzene [48]) [48] 8 ( phosphate buffer [57]) [57] 8.5-9.5 [9] 8.7 ( Tris buffer [57]) [57] 8.8 [6] 9.3-9.5 ( cleavage of fluorodifen [54]) [54] pH-Range 5-8.5 ( effect of pH on binding of glutathione [61]) [61] 6-8 ( pH 6: about 40% of activity maximum, pH 8: about 55% of activity maximum [10]) [10] 6-8.1 ( at pH 6.0 and 8.1 about 50% of activity maximum [41]) [41] Temperature optimum ( C) 30 ( assay at [15,23,25]) [15, 23, 25]

4 Enzyme Structure Molecular weight 14000 ( SDS-PAGE [3]) [3] 43000 ( gel filtration [41]) [41] 45000 ( gel filtration [1, 6, 45, 47]; erythrocytes, minor form [29]; isoenzymes 1 and 2.2 [47]) [1, 6, 14, 29, 45, 47] 45000-47000 ( sedimentation equilibrium analysis, gel filtration [40]) [40] 45000-49000 [12] 45000-50000 [32] 45000-60000 ( gel filtration [52]) [52] 47000 ( erythrocytes, major form [29]) [15, 29] 48000 ( isoenzyme [48]) [48] 48000-54000 ( gel filtration [54]) [54] 49000 ( gel filtration [10,11,55]) [10, 11, 55] 49000-54000 ( gel filtration [43]) [43] 49500 ( native PAGE [62]) [62] 50000 ( gel filtration [9,44,64]) [9, 44, 64] 88000 ( gel filtration, isoenzyme 2.1 [47]) [47] Subunits dimer ( 2 * 23000, acidic isoenzyme, SDS-PAGE [16]; 2 * 23000, enzyme MII, SDSPAGE [1]; 2 * 23000, SDS-PAGE [6]; 2 * 25000, enzyme MI, SDSPAGE [1]; 2 * 25000, basic isoenzyme, SDS-PAGE [16]; 2 * 26500, enzyme MIII, SDS-PAGE [1]; 2 * 26000, SDS-PAGE [18]; 2 * 27000, SDS-PAGE [10]; 1 * 27100 + 1 * 26800, isoenzyme IV, SDS-PAGE [21];

536

2.5.1.18


1 * 27100 + 1 * 28700, isoenzyme I, SDS-PAGE [21]; 1 * 26800 + 1 * 26000, isoenzyme V, SDS-PAGE [21]; 2 * 26400, isoenzyme VI, SDSPAGE [21]; 2 * 26000, isoenzyme VII, SDS-PAGE [21]; 2 * 25200, form I-XII, SDS-PAGE [22]; 2 * 25600, form XIII, SDS-PAGE [22]; 2 * 26000, SDS-PAGE [24]; 2 * 25500 [25]; 2 * 24400, SDS-PAGE [27]; 2 * 22000, SDS-PAGE [36]; 2 * 25000, SDS-PAGE [40]; 2 * 22000, SDS-PAGE [41]; 2 * 22800 or 2 * 24600, SDS-PAGE [32]; 2 * 26000, SDS-PAGE [43]; 2 * 29000, enzyme GST I, SDS-PAGE [44]; 2 * 29000, enzyme GST II, SDS-PAGE [44]; 2 * 23500, SDS-PAGE [45]; 2 * 24000, cationic isoenzyme [46]; 2 * 26000, anionic isoenzyme [46]; 2 * 24000, SDS-PAGE [48]; dimeric forms: B1B1, B2B2 and B1B2 [20]; dimer composed of two of the following subunits: Ya MW 23000, Yb MW 23500, Yc MW 25000, SDS-PAGE [38]; 2 * 30000, GST I, SDS-PAGE [52]; 1 * 25000 + 1 * 28000, GST II-III, SDS-PAGE [52]; pi and theta class enzymes are homodimers, a and mu class enzymes can be homo- or heterodimeric [53]; 1 * 27500 + 1 * 29000, SDS-PAGE, GSTC and GPOX [54]; 1 * 30000 + 1 * 27500 or 1 * 30000 + 1 * 29000, SDS-PAGE, GSTF [54]; 2 * 26000, SDS-PAGE [55]; 2 * 2600028000 [58]; 2 * 26000 [61]; 2 * 24500, SDS-PAGE [62]; 2 * 27328, electrospray ionization MS [64]; homodimer [65]) [1, 6, 10, 1316, 18, 20, 21, 22, 24, 25, 27, 36, 38, 40, 41, 43-46, 48, 52-55, 58, 60-62, 64, 65, 66] Additional information ( more than one subunit [3]) [3]

5 Isolation/Preparation/Mutation/Application Source/tissue brain ( distribution in brain [41,42]) [41, 42] diaphragm [64] epididymis [18] erythrocyte [8, 12, 13, 22, 28, 29] hepatoma [18, 30] intestine [60] kidney [18, 30, 42] larva [32] latex [9] leaf ( etiolated [44]) [44] liver ( foetal [6]; no activity in liver [18]) [1-6, 8-10, 12, 16, 17, 18, 19-23, 25-27, 30, 34, 35, 37, 39, 40, 42, 43, 45-48, 59, 64] lung [17, 30, 39, 42] muscle [42, 64] pancreas [30] placenta [7, 8, 15, 22, 30, 36, 42, 50, 63, 67] platelet [13, 28] retina [11]

537


2.5.1.18

root [54, 62] seedling ( etiolated [54]) [54] shoot ( etiolated [52]) [52, 62] skeletal muscle [18] skin [67] spleen [42, 64] testis [18, 24] Additional information ( different isoenzyme patterns in rat lung and liver [17]; distribution in various tissues [64]) [17, 64] Localization cytosol [1, 6, 15, 16, 17, 19, 20, 23, 26, 27, 37, 39, 41, 43, 45, 53, 64] endoplasmic reticulum ( exposed on cytoplasmic surface [4]; highest activity in rough endoplasmic reticulum [5]) [4, 5] microsome ( highest activity in rough endoplasmic reticulum [5]) [2-5, 41, 53] mitochondrion [41, 47] Additional information ( microsomal enzyme is clearly distinct from cytosolic [2-4]) [2-4] Purification [1] (2 isozymes [11]) [10, 11] (36fold [3]; isoenzyme A, AA, B, C and E [12]; isoenzyme 7-7 [18]; 7 major isoenzymes [23]; 90% pure [24]; 79fold [30]; isoenzyme A and C [37]; 200fold, isoenzyme A, B and C [39]; homogeneity, anionic enzyme [43]; homogeneity [45]; transferase 1, 2.1 and 2.2 [47]) [2, 3, 12, 14, 17, 18, 23, 24, 26, 30, 37, 39, 43, 45, 47] [6] (3 forms, homogeneity [7]; forms: a, b, g, d, e and a species with a very low isoelectric point [12]; an acidic and a basic form [16]; 13 isoforms [22]; multiple forms [35]; wild type and mutants [50]; P1-1, A1-1 and M1-1 [51]; isoenzyme P1-1 [63]; GST pi [67]) [7, 12, 14, 15, 16, 22, 25, 29, 34, 35, 36, 50, 51, 63, 67] (basic and acidic isoenzymes [19]) [19] (5 major: I, IV, V, VI, VII and 3 minor forms [21]; homogeneity, 4 enzyme fractions [41]; w isoenzyme [64]) [21, 41, 64] [32] [40] [46] (homogeneity [42]) [42, 48] (2 forms: GST I, GST II [44]) [14, 44] (GSTC: activity towards 1-chloro-2,4-dinitrobenzene, GSTF: activity towards the herbicide fluorodifen, GPOX: peroxidase activity [54]) [54] [49] (homogeneity, 2 isoenzymes: GST I-I and GST II-III [52]) [52] [57] 538

2.5.1.18


(48fold [62]) [62] (near homogeneity, 2,4-dichloro-phenoxyacetic acid-inducible GST isoenzyme [55]) [55] (four isoenzymes [58]) [58] (recombinant protein after expression in E. coli [60]) [60] [61] (most abundant inducible GST isoenzyme after treatment with safener [65]) [65] [66] Renaturation (partial regeneration after urea and guanidine hydrochloride denaturation [48]) [48] Crystallization (complexed with glutathione or its analogues [53]) [53] (hanging drop vapor diffusion method [53]) [53] Cloning [51, 63, 67] [49] [57] [60] [61] [65] [66] Engineering C47S ( decreased kcat [63]) [50, 63] C47S/C101S ( decreased kcat [63]) [50, 63] S9A ( decreased kcat [49]) [49] T13A ( reduced hydrogen bonding strength to the glutathione's sulfur and decreased stability of the thiolate anion [53]) [53] T13V ( reduced hydrogen bonding strength to the glutathione's sulfur and decreased stability of the thiolate anion [53]) [53] Y113F ( increased kcat [49]) [49]

6 Stability pH-Stability 3.5 ( 10 min, 37 C, 50% loss of activity, 18 h at 4 C, 50% loss of activity [10]) [10] 4.2 ( 10 min at 37 C, complete loss of activity, at lower pH values somewhat more stable [48]) [48] 6-10 ( 10 min at 37 C, stable, 18 h at 4 C, stable [10]) [10] 7-10 ( 10 min, 37 C, stable [48]) [48]

539


2.5.1.18

Temperature stability 30 ( 3 h, stable [10]) [10] 35 ( 1 h, stable [10]) [10] 37 ( fairly stable between pH 6 and 10 [10]; pH 7.5, 3 h, stable [48]) [10, 48] 40 ( 10 min, pH 7.5, stable up to 40 C [48]) [48] 40 ( 10 min, stable [10]; moderately stable below [48]) [10, 48] 45 ( 10 min, 50% loss of activity [6]) [6] 50 ( complete inactivation within 1 h [48]) [48] 55 ( above, 10 min, 50% inactivation [10]) [10] 80 ( complete inactivation after 10 min [6]) [6] 100 ( complete inactivation after 10 min [5]) [5] Organic solvent stability acetone ( 5% acetone: stable [10]; 25% acetone: 70-80% inactivation [10]; stable for 2 h at 5% and 25% acetone [48]) [10, 48] butanol ( stable at 5% butanol [10]; 90% inactivation at 25% butanol, stable for 2 h at 5% butanol [48]) [10, 48] dimethyl sulfoxide ( stable at 5% and 25% dimethyl sulfoxide [10]; 5% and 25% dimethyl sulfoxide: stable for 2 h [48]) [10, 48] dimethylformamide ( stable for 2 h at 5% and 25% dimethylformamide [48]; 5% dimethylformamide: stable, 25% dimethylformamide: 70-80% inactivation [10]) [10, 48] dioxane ( 5% dioxane: stable [10]; 25% dioxane: 70-80% inactivation [10]; 5% dioxane: stable for 2 h [48]; 25% dioxane: 25% loss of activity [48]) [10, 48] ethanol ( 5% ethanol: stable [10]; 25% ethanol: 70-80% inactivation [10]; 5% and 25% ethanol: stable for 2 h [48]) [10, 48] methanol ( 5% methanol: stable [10]; 25% methanol: 7080% inactivation [10]; stable at 5% and 25% methanol for 2 h [48]) [10, 48] methyl cellosolve ( stable at 5% and 25% methyl cellosolve for 2 h [10,48]) [10, 48] propanol ( 5% propanol: stable [10]; 25% propanol: 70-80% inactivation [10]; stable for 2 h at 5% propanol [48]; 90% loss of activity at 25% propanol [48]) [10, 48] General stability information , glycerol, 30%, protects against thermal inactivation [10] , sucrose, 0.5 M, protects against thermal inactivation [10] , loss of activity during purification due to irreversible inactivation [43] , basic forms are more stable than acidic ones [19] , loss of activity during dialysis [41] , guanidine hydrochloride, 6 M, denaturation [14]

540

2.5.1.18


Storage stability , 4 C, 50% loss of activity after 18 h [10] , -70 C, pH 7, 10% glycerol [45] , -70 C, stable for several months [12] , -20 C, 6 months [9] , 4 C, 30% loss of activity after 16 h [54]

References [1] Warholm, M.; Jensson, H.; Tahir, M.K.; Mannervik, B.: Purification and characterization of three distinct glutathione transferases from mouse liver. Biochemistry, 25, 4119-4125 (1986) [2] Morgenstern, R.; DePierre, J.W.: Microsomal glutathione transferase. Purification in unactivated form and further characterization of the activation process, substrate specificity and amino acid composition. Eur. J. Biochem., 134, 591-597 (1983) [3] Morgenstern, R.; Guthenberg, C.; DePierre, J.W.: Microsomal glutathione Stransferase. Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione S-transferases A, B and C. Eur. J. Biochem., 128, 243-248 (1982) [4] Morgenstern, R.; Meijer, J.; DePierre, J.W.; Ernster, L.: Characterization of rat-liver microsomal glutathione S-transferase activity. Eur. J. Biochem., 104, 167-174 (1980) [5] Friedberg, T.; Bentley, P.; Stasiecki, P.; Glatt, H.R.; Raphael, D.; Oesch, F.: The identification, solubilization, and characterization of microsome-associated glutathione S-transferases. J. Biol. Chem., 254, 12028-12033 (1979) [6] Yeung, T.-C.; Gidari, A.S.: Purification and properties of a chicken liver glutathione S-transferase. Arch. Biochem. Biophys., 205, 404-411 (1980) [7] Radulovic, L.L.; Kulkarni, A.P.: A rapid, novel high performance liquid chromatography method for the purification of glutathione S-transferase: an application to the human placental enzyme. Biochem. Biophys. Res. Commun., 128, 75-81 (1985) [8] Jakoby, W.B.: Glutathione transferases: an overview. Methods Enzymol., 113, 495-499 (1985) [9] Balabaskaran, S.; Muniandy, N.: Glutathione S-transferase from Heva Brasiliensis. Phytochemistry, 23, 251-256 (1984) [10] Asaoka, K.: Affinity purification and characterization of glutathione Stransferases from bovine liver. J. Biochem., 95, 685-696 (1984) [11] Saneto, R.P.; Awasthi, Y.C.; Srivastava, S.K.: Glutathione S-transferases of the bovine retina. Evidence that glutathione peroxidase activity is the result of glutathione S-transferase. Biochem. J., 205, 213-217 (1982) [12] Habig, W.H.; Jakoby, W.B.: Glutathione S-transferases (rat and human). Methods Enzymol., 77, 218-231 (1981) [13] Rogerson, K.S.; Mitchell, A.L.; Ibbotson, R.; Cotton, W.; Strange, R.C.: Studies on the glutathione S-transferase of human platelets and erythrocytes. Biochem. Soc. Trans., 13, 200-201 (1985) 541


2.5.1.18

[14] Jakoby, W.B.; Keen, J.H.: A triple-threat in detoxification: the glutathione Stransferases. Trends Biochem. Sci., 2, 229-231 (1977) [15] Mannervik, B.; Guthenberg, C.: Glutathione transferase (human placenta). Methods Enzymol., 77, 231-235 (1981) [16] Guthenberg, C.; Warholm, M.; Rane, A.; Mannervik, B.: Two distinct forms of glutathione transferase from human foetal liver. Purification and comparison with isoenzymes isolated from adult liver and placenta. Biochem. J., 235, 741-745 (1986) [17] Robertson, I.G.C.; Jensson, H.; Guthenberg, C.; Tahir, M.K.; Jernström, B.; Mannervik, B.: Differences in the occurrence of glutathione transferase isoenzymes in rat lung and liver. Biochem. Biophys. Res. Commun., 127, 80-86 (1985) [18] Meyer, D.J.; Beale, D.; Tan, K.H.; Coles, B.; Ketterer, B.: Glutathione transferases in primary rat hepatomas: the isolation of a form with GSH peroxidase activity. FEBS Lett., 184, 139-143 (1985) [19] Ramage, P.I.N.; Nimmo, I.A.: The purification of the hepatic glutathione Stransferases of rainbow trout by glutathione affinity chromatography alters their isoelectric behaviour. Biochem. J., 211, 523-526 (1983) [20] Stockman, P.K.; McLellan, L.I.; Hayes, J.D.: Characterization of the basic glutathione S-transferase B1 and B2 subunits from human liver. Biochem. J., 244, 55-61 (1987) [21] Williamson, G.; Ball, S.K.M.; Chan, H.W.-C.: The purification of multiple forms of glutathione S-transferase from pig liver and their reaction with individual methyl linoleate hydroperoxides. Biochem. Soc. Trans., 14, 1278-1279 (1986) [22] Vander Jagt, D.L.; Hunsaker, L.A.; Garcia, K.B.; Royer, R.E.: Isolation and characterization of the multiple glutathione S-transferases from human liver. Evidence for unique heme-binding sites. J. Biol. Chem., 260, 1160311610 (1985) [23] Alin, P.; Jensson, H.; Guthenberg, C.; Danielson, U. H.; Tahir, M.K.; Mannervik, B.: Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing. Anal. Biochem., 146, 313-320 (1985) [24] Guthenberg, C.; Alin, P.; Mannervik, B.: Glutathione transferase from rat testis. Methods Enzymol., 113, 507-510 (1985) [25] Warholm, M.; Guthenberg, C.; van Bahr, C.; Mannervik, B.: Glutathione transferases from human liver. Methods Enzymol., 113, 499-504 (1985) [26] Jensson, H.; Alin, P.; Mannervik, B.: Glutathione transferase isoenzymes from rat liver cytosol. Methods Enzymol., 113, 504-507 (1985) [27] Reddy, C.C.; Li, N.-Q.; Tu, C.-P. D.: Identification of a new glutathione Stransferase from rat liver cytosol. Biochem. Biophys. Res. Commun., 121, 1014-1020 (1984) [28] Rogerson, K.S.; Mitchell, D.; Lawton, A.; Ibbotson, R.; Cotton, W.; Strange, R.C.: Studies on the glutathione S-transferase of human platelets. Biochem. Biophys. Res. Commun., 122, 407-412 (1984)

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[29] Awasthi, Y.C.; Singh, S.V.: Purification and characterization of a new form of glutathione S-transferase from human erythrocytes. Biochem. Biophys. Res. Commun., 125, 1053-1060 (1984) [30] Satoh, K.; Kitahara, A.; Soma, Y.; Inaba, Y.; Hatayama, I.; Sata, K.: Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis. Proc. Natl. Acad. Sci. USA, 82, 3964-3968 (1985) [31] Habig, W.H.; Jakoby, W.B.: Assays for differentiation of glutathione S-transferases. Methods Enzymol., 77, 398-405 (1981) [32] Clark, A.G.; Drake, B.: Purification and properties of glutathione S-transferases from larvae of Wiseana cervinata. Biochem. J., 217, 41-50 (1984) [33] Lee, C.-Y.G.: Multiple forms of mouse glutathione S-transferases. Biochem. Soc. Trans., 12, 30-33 (1984) [34] Simons, P.C.; Vander Jagt, D.L.: Purification of glutathione S-transferases by glutathione-affinity chromatography. Methods Enzymol., 77, 235-237 (1981) [35] Simons, P.C.; Vander Jagt, D.L.: Purification of glutathione S-transferases from human liver by glutathione-affinity chromatography. Anal. Biochem., 82, 334-341 (1977) [36] Vander Jagt, D.L.; Wilson, S.P.; Heidrich, J.E.: Purification and bilirubin binding properties of glutathione S-transferase from human placenta. FEBS Lett., 136, 319-321 (1981) [37] Pattinson, N.: Purification by affinity chromatography of glutathione Stransferases A and C from rat liver cytosol. Anal. Biochem., 115, 424-427 (1981) [38] Ketterer, B.; Beale, D.; Meyer, D.: The structure and multiple functions of glutathione transferases. Biochem. Soc. Trans., 10, 82-84 (1982) [39] Guthenberg, C.; Mannervik, B.: Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver. Biochem. Biophys. Res. Commun., 86, 1304-1310 (1979) [40] Hayakawa, T.; Myokei, Y.; Yagi, H.; Jerina, D.M.: Purification and some properties of glutathione-S-epoxide transferase from guinea pig liver. J. Biochem., 82, 407-415 (1977) [41] Asaoka, K.; Takahashi, K.: Purification and properties of porcine brain glutathione S-transferases. J. Biochem., 94, 1191-1199 (1983) [42] Asaoka, K.; Ito, H.; Takahashi, K.: Monkey glutathione S-aryltransferases. I. Tissue distribution and purification from the liver. J. Biochem., 82, 973-981 (1977) [43] Reddy, C.C.; Burgess, J.R.; Tu, C.-P.D.: Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol. Biochem. Biophys. Res. Commun., 111, 840-846 (1983) [44] Mozer, T.J.; Tiemeier, D.C.; Jaworski, E.G.: Purification and characterization of corn glutathione S-transferase. Biochemistry, 22, 1068-1072 (1983) [45] Friedberg, T.; Milbert, U.; Bentley, P.; Guenther, T.M.; Oesch, F.: Purification and characterization of a new cytosolic glutathione S-transferase (glutathione S-transferase X) from rat liver. Biochem. J., 215, 617-625 (1983)

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[46] Reddy, C.C.; Burgess, J.R.; Gong, Z.-Z.; Massaro, E. J.; Tu, C.-P.D.: Purification and characterization of the individual glutathione S-transferases from sheep liver. Arch. Biochem. Biophys., 224, 87-101 (1983) [47] Kraus, P.: Resolution, purification and some properties of three glutathione transferases from rat liver mitochondria. Hoppe-Seyler's Z. Physiol. Chem., 361, 9-15 (1980) [48] Asaoka, K.; Takahashi, K.: Monkey glutathione S-aryltransferases. II. Properties of the major enzyme purified from the liver. J. Biochem., 82, 13131323 (1977) [49] Caccuri, A.M.; Antonini, G.; Nicotra, M.; Battistoni, A.; Lo Bello, M.; Board, P.G.; Parker, M.W.; Ricci, G.: Catalytic mechanism and role of hydroxyl residues in the active site of theta class glutathione S-transferases. Investigation of Ser-9 and Tyr-113 in a glutathione S-transferase from the Australian sheep blowfly, Lucilia cuprina. J. Biol. Chem., 272, 29681-29686 (1997) [50] Caccuri, A.M.; Ascenzi, P.; Antonini, G.; Parker, M.W.; Oakley, A.J.; Chiessi, E.; Nuccetelli, M.; Battistoni, A.; Bellizia, A.; Ricci, G.: Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase. J. Biol. Chem., 271, 16193-16198 (1996) [51] Berhane, K.; Widersten, M.; Engstroem, A.; Kozarich, J.W.; Mannervik, B.: Detoxication of base propenals and other a,b-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases. Proc. Natl. Acad. Sci. USA, 91, 1480-1484 (1994) [52] Deng, F.; Hatzios, K.K.: Purification and characterization of two glutathione S-transferase isozymes from Indica-type rice involved in herbicide detoxification. Pestic. Biochem. Physiol., 72, 10-23 (2002) [53] Dirr, H.; Reinemer, P.; Huber, R.: X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur. J. Biochem., 220, 645-661 (1994) [54] Edwards, R.: Characterization of glutathione transferases and glutathione peroxidases in pea (Pisum sativum). Physiol. Plant., 98, 594-604 (1996) [55] Flury, T.; Daam, D.; Kreuz, K.: A 2,4-D-inducible glutathione S-transferase from soybean (Glycine max). Purification, characterization and induction. Physiol. Plant., 94, 312-318 (1995) [56] Fujita, M.; Adachi, Y.: Effects of chemical structure of 2,4-dichlorophenoxyacetic acid derivatives on the accumulation of glutathione S-transferases in cultured pumpkin cells. Biosci. Biotechnol. Biochem., 60, 128-130 (1996) [57] Girardini, J.; Amirante, A.; Zemzoumi, K.; Serra, E.: Characterization of an w-class glutathione S-transferase from Schistosoma mansoni with glutaredoxin-like dehydroascorbate reductase and thiol transferase activities. Eur. J. Biochem., 269, 5512-5521 (2002) [58] Hatton, P.J.; Cummins, I.; Cole, D.J.; Edwards, R.: Glutathione transferases involved in herbicide detoxification in the leaves of Setaria faberii (giant foxtail). Physiol. Plant., 105, 9-16 (1999) [59] Kitteringham, N.R.; Powell, H.; Jenkins, R.E.; Hamlett, J.; Lovatt, C.; Elsby, R.; Henderson, C.J.; Wolf, C.R.; Pennington, S.R.; Park, B.K.: Protein expression profiling of glutathione S-transferase p null mice as a strategy to iden544

2.5.1.18

[60]

[61]

[62] [63]

[64]

[65] [66]

[67]


tify potential markers of resistance to paracetamol-induced toxicity in the liver. Proteomics, 3, 191-207 (2003) Liebau, E.; Eckelt, V.H.O.; Wildenburg, G.; Teesdale-Spittle, P.; Brophy, P.M.; Walter, R.D.; Henkle-Duhrsen, K.: Structural and functional analysis of a glutathione S-transferase from Ascaris sum. Biochem. J., 324, 659-666 (1997) Ortiz-Salmeron, E.; Yassin, Z.; Clemente-Jimenez, M.J.; Heras-Vazquez, F.J.L.; Rodriguez-Vico, F.; Baron, C.; Garcia-Fuentes, L.: Thermodynamic analysis of the binding of glutathione to glutathione S-transferase over a range of termeratures. Eur. J. Biochem., 268, 4307-4314 (2001) Pascal, S.; Scalla, R.: Purification and characterization of a safener-induced glutathione S-transferase from wheat (Triticum aestivum). Physiol. Plant., 106, 17-27 (1999) Ricci, G.; Caccuri, A.M.; Lo Bello, M.; Rosato, N.; Mei, G.; Nicotra, M.; Chiessi, E.; Mazzetti, A.P.; Federici, G.: Structural flexibility modulates the activity of human glutathione transferase P1-1. Role of helix 2 flexibility in the catalytic mechanism. J. Biol. Chem., 271, 16187-16192 (1996) Rouimi, P.; Anglade, P.; Benzekri, A.; Costet, P.; Debrauwer, L.; Pineau, T.; Tulliez, J.: Purification and characterization of a glutathione S-transferase w in pig: evidence for two distinct organ-specific transcripts. Biochem. J., 358, 257-262 (2001) Scalla, R.; Roulet, A.: Cloning and characterization of a glutathione S-transferase induced by a herbicide safener in barley (Hordeum vulgare). Physiol. Plant., 116, 336-344 (2002) Stevens, J.M.; Hornby, J.A.T.; Armstrong, R.N.; Dirr, H.W.: Class sigma glutathione transferase unfolds via a dimeric and a monomeric intermediate: Impact of subunit interface on conformational stability in the superfamily. Biochemistry, 37, 15534-15541 (1998) Whalen, R.; Kempner, E.S.; Boyer, T.D.: Structural studies of a human pi class glutathione S-transferase. Photoaffinity labeling of the active site and target size analysis. Biochem. Pharmacol., 52, 281-288 (1996)

545

3-Phosphoshikimate 1-carboxyvinyltransferase

2.5.1.19

1 Nomenclature EC number 2.5.1.19 Systematic name phosphoenolpyruvate:3-phosphoshikimate 5-O-(1-carboxyvinyl)transferase Recommended name 3-phosphoshikimate 1-carboxyvinyltransferase Synonyms 3-enolpyruvylshikimate 5-phosphate synthase 3-enolpyruvylshikimic acid-5-phosphate synthetase 3-phosphoshikimate 1-carboxyvinyltransferase [24] 5'-enolpyruvylshikimate-3-phosphate synthase 5-enolpyruvyl-3-phosphoshikimate synthase 5-enolpyruvylshikimate-3-phosphate synthase 5-enolpyruvylshikimate-3-phosphate synthetase 5-enolpyruvylshikimate-3-phosphoric acid synthase 5-enolpyruvylshikimic acid-3-phosphate synthase EPSP synthase enolpyruvylshikimate phosphate synthase synthase, 5-enolpyruvoylshikimate 3-phosphate CAS registry number 9068-73-9

2 Source Organism no activity in mammals [26] Corydalis sempervirens [19] Fagopyrum esculentum [20] Lactuca sativa (lettuce [13]) [13] Nicotiana sylvestris (cv. Speg et Comes, aneuploid cell line ANS-1 [16]) [16] Petunia hybrida [14, 26] Vigna radiata (mung bean [23]) [23] Pisum sativum (pea, cv. Onward [13]) [1, 12, 13] Sorghum bicolor [15] Spinacia oleracea (spinach [13]) [13]

546

2.5.1.19


Triticum vulgare (wheat, cv. Maris Dove [13]) [13] Zea mays (maize [13]; var. Black Mexican Sweet, isoenzymes I and II [26]; isoenzymes I and II [36]) [13, 26, 36] Aspergillus nidulans [24] Neurospora crassa (strain 74-OR23-1A [17]) [1, 17, 18, 24] Saccharomyces cerevisiae [24] Anabaena variabilis [11] Escherichia coli (K-12 [1,2,5,6]; strain N5259 [7]; overproducing recombinant strain AB2829pKD501 [1,3,4]) [1-7, 22, 24, 28, 29, 31, 34, 35, 37, 39] Klebsiella pneumoniae (formerly Aerobacter aerogenes, glyphosate sensitive and resistant strains [8]) [1, 8-10, 37] Pseudomonas sp. [21] Salmonella typhimurium [1, 24, 31, 38] Euglena gracilis (enzyme exists in 2 forms: one form is a monofunctional 59000 Da enzyme, the other form constitutes a single domain of the multifunctional 165000 Da arom protein [25]) [25] Bacillus subtilis [27] Streptococcus pneumoniae [30] Spirulina platensis (cyanobacterium [32]) [32] Mycobacterium tuberculosis [33, 40] Eleusine indica (goosegrass [38]) [38]

3 Reaction and Specificity Catalyzed reaction phosphoenolpyruvate + 3-phosphoshikimate = phosphate + 5-O-(1-carboxyvinyl)-3-phosphoshikimate ( proposed mechanism [4,5,29]; binding of shikimate 3-phosphate leads to a saturable and stable conformational change in the isolated N-terminal domain [34]) Reaction type enolpyruvate group transfer Natural substrates and products S phosphoenolpyruvate + 3-phosphoshikimate ( involved in chorismate biosynthesis [2]; involved in aromatic acid biosynthesis [3,27,32]; part of shikimate pathway [13,24,25]; sixth reaction of chorismate pathway [1]) [1, 2, 3, 13, 24, 25, 27, 32] P phosphate + 5-enolpyruvylshikimate 3-phosphate Substrates and products S phosphoenolpyruvate + 3-phosphoshikimate ( transfers enolpyruvate group from phosphoenolpyruvate to 5-hydroxyl group of 3phosphoshikimate [2]) (Reversibility: r [1-40]) [1-40] P phosphate + 5-enolpyruvylshikimate 3-phosphate ( i.e. 5O-(1-carboxyvinyl)-3-phosphoshikimate [1-27]) [1-40]

547


2.5.1.19

Inhibitors (Z)-3-fluorophosphoenolpyruvate ( competitive vs. phosphoenolpyruvate at saturated shikimate 3-phosphate concentration and vice versa [4]) [4] 3-bromopyruvate ( 0.1 mM, approx. 80% inactivation after 5 min, maximum rate-constant: 0.31/min, substrates or a combination of shikimate 3-phosphat and glyphosate protect from inactivation, bromopyruvate modifies residues C408 and L411 [7]; 1 mM, 50% inactivation after 3.4 min [8]; glyphosate protects [10]) [7, 8, 10] 5-deoxy-shikimate 3-phosphate ( competitive vs. shikimate 3phosphate [4]) [4] 5-enolpyruvylshikimate 3-phosphate ( product inhibition [3,9]) [3, 9] Al3+ ( 10 mM, 37% inhibition [9]) [9] Br- ( inhibition above 200 mM, stimulation below [9]) [9] CaCl2 ( above 100 mM [15]) [15] Cl- ( inhibition above 400 mM, stimulation below [9]) [9] Co3+ ( 10 mM, 25% inhibition [9]) [9] Cu2+ ( 10 mM, 90-95% inhibition [9]; 5 mM, complete inhibition [15]) [9, 15] Fe2+ ( 10 mM, complete inhibition [9]; 5 mM, 42% inhibition [15]) [9, 15] Fe3+ ( 10 mM, 60% inhibition [9]; 5 mM, 68% inhibition [15]) [9, 15] I- ( inhibition above 100 mM, stimulation below [9]) [9] La3+ ( 10 mM, complete inhibition [9]) [9] MgCl2 ( above 100 mM [15]) [15] Mn2+ ( 10 mM, 16% inhibition [9]; 5 mM, 42% inhibition [15]) [9, 15] N-ethylmaleimide ( 1 mM, 50% inactivation after approx. 3 min [8]; glyphosate protects [10]) [8, 10] N-phosphonomethylglycine ( trivial name glyphosate, non-selective herbicide ªRound upª, free acid or monoisopropylamine salt [11]; pH-dependent [10,16]; wildtype enzyme [8]; 0.0183 mM, 50% inhibition, 0.5 mM 96% inhibition, competitive vs. phosphoenolpyruvate, uncompetitive vs. shikimate 3-phosphate at pH 7.0, mechanism [16]; competitive vs. phosphoenolpyruvate, noncompetitive vs. shikimate 3-phosphate, 5-enolpyruvylshikimate 3phosphate and phosphate, not inhibited by non-herbicidal analogues of glyphosate, e.g. aminomethylphosphonic acid, glyphosine, i.e. bis-N-(phosphonomethyl)glycine, or iminodiacetic acid [10]; inhibition of enolpyruvate transfer, competitive vs. phosphoenolpyruvate [1]; noncompetitive vs. phosphate, competitive vs. phosphoenolpyruvate, uncompetitive vs. shikimate 3-phosphate [13]; 0.01 mM, 50% inhibition [5]; 3 mM, 50% inhibition, 25 mM, 90% inhibition [11]; 0.006-0.008 mM, 50% inhibition, 1 mM, complete inhibition [26]; 0.25 mM, 50% inhibition of NH4 Cl activated enzyme, 75 mM, 50% inhibition of nonactivated enzyme 548

2.5.1.19


[27]; mechanism of inhibition in atomic detail [35]; 0.006 mM, 50% inhibition of glyphosate-sensitve strain, 0.03 mM, 50% inhibition of glyphosate-resistant strain [38]) [1, 3-8, 10-13, 15, 16, 26, 27, 30, 35, 38] NH4 Cl ( inhibition above 300 mM, stimulation below [9]) [9] NO3- ( inhibition above 150 mM, stimulation below [9]) [9] Pb2+ ( 10 mM, 90-95% inhibition [9]; 5 mM, 97% inhibition [15]) [9, 15] SO24- ( inhibition above 50 mM, slight stimulation below [15]) [15] Zn2+ ( 10 mM, 90-95% inhibition [9]; 5 mM, complete inhibition [15]) [9, 15] ammonium heptamolybdate ( 1.1 mM, 50% inhibition, no inhibition below 0.1 mM [15]) [15] carboxyallenyl phosphate ( strong [4]) [4] diethyldicarbonate ( inactivation with a second-order rate constant of 220/M/min, subtstrates protect from inactivation, enzyme activity is recovered by treatment with hydroxylamine [5]) [5] molybdate ( 10 mM, complete inhibition [9]) [9] phenylglyoxal ( 2 mM, 50% inactivation after 32 min [8]; glyphosate protects [10]) [8, 10] phosphoenolpyruvate ( substrate inhibition [4]) [4] pyruvate ( 20 mM, 85% inactivation after 1 h in the presence of cyanoborhydride, no inactivation in the absence of cyanoborhydride, preincubation with 5-enolpyruvylshikimate or a combination of shikimate 3-phosphate and glyphosate prevents from inactivation [6]) [6] reaction intermediate analogue [14] shikimate 3-phosphate ( product inhibition [9]) [9] shikimate 3-phosphate-5-carboxymethyl-(2R)-phosphonate ( competitive vs. 5-enolpyruvylshikimate-3-phosphate and phosphate [14]) [14] shikimate 3-phosphate-5-carboxymethyl-(2S)-phosphonate ( competitive vs. 5-enolpyruvylshikimate-3-phosphate and phosphate [14]) [14] Cofactors/prosthetic groups Additional information ( no known cofactor [13]) [13] Activating compounds Br- ( stimulation at low concentrations, inhibition above 200 mM [9]) [9] Cl- ( activation up to 100 mM, inhibition above [9]; at about 100 mM [15]) [9, 15] F- ( stimulation [9,15]) [9, 15] I- ( stimulation at low concentrations, inhibition above 100 mM [9]) [9] NH4 Cl ( stimulation, inhibition above 300 mM [9]; maximal activation at 100 mM [27]; strong activation of forward reaction, effectiveness of activation in descending order: NH4 Cl, Rb+, K+ [30]) [9, 27, 30]

549


2.5.1.19

NO3- ( stimulation at low concentrations, inhibition above 15 mM [9,15]) [9, 15] SO24- ( slight stimulation, inhibition above 50 mM [15]) [15] Metals, ions CaCl2 ( stimulation at 20 mM, inhibition above 100 mM [15]) [15] K+ ( strong activation of forward reaction, effectiveness of activation in descending order: NH4 Cl, Rb+, K+ [30]) [30] MgCl2 ( stimulation at 20 mM, inhibition above 100 mM [15]) [15] Rb+ ( strong activation of forward reaction, effectiveness of activation in descending order: NH4 Cl, Rb+, K+ [30]) [30] Additional information ( not activated by Ba2+ , Ca2+ , Mg2+ , Ni2+ or Sr2+ [9]) [9] Turnover number (min±1) 13.2 (phosphate, reverse reaction, in the presence of 50 mM KCl [30]) [30] 13.8 (5-enolpyruvylshikimate 3-phosphate, reverse reaction, in the presence of 50 mM KCl [30]) [30] 120 (phosphoenolpyruvate, in the presence of 1 mM NH4 Cl and 10 mM KCl [30]) [30] 126 (phosphoenolpyruvate, in the presence of 1 mM NH4 Cl [30]) [30] 168 (phosphoenolpyruvate, in the presence of 10 mM NH4 Cl [30]) [30] 180 (phosphoenolpyruvate, in the presence of 100 mM NH4 Cl [30]) [30] 768 (phosphoenolpyruvate, in the presence of 1 mM NH4 Cl and 100 mM KCl [30]) [30] 2016 (3-phosphoshikimate, cosubstrate phosphoenolpyruvate [17]) [17] 2016 (phosphoenolpyruvate, cosubstrate 3-phosphoshikimate [17]) [17] 3396 (3-phosphoshikimate) [4] Specific activity (U/mg) 0.0058 ( recombinant enzyme, activity in crude extracts [31]) [31] 0.0064 ( recombinant enzyme, activity in crude extracts [31]) [31] 1.26 ( reverse reaction, i.e. phosphate + 5-enolpyruvylshikimate 3phosphate [11]) [11] 4.07 ( forward reaction, i.e. phosphoenolpyruvate + 3-phosphoshikimate [11]) [11] 5.67 ( phosphate + 5-enolpyruvylshikimate 3-phosphate [12]) [12] 5.68 ( activity of recombinant enzyme in crude extract [33]) [33] 6.12 ( phosphate + 5-enolpyruvylshikimate 3-phosphate [13]) [13] 10 [28] 10.4 [27] 10.86 ( glyphosate insensitive strain [8]) [8]

550

2.5.1.19


11.52 ( forward reaction, i.e. phosphoenolpyruvate + 3-phosphoshikimate [12]) [12] 12 ( phosphoenolpyruvate + 3-phosphoshikimate [13]) [13] 16.9 [16] 18 ( phosphate + 5-enolpyruvylshikimate 3-phosphate, native enzyme [1]) [1, 2] 18.13 ( recombinant enzyme [40]) [40] 20 ( recombinant His-tagged wild-type enzyme [39]) [39] 21.1 ( phosphate + 5-enolpyruvylshikimate 3-phosphate, recombinant enzyme [3]) [3, 15] 24 ( recombinant wild-type enzyme [39]) [39] 37.8 [9] 61 ( isoenzyme I [26]) [26] 62 ( phosphoenolpyruvate + 3-phosphoshikimate [1]) [1, 2] 94.7 ( isoenzyme II [26]) [26] Additional information ( activity of isoenzyme II increases in elicited cells [36]) [36] Km-Value (mM) 0.00025 (5-enolpyruvylshikimate 3-phosphate) [1] 0.00036 (3-phosphoshikimate) [1] 0.001 (5-enolpyruvylshikimate 3-phosphate) [11] 0.0018 (phosphate) [1] 0.00195 (5-enolpyruvylshikimate 3-phosphate, reverse reaction, in the presence of 50 mM KCl [30]) [30] 0.0025 (3-phosphoshikimate, recombinant overproducing strain [1,3]) [1, 3] 0.0027 (phosphoenolpyruvate) [1] 0.003 (5-enolpyruvylshikimate 3-phosphate, recombinant overproducing strain [1,3]) [1, 3] 0.0032-0.0036 (3-phosphoshikimate, wild-type enzyme [3]) [3, 4] 0.0038 (phosphoenolpyruvate, glyphosate-sensitive strain [38]) [38] 0.0052 (5-enolpyruvylshikimate 3-phosphate) [12] 0.0052 (phosphoenolpyruvate, cosubstrate 5-enolpyruvylshikimate 3-phosphate [1]) [1, 12] 0.007 (3-phosphoshikimate) [15] 0.007 (5-enolpyruvylshikimate 3-phosphate, H385N mutant enzyme [28]) [28] 0.007 (phosphoenolpyruvate, glyphosate-resistant strain [38]) [38] 0.0077 (3-phosphoshikimate) [1, 12] 0.008 (phosphoenolpyruvate) [15] 0.009 (phosphoenolpyruvate, glyphosate-sensitive enzyme [8]) [8] 0.011 (5-enolpyruvylshikimate 3-phosphate, wild-type enzyme [28]) [28]

551


2.5.1.19

0.015 (phosphoenolpyruvate, wild-type enzyme [3]) [3] 0.016 (phosphoenolpyruvate, recombinant overproducing strain [1,3]) [1, 3] 0.017 (phosphoenolpyruvate, in the presence of glyphosate [10]) [10] 0.02 (shikimate 3-phosphate) [5] 0.021-0.023 (phosphoenolpyruvate) [4] 0.022 (phosphoenolpyruvate, in the presence of 100 mM NH4 Cl [30]) [30] 0.023 (phosphoenolpyruvate, in the presence of 1 mM NH4 Cl and 100 mM KCl [30]) [30] 0.025 (phosphoenolpyruvate) [5] 0.028 (phosphoenolpyruvate, isoenzyme II [26]) [26] 0.029 (shikimate 3-phosphate, isoenzyme II [26]) [26] 0.031 (shikimate 3-phosphate, in the presence of 100 mM NH4 Cl [30]) [30] 0.033 (phosphoenolpyruvate, isoenzyme I [26]) [26] 0.033 (shikimate 3-phosphate, isoenzyme I [26]) [26] 0.034 (phosphoenolpyruvate) [11] 0.045 (3-phosphoshikimate, glyphosate-sensitive enzyme [8]) [8] 0.049 (shikimate 3-phosphate, in the presence of 1 mM NH4 Cl and 10 mM KCl [30]) [30] 0.07 (shikimate 3-phosphate, H385 mutant enzyme [28]) [28] 0.085 (phosphoenolpyruvate, in the presence of 1 mM NH4 Cl and 10 mM KCl [30]) [30] 0.088 (phosphoenolpyruvate, wild-type enzyme [37]) [37] 0.09 (phosphoenolpyruvate, wild-type enzyme [27]) [27] 0.091 (phosphoenolpyruvate, in the presence of 10 mM NH4 Cl [30]) [30] 0.1 (phosphoenolpyruvate, wild-type enzyme [28]) [28] 0.1 (phosphoenolpyruvate, in the presence of 1 mM NH4 Cl [30]) [30] 0.106 (phosphoenolpyruvate, H385N mutant enzyme [28]) [28] 0.109 (shikimate 3-phosphate, in the presence of 1 mM NH4 Cl and 100 mM KCl [30]) [30] 0.118 (shikimate 3-phosphate, in the presence of 10 mM NH4 Cl [30]) [30] 0.12 (shikimate 3-phosphate, wild-type and G96A mutant enzyme [37]) [37] 0.135 (shikimate 3-phosphate, wild-type enzyme [28]) [28] 0.14 (phosphoenolpyruvate, glyphosate-resistant enzyme [8]) [8] 0.145 (shikimate 3-phosphate, in the presence of 1 mM NH4 Cl [30]) [30] 0.16 (shikimate 3-phosphate, H385 mutant enzyme [27]) [27] 0.193 (3-phosphoshikimate, glyphosate-resistant enzyme [8]) [8] 0.2 (phosphoenolpyruvate, R24D mutant enzyme [27]) [27] 0.2 (shikimate 3-phosphate, R24D mutant enzyme [27]) [27] 552

2.5.1.19


0.33 (shikimate 3-phosphate, wild-type enzyme [27]) [27] 0.343 (phosphate, reverse reaction, in the presence of 50 mM KCl [30]) [30] 0.43 (shikimate 3-phosphate, P105S mutant enzyme [27]) [27] 0.59 (phosphoenolpyruvate, P105S mutant enzyme [27]) [27] 1 (phosphoenolpyruvate, H385L mutant enzyme [27]) [27] 1.2 (phosphate, H385N mutant enzyme [28]) [28] 2.5 (phosphate) [1, 3] 2.8 (phosphoenolpyruvate, G96A mutant enzyme [37]) [37] 3.7 (phosphate) [12] 4 (phosphate) [1] 4.6 (phosphate, wild-type enzyme [28]) [28] 22.5 (phosphoenolpyruvate, recombinant enzyme, in crude extracts [31]) [31] 43.6 (phosphoenolpyruvate, recombinant enzyme, in crude extracts [31]) [31] Additional information ( kinetic study [4,9]; comparison of kinetic parameters from a variety of organism [11]; kinetic parameters of native and diethyldicarbonate-inactivated enzyme [5]) [4, 5, 9, 11] Ki-Value (mM) 0.000015 (shikimate 3-phosphate-5-carboxymethyl-(2R)-phosphonate, vs. 5-enolpyruvylshikimate-3-phosphate [14]) [14] 0.00008 (N-phosphonomethylglycine) [12] 0.00009 (shikimate 3-phosphate-5-carboxymethyl-(2R)-phosphonate, vs. phosphate [14]) [14] 0.00012-0.00013 (N-phosphonomethylglycine, isoenzymes I and II, vs. phosphoenolpyruvate [26]) [26] 0.00016 (N-phosphonomethylglycine, vs. phosphoenolpyruvate [15]) [15] 0.0003 (N-phosphonomethylglycine, in the presence of 100 mM NH4 Cl [30]) [30] 0.00076 (N-phosphonomethylglycine, glyphosate-resistant strain [38]) [38] 0.0008 (N-phosphonomethylglycine, H385N mutant enzyme [28]) [28] 0.0009 (N-phosphonomethylglycine, vs. shikimate 3-phosphate, recombinant enzyme [3]) [3] 0.001 (N-phosphonomethylglycine, vs. shikimate 3-phosphate, native enzyme [3]) [3] 0.001 (N-phosphonomethylglycine, vs. phosphoenolpyruvate [10]) [10] 0.001 (N-phosphonomethylglycine, P106S mutant enzyme [38]) [38] 0.0011 (shikimate 3-phosphate-5-carboxymethyl-(2S)-phosphonate, vs. 5-enolpyruvylshikimate-3-phosphate [14]) [14]

553


2.5.1.19

0.0012 (N-phosphonomethylglycine, wild-type enzyme [28]) [28] 0.00125 (N-phosphonomethylglycine, vs. phosphoenolpyruvate [16]) [16] 0.0013 (5-enolpyruvylshikimate 3-phosphate) [9] 0.0021 (shikimate 3-phosphate-5-carboxymethyl-(2S)-phosphonate, vs. phosphate [14]) [14] 0.0029 ((Z)-3-fluorophosphoenolpyruvate, vs. shikimate 3-phosphate [4]) [4] 0.005 (N-phosphonomethylglycine, in the presence of 1 mM NH4 Cl and 100 mM K+ [30]) [30] 0.0064 ((Z)-3-fluorophosphoenolpyruvate, vs. phosphoenolpyruvate [4]) [4] 0.008 (N-phosphonomethylglycine, vs. 5-enolpyruvylshikimate 3phosphate [10]) [10] 0.0094 (N-phosphonomethylglycine, isoenzyme I, vs. shikimate 3-phosphate [26]) [26] 0.01 (N-phosphonomethylglycine) [37] 0.012 (N-phosphonomethylglycine, isoenzyme II, vs. shikimate 3phosphate [26]) [26] 0.0126 (5-deoxy-shikimate 3-phosphate) [4] 0.0183 (N-phosphonomethylglycine, vs. shikimate 3-phosphate [16]) [16] 0.023 (shikimate 3-phosphate) [9] 0.027 (N-phosphonomethylglycine, vs. phosphate [10]) [10] 0.029 (N-phosphonomethylglycine, P381L mutant enzyme [38]) [38] 0.04 (N-phosphonomethylglycine, H385L mutant enzyme [27]) [27] 0.044 (phosphoenolpyruvate) [9] 0.048 (N-phosphonomethylglycine, glyphosate-sensitive strain [38]) [38] 0.05 (N-phosphonomethylglycine, R24D mutant enzyme [27]) [27] 0.06 (N-phosphonomethylglycine, wild-type enzyme [27]) [27] 0.06-0.08 (N-phosphonomethylglycine, vs. shikimate 3-phosphate [13]) [13] 0.07-0.1 (N-phosphonomethylglycine, vs. phosphate [13]) [13] 0.08-0.1 (N-phosphonomethylglycine, vs. enolpyruvate [13]) [13] 0.098 (N-phosphonomethylglycine, in the presence of 1 mM NH4 Cl and 10 mM K+ [30]) [30] 0.185 (N-phosphonomethylglycine, in the presence of 1 mM NH4 Cl [30]) [30] 0.21 (N-phosphonomethylglycine, P105S mutant enzyme [27]) [27] 0.22 (N-phosphonomethylglycine, vs. shikimate 3-phosphate [10]) [10] 0.35 (N-phosphonomethylglycine) [11] 554

2.5.1.19


0.54 (N-phosphonomethylglycine, recombinant enzyme, in crude extracts [31]) [31] 0.96 (N-phosphonomethylglycine, recombinant enzyme, in crude extracts [31]) [31] pH-Optimum 5.4 ( wild-type enzyme, 2 maxima at pH 5.4 and pH 7.0 [8]) [8] 5.6 ( phosphate + 5-enolpyruvylshikimate 3-phosphate, 2 maxima at pH 5.6 and pH 7.6 [9]) [9] 6 ( glyphosate-resistant enzyme, 2 maxima at pH 6 and pH 7.3 [8]) [8] 7 ( wild-type enzyme, 2 maxima at pH 5.4 and pH 7.0 [8]; H385N mutant enzyme [28]) [8, 28] 7-7.2 ( 50% of maximal activity between pH 6.2 and pH 8.5 [16]) [16] 7.3 ( glyphosate-resistant enzyme, 2 maxima at pH 6 and pH 7.3 [8]) [8] 7.4 ( Tris-HCl buffer preferred [15]) [15] 7.4-7.5 ( isoenzymes I and II [26]) [26] 7.6 ( phosphate + 5-enolpyruvylshikimate 3-phosphate, 2 maxima, at pH 5.6 and pH 7.6 [9]) [9] 7.8 ( wild-type enzyme [28]) [28] Additional information ( pI: 4.1, glyphosate-resistant enzyme [8]; pI: 4.6, wild-type enzyme [8,9]) [8, 9] pH-Range 5-8.5 ( approx. half-maximal activity at pH 5.0 and 8.5 [9]) [9] 5.5-8.7 ( approx. half-maximal activity at pH 5.5 and 8.7, phosphate + 5-enolpyruvylshikimate 3-phosphate [9]) [9] 5.6-8.4 ( H385N mutantt enzyme [28]) [28] 6-8.4 ( wild-type enzyme [28]) [28] 6.2-8.5 ( approx. half-maximal activity at pH 6.2 and 8.5 [16]) [16] 6.5-8.5 ( approx. half-maximal activity at pH 6.5 and 8.5 [15]) [15] Temperature optimum ( C) 25 ( assay at [1-3,5-7,11]) [1-3, 5-7, 11] 30 ( assay at [4,8-10,15]) [4, 8-10, 15] 55 ( isoenzymes I and II [26]) [26] 60 ( wild-type enzyme [8,9]) [8, 9] Temperature range ( C) 45-62 ( approx. half-maximal activity at 45 C and 62 C [9]) [9]

4 Enzyme Structure Molecular weight 32400 ( gel filtration [9]) [9] 42000 ( gel filtration [1,3]) [1, 3] 555


2.5.1.19

44000 ( gel filtration [12]) [12] 46110 ( calculated from polypeptide sequence of aroA gene product [1]) [1] 48000 ( mixture of monomers and dimers in the absence of NH4 Cl, only monomers in the presence of NH4 Cl, gel filtration [27]) [27] 55000 ( gel filtration [1,2]) [1, 2] 57000 ( gel filtration [15]) [15] 58100 ( isoenzyme II, gel filtration [26]) [26] 58700 ( isoenzyme I, gel filtration [26]) [26] 59000 ( monofunctional enzyme [25]) [25] 91000 ( gel filtration [32]) [32] 159000 ( multifunctional enzyme called arom [25]) [25] 290000 ( aromatic multifunctional enzyme protein, sedimentation equilibrium analysis [18]) [18] Subunits ? ( x * 46458, electrospray ionization mass spectrometry [40]; x * 46425, deduced from nucleotide sequence [40]) [40] dimer ( 2 * 49000, SDS-PAGE [32]) [32] monomer ( 1 * 42500, wild-type enzyme, SDS-PAGE [8]; 1 * 42900, glyphosate-resistant enzyme, SDS-PAGE [8,9]; 1 * 49000, [1,2]; SDS-PAGE [1,2,11]; 1 * 50000, SDS-PAGE [12]; 1 * 50600-51900, 3 isozymes, SDS-PAGE [15]; 1 * 46112, deduced from nucleotide sequence [24]; 1 * 53600, isoenzyme II, SDS-PAGE [26]; 1 * 54900, isoenzyme I, SDS-PAGE [26]) [1, 2, 8, 9, 11, 12, 15, 24, 26]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture [16] leaf [13] seedling ( shoot tissue [12,13]) [12, 13] Localization chloroplast ( monofunctional enzyme form [25]; isoenzymes I and II [26]) [13, 25, 26] cytosol ( multifunctional enzyme form i.e. single domain of arom protein [25]) [25] soluble [1, 2, 9, 11] Purification (DEAE-cellulose, CM-cellulose, partial purification [16]) [16] (ammonium sulfate, DEAE-Sephacel, phenyl-Sepharose, cellulose phosphate [13]) [12, 13] (ammonium sulfate, DEAE-cellulose, hydroxyapatite, phenylaggarose, Sephacryl S-200, isozymes I, II and III [15]) [15]

556

2.5.1.19


(ammonium sulfate, DEAE-Sephacel, cellulose phosphate column, isoenzymes I and II [26]) [26] (enzyme is part of multifunctional protein with distinct domains [17,18]) [17, 18] (ammonium sulfate, DEAE-Sephacel, Mono Q, cellulose phosphate [11]) [11] (recombinant enzyme, ammonium sulfate, DEAE-Sephacel, phenyl-Sepharose, phosphocellulose chromatography [1]; recombinant enzyme [34]; recombinant wild-type and G96A mutant enzyme [37]) [1-3, 34, 35, 37] (glyphosate insensitve strain, ammonium sulfate, heat treatment, DEAE-cellulose, Sephadex G-75, cellulose phosphate, chromatofocusing [8]) [8, 9] (recombinant enzyme, ammonium sulfate [27]) [27] (recombinant enzyme, ammonium sulfate, hydrophobic interaction chromatography, anion-exchange chromatography [30]) [30] (ammonium sulfate, anion exchange chromatography, gel filtration, chromatofocusing [32]) [32] (recombinant enzyme, Q-Sepharose, one step purification [40]) [40] Cloning (Escherichia coli structural gene aroA [1,3]; subcloned from bacteriophage lambdapserC into multicopy plasmid pAT153 with subsequent transformation of Escherichia coli AB2829 CGSC2829 cells [3]; overexpression in Escherichia coli [29]; expression of wild-type and chimeras of Salmonella and Escherichia coli mutant enzymes in Escherichia coli [31]; expression of wildtype and G96A mutant enzyme in Escherichia coli [37]; overexpression of wild-type, His-tagged wild-type and several mutant enzymes in Escherichia coli [39]) [1, 3, 28, 29, 31, 34, 37, 39] (expression of wild-type and chimeras of Salmonella typhimurium and Escherichia coli mutant enzymes in Escherichia coli [31]) [31] (expression of wild-type and R24D, P105S, H385L mutant enzymes in Escherichia coli [27]) [27] (expression in Escherichia coli [30]) [30] (overexpression in Escherichia coli [33]) [33, 40] Engineering D313A ( 0.42% of wild-type activity [39]) [39] D313N ( 5% of wild-type activity [39]) [39] D49A ( 41% of wild-type activity [39]) [39] E341A ( 0.3% of wild-type activity [39]) [39] E341Q ( 10% of wild-type activity [39]) [39] G96A ( glyphosate-insensitive [37]) [37] G96A ( glyphosate-insensitive [37]) [37] H385A ( 0.08% of wild-type activity [39]) [39] H385L ( 0.2% of wild-type activity, 2fold activation at 100 mM NH4 Cl [27]) [27] H385N ( 6% of wild-type activity [27]) [27] K22A ( 0.7% of wild-type activity [39]) [39] 557


2.5.1.19

K22R ( 3% of wild-type activity [39]) [39] K340A ( 2.4% of wild-type activity [39]) [39] K411A ( 10.4% of wild-type activity [39]) [39] N94A ( 50% of wild-type activity [39]) [39] P105S ( 69% of wild-type activity, 8fold activation at 100 mM NH4 Cl [27]) [27] P106S ( glyphosate-insensitive [38]) [38] P381L ( similiar glyphosate sensitivity like wild-type [38]) [38] Q171A ( 1.7% of wild-type activity [39]) [39] R100M ( 0.2% of wild-type activity [39]) [39] R124A ( 19.6% of wild-type activity [39]) [39] R24D ( 0.8% of wild-type activity, 2fold activation at 100 mM NH4 Cl [27]) [27] R27A ( binding of shikimate 3-phosphate is abolished [34]) [34] R344K ( 31.7% of wild-type activity [39]) [39] R344M ( 16.3% of wild-type activity [39]) [39] R386M ( 15.8% of wild-type activity [39]) [39] Y200F ( 1% of wild-type activity [39]) [39]

6 Stability Temperature stability 40 ( rapid inactivation above, glyphosate resistant enzyme [8]) [8] 45 ( isoenzyme I, 50% loss of activity after 20 min, isoenzyme II, 50% loss of activity after 60 min [26]) [26] 60 ( rapid inactivation above [9]) [9] General stability information , prolonged dialysis against 50 mM Tris-HCl buffer, pH 7.5, 0.1 mM DTT, 1 mM benzamidine hydrochloride, 5 mM EDTA, 1 mM PMSF, stable to [13] , dithiothreitol reverses aggregation which sometimes occurs after storage at -20 C [1] , dithiothreitol, 0.4 mM, stabilizes during purification [2] , glycerol, 50% v/v, stabilizes [1, 2, 11] , EDTA stabilizes [9] , freezing inactivates, 1 mM dithioerythritol stabilize [9] , glyphosate-sensitive enzyme is more stable than wild-type enzyme [8] Storage stability , -20 C, 50% glycerol, 10-15% loss of activity per month [12, 13] , -20 C, 40% v/v glycerol, at least 1 year, no loss of activity [15] , -20 C, 3 months, 15% loss of activity [26] , 0 C, 30 d, 25% loss of activity [26] , -20 C, more than a year [17] , -20 C, 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 0.4 mM dithiothreitol, 50% v/v glycerol, at least 4 months, no loss of activity [2]

558

2.5.1.19


, -20 C, in 50% v/v glycerol, enzyme concentration 5 mg/ml, at least 1 year [1] , -18 C, pH 7-4, 1 mM dithioerythritol, at least 9 months [9] , 0-4 C, 2 months [9] , 4 C, ammonium sulfate precipitate, 4 months, no loss of activity [40]

References [1] Lewendon, A.; Coggins, J.R.: 3-Phosphoshikimate 1-carboxyvinyltransferase from Escherichia coli. Methods Enzymol., 142, 342-348 (1987) [2] Lewendon, A.; Coggins, J.R.: Purification of 5-enolpyruvylshikimate 3phosphate synthase from Escherichia coli. Biochem. J., 213, 187-191 (1983) [3] Duncan, K.; Lewendon, A.; Coggins, J.R.: The purification of 5-enolpyruvylshikimate 3-phosphate synthase from an overproducing strain of Escherichia coli. FEBS Lett., 165, 121-127 (1984) [4] Gruys, K.J.; Walker, M.C.; Sikorski, J.A.: Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli. Biochemistry, 31, 5534-5544 (1992) [5] Huynh, Q.K.: Reaction of 5-enol-pyruvoylshikimate-3-phosphate synthase with diethyl pyrocarbonate: evidence for an essential histidine residue. Arch. Biochem. Biophys., 258, 233-239 (1987) [6] Huynh, Q.K.: Inactivation of 5-enolpyruvylshikimate 3-phosphate synthase by its substrate analogue pyruvate in the presence of sodium cyanoborohydride. Biochem. Biophys. Res. Commun., 185, 317-322 (1992) [7] Huynh, Q.K.: 5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli±the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411. Arch. Biochem. Biophys., 284, 407-412 (1991) [8] Sost, D.; Chulz, A.; Amrhein, N.: Characterizaation of a glyphosphate-insensitive 5-enolpyruvylshikimic acid-3-phosphate synthase. FEBS Lett., 173, 238-242 (1984) [9] Steinrücken, H.C.; Amrhein, N.: 5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae. 1. Purification and properties. Eur. J. Biochem., 143, 341-349 (1984) [10] Steinrücken, H.C.; Amrhein, N.: 5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae 2. Inhibition by glyphosate [N-(phosphonomethyl)glycine]. Eur. J. Biochem., 143, 351-357 (1984) [11] Powell, H.A.; Kerby, N.W.; Rowell, P.; Mousdale, D.M.; Coggins, J.R.: Purification and properties of a glyphosphat-tolerant 5-enolpyruvylshikimate 3phosphate synthase from the cyanobacterium Anabaena variabilis. Planta, 188, 484-490 (1992) [12] Mousdale, D.M.; Coggins, J.R.: Purification and properties of 5-enolpyruvylshikimate 3-phosphate synthase from sedlings of Pisum sativum L.. Planta, 160, 78-83 (1984) [13] Mousdale, D.M.; Coggins, J.R.: 3-phosphoshikimate 1-carboxyvinyltransferase from Pisum sativum. Methods Enzymol., 142, 348-354 (1987) 559


2.5.1.19

[14] Alberg, D.G.; Bartlett, P.A.: Potent inhibition of 5-enolpyruvylshikimate-3phosphate synthase by a reaction intermediate analogue. J. Am. Chem. Soc., 111, 2337-2338 (1989) [15] Ream, J.E.; Steinrücken, H.C.; Porter, C.A.; Sikorski, J.A.: Purificatioin and properties of 5-enolpyruvylshikimate-3-phosphate synthase from darkgrown seedlings of Sorghum bicolor. Plant Physiol., 87, 232-238 (1988) [16] Rubin, J.L.; Gaines, C.G.; Jensen, R.A.: Glyphosphate inhibition of 5-enolpyruvylshikimate 3-phosphate synthase from suspension-cultured cells of Nicotiana silvestris. Plant Physiol., 75, 839-845 (1984) [17] Lambert, J.M.; Boocock, M.R.; Coggins, J.R.: The 3-dehydroquinate synthase activity of the pentafunctional arom enzyme complex of Neurospora crassa is Zn2+ -dependent. Biochem. J., 226, 817-829 (1985) [18] Gaertner, F.H.: Purification of two multienzyme complexes in the aromatictryptophan pathway of Neurospora crassa. Arch. Biochem. Biophys., 151, 277-284 (1972) [19] Smart, C.C.; Johänning, D.; Müller, G.; Amrhein, N.: Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate. J. Biol. Chem., 260, 16338-16346 (1985) [20] Smart, C.C.; Steinrücken, H.C.: Overproduction of 5-enolpyruvylshikimate 3-phosphate synthase in glyphosate-tolerant plant cell cultures. Plant Biol., 3, 119-133 (1987) [21] Kishore, G.M.; Shah, D.M.: Amino acid biosynthesis inhibitors as herbicides. Annu. Rev. Biochem., 57, 627-663 (1988) [22] Abdel-Meguid, S.S.; Smith, W.W.; Bild, G.S.: Crystallization of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli. J. Mol. Biol., 186, 673 (1985) [23] Koshiba, T.: Organization of enzymes in the shikimate pathway of Phaseolus mungo seedlings. Plant Cell Physiol., 20, 667-670 (1979) [24] Coggins, J.R.; Duncan, K.; Anton, I.A.; Boocock, M.R.; Chaudhuri, S.; Lambert, J.M.; Lewendon, A.; Millar, G.; Mousdale, D.M.; Smith, D.D.S.: The anatomy of a multifunctional enzyme. Biochem. Soc. Trans., 15, 754-759 (1987) [25] Reinbothe, C.; Ortel, B.; Parthier, B.; Reinbothe, S.: Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development. Mol. Gen. Genet., 245, 616-622 (1994) [26] Forlani, G.; Parisi, B.; Nielsen, E.: 5-Enol-pyruvyl-shikimate-3-phosphate synthase from Zea mays cultured cells. Purification and properties. Plant Physiol., 105, 1107-1114 (1994) [27] Majumder, K.; Selvapandiyan, A.; Fattah, F.A.; Arora, N.; Ahmad, S.; Bhatnagar, R.K.: 5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis is an allosteric enzyme. Analysis of Arg24 ! Asp, Pro105 ! Ser and His385 ! Lys mutations suggests a hidden phosphoenolpyruvate-binding site. Eur. J. Biochem., 229, 99-106 (1995)

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[28] Shuttleworth, W.A.; Evans, J.N.S.: The H385N mutant of 5-enolpyruvylshikimate-3-phosphate synthase: kinetics, fluorescence, and nuclear magnetic resonance studies. Arch. Biochem. Biophys., 334, 37-42 (1996) [29] Jakeman, D.L.; Mitchell, D.J.; Shuttleworth, W.A.; Evans, J.N.: On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase. Biochemistry, 37, 12012-12019 (1998) [30] Du, W.; Wallis, N.G.; Mazzulla, M.J.; Chalker, A.F.; Zhang, L.; Liu, W.-S.; Kallender, H.; Payne, D.J.: Characterization of Streptococcus pneumoniae 5-enolpyruvylshikimate 3-phosphate synthase and its activation by univalent cations. Eur. J. Biochem., 267, 222-227 (2000) [31] He, M.; Yang, Z.-Y.; Nie, Y.-F.; Wang, J.; Xu, P.: A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate. Biochim. Biophys. Acta, 1568, 1-6 (2001) [32] Forlani, G.; Campani, A.: A dimeric 5-enol-pyruvyl-shikimate-3-phosphate synthase from the cyanobacterium Spirulina platensis. New Phytol., 151, 443-450 (2001) [33] Oliveira, J.S.; Pinto, C.A.; Basso, L.A.; Santos, D.S.: Cloning and overexpression in soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3-phosphate synthase enzymes from Mycobacterium tuberculosis. Protein Expr. Purif., 22, 430-435 (2001) [34] Stauffer, M.E.; Young, J.K.; Evans, J.N.S.: Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase. Biochemistry, 40, 3951-3957 (2001) [35] Schonbrunn, E.; Eschenburg, S.; Shuttleworth, W.A.; Schloss, J.V.; Amrhein, N.; Evans, J.N.S.; Kabsch, W.: Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. Proc. Natl. Acad. Sci. USA, 98, 1376-1380 (2001) [36] Forlani, G.: Differential expression of 5-enol-pyruvyl-shikimate-3-phosphate synthase isoforms in elicitor-treated, cultured maize cells. Funct. Plant Biol., 29, 1483-1490 (2002) [37] Eschenburg, S.; Healy, M.L.; Priestman, M.A.; Lushington, G.H.; Schonbrunn, E.: How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli. Planta, 216, 129-135 (2002) [38] Baerson, S.R.; Rodriguez, D.J.; Tran, M.; Feng, Y.; Biest, N.A.; Dill, G.M.: Glyphosate-resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol., 129, 1265-1275 (2002) [39] Mizyed, S.; Wright, J.E.; Byczynski, B.; Berti, P.J.: Identification of the catalytic residues of AroA (Enolpyruvylshikimate 3-phosphate synthase) using partitioning analysis. Biochemistry, 42, 6986-6995 (2003) [40] Oliveira, J.S.; Mendes, M.A.; Palma, M.S.; Basso, L.A.; Santos, D.S.: One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis. Protein Expr. Purif., 28, 287-292 (2003)

561

Rubber cis-polyprenylcistransferase

2.5.1.20

1 Nomenclature EC number 2.5.1.20 Systematic name poly-cis-polyprenyl-diphosphate:isopentenyl-diphosphate polyprenylcistransferase Recommended name rubber cis-polyprenylcistransferase Synonyms allyltransferase, rubber cis-prenyl transferase isopentenyl pyrophosphate cis-1,4-polyisoprenyl transferase rubber allyltransferase rubber polymerase rubber prenyltransferase rubber transferase Additional information ( enzyme has activities of EC 2.5.1.20 and EC 2.5.1.10 [4]) [4] CAS registry number 62213-41-6

2 Source Organism Hevea brasiliensis [1-6, 9, 12, 13] Parthenium argentatum Gray (guayule [7,8]; var N565II [8]) [7, 8, 10, 11, 14] Parthenium husterophorus [7] Ficus larata [7] Ficus elastica [7, 10, 11, 12] Ficus benjamina [7] Ficus deltoidea [7] Ficus decora [7] Ficus indica [7] Euphorbia tirucalli [7] Ficus carica [13]

562

2.5.1.20


3 Reaction and Specificity Catalyzed reaction poly-cis-polyprenyl diphosphate + isopentenyl diphosphate = diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit Reaction type alkenyl group transfer Natural substrates and products S isopentenyl diphosphate + cis-1,4-polyisoprene ( incorporation of isopentenyl diphosphate into rubber [2]) [2] P diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit Substrates and products S dimethylallyl diphosphate + isopentenyl diphosphate (Reversibility: ? [3,4]) [3, 4] P (E,E)-farnesyl diphosphate + (E)-geranyl diphosphate ( in the absence of rubber [3]; in the absence of rubber elongation factor bound to rubber particles [4]) [3, 4] S isopentenyl diphosphate + cis-1,4-polyisoprene ( of rubber particles [1-8]; allylic diphosphate [8]; reaction of the isopentenyl diphosphate occurs with existing rubber molecules possessing a terminal allylic diphosphate group [1]; strictly stereospecific, yields exclusively cis-polyisoprene from isopentenyl diphosphate [2]; rubber particles have the ability to alter the stereoselective removal of the 2Rprochiral proton in favor of the removal of the 2S-prochiral proton [4]; no elongation can take place without rubber molecule initiation by an allylic diphosphate i.e. geranyl diphosphate, farnesyl diphosphate or geranyl geranyl diphosphate [9]; effectivity of initiator molecule in descending order: geranyl geranyl diphosphate, farnesyl diphosphate, geranyl diphosphate, dimethylallyl diphosphate [14]) (Reversibility: ? [1-13]) [1-14] P diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit ( the peak molecular weight of the radioactive polymer increases from 70000 Da in 15 min to 750000 Da in 3 h, the weight average molecular weight of the polymer synthesized over a 3 h period is 117000 Da compared to 149000 Da of the natural rubber polymer [8]) [1-14] S isopentenyl diphosphate + farnesyl diphosphate + cis-1,4-polyisoprene ( an allylic diphosphate e.g. farnesyl diphosphate is required to initiate the biosynthesis of new rubber molecules [11, 12, 13]) (Reversibility: ? [11, 12, 13]) [11, 12, 13] P diphosphate + a poly-cis-polyprenyl diphosphate longer by one C5 unit [11, 12, 13] Inhibitors Co2+ (0.1-1 mM, complete inhibition [3]) [3] EDTA ( 50 mM, strong inhibition [2]) [2] N-ethylmaleimide [2] 563


2.5.1.20

Zn2+ (0.1-1 mM, complete inhibition [3]) [3] farnesyl diphosphate [2] iodoacetamide ( complete inhibition [3]) [2, 3] iodoacetic acid [3] p-chloromercuribenzoate ( 50 mM, strong inhibition [2]) [2] sodium diphosphate [3] sodium phosphate [3] thiol alkylating agents [3] Cofactors/prosthetic groups Additional information ( ascorbic acid is not cofactor [2]) [2] Activating compounds 2-mercaptoethanol ( 10 mM, not as effective as dithiothreitol [3]) [3] EDTA ( approx. 3.5fold activation at 20 mM [13]) [13] dithiothreitol ( 10 mM, required for maximal stability [3]) [3] glutathione ( required for maximal activity [7,8]) [7, 8] neryl diphosphate ( accelerates activity [2]) [2] rubber elongation factor ( i.e.REF, protein tightly bound to serumfree rubber particles, required for prenyltransferase activity, no requirement for farnesyl diphosphate synthase activity [5]; amino acid sequence of REF [6]) [5, 6] rubber particle [7] sulfhydryl compound ( essential for full activity [2]) [2] Metals, ions Ca2+ ( maximal stimulation at 5 mM [3]) [3] Mg2+ ( required for activity [3,7,8,13]; maximal stimulation at 1 mM [3]; Km : 0.52 mM [8]; magnesium compounds essential for full activity, cannot be replaced by manganese, cobalt or iron [2]; maximal activity at 1 mM, inhibition above 10 mM [13]; maximal activity at 2 mM, inhibition above 10 mM [13]) [2, 3, 7, 8, 13, 14] Mn2+ ( stimulates, maximal activity at 1 mM [3]) [3] Pb2+ ( maximal stimulation at 5 mM [3]) [3] Specific activity (U/mg) 0.000695 ( partially purified enzyme [7]) [7] Km-Value (mM) 0.0003 (farnesyl diphosphate) [12] 0.0028 (farnesyl diphosphate) [13] 0.083 (isopentenyl diphosphate) [8] 0.096 (dimethylallyl diphosphate) [8] 0.16 (isopentenyl diphosphate) [12] 0.228 (isopentenyl diphosphate) [13] Ki-Value (mM) 5 (sodium diphosphate) [3] 200 (diphosphate) [3]

564

2.5.1.20


Temperature optimum ( C) 25 ( assay at [3]) [3] 30 ( assay at [1,2,7]) [1, 2, 7]

4 Enzyme Structure Molecular weight 60000 ( gel filtration [2]) [2] 70000 ( gel filtration [3]) [3] Additional information ( a large protein complex of 450000 Da may play a role in rubber transferase activity [11]) [11] Subunits dimer ( 2 * 38000, SDS-PAGE [3]) [3] Additional information ( glycoprotein LPR may contain the cistransferase active site [10]; ) [10]

5 Isolation/Preparation/Mutation/Application Source/tissue latex ( surface of rubber particles [1]; cis-1,4-prenyl transferase is firmly associated with the rubber particle [9]; buoyant particle-bound rubber transferase [12]; associated with rubber particles [13]) [1, 9, 10, 12, 13] latex leaf ( serum [1]) [1-3, 5, 7] mesophyll [7] stem ( stem bark tissue [14]) [7, 14] Localization particle-bound ( surface of rubber particles [1]; cis1,4-prenyl transferase is firmly associated with the rubber particle [9]; buoyant particle-bound rubber transferase [12]; associated with rubber particles [13]) [1, 9, 10, 12, 13] Purification (partial [1]; pH 5.4, ammonium sulfate, Sephadex G-100, DEAE-Sephadex, partial purification [2]; ammonium sulfate, DE52, Sephacryl S-200, hexyl-Sepharose, Mono Q [3]) [1, 2, 3, 4] (ammonium sulfate, Sephadex G-25, DEAE-cellulose, partial purification [7]) [7]

6 Stability pH-Stability 4-9 ( maximal stability at pH 7 [12]) [12]

565


2.5.1.20

Temperature stability 40 ( 10 min, partial denaturation [2]) [2] 60 ( 10 min, complete inactivation [2]) [2] Oxidation stability , thiols stabilize [3] Storage stability , -80 C, 25 mM Tris-HCl, pH 8, 10 mM dithiothreitol, 1 mM MgSO4, 30% glycerol, 0.1% sodium azide, at least 6 months, no loss of activity [3]

References [1] McMullen, A.I.; McSweeney, G.P.: The biosynthesis of rubber. Incorporation of isopentenyl pyrophosphate into purfied rubber particles by a soluble latex-serum enzyme. Biochem. J., 101, 42-47 (1966) [2] Archer, B.L.; Cockbain, E.G.: Rubber transferase from hevea brasiliensis latex. Methods Enzymol., 15, 476-480 (1969) [3] Light, D.R.; Dennis, M.S.: Purification of a prenyltransferase that elongates cis-polyisoprene rubber from the latex of Hevea brasiliensis. J. Biol. Chem., 264, 18589-18597 (1989) [4] Light, D.R.; Lazarus, R.A.; Dennis, M.S.: Rubber elongation by farnesyl pyrophosphate synthases involves a novel switch in enzyme stereospecificity. J. Biol. Chem., 264, 18598-18607 (1989) [5] Dennis, M.S.; Light, D.R.: Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis. J. Biol. Chem., 264, 18608-18617 (1989) [6] Dennis, M.S.; Henzel, W.J.; Bell, J.; Kohr, W.; Light, D.R.: Amino acid sequence of rubber elongation factor protein associated with rubber particles in Hevea latex. J. Biol. Chem., 264, 18618-18626 (1989) [7] Madhavan, S.; Benedict, C.R.: Isopentenyl pyrophosphate cis-1,4-polyisoprenyl transferase from guayule (Parthernium argentatum gray). Plant Physiol., 75, 908-913 (1984) [8] Benedict, C.R.; Madhavan, S.; Greenblatt, G.A.; Venkatachalam, K.V.; Foster, M.A.: Plant Physiol., 92, 816-821 (1989) [9] Cornish, K.: The separate roles of plant cis and trans prenyl transferases in cis-1,4-polyisoprene biosynthesis. Eur. J. Biochem., 218, 267-271 (1993) [10] Cornish, K.; Siler, D.J.; Grosjean, K.K.: Immunoinhibition of rubber particle-bound cis-prenyl transferases in Ficus elastica and Parthenium argentatum. Phytochemistry, 35, 1425-1428 (1994) [11] Siler, D.J.; Cornish, k.: Identification of Parthenium argentatum rubber particle proteins immunoprecipitated by an antibody that specifically inhibits rubber transferase activity. Phytochemistry, 36, 623-627 (1994) [12] Cornish, K.; Siler, D.J.: Characterization of cis-prenyl transferase activity localized in a buoyant fraction of rubber particles from Ficus elastica latex. Plant Physiol. Biochem., 34, 377-384 (1996)

566

2.5.1.20


[13] Kang, H.; Kang, M.Y.; Han, K.-H.: Identification of natural rubber and characterization of rubber biosynthetic activity in fig tree. Plant Physiol., 123, 1133-1142 (2000) [14] Sundar, D.; Reddy, A.R.: Low night temperature-induced changes in photosynthesis and rubber accumulation in guayule (Parthenium argentatum Gray). Photosynthetica, 38, 421-427 (2000)

567

Squalene synthase

2.5.1.21

1 Nomenclature EC number 2.5.1.21 Systematic name farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase Recommended name squalene synthase Synonyms farnesyldiphosphate:farnesyldiphosphate farnesyltransferase farnesyltransferase presqualene synthase presqualene-diphosphate synthase squalene synthase squalene synthetase synthase, squalene CAS registry number 9077-14-9

2 Source Organism

568

Sus scrofa [11, 12, 22] Nicotiana tabacum [16, 23, 27] Narcissus pseudonarcissus (daffodil [10]) [10] Saccharomyces cerevisiae (recombinant enzyme [13,28,35]) [1-8, 12, 13, 18-21, 28, 31, 35] yeast (overexpressed in E. coli [14]) [14] Rattus norvegicus (expressed in E. coli [9]) [8, 9, 15, 17, 29, 31] Homo sapiens (recombinant enzyme [32]) [24, 26, 31, 32] Botryococcus braunii (green microalga, race B [25]) [25] Glycyrrhiza glabra (licorice [30]) [30] Arabidopsis thaliana [33] Tabernaemontana divarctica [34]

2.5.1.21

Squalene synthase

3 Reaction and Specificity Catalyzed reaction 2 farnesyl diphosphate = diphosphate + presqualene diphosphate ( mechanism [7,28,32]; enzyme also catalyses the reduction of presqualene diphosphate by NADPH to squalene [20,28,33,34]; overview mechanism, kinetics [31]) presqualene diphosphate + NAD(P)H + H+ = squalene + diphosphate + NAD(P)+ Reaction type alkenyl group transfer Natural substrates and products S farnesyl diphosphate + farnesyl diphosphate ( first pathwayspecific enzyme in cholesterol biosynthesis [13, 16]; squalene is the first sterol intermediate in cholesterol biosynthesis [9]; essential enzyme in cholesterol biosynthetic pathway [14]; enzyme may be important during response to infection and inflammation [31]) [9, 13, 14, 16, 31] P diphosphate + presqualene diphosphate Substrates and products S (2E,6E)-3,7,10-trimethylundeca-2,6-dienyl diphosphate + NADPH ( weak [11]) (Reversibility: ? [11]) [11] P diphosphate + ? S (2E,6E)-3,7,11-trimethyldodeca-2,6-dienyl diphosphate + NADPH (Reversibility: ? [11]) [11] P diphosphate + ? S (2E,6E)-3,7,12-trimethyltrideca-2,6-dienyl diphosphate + NADPH ( weak [11]) (Reversibility: ? [11]) [11] P diphosphate + ? S (2E,6E)-3,7-dimethyldodeca-2,6-dienyl diphosphate + NADPH (Reversibility: ? [11]) [11] P diphosphate + ? S (2E,6E)-3,7-dimethyltetradeca-2,6-dienyl diphosphate + NADPH ( weak [11]) (Reversibility: ? [11]) [11] P diphosphate + ? S (2E,6E)-3,7-dimethyltrideca-2,6-dienyl diphosphate + NADPH ( weak [11]) (Reversibility: ? [11]) [11] P diphosphate + ? S (2E,6E)-3,7-dimethylundeca-2,6-dienyl diphosphate + NADPH ( weak [11]) (Reversibility: ? [11]) [11] P diphosphate + ? S (E,E)-7-desmethylfarnesyl diphosphate + NADPH ( at 60% of the efficiency that farnesyl diphosphate is converted to squalene [6]) (Reversibility: ? [6]) [6] P 6,19-didesmethylsqualene + NADP+ + diphosphate ( in absence of farnesyl diphosphate, 6-desmethylsqualene is produced in presence of farnesyl diphosphate [6]) [6]

569

Squalene synthase

2.5.1.21

S 10,11-dihydrofarnesyl diphosphate + NADPH ( at 60% of the efficiency that farnesyl diphosphate is converted to squalene [6]) (Reversibility: ? [6]) [6] P 2,3,22,23-tetrahydrosqualene + NADP+ + diphosphate [6] S 2-methylfarnesyl diphosphate + NADPH (Reversibility: ? [8]) [8] P 11-methylsqualene + NADP+ + diphosphate [8] S 3-demethylfarnesyl diphosphate + NADPH (Reversibility: ? [8]) [8] P 10-demethylsqualene + NADP+ + diphosphate [8] S farnesyl diphosphate ( in presence of Mn2+ [3]) (Reversibility: ? [3,12]) [3, 12] P 12,13-cis-dehydrosqualene + diphosphate ( 12-cis-dehydrosqualene + diphosphate [3]) [3, 12] S farnesyl diphosphate + NAD(P)H (Reversibility: ? [36,8,10,12,15-18,21]) [3-6, 8, 10, 12, 15-18, 21] P squalene + diphosphate + NAD(P)+ ( presqualene diphosphate and squalene are produced in a ratio of 6:1 [5]) [3-6, 8, 10, 12, 1518, 21] S farnesyl diphosphate + farnesyl diphosphate (Reversibility: ? [1,2,4,5,7,9,13-15,20,24]) [1, 2, 4, 5, 7, 9, 13-15, 20, 24] P diphosphate + presqualene diphosphate [1, 2, 4, 5, 7, 9, 13-15, 20] S geranylgeranyl diphosphate + NAD(P)H (Reversibility: ? [19]) [19] P lycopersene + NAD(P)+ + diphosphate [19] S geranylgeranyl diphosphate + geranylgeranyl diphosphate (Reversibility: ? [19]) [19] P prelycopersene diphosphate + diphosphate [19] S presqualene diphosphate + NAD(P)H ( the polymeric form of the enzyme also catalyzes the reduction of presqualene diphosphate by NADPH to squalene [4]; presqualene diphosphate synthetase and squalene synthetase are copurified during isolation [1]; in presence of reducing pyridine nucleotide, preferably NADPH, squalene is formed, in absence of reducing cofactor the rate of the condensation reaction is lower and all of the product accumulates as presqualene diphosphate [2, 12, 15]; one protein with 2 catalytic sites may be involved in synthesis of presqualene diphosphate and for its reduction to squalene [5]; a single active site catalyzes both reactions [7]) (Reversibility: ? [1,2,4,5,7,9,12-15,20]) [1, 2, 4, 5, 7, 9, 12-15, 20] P squalene + NAD(P)+ + diphosphate [1, 2, 7, 9, 12-14, 20] S Additional information ( specificity overview [6, 8, 11]; not metabolized: (E)-6,7,10,11-tetrahydrofarnesyl diphosphate [6]; no substrate: 6,7-dihydrofarnesyl diphosphate, 3-desmethylfarnesyl diphosphate [6]; none of the following analogues gives nonpolar products: 7,11-dimethyl-3-ethyl-2,6,10-dodecatrienyl diphosphate, 6,7,10,11-tetrahydrofarnesyl diphosphate, 4-methylthiofarnesyl diphosphate, 4-fluorofarnesyl diphosphate [8]; replacement of 3-methyl of 570

2.5.1.21

Squalene synthase

farnesyl diphosphate by an ethyl group or introduction of a methyl group at C-4 results in a complete loss of activity [11]; reaction is completely regioselective [35]) [6, 8, 11, 35] P ? Inhibitors CP-294838 ( a benzoxazepinone IC50 130 nM [24]) [24] CP-295697 ( a biphosphonate, IC50 20 nM [24]) [24] Mg2+ ( above 35 mM [34]) [34] N-ethylmaleimide [13] NADP+ ( inhibitor of squalene synthesis [5]) [5] ammonium analogues ( overview [7]) [7] deoxycholate [21] farnesyl diphosphate ( substrate inhibitor of squalene synthase reaction [2,5]; no inhibition of presqualene diphosphate synthase reaction [2]) [2, 5] zaragozic acid ( IC50 0.7 nM [24]; competitive to farnesyl diphosphate [26]) [24, 26] Additional information ( peroxisomal squalene synthase is inhibited by sonication, microsomal enzyme not [15]) [15] Cofactors/prosthetic groups NADH ( NADH or NADPH required for squalene synthase reaction [2]; preference for NADPH over NADH [14, 16, 19]) [1, 2, 14, 16, 18-20, 22] NADPH ( NADPH or NADH required for squalene synthase reaction [2]; preference for NADPH over NADH [1,14,16,19]) [1-7, 12, 14, 16, 18-20, 22] Activating compounds NADPH ( stimulates [15,20,28]) [15, 20, 28] Tween 20 ( stimulates recombinant enzyme [13]) [13] Tween 40 ( stimulates recombinant enzyme [13]) [13] Tween 80 ( stimulates recombinant enzyme [13]) [13] W-1 ( detergent, stimulates recombinant enzyme [13]) [13] bovine serum albumin ( activates recombinant enzyme [13]) [13] Additional information ( absolute requirement for phospholipid [21]; overview on regulation [31]) [21, 31] Metals, ions Mg2+ ( most effective in stimulation of squalene formation, half-maximal activity at 2.5 mM [3]; Mn2+ is more effective than Mg2+ in activation of dehydrosqualene formation [3, 13]; required for squalene synthase reaction [1,13]; Mn2+ is six times more effective in activation than Mg2+ [12]; no effect [12]; Mn2+ or Mg2+ are effective at low concentration as cofactors for synthesis of lycopersene, for the synthesis of prelycopersene diphosphate, Mn2+ is most effective at very low concentrations, Mg2+ is more effective at higher concentrations [19]; required, best at 10 mM, inhibition above 35 mM [34]) [1, 3, 12, 13, 19, 33, 34] 571

Squalene synthase

2.5.1.21

Mn2+ ( more effective than Mg2+ in activation of dehydrosqualene formation [3, 13]; six times more effective in activation than Mg2+ [12]; in presence of Mn2+ and Mg2+ the activity is reduced to one-third of that in presence of Mn2+ alone [12]; stimulation [12]; stimulates, maximum stimulation at 0.1 mM [13]; Mn2+ or Mg2+ are effective at low concentration as cofactors for synthesis of lycopersene, for the synthesis of prelycopersene diphosphate, Mn2+ is most effective at very low concentrations, Mg2+ is more effective at higher concentrations [19]) [3, 12, 13, 19] Turnover number (min±1) 31.8 (farnesyl diphosphate, cosubstrate + NAD(P)H [14]) [14] 198 (farnesyl diphosphate, in presence of 1% Tween 80 and Mg2+ [13]) [13] Specific activity (U/mg) 0.03 [10] 0.16 [15] 0.164 [16] 0.816 ( presqualene [5]) [5] 0.95 ( squalene [1]) [1] 1.2 [17] 15.6 ( presqualene [1]) [1] Additional information ( assay methods [2]) [2, 12-14] Km-Value (mM) 0.0004 (farnesyl diphosphate, plus NADPH [22]) [22] 0.001 (trans-farnesyl diphosphate, plus NADPH [17]) [17] 0.0023 (farnesyl diphosphate) [24] 0.0025 (farnesyl diphosphate) [14] 0.004 (NADH) [5] 0.004 (NADPH) [5] 0.0078 (farnesyl diphosphate) [34] 0.0095 (farnesyl diphosphate) [16] 0.045 (NADPH) [34] 0.5 (NADPH) [14] 3.6 (NADH) [14] Additional information [18] Ki-Value (mM) 0.00000025 (zaragozic acid) [26] pH-Optimum 6 [16] 7 [13] 7-8 [34] pH-Range 5.2-8.2 ( pH 5.2: about 45% of maximum activity, pH 8.2: about 75% of maximum activity [13]) [13]

572

2.5.1.21

Squalene synthase

Temperature optimum ( C) 25-30 ( formation of lycopersene [19]) [19] 28 ( assay at [10]) [10] 30 ( assay at [12]; formation of prelycopersene diphosphate [19]) [12, 19] 35 ( assay at [16]) [16] 37 ( assay at [6,11]) [6, 11]

4 Enzyme Structure Molecular weight 32000-35000 ( gel filtration [17]) [17] 42600 ( sucrose density gradient centrifugation, gel filtration [18]) [18] 54500 ( gel filtration, sucrose gradient sedimentation [21]) [21] 55000 ( gel filtration [1]) [1] 55000-60000 ( gel filtration [34]) [34] 68000 ( gel filtration [12]) [12] Subunits ? ( x * 47000, SDS-PAGE [5]; x * 42000-44000, enzyme expressed in E. coli, SDS-PAGE, carboxy-terminal truncated enzyme [14]; x * 47000, immunoblot analysis of enzyme separated by SDS-PAGE [16]; x * 32000-33000, trypsin-truncated enzyme, SDS-PAGE [17]; x * 44000, expressed in E. coli, SDS-PAGE [13]; x * 46700, deduced from gene sequence [23]; x * 52500, deduced from gene sequence [25]; x * 47000, deduced from gene sequence [33]) [5, 13, 14, 16, 17, 23, 25, 33] monomer ( 1 * 64000, SDS-PAGE [34]) [34]

5 Isolation/Preparation/Mutation/Application Source/tissue cell suspension culture [16] corona [10] leaf ( low activity [27]) [27] liver [8, 9, 11, 12, 15, 17, 22] meristem ( main source [27]) [27] root ( low activity [27]) [27] Localization membrane ( model of the secondary structure and its possible membrane orientation [9]) [9, 10, 20, 34] microsome ( integral membrane protein [16]; membranes [10]) [1, 3, 10-12, 16, 17, 20, 21] peroxisome [15]

573

Squalene synthase

2.5.1.21

Purification [22] (partial) [16] (partial) [10] (expressed in E. coli [13]; partial [1,12]) [1, 4, 5, 12, 13, 18, 19] (overexpressed in E. coli [14]) [14] (trypsin-truncated enzyme [17]) [17] (recombinant protein [26]) [26] (partial [34]) [34] Crystallization (mutant with deletion of 47 C-terminal amino acids [24]; in complex with different inhibitors [32]) [24, 32] Cloning [23] (development of a plasmid for heterologous expression of yeast squalene synthase in Escherichia coli [13]; overview [31]) [13, 31] (yeast enzyme overexpressed in Escherichia coli [14]) [14] (enzyme expressed in Escherichia coli [9]; overview [31]) [9, 31] (overview [31]) [26, 31] [25] (3 genes [30]) [30] [33] Engineering Additional information ( deletion of 30 N-terminal amino acids without effect to activity, additional deletion of 81 to 97 C-terminal amino acids abolishes activity, deletion of only 47 C-terminal amino acids retains activity [24]; point mutations in conserved regions A, B, C indicate that Tyr171, Asp219, Asp223 are essential for activity and Phe288 may be involved in second step of catalysis [29]) [24, 29] Application medicine ( induction of enzyme by a diet containing fluvastatin and cholestyramine [17]; enzyme may be important during response to infection and inflammation [31]) [17, 31]

6 Stability pH-Stability 8.5 ( irreversible denaturation above [13]) [13] Temperature stability 25 ( in presence of deoxycholate, 5 min, 50% loss of activity [21]) [21] 41 ( without deoxycholate, 5 min, 50% loss of activity [21]) [21]

574

2.5.1.21

Squalene synthase

General stability information , detergent, e.g. deoxycholate, destabilizes [11] , NADPH has no effect on stability [21] , markedly stabilized by 30% glycerol and 0.02 M 2-mercaptoethanol [18] , methanol stabilizes [1] , octylglucoside, 5 mM, causes rapid loss of activity [13] , stable in buffer containing 5 mM octylglucoside and 15 mM CHAPS [13] , sucrose stabilizes [1] , a combination of 10% methanol, 10% glycerol, 30 mM octyl-b-d-glucopyranoside, 0.4% Brij-58, and 1 mM DTT in 25 mM sodium phosphate, pH 7.4, essential for stability and maximal activity [14] , methanol, 10%, does not stabilize [10, 13] , sucrose, 10%, does not stabilize [10, 13] Storage stability , -70 C, stable [13]

References [1] Kuswik-Rabiega, G.; Rilling, H.C.: Squalene synthetase. Solubilization and partial purification of squalene synthetase, copurification of presqualene pyrophosphate and squalene synthetase activities. J. Biol. Chem., 262, 1505-1509 (1987) [2] Agnew, W.S.: Squalene synthetase. Methods Enzymol., 110, 359-373 (1985) [3] Nishino, T.; Katsuki, H.: Formation of 12-cis-dehydrosqualene catalyzed by squalene synthetase. Methods Enzymol., 110, 373-375 (1985) [4] Qureshi, A.A.; Beytia, E.D.; Porter, J.W.: Squalene synthetase. I. Dissociation and reassociation of enzyme complex. Biochem. Biophys. Res. Commun., 48, 1123-1128 (1972) [5] Sasiak, K.; Rilling, H.C.: Purification to homogeneity and some properties of squalene synthetase. Arch. Biochem. Biophys., 260, 622-627 (1988) [6] Washburn, W.N.; Kow, R.: Investigations of substrate specificity of squalene synthase. Tetrahedron Lett., 18, 1555-1558 (1977) [7] Poulter, C.D.; Capson, T.L.; Thompson, M.D.; Bard, R.S.: Squalene synthetase. Inhibition by ammonium analogues of carbocationic intermediates in the conversion of presqualene diphosphate to squalene. J. Am. Chem. Soc., 111, 3734-3739 (1989) [8] Ortiz de Montellano, P.R.; Wei, J.S.; Vinson, W.A.; Castillo, R.; Boparai, A.S.: Substrate selectivity of squalene synthetase. Biochemistry, 16, 2680-2685 (1977) [9] McKenzie, T.L.; Jiang, G.; Straubhaar, J.R.; Conrad, D.G.; Shechter, I.: Molecular cloning, expression, and characterization of the cDNA for the rat hepatic squalene synthase. J. Biol. Chem., 267, 21368-21374 (1992) [10] Belingheri, L.; Beyer, P.; Kleinig, H.; Gleizes, M.: Solubilization and partial purification of squalene synthase from daffodil microsomal membranes. FEBS Lett., 292, 34-36 (1991)

575

Squalene synthase

2.5.1.21

[11] Koyama, T.; Ogura, K.; Seto, S.: Substrate specificity of squalene synthetase. Biochim. Biophys. Acta, 617, 218-224 (1980) [12] Takatsuji, H.; Nishino, T.; Izui, K.; Katsuki, H.: Formation of dehydrosqualene catalyzed by squalene synthetase in Saccharomyces cerevisiae. J. Biochem., 91, 911-921 (1982) [13] Zhang, D.; Jennnings, S.M.; Robinson, G.W.; Poulter, C.D.: Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme [published erratum appears in Arch Biochem Biophys 1993 Sep;305(2):622]. Arch. Biochem. Biophys., 304, 133-143 (1993) [14] LoGrasso, P.V.; Soltis, D.A.; Boettcher, B.R.: Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase. Arch. Biochem. Biophys., 307, 193-199 (1993) [15] Ericsson, J.; Appelkvist, E.L.; Thelin, A.; Chojnacki, T.; Dallner, G.: Isoprenoid biosynthesis in rat liver peroxisomes. Characterization of cis-prenyltransferase and squalene synthetase. J. Biol. Chem., 267, 18708-18714 (1992) [16] Hanley, K.; Chappell, J.: Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures. Plant Physiol., 98, 215-220 (1992) [17] Shechter, I.; Klinger, E.; Rucker, M.L.; Engstrom, R.G.; Spirito, J.A.; Islam, M.A.; Boettcher, B.R.; Weinstein, D.B.: Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase. J. Biol. Chem., 267, 8628-8635 (1992) [18] Shechter, I.; Bloch, K.: Solubilization and purification of trans-farnesyl pyrophosphate-squalene synthetase. J. Biol. Chem., 246, 7690-7696 (1971) [19] Qureshi, A.A.; Barnes, F.J.; Semmler, E.J.; Porter, J.W.: Biosynthesis of prelycopersene pyrophosphate and lycopersene by squalene synthetase. J. Biol. Chem., 248, 2755-2767 (1973) [20] Agnew, W.S.; Popjak, G.: Squalene synthetase. Stoichiometry and kinetics of presqualene pyrophosphate and squalene synthesis by yeast microsomes. J. Biol. Chem., 253, 4566-4573 (1978) [21] Agnew, W.S.; Popjak, G.: Squalene synthetase. Solubilization from yeast microsomes of a phospholipid-requiring enzyme. J. Biol. Chem., 253, 45744583 (1978) [22] Dugan, R.E.; Porter, J.W.: Hog liver squalene synthetase: the partial purification of the particulate enzyme and kinetic analysis of the reaction. Arch. Biochem. Biophys., 152, 28-35 (1972) [23] Devarenne, T.P.; Shin, D.H.; Back, K.; Yin, S.; Chappell, J.: Molecular characterization of tobacco squalene synthase and regulation in response to fungal elicitor. Arch. Biochem. Biophys., 349, 205-215 (1998) [24] Thompson, J.F.; Danley, D.E.; Mazzalupo, S.; Milos, P.M.; Lira, M.E.; Harwood, H.J., Jr.: Truncation of human squalene synthase yields active, crystallizable protein. Arch. Biochem. Biophys., 350, 283-290 (1998) [25] Okada, S.; Devarenne, T.P.; Chappell, J.: Molecular characterization of squalene synthase from the green microalga Botryococcus braunii, race B. Arch. Biochem. Biophys., 373, 307-317 (2000)

576

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Squalene synthase

[26] Soltis, D.A.; McMahon, G.; Caplan, S.L.; Dudas, D.A.; Chamberlin, H.A.; Vattay, A.; Dottavio, D.; Rucker, M.L.; Engstrom, R.G.; et al.: Expression, purification, and characterization of the human squalene synthase: use of yeast and baculoviral systems. Arch. Biochem. Biophys., 316, 713-723 (1995) [27] Devarenne, T.P.; Ghosh, A.; Chappell, J.: Regulation of squalene synthase, a key enzyme of sterol biosynthesis, in tobacco. Plant Physiol., 129, 10951106 (2002) [28] Mookhtiar, K.A.; Kalinowski, S.S.; Zhang, D.; Poulter, C.D.: Yeast squalene synthase. A mechanism for addition of substrates and activation by NADPH. J. Biol. Chem., 269, 11201-11207 (1994) [29] Gu, P.; Ishii, Y.; Spencer, T.A.; Shechter, I.: Function-structure studies and identification of three enzyme domains involved in the catalytic activity in rat hepatic squalene synthase. J. Biol. Chem., 273, 12515-12525 (1998) [30] Hayashi, H.; Hirota, A.; Hiraoka, N.; Ikeshiro, Y.: Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase. Biol. Pharm. Bull., 22, 947-950 (1999) [31] Tansey, T.R.; Shechter, I.: Structure and regulation of mammalian squalene synthase. Biochim. Biophys. Acta, 1529, 49-62 (2000) [32] Pandit, J.; Danley, D.E.; Schulte, G.K.; Mazzalupo, S.; Pauly, T.A.; Hayward, C.M.; Hamanaka, E.S.; Thompson, J.F.; Harwood, H.J., Jr.: Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis. J. Biol. Chem., 275, 30610-30617 (2000) [33] Nakashima, T.; Inoue, T.; Oka, A.; Nishino, T.; Osumi, T.; Hata, S.: Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase. Proc. Natl. Acad. Sci. USA, 92, 2328-2332 (1995) [34] Kroon, P.A.; Threlfall, D.R.: Squalene synthase from cell suspension cultures of Tabernaemontana divaricata. Phytochemistry, 45, 1157-1163 (1997) [35] Zhang, D.l.; Poulter, C.D.: Biosynthesis of non-head-to-tail isoprenoids. Synthesis of 1'-1 and 1'-3 structures by recombinant yeast squalene synthase. J. Am. Chem. Soc., 117, 1641-1642 (1995)

577

Spermine synthase

2.5.1.22

1 Nomenclature EC number 2.5.1.22 Systematic name S-adenosylmethioninamine:spermidine 3-aminopropyltransferase Recommended name spermine synthase Synonyms aminopropyltransferase, spermidine spermidine aminopropyltransferase spermine synthetase synthase, spermine Additional information (not identical with EC 2.5.1.16 or EC 2.5.1.23) CAS registry number 74812-43-4

2 Source Organism Rattus norvegicus (Wistar [2,12]; Sprague-Dawley [10]) [1, 2, 6, 7, 9, 10, 12, 16, 18] Homo sapiens [8, 19] Bos taurus [3-5, 6, 7, 9, 13-15] Brassica pekinensis (Chinese cabbage, var. Pak Choy [11]) [11] Mus musculus [17]

3 Reaction and Specificity Catalyzed reaction S-adenosylmethioninamine + spermidine = 5'-methylthioadenosine + spermine ( mechanism [13]) Reaction type aminopropyl group transfer

578

2.5.1.22

Spermine synthase

Natural substrates and products S S-adenosylmethioninamine + spermidine ( involved in polyamine biosynthetic pathway [3]) (Reversibility: ? [3]) [3] P 5'-methylthioadenosine + spermine Substrates and products S S-adenosylmethioninamine + 1,8-diaminooctane ( poor substrate [1]) (Reversibility: ? [1]) [1] P 5'-methylthioadenosine + N-(3-aminopropyl)octane-1,8-diamine S S-adenosylmethioninamine + 6,6-difluorospermidine (Reversibility: ? [9]) [9] P 5'-methylthioadenosine + 6,6-difluorospermine S S-adenosylmethioninamine + 6-monofluorospermidine (Reversibility: ? [9]) [9] P 5'-methylthioadenosine + 6-monofluorospermine S S-adenosylmethioninamine + 7,7-difluorospermidine (Reversibility: ? [9]) [9] P 5'-methylthioadenosine + 7,7-difluorospermine S S-adenosylmethioninamine + 7-monofluorospermidine (Reversibility: ? [9]) [9] P 5'-methylthioadenosine + 7-monofluorospermine S S-adenosylmethioninamine + N-(3-aminopropyl)cadaverine ( poor substrate [1]) (Reversibility: ? [1]) [1] P 5'-methylthioadenosine + ? S S-adenosylmethioninamine + spermidine ( strictly specific for spermidine [3, 4]; best substrate [1]; mono and geminal difluorinated analogues of spermidine [9]) (Reversibility: ? [1, 3, 4, 10, 11, 13, 15]) [1, 3, 4, 10, 11, 13, 15] P 5'-methylthioadenosine + spermine [1, 3, 4, 10, 11, 13] S S-adenosylmethioninamine + sym-homospermidine ( 17% of the activity with spemidine [1]) (Reversibility: ? [1]) [1] P 5'-methylthioadenosine + ? S S-adenosylmethioninamine + sym-norspermidine ( poor substrate [1]) (Reversibility: ? [1]) [1] P 5'-methylthioadenosine + ? S Additional information ( synthetic substrates [6]; S-adenosylmethionine cannot act as aminopropyl donor [4]; no substrates are putrescine, cadaverine, spermine, sym-norspermine, spermidine monoacetyl derivatives [1]; overview [1,3]) [1, 3, 4, 6] P ? Inhibitors 1,3-diaminopropane [7] 1,8-diaminooctane [7] 1,9-diamino-4-azanonane ( weak [7]) [7] 1,9-diamino-5-azanonane ( weak [7]) [7] 1-aminooxy-3-N-[3-aminopropyl]-aminopropane ( aminooxy analogue of spermidine, kinetics [5]) [5] 579

Spermine synthase

2.5.1.22

3,3'-diaminodipropylamine ( weak [7]) [7] 5'-ethylthioadenosine ( strong [10]) [7, 10] 5'-methylthioadenosine ( strong [3,4,7,10]; kinetics [13]; partially reversible by adenosine [15]) [3, 4, 7, 10, 13, 15] 5'-methylthiotubercidine ( strong, in vivo and in vitro, kinetics [7,10]) [7, 10] 5-[5'-deoxy-5'-(C)-4',5'-didehydroadenosyl]-l-ornithine ( i.e. compound A9154C, prostate [7]) [7] 7,7-difluorospermidine ( pH-dependent, substrate inhibition [9]) [9] 7-monofluorospermidine ( pH-dependent, substrate inhibition [9]) [9] N-(3-aminopropyl)cyclohexylamine ( strong, selective [16]) [16] N-[2-aminooxyethyl]-1,4-diaminobutane ( aminooxy analogue of spermidine, kinetics [5]) [5] N-ethylmaleimide ( in the absence of sulfhydryl compounds [3,4]) [3, 4] PCMB ( in the absence of sulfhydryl compounds [3,4]) [3, 4] S-5'-deoxyadenosyl-(5')-1-methyl-3-(methylthio)propylamine [7] S-adenosyl-1,8-diamino-3-thiooctane ( weak, prostate [7]) [7] S-adenosyl-4-methylthiobutyrate ( prostate [7]; 70-98% inhibition at 1 mm [10]) [7, 10] S-adenosyl-4-thiobutyrate methylester ( prostate [7]) [7] S-adenosyl-d-methionine ( 70-98% inhibition at 1 mM [10]) [7, 10] S-adenosyl-l-ethionine ( 70-98% inhibition at 1 mM [10]) [10] S-adenosyl-l-homocysteine ( weak [10]; not [3,15]) [10] S-adenosyl-l-methionine ( prostate [7,10]; not [3,15]) [7, 10] S-tubercidinylmethionine ( 70-98% inhibition at 1 mM [10]) [10] putrescine ( weak [4]) [2-4, 7] spermine ( product inhibition, weak [3,4]; kinetics [13]) [2-4, 13] Additional information ( no inhibition by erythro-9-(2-hydroxy3-nonyl)adenine, methylglyoxal-bis-(guanylhydrazone), aminoguanidine, guanethidine, l-canaline, MgCl2 , KCl, diamines, polyamines, polyamine derivatives [3]; not inhibitory: decarboxylated adenosylmethionine [4,13]; not inhibitory: cyclohexylamine or dicyclohexylamine [7]; overview on inhibitors [7]) [3, 4, 7, 13] Cofactors/prosthetic groups Additional information ( no cofactor requirement [2,3]) [2, 3] Metals, ions Additional information ( no metal ion requirement [2,3]) [2, 3] Specific activity (U/mg) 0.0062 [10] 0.306 [2] 0.396 [3, 4]

580

2.5.1.22

Spermine synthase

0.403 [9] 0.455 ( placenta [8]) [8] 0.579 [14] Additional information ( assay method [12]; assay method using antibody against 5'-deoxy-5'-methylthioadenosine [18]) [12, 18] Km-Value (mM) 0.0001 (S-adenosylmethioninamine) [13] 0.00054 (7,7-difluorospermidine) [9] 0.0006 (S-adenosylmethioninamine) [3, 4, 15] 0.001 (S-adenosylmethioninamine) [8] 0.0011 (7-monofluorospermidine) [9] 0.005 (S-adenosylmethioninamine) [2] 0.006 (spermidine) [3, 4, 13] 0.02 (spermidine) [1] 0.07 (spermidine) [2] 0.084-0.088 (spermidine) [9] 0.093 (6-monofluorospermidine) [9] 0.2 (spermidine, placenta [8]) [8] 0.458 (6,6-difluorospermidine) [9] Ki-Value (mM) 0.0003 (5'-methylthioadenosine) [3] 0.0015 (1-aminooxy-3-N-[3-aminopropyl]-aminopropane) [5] 0.0017 (putrescine) [3] 0.186 (N-[2-aminooxyethyl]-1,4-diaminobutane) [5] pH-Optimum 7-8 ( broad [3]) [3] 7.5-8.1 [2] Additional information ( pI: 5 [2]; pI: 5.1, brain [3]) [2, 3] Temperature optimum ( C) 37 [3-5, 8, 10-13]

4 Enzyme Structure Molecular weight 78000 ( kidney, pore-gradient electrophoresis [8]) [8] 88000-90000 ( brain, gel filtration, sedimentation equilibrium centrifugation [3,4]) [3, 4] Subunits dimer ( 2 * 45000, SDS-PAGE [3,4]; 2 * 45000, kidney, SDSPAGE [8]) [3, 4, 8]

581

Spermine synthase

2.5.1.22

5 Isolation/Preparation/Mutation/Application Source/tissue brain ( high activity [18]) [2-4, 7, 8, 12-15, 18] fibroblast ( primary culture of embryonic fibroblast [17]) [17] hepatoma cell [9] kidney ( low activity [18]) [18, 8] leaf [11] liver ( low activity [18]) [5, 18] placenta [8] prostate gland [1, 7, 10, 12] spleen [5, 8, 9] thymus [12] Additional information ( tissue distribution [8,12]) [8, 12] Localization cytosol [2, 8, 13] Purification (partial [2]) [2] [8] (spermine-Sepharose affinity chromatography [3,14]; partial [14]) [3, 4, 14] Cloning [19] Engineering Additional information ( enzyme null mutant, lack of spermine increases sensitivity of cells to anti-tumor agents [17]) [17] Application medicine ( specific and marked decrease in spermine after diet containing N-(3-aminopropyl)cyclohexylamine [16]; enzyme null mutant, lack of spermine increases sensitivity of cells to anti-tumor agents [17]) [16, 17]

6 Stability General stability information , bovine serum albumin stabilizes enzyme in dilute solutions [13] Storage stability , 0 C, in buffer containing 0.3 M NaCl and 1 mM spermidine, at least 6 months [4]

582

2.5.1.22

Spermine synthase

References [1] Pegg, A.E.; Shuttleworth, K.; Hibasami, H.: Specificity of mammalian spermidine synthase and spermine synthase. Biochem. J., 197, 315-320 (1981) [2] Hannonen, P.; Jänne, J.; Raina, A.: Partial purification and characterization of spermine synthase from rat brain. Biochim. Biophys. Acta, 289, 225-231 (1972) [3] Pajula, R.L.; Raina, A.; Eloranta, T.: Polyamine synthesis in mammalian tissues. Isolation and characterization of spermine synthase from bovine brain. Eur. J. Biochem., 101, 619-626 (1979) [4] Raina, A.; Pajula, R.L.; Eloranta, T.: Purification of spermidine aminopropyltransferase (spermine synthase) from bovine brain. Methods Enzymol., 94, 276-279 (1983) [5] Eloranta, T.; Khomutov, A.R.; Khomutov, R.M.; Hyvönen, T.: Aminooxy analogues of spermidine as inhibitors of spermine synthase and substrates of hepatic polyamine acetylating activity. J. Biochem., 108, 593-598 (1990) [6] Coward, J.K.; Anderson, G.L.; Tang, K.C.: Aminopropyltransferase substrates and inhibitors. Methods Enzymol., 94, 286-294 (1983) [7] Pegg, A.E.: Inhibition of aminopropyltransferases. Methods Enzymol., 94, 294-297 (1983) [8] Kajander, E.O.; Kauppinen, L.I.; Pajula, R.L.; Karkola, K.; Eloranta, T.O.: Purification and partial characterization of human polyamine synthases. Biochem. J., 259, 879-886 (1989) [9] Baillon, J.G.; Mamont, P.S.; Wagner, J.; Gerhart, F.; Lux, P.: Fluorinated analogues of spermidine as substrates of spermine synthase. Eur. J. Biochem., 176, 237-242 (1988) [10] Hibasami, H.; Borchardt, R.T.; Chen, S.Y.; Coward, J.K.; Pegg, A.E.: Studies of inhibition of rat spermidine synthase and spermine synthase. Biochem. J., 187, 419-428 (1980) [11] Sindhu, R.K.; Cohen, S.S.: Propylaminetransferases in chinese cabbage leaves. Plant Physiol., 74, 645-649 (1984) [12] Raina, A.; Pajula, R.L.; Eloranta, T.: A rapid assay method for spermidine and spermine synthases. Distribution of polyamine-synthesizing enzymes and methionine adenosyltransferase in rat tissues. FEBS Lett., 67, 252-255 (1976) [13] Pajula, R.L.: Kinetic properties of spermine synthase from bovine brain. Biochem. J., 215, 669-676 (1983) [14] Pajula, R.L.; Raina, A.; Kekoni, J.: Purification of spermine synthase from bovine brain by spermine-Sepharose affinity chromatography. FEBS Lett., 90, 153-156 (1978) [15] Pajula, R.L.; Raina, A.: Methylthioadenosine, a potent inhibitor of spermine synthase from bovine brain. FEBS Lett., 99, 343-345 (1979) [16] Shirahata, A.; Takahashi, N.; Beppu, T.; Hosoda, H.; Samejima, K.: Effects of inhibitors of spermidine synthase and spermine synthase on polyamine synthesis in rat tissues. Biochem. Pharmacol., 45, 1897-1903 (1993)

583

Spermine synthase

2.5.1.22

[17] Ikeguchi, Y.; Mackintosh, C.A.; McCloskey, D.E.; Pegg, A.E.: Effect of spermine synthase on the sensitivity of cells to anti-tumour agents. Biochem. J., 373, 885-892 (2003) [18] Lee, S.H.; Cho, Y.D.: A new assay method for spermidine and spermine synthases using antibody against MTA. J. Biochem. Mol. Biol., 30, 443-447 (1997) [19] Korhonen, V.P.; Halmekyto, M.; Kauppinen, L.; Myohanen, S.; Wahlfors, J.; Keinanen, T.; Hyvonen, T.; Alhonen, L.; Eloranta, T.; Janne, J.: Molecular cloning of a cDNA encoding human spermine synthase. DNA Cell Biol., 14, 841-847 (1995)

584

sym-Norspermidine synthase

2.5.1.23

1 Nomenclature EC number 2.5.1.23 Systematic name S-adenosylmethioninamine:propane-1,3-diamine 3-aminopropyltransferase Recommended name sym-norspermidine synthase Synonyms Additional information (not identical with EC 2.5.1.16 or EC 2.5.1.22)

2 Source Organism Euglena gracilis (Z, strain 1224-5/25 [1]) [1]

3 Reaction and Specificity Catalyzed reaction S-adenosylmethioninamine + propane-1,3-diamine = 5'-methylthioadenosine + bis(3-aminopropyl)amine Reaction type aminopropyl group transfer Natural substrates and products S S-adenosylmethioninamine + propane-1,3-diamine [1] P 5'-methylthioadenosine + N-(3-aminopropyl)-1,3-diaminopropane Substrates and products S S-adenosylmethioninamine + propane-1,3-diamine (i.e. decarboxylated S-adenosylmethionine + 1,3-diaminopropane [1]) (Reversibility: ? [1]) [1] P 5'-methylthioadenosine + N-(3-aminopropyl)-1,3-diaminopropane (, i.e. sym-norspermidine [1]) [1]

585

sym-Norspermidine synthase

2.5.1.23

5 Isolation/Preparation/Mutation/Application Source/tissue cell culture [1]

References [1] Aleksijevic, A.; Grove, J.; Schuber, F.: Studies on polyamine biosynthesis in Euglena gracilis. Biochim. Biophys. Acta, 565, 199-207 (1979)

586

Discadenine synthase

2.5.1.24

1 Nomenclature EC number 2.5.1.24 Systematic name S-adenosyl-l-methionine:6-N-(D2 -isopentenyl)-adenine 3-(3-amino-3-carboxypropyl)-transferase Recommended name discadenine synthase Synonyms discadenine synthetase synthase, discadenine CAS registry number 74082-52-3

2 Source Organism no activity in Polysphondylium pallidum [3] no activity in Polysphondylium violaceum [3] no activity in Dictyostelium minutum [3] Dictyostelium discoideum [1-3] Dictyostelium purpureum [3] Dictyostelium mucoroides [3]

3 Reaction and Specificity Catalyzed reaction S-adenosyl-l-methionine + 6-N-(D2 -isopentenyl)-adenine = 5'-methylthioadenosine + discadenine Reaction type aminocarboxypropyl group transfer Natural substrates and products S S-adenosyl-l-methionine + N6 -(D2 -isopentenyl)-adenine (, biosynthesis of discadenine, an inhibitor of spore germination and a potent cytokinin [1]; , discadenine is produced as an inhibitor of spore

587


2.5.1.24

germination in the species of cellular slime molds in which in which acrasin is cyclic adenosine 5'-monophosphate [3]) [1, 3] P 5'-methylthioadenosine + discadenine Substrates and products S S-adenosyl-l-methionine + N6 -(D2 -isopentenyl)-adenine (Reversibility: ? [1-3]) [1-3] P 5'-methylthioadenosine + discadenine (, discadenine is 3-(3amino-3-carboxypropyl)-N6 -(D2 -isopentenyl)-adenine [1,2]) [1, 2] S S-adenosylmethionine + N6 -benzyladenine (Reversibility: ? [2]) [2] P 3-(3-amino-3-carboxypropyl)-N6 -benzyladenine + 5'-methylthioadenosine [2] Inhibitors Ca2+ [2] Mg2+ (, 100 mM, 30% inhibition [2]) [2] Mn2+ [2] Zn2+ (, 100 mM, 80% inhibition [2]) [2] Specific activity (U/mg) 0.0123 [2] Km-Value (mM) 0.0007 (N6 -benzyladenine) [2] 0.0185 (S-adenosyl-l-methionine) [2] pH-Optimum 7.5 [1, 2] pH-Range 6-8.5 (, pH 6.0: about 55% of activity maximum, pH 8.5: about 60% of activity maximum [2]) [2] Temperature optimum ( C) 35 [2]

4 Enzyme Structure Molecular weight 82000 (, gel filtration [2]) [2]

5 Isolation/Preparation/Mutation/Application Source/tissue fruiting body [2] Purification [2] 588

2.5.1.24


References [1] Taya, Y.; Tanaka, Y.; Nishimura, S.: Cell-free biosynthesis of discadenine, a spore germination inhibitor of Dictyostelium discoideum. FEBS Lett., 89, 326-328 (1978) [2] Ihara, M.; Tanaka, Y.; Yanagisawa, K.; Taya, Y.; Nishimura, S.: Purification and some properties of discadenine synthase from Dictyostelium discoideum. Biochim. Biophys. Acta, 881, 135-140 (1986) [3] Taya, Y.; Yamada, T.; Nishimura, S.: Correlation between acrasins and spore germination inhibitors in cellular slime molds. J. Bacteriol., 143, 715-719 (1980)

589

tRNA-uridine aminocarboxypropyltransferase

2.5.1.25

1 Nomenclature EC number 2.5.1.25 Systematic name S-adenosyl-l-methionine:tRNA-uridine 3-(3-amino-3-carboxypropyl)transferase Recommended name tRNA-uridine aminocarboxypropyltransferase

2 Source Organism Escherichia coli (strain B [1]) [1]

3 Reaction and Specificity Catalyzed reaction S-adenosyl-l-methionine + tRNA uridine = 5'-methylthioadenosine + tRNA 3-(3-amino-3-carboxypropyl)-uridine Reaction type aminocarboxypropyl group transfer Substrates and products S S-adenosyl-l-methionine + tRNAPhe (, methyl-deficient tRNAPhe , extra-region contains G-U-C instead of m7 G-X-C, in vitro [1]) (Reversibility: ? [1]) [1] P 5'-methylthioadenosine + tRNA 3-(3-amino-3-carboxypropyl) uridine [1] Metals, ions Mg2+ (, required [1]) [1] pH-Optimum 8.7 [1]

590

2.5.1.25

tRNA-uridine aminocarboxypropyltransferase

5 Isolation/Preparation/Mutation/Application Purification (strain B, partial [1]) [1]

References [1] Nishimura, S.; Taya, Y.; Kuchino, Y.; Ohashi, Z.: Enzymatic synthesis of 3-(3amino-3-carboxypropyl)uridine in Escherichia coli phenylalanine transfer RNA: transfer of the 3-amino-acid-3-carboxypropyl group from S-adenosylmethionine. Biochem. Biophys. Res. Commun., 57, 702-708 (1974)

591

Alkylglycerone-phosphate synthase

2.5.1.26

1 Nomenclature EC number 2.5.1.26 Systematic name 1-acyl-glycerone-3-phosphate:long-chain-alcohol O-3-phospho-2-oxopropanyltransferase Recommended name alkylglycerone-phosphate synthase Synonyms alkyl DHAP synthetase alkyl dihydroxyacetone phosphate synthase alkyl dihydroxyacetone phosphate synthetase alkyl-DHAP alkyldihydroxyacetone phosphate synthase alkyldihydroxyacetonephosphate synthase alkyldihydroxyacetonephosphate synthetase alkylglycerone phosphate synthase synthase, alkylglycerone phosphate synthetase, alkyldihydroxyacetone phosphate CAS registry number 102484-74-2 64060-42-0

2 Source Organism

592

Mus musculus [1, 2, 4, 11] Homo sapiens [6, 11, 13, 14, 16] Bos taurus [6] Cavia porcellus (recombinant enzyme [18]) [3, 6, 9-11, 13, 15, 17, 18] Rattus norvegicus [5-8] Pan troglodytes (chimpanzee [6]) [6] Trypanosoma brucei [12] Caenorhabditis elegans [13]

2.5.1.26


3 Reaction and Specificity Catalyzed reaction 1-acyl-glycerone 3-phosphate + a long-chain alcohol = an alkyl-glycerone 3phosphate + a long-chain acid anion ( ping-pong mechanism [1,4,10]; mechanism [2,3,18]; enzyme also catalyzes the following reactions: 1. acyl exchange reaction in which palmitic acid is incorporated into palmitoyldihydroxyacetone phosphate, 2. alkyl exchange reaction in which hexadecanol is incorporated into hexadecyldihydroxyacetone phosphate [1]) Reaction type ether formation Natural substrates and products S 1-acylglycerone 3-phosphate + long-chain alcohol ( first committed step in biosynthesis of ether-linked glycerolipids [4]; forms the ether bond found in alkyl and alk-1-enyl glycerolipids [1, 2]; 2 separate alkylglycerone-phosphate synthases, one in microsomes and another in peroxisomes might be engaged in biosynthesis of 1-O-alkylglycerolipids in rat liver [7]) [1, 2, 4, 7] P alkylglycerone 3-phosphate + long-chain acid anion Substrates and products S 1-O-hexadecyldihydroxyacetone phosphate + hexadecanol (Reversibility: ? [6]) [6] P long-chain acid anion + 1-O-hexadecyldihydroxyacetone phosphate S 1-acylglycerone 3-phosphate + long-chain alcohol ( 1-acylglycerone 3-phosphate is acyldihydroxyacetone phosphate, the ester-linked fatty acid of the substrate is cleaved and replaced by a long-chain alcohol in the ether [1]) (Reversibility: ? [1,13]) [1, 13] P alkylglycerone 3-phosphate + long-chain acid anion [1] S oleoyldihydroxyacetone phosphate + hexadecanol (Reversibility: ? [8]) [8] P oleate + 1-O-hexadecyldihydroxyacetone phosphate S palmitoyldihydroxyacetone phosphate + hexadecanol (Reversibility: ir [3]; ? [1,2,4,6-8]) [1-4, 6-8] P 1-O-hexadecyldihydroxyacetone phosphate + palmitate [3, 4] S palmitoyldihydroxyacetone phosphate + octadecanol (Reversibility: ? [8,10,12,13]) [8, 10, 12, 13] P palmitate + 1-O-octadecyldihydroxyacetone phosphate S stearoyldihydroxyacetone phosphate + hexadecanol (Reversibility: ? [8,12]) [8, 12] P stearate + 1-O-hexadecyldihydroxyacetone phosphate S stearoyldihydroxyacetone phosphate + octadecanol (Reversibility: ? [8]) [8] P stearate + 1-O-octadecyldihydroxyacetone phosphate S Additional information ( alkyldihydroxyacetone phosphate synthase also catalyzes the following reactions: 1. acyl exchange re-

593


2.5.1.26

action in which palmitic acid is incorporated into palmitoyldihydroxyacetone phosphate, 2. alkyl exchange reaction in which hexadecanol is incorporated into hexadecyldihydroxyacetone phosphate [1]; specific for acyldihydroxyacetone phosphate, acyldihydroxyacetone or 1-acyl-sn-glycerol 3-phosphate cannot substitute for acyldihydroxyacetone phosphate, the acyl group chain length in acyldihydroxyacetone phosphate should be C12 or longer [2]; overview on substrate specificity [11]) [1, 2, 7, 11] P Additional information ( two reaction products: alkylglycerone phosphate and alkylglycerone, alkylglycerone phosphate is mainly synthesized by the peroxisomal synthase, whereas the inverse proportion is observed with the microsomal enzyme [7]) [7] Inhibitors 2,4-dinitrofluorobenzene ( 1 mM, 2% activity [10]) [10] 3-bromo-2-oxoheptadecyl phosphate [3] Mg2+ ( 5 mM, 70% activity [10]) [10] Mn2+ ( 5 mM, 6% activity [10]) [10] N-ethylmaleimide ( 5 mM, 15% activity [10]) [10] NaCl ( 50% inhibition at 0.6 M [12]) [12] Zn2+ ( 5 mM, 8% activity [10]) [10] acyldihydroxyacetone phosphate ( high concentration, substrate inhibition [2]) [2] fatty acids ( competitive to fatty alcohols [2]) [2, 3] hexadecanol ( inhibitory above 0.3 mM [12]) [12] p-bromophenacylbromide ( 1 mM, 55% activity [10]) [10] palmitoyl-dihydroxyacetonphosphate ( inhibitory above 0.1 mM [12]) [12] phenylglyoxal ( inactivation [16]) [16] Additional information ( not inhibitory: Ca2+ [10]) [10] Cofactors/prosthetic groups FAD ( involved in initial oxidation of substrate [18]) [18] Specific activity (U/mg) 0.00714 [3] 0.0208 [1] 0.067 [2, 4] 0.35 [6] Km-Value (mM) 0.038 (octadecanol) [8] 0.04 (hexadecanol, plus palmitoyldihydroxyacetone phosphate [3]) [3] 0.042 (hexadecanol) [12] 0.044 (hexadecanol) [8] 0.045 (palmitoyldihydroxyacetone phosphate, plus hexadecanol [3]) [3] 0.068 (palmitoyldihydroxyacetone phosphate, plus hexadecanol [6]) [6] 594

2.5.1.26


0.072 (hexadecanol, plus palmitoyldihydroxyacetone phosphate [6]) [6] 0.1 (hexadecanol, plus hexadecyldihydroxyacetone phosphate [6]) [6] 0.1 (palmitoyldihydroxyacetone phosphate) [12] 0.115 (hexadecyldihydroxyacetone phosphate, plus hexadecanol, 0.4 mM [6]) [6] 0.192 (hexadecyldihydroxyacetone phosphate, plus hexadecanol, 0.05 mM [6]) [6] pH-Optimum 7.5 [6, 8] 8 ( assay at [1,3]) [1, 3] 8-9 [3] pH-Range 6.5-9 ( pH 6.5: about 50% of maximum activity, pH 9.0: about 30% of maximum activity [6]) [6] Temperature optimum ( C) 37 ( assay at [1,3,7]) [1, 3, 7]

4 Enzyme Structure Molecular weight 65000 ( gel filtration, phosphocellulose chromatography [6]) [6] 79000 ( radiation inactivation [9]) [9] Subunits ? ( x * 68900, deduced from gene sequence [12]; x * 65000, SDS-PAGE [15]) [12, 15] monomer ( 1 * 65000, SDS-PAGE [6]) [6] Additional information ( enzyme interacts with dihydroxyacetone phosphate acyltransferase in a heterotrimeric complex [17]) [17]

5 Isolation/Preparation/Mutation/Application Source/tissue Ehrlich ascites carcinoma cell [1, 2, 4] brain [6] fibroblast [13, 14] kidney [6] liver [5-9, 15, 17] placenta ( low activity [6]) [6] Additional information ( enzyme is present in all tissues examined, overview [11, 15]) [11, 15]

595


2.5.1.26

Localization glycosome [13] microsome [1, 2, 4, 7] peroxisome ( at least the active site of the integral membrane protein is localized exclusively at the inner surface of the peroxisomal membrane [5]; membrane [6]; entire enzyme is located inside peroxisomes [11]; type-1 peroxisomal targeting signal [12,13]; type-2 peroxisomal targeting signal [13]; enzyme is not an integral membrane protein [15]) [3, 5-9, 12, 13, 15, 17] Purification (partial [1]) [1, 2] [3, 6, 13] (overview [11]) [11] Cloning [13] [13] [12] (overview [11]) [11] Engineering C576A ( 187% of wild type activity [10]) [10] H617A ( complete loss of activity [10]) [10] R419H ( inactive enzyme [16]) [16] R419K ( 1% of wild type activity [10]) [10] S367A ( 4% of wild type activity [10]) [10] Additional information ( deletion of C-terminal 5 amino acids, inactive enzyme [10]) [10] Application medicine ( enzyme levels are strongly reduced in fibroblasts derived from Zellweger syndrome and rhizomelic chondrodysplasia punctata patients [13,16]; in fibroblast cell lines derived from Zellweger syndrome and rhizomelic chondrodysplasia punctata patients enzyme is mainly present as precursor form [14]) [13, 14, 16]

6 Stability General stability information , sensitive to sulfhydryl reagents, dithiothreitol prevents oxidation [2] , loss of activity on freezing and thawing [3] Storage stability , 4 C, 20% ethylene glycol, solubilized and delipidated enzyme is stable for 1 month [2] , in liquid N2 , stable for 1 year [2] , 4 C, 50% loss of activity after 10 days [3]

596

2.5.1.26


References [1] Brown, A.J.; Snyder, F.: Alkyldihydroxyacetone-P synthase. Solubilization, partial purification, new assay method, and evidence for a ping-pong mechanism. J. Biol. Chem., 257, 8835-8839 (1982) [2] Brown, A.; Snyder, F.: Alkyldihydroxyacetonephosphate synthase. Methods Enzymol., 209, 377-384 (1992) [3] Horie, S.; Das, A.K.; Hajra, A.K.: Alkyldihydroxyacetonephosphate synthase from guinea pig liver peroxisomes. Methods Enzymol., 209, 385-390 (1992) [4] Brown, A.J.; Snyder, F.: The mechanism of alkyldihydroxyacetone-P synthase. Formation of [3 H]H2 O from acyl[1-R-3 H]dihydroxyacetone-P by purified alkyldihydroxyacetone-P synthase in the absence of acylhydrolase activity. J. Biol. Chem., 258, 4184-4189 (1983) [5] Hardeman, D.; van den Bosch, H.: Rat liver dihydroxyacetone-phosphate acyltransferase: enzyme characteristics and localization studies. Biochim. Biophys. Acta, 963, 1-9 (1988) [6] Zomer, A.W.M.; de Weerd, W.F.C.; Langeveld, J.; van den Bosch, H.: Ether lipid synthesis: purification and identification of alkyl dihydroxyacetone phosphate synthase from guinea-pig liver. Biochim. Biophys. Acta, 1170, 189-196 (1993) [7] Rabert, U.; Völkl, A.; Debuch, H.: Distribution of alkylglycerone-phosphate synthase in subcellular fractions of rat liver. Biol. Chem. Hoppe-Seyler, 367, 215-222 (1986) [8] Gunawan, J.; Rabert, U.; Völkl, A.; Debuch, H.: Kinetic studies of alkyl-dihydroxyacetone-phosphate (alkyl-glycerone-phosphate) synthase in peroxisomes of rat liver. Biol. Chem. Hoppe-Seyler, 371, 339-344 (1990) [9] Biermann, J.; Schoonderwoerd, K.; Hom, M.L.; Luthjens, L.H.; Van den Bosch, H.: The native molecular size of alkyl-dihydroxyacetonephosphate synthase and dihydroxyacetone phosphate acyltransferase. Biochim. Biophys. Acta, 1393, 137-142 (1998) [10] De Vet, E.C.J.M.; Van Den Bosch, H.: Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli. Kinetics, chemical modification and mutagenesis. Biochim. Biophys. Acta, 1436, 299-306 (1999) [11] Van den Bosch, H.; de Vet, E.C.J.M.: Alkyl-dihydroxyacetonephosphate synthase. Biochim. Biophys. Acta, 1348, 35-44 (1997) [12] Zomer, A.W.M.; Opperdoes, F.R.; van den Bosch, H.: Alkyl dihydroxyacetone phosphate synthase in glycosomes of Trypanosoma brucei. Biochim. Biophys. Acta, 1257, 167-173 (1995) [13] De Vet, E.C.J.M.; Van den Bosch, H.: Alkyl-dihydroxyacetonephosphate synthase. Cell Biochem. Biophys., 32, 117-121 (2000) [14] Biermann, J.; Gootjes, J.; Wanders, R.J.A.; Van den Bosch, H.: Stability of alkyl-dihydroxyacetonephosphate synthase in human control and peroxisomal disorder fibroblasts. IUBMB Life, 48, 635-639 (1999) [15] De Vet, E.C.J.M.; Biermann, J.; Van Den Bosch, H.: Immunological localization and tissue distribution of alkyldihydroxyacetonephosphate synthase

597


2.5.1.26

and deficiency of the enzyme in peroxisomal disorders. Eur. J. Biochem., 247, 511-517 (1997) [16] De Vet, E.C.J.M.; Ijlst, L.; Oostheim, W.; Wanders, R.J.A.; Van Den Bosch, H.: Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency. J. Biol. Chem., 273, 10296-10301 (1998) [17] Biermann, J.; Just, W.W.; Wanders, R.J.A.; Van den Bosch, H.: Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes. Eur. J. Biochem., 261, 492-499 (1999) [18] De Vet, E.C.J.M.; Hilkes, Y.H.A.; Fraaije, M.W.; Van den Bosch, H.: Alkyldihydroxyacetonephosphate synthase: Presence and role of flavin adenine dinucleotide. J. Biol. Chem., 275, 6276-6283 (2000)

598

Adenylate dimethylallyltransferase

2.5.1.27

1 Nomenclature EC number 2.5.1.27 Systematic name dimethylallyl-diphosphate:AMP dimethylallyltransferase Recommended name adenylate dimethylallyltransferase Synonyms 2-isopentenyl-diphosphate:AMP D2 -isopentenyltransferase DMA transferase cytokinin synthase dimethylallyl-diphosphate:AMP D2 -isopentenyltransferase dimethylallylpyrophosphate:5'-AMP transferase isopentenyltransferase isopentenyltransferase, adenylate CAS registry number 72840-95-0

2 Source Organism Nicotiana tabacum (tobacco, crown gall tumor line 15055/01 [2]) [1, 2] Agrobacterium tumefaciens [3] Arabidopsis thaliana [4]

3 Reaction and Specificity Catalyzed reaction dimethylallyl diphosphate + AMP = diphosphate + 6-N-(dimethylallyl)adenosine 5'-phosphate Reaction type alkenyl group transfer Natural substrates and products S dimethylallyl diphosphate + AMP ( biosynthesis of cytokinins [1,4]) (Reversibility: ? [1, 4]) [1, 4] P diphosphate + N6 -(dimethylallyl)-adenosine 5'-monophosphate 599


2.5.1.27

Substrates and products S dimethylallyl diphosphate + AMP (Reversibility: ? [1-4]) [14] P diphosphate + N6 -(dimethylallyl)adenosine 5'-monophosphate [1] S Additional information ( strict requirement for AMP, no substrate: adenine, adenosine [2]; no substrate: adenine, adenosine, isopentenyldiphosphate [4]) [2, 4] P ? Inhibitors ADP ( strong [4]) [4] ATP ( strong [4]) [4] GDP ( strong [4]) [4] GTP ( strong [4]) [4] Cofactors/prosthetic groups AMP ( strict requirement [2]) [2] Specific activity (U/mg) Additional information ( assay principle [2]) [2] Km-Value (mM) 0.000008 (dimethylallyl diphosphate) [3] 0.000086 (AMP) [3] 0.05 (dimethylallyl diphosphate) [4] 0.185 (AMP) [4] pH-Optimum 8 [3, 4] Temperature optimum ( C) 30 [3] 37 ( assay at [1]) [1]

5 Isolation/Preparation/Mutation/Application Source/tissue callus ( cytokinin-autotrophic [1]) [1] crown gall (crown gall tumor line 15055/01 [2]) [2] Purification (partial [1,2]) [1, 2] (recombinant enzyme [3]) [3] Cloning [4]

600

2.5.1.27


6 Stability Temperature stability Additional information ( low thermal stability [3]) [3] Storage stability , -70 C, about 90% loss of activity after 3 weeks [1]

References [1] Chen, C.; Melitz, D.K.: Cytokinin biosynthesis in a cell-free system from cytokinin-autotrophic tobacco tissue cultures. FEBS Lett., 107, 15-20 (1979) [2] Hommes, B.G.; Akiyosh, D. E.; Morris, R.O.: Assay and partial purification of the cytokinin biosynthetic enzyme dimethylallylpyrophosphate:5'-AMP transferase. Methods Enzymol., 110, 340-347 (1985) [3] Blackwell, J.R.; Horgan, R.: Cloned Agrobacterium tumefaciens ipt1 gene product, DMAPP:AMP isopentenyl transferase. Phytochemistry, 34, 14771481 (1993) [4] Takei, K.; Sakakibara, H.; Sugiyama, T.: Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana. J. Biol. Chem., 276, 26405-26410 (2001)

601

Dimethylallylcistransferase

2.5.1.28

1 Nomenclature EC number 2.5.1.28 Systematic name dimethylallyl-diphosphate:isopentenyl-diphosphate dimethylallylcistransferase Recommended name dimethylallylcistransferase Synonyms neryl-diphosphate synthase CAS registry number 9032-79-5

2 Source Organism Pinus radiata [1] Tanacetum vulgare [2]

3 Reaction and Specificity Catalyzed reaction dimethylallyl diphosphate + isopentenyl diphosphate = diphosphate + neryl diphosphate Reaction type alkenyl group transfer Natural substrates and products S dimethylallyl diphosphate + isopentenyl diphosphate (, the product neryl diphosphate may be the precursor of cyclic monoterpenes [1]) [1] P diphosphate + neryl diphosphate Substrates and products S dimethylallyl diphosphate + isopentenyl diphosphate (Reversibility: ? [1,2]) [1, 2] P diphosphate + neryl diphosphate [1, 2]

602

2.5.1.28

Dimethylallylcistransferase

5 Isolation/Preparation/Mutation/Application Source/tissue leaf [2] seedling [1] Localization soluble [1]

References [1] Beytia, E.; Valenzuela, P.; Cori, O.: Terpene biosynthesis: formation of nerol, geraniol, and other prenols by an enzyme system from Pinus radiata seedlings. Arch. Biochem. Biophys., 129, 346-356 (1969) [2] Banthorpe, D.V.; Bucknall, G.A.; Doonan, H.J.; Doonan, S.; Rowan, M.G.: Biosynthesis of geraniol and nerol in cell-free extracts of Tanatecum vulgare. Phytochemistry, 15, 91-100 (1976)

603

Farnesyltranstransferase

2.5.1.29

1 Nomenclature EC number 2.5.1.29 Systematic name trans,trans-farnesyl-diphosphate:isopentenyl-diphosphate farnesyltranstransferase Recommended name farnesyltranstransferase Synonyms farnesyltransferase geranylgeranyl diphosphate synthase geranylgeranyl pyrophosphate synthase geranylgeranyl pyrophosphate synthetase geranylgeranyl-PP synthetase synthetase, geranylgeranyl pyrophosphate CAS registry number 9032-58-0

2 Source Organism 604

Erwinia uredovora (overexpressed in E. coli [10]) [10] Phycomyces blakesleeanus [11] Bacillus subtilis [12] Capsicum annuum [3] Sus scrofa [1, 4] Cucurbita pepo (pumpkin [2]) [2, 13, 14, 17] Sinapis alba (mustard [5]) [5] Rattus norvegicus [6] Methanobacterium thermoformicicum (SF-4 [7,8]) [7, 8] Micrococcus luteus (the activities of EC 2.5.1.1 and EC 2.5.1.29 may be a mixture of 2 enzymes or a single enzyme with two independent catalytic sites, see EC 2.5.1.1 [15,16]) [15, 16] Bos taurus [9, 18] Micrococcus lysodeikticus [17] Sulfolobus acidocaldarius [19, 20, 21, 22] Taxus canadensis (canadian yew [23] SwissProt-ID: Q9ZPM3) [23] Taxus baccata [24]

2.5.1.29


Ricinus communis [24] Arabidopsis thaliana [25] Homo sapiens [26] Abies grandis (farnesyldiphosphate synthase, SwissProt-ID: Q94F74 [27]) [27] Abies grandis (geranylgeranyldiphosphate synthase, SwissProt-ID: Q8W1R9 [28]) [28] Taxus canadensis [28] Hevea brasiliensis [29] Streptomyces griseolosporeus (MF730-N6 [30]) [30]

3 Reaction and Specificity Catalyzed reaction trans,trans-farnesyl diphosphate + isopentenyl diphosphate = diphosphate + geranylgeranyl diphosphate ( ordered-sequential bi bi mechanism [8]) Reaction type alkenyl group transfer Natural substrates and products S (E,E)-farnesyl diphosphate + isopentenyl diphosphate ( no activity with dimethylallyl diphosphate [4]; preferred allylic substrate [11]; 66% of activity with geranyl diphosphate [23]; no activity with dimethylallyl diphosphate [24]; enzyme regulates taxane biosynthesis [24]) [1-18, 23, 24, 26, 28, 29, 30] P geranylgeranyl diphosphate + diphosphate S dimethylallyl diphosphate + isopentenyl diphosphate ( 9% of the activity with farnesyl diphosphate [6]; 0.3% of the activity with farnesyl diphosphate [9]; less than 5% of the activity with farnesyl diphosphate [11]; preferred substrate [17]; 33% of activity with geranyl diphosphate [23]; low activity [24]) [2-7, 9-12, 14, 17, 23, 24, 28, 29, 30] P geranylgeranyl diphosphate + diphosphate S geranyl diphosphate + isopentenyl diphosphate ( 15% of the activity with farnesyl diphosphate [6]; 3% of the activity with farnesyl diphosphate [9]; less than 5% of the activity with farnesyl diphosphate [11]; preferred substrate [17]; preferred substrate [23]; involved in taxol biosynthesis [23]) [1-7, 9-12, 14-18, 20, 21, 23, 24, 26, 28, 29, 30] P geranylgeranyl diphosphate + diphosphate Substrates and products S (2E)-3,7-dimethyl-2-octenyl diphosphate + isopentenyl diphosphate ( 62% of activity with geranyl diphosphate [20]) (Reversibility: ? [20]) [20] P ?

605


2.5.1.29

S (2E)-3-methyl-2,6-heptadienyl diphosphate + isopentenyl diphosphate ( 24% of activity with geranyl diphosphate [20]) (Reversibility: ? [20]) [20] P ? S (2E)-3-methyl-2-butenyl diphosphate + isopentenyl diphosphate ( 21.5% of activity with geranyl diphosphate [20]) (Reversibility: ? [20]) [20] P ? S (2E)-3-methyl-2-hexenyl diphosphate + isopentenyl diphosphate ( 95% of activity with geranyl diphosphate [20]) (Reversibility: ? [20]) [20] P ? S (2E)-3-methyl-2-pentenyl diphosphate + isopentenyl diphosphate ( 63% of activity with geranyl diphosphate [20]) (Reversibility: ? [20]) [20] P ? S (E,E)-farnesyl diphosphate + isopentenyl diphosphate ( no activity with dimethylallyl diphosphate [4]; preferred allylic substrate [11]; 66% of activity with geranyl diphosphate [23]; no activity with dimethylallyl diphosphate [24]) (Reversibility: ? [1-17, 23, 24, 26, 28, 29, 30]) [118, 23, 24, 26, 28, 29, 30] P geranylgeranyl diphosphate + diphosphate ( (E,E,E)-geranylgeranyl diphosphate [2, 6, 12]; exclusive product [18]) [1-18, 23, 24, 26, 28, 29, 30] S 3,8-dimethyl-2-nonenyl diphosphate + isopentenyl diphosphate ( 59% of activity with dimethylallyl diphosphate [17]) (Reversibility: ? [17]) [17] P ? S 3-methyl-2-alkenyl diphosphate + isopentenyl diphosphate ( a number of 3-methyl-2-alkenyl diphosphates ranging in carbon number from 6 to 13 act as substrates [2]) (Reversibility: ? [2]) [2] P ? S 6,7-dihydrogeranyl diphosphate + isopentenyl diphosphate ( 87% of activity with dimethylallyl diphosphate [17]) (Reversibility: ? [17]) [17] P ? S 8,8'-bisnorgeranyl diphosphate + isopentenyl diphosphate ( 107% of activity with dimethylallyl diphosphate [17]) (Reversibility: ? [17]) [17] P ? S (2E)-3-methyl-2-undecenyl diphosphate + isopentenyl diphosphate ( 53% of the activity with farnesyl diphosphate [16]) (Reversibility: ? [16]) [16] P ? S cis-3-methyl-2-hexenyl diphosphate + isopentenyl diphosphate ( 56% of activity with dimethylallyl diphosphate [17]; 14% of 606

2.5.1.29

P S

P S

P S

P

S P S P


activity with dimethylallyl diphosphate [17]) (Reversibility: ? [17]) [17] ? cyclopentylideneethyl diphosphate + isopentenyl diphosphate ( 13% of activity with dimethylallyl diphosphate [17]; 13% of activity with dimethylallyl diphosphate [17]) (Reversibility: ? [17]) [17] ? dimethylallyl diphosphate + isopentenyl diphosphate ( 9% of the activity with farnesyl diphosphate [6]; 0.3% of the activity with farnesyl diphosphate [9]; less than 5% of the activity with farnesyl diphosphate [11]; preferred substrate [17]; 33% of activity with geranyl diphosphate [23]; low activity [24]) (Reversibility: ? [2-7, 9-12, 14, 17, 23, 24, 28, 29, 30]) [2-7, 9-12, 14, 17, 23, 24, 28, 29, 30] geranylgeranyl diphosphate + diphosphate ( (E,E,E)-geranylgeranyl diphosphate [2, 14]; exclusively geranylgeranyl diphosphate [14]) [2-7, 9-12, 14, 17, 23, 24, 28, 29, 30] geranyl diphosphate + isopentenyl diphosphate ( 15% of the activity with farnesyl diphosphate [6]; 3% of the activity with farnesyl diphosphate [9]; less than 5% of the activity with farnesyl diphosphate [11]; preferred substrate [17]; preferred substrate [23]) (Reversibility: ? [1-7, 9-12, 14-17, 20, 23, 24, 26, 28, 29, 30]) [1-7, 9-12, 14-18, 20, 21, 23, 24, 26, 28, 29, 30] geranylgeranyl diphosphate + diphosphate ( (E,E,E)-geranylgeranyl diphosphate [2]; exclusively geranylgeranyl diphosphate [14]; geranyl diphosphate + farnesyl diphosphate, ratio of farnesyl diphosphate to geranylgeranyl diphosphate increases to more than 2 under both conditions of substrate and product inhibition [7]; (E,E)-farnesyl diphosphate + (E,E,E)-geranylgeranyl diphosphate [12]; approx. 30% of activity with dimethylallyl diphosphate [17]; geranylgeranyl diphosphate + farnesyl diphosphate [18]; main product [21]) [1-7, 9-12, 14-18, 20, 21, 23, 24, 26, 28, 29, 30] trans-3-methyl-2-octenyl diphosphate + isopentenyl diphosphate ( similar activity as with dimethylallyl diphosphate [15]) (Reversibility: ? [15]) [15] ? trans-3-methyl-2-pentenyl diphosphate + isopentenyl diphosphate ( 33% of activity with dimethylallyl diphosphate [15]) (Reversibility: ? [15]) [15] ?

Inhibitors (E,E,E)-geranylgeranyl diphosphate ( 0.005 mM, 50% inhibition, competitive vs. farnesyl diphosphate [18]) [6, 18] (Z,E,E)-geranylgeranyl diphosphate ( 0.005 mM, approx. 50% inhibition, competitive vs. farnesyl diphosphate [18]) [6, 18]

607


2.5.1.29

3-azafarnesyl diphosphate ( 0.00074 mM, 50% inhibition [26]) [26] 3-azageranyl diphosphate ( 0.24 mM, 50% inhibition [26]) [26] 3-azageranylgeranyl diphosphate ( 0.0009 mM, 90% inhibition [6]; 0.00014 mM, 50% inhibition [26]) [6, 18, 26] 3-azahomofarnesyl diphosphate ( 0.00031 mM, 50% inhibition [26]) [26] 3-azahomogeranylgeranyl diphosphate ( 0.00037 mM, 50% inhibition [26]) [26] Li+ ( 400 mM, 72% inhibition, slight activation at 50 mM [8]) [8] NH+4 ( 20 mM, 20% inhibition [12]) [12] SDS ( 1%, 97% inhibition [7]) [7] Triton X-100 ( stimulates farnesyl-transferring activity, inhibits dimethylallyl-transferring activity [15,16]) [15, 16] aminophenylethyl diphosphate [5] diphosphate ( 10 mM, more than 90% inhibition [4]; 10 mM, 97% inhibition [7]; 10 mM, complete inhibition [15]) [4, 7, 8, 15, 16] farnesyl diphosphate ( above 0.025 mM [4]) [4, 16] geranylgeranyl diphosphate [8] homorisedronate ( i.e. NE58051, 0.41 mM, 50% inhibition [26]) [26] ibandronate ( 0.083 mM, 50% inhibition [26]) [26] p-chloromercuribenzoate ( 1 mM, 90% inhibition, 30% inhibition in the presence of 800 mM KCl [7]) [7] pamidronate ( 0.18 mM, 50% inhibition [26]) [26] risedronate ( i.e. NE58095, 0.35 mM, 50% inhibition [26]) [26] Additional information ( inhibition by irradiation with the photolabile analog of geranyl diphosphate [3]; monovalent cations at low concentrations, i.e. 50 mM, enhance activity, at high concentrations, i.e. 400 mM, they are inhibitory, except for K+ [8]; geranylgeranyl diphosphate synthase is inhibited by a variety of bisphosphonates [26]) [3, 8, 26] Activating compounds NH+4 ( 50 mM, enhances activity approx. 13fold [8]) [8] Triton X-100 ( stimulates farnesyl-transferring activity, inhibits dimethylallyl-transferring activity [15,16]) [12, 15, 16] Tween 80 ( stimulates [12,15,16]) [12, 15, 16] octyl glucoside ( 0.8%, 5fold activation [9]; 0.8%, 1.2fold activation [18]) [9, 18] Metals, ions Co2+ ( activates, less effective than Mg2+ [4]) [4] KCl ( 800 mM, activates [7]; 200-2300 mM, enhances activity approx. 10fold [8]) [7, 8] Mg2+ ( required for activity [2, 4, 9, 12-16, 18, 23, 24, 29]; less effective than Mn2+ [2, 4, 13]; optimal activity at 5-10 mM [9]; optimal activity at 5 mM [12]; 608

2.5.1.29


optimal activation of farnesyl-transferring activity at 1 mM, optimal activation of dimethylallyl-transferring activity at 3 mM [15, 16]; maximal activity at 1 mM [18]; maximal activity at 2 mM together with Mn2+ at 3 mM [24]; maximal activity at 5 mM [29]) [2, 4, 9, 12-16, 18, 23, 24, 29] Mn2+ ( required for activity [1, 4, 9, 14, 18, 24, 29]; optimal activity at 0.5-1 mM [9]; optimal activity at 0.5 mM [12]; activates much more effectively than Mg2+ [2]; activates [12]; slight activation [15, 16]; maximal activity at 1 mM, less effective than Mg2+ [18]; most effective divalent cation, maximal activity at 3 mM [24]; recombinant enzyme, maximal activity at 0.1 mM, inhibition at high concentrations [29]) [1, 2, 4, 9, 12, 14-16, 18, 24, 29] Zn2+ ( maximal activity at 1 mM, approx. 30% of activation with Mg2+ [18]; least effective divalent cation [24]) [18, 24] Additional information ( monovalent cations at low concentrations, i.e. 50 mM, enhance activity, at high concentrations, i.e. 400 mM, they are inhibitory, except for K+ [8]) [8] Turnover number (min±1) 60 (dimethylallyl diphosphate) [28] 126 (geranyl diphosphate) [28] 198 (farnesyl diphosphate) [28] Specific activity (U/mg) 0.0000279 [4] 0.00461 [9] 0.017 [3] 0.0182 [15] 0.098 [18] 0.123 ( recombinant enzyme [29]) [29] 0.14 ( with farnesyl diphosphate [7]) [7] 0.289 ( with geranyl diphosphate [7]) [7] 0.3 [11] 13.8 [24] Additional information ( 4300000 cpm/mg [5]) [5] Km-Value (mM) 0.0005 (farnesyl diphosphate) [24] 0.00074 (farnesyl diphosphate) [18] 0.0008 (geranyl diphosphate) [18] 0.00095 (dimethylallyl diphosphate) [3] 0.001 (geranyl diphosphate) [3] 0.0012 (farnesyl diphosphate) [3] 0.0015 (isopentenyl diphosphate) [24] 0.0017 (farnesyl diphosphate) [2] 0.002 (dimethylallyl diphosphate) [5] 0.002 (isopentenyl diphosphate) [18]

609


2.5.1.29

0.00234 (geranyl diphosphate) [29] 0.00246 (farnesyl diphosphate) [24] 0.0026 (farnesyl diphosphate) [28] 0.003 (isopentenyl diphosphate) [3] 0.0034 (Mg2+ ) [28] 0.0035 (isopentenyl diphosphate) [24] 0.006 (farnesyl diphosphate, recombinant enzyme [23]) [23] 0.00678 (farnesyl diphosphate) [29] 0.007 (isopentenyl diphosphate, recombinant enzyme [23]) [23] 0.0077 (geranyl diphosphate) [28] 0.008 (isopentenyl diphosphate, cosubstrate dimethylallyl diphosphate at 0.175 mM [15]) [15] 0.009 (geranyl diphosphate) [10] 0.009 (isopentenyl diphosphate) [11] 0.011 (farnesyl diphosphate) [10] 0.0115 (dimethylallyl diphosphate) [29] 0.0126 (geranyl diphosphate) [7] 0.0127 (dimethylallyl diphosphate) [24] 0.0133 (geranyl diphosphate) [12] 0.0147 (farnesyl diphosphate) [7] 0.0168 (dimethylallyl diphosphate) [7] 0.024 (isopentenyl diphosphate) [29] 0.0308 (isopentenyl diphosphate) [7] 0.033 (dimethylallyl diphosphate) [18] 0.033 (isopentenyl diphosphate) [5] 0.035 (Mg2+ , recombinant enzyme [23]) [23] 0.036 (isopentenyl diphosphate) [10] 0.036 (isopentenyl diphosphate, cosubstrate farnesyl diphosphate [30]) [30] 0.039 (isopentenyl diphosphate, cosubstrate farnesyl diphosphate [28]) [28] 0.06 (farnesyl diphosphate) [11] 0.062 (dimethylallyl diphosphate) [15] 0.066 (dimethylallyl diphosphate) [30] 0.088 (isopentenyl diphosphate, cosubstrate dimethylallyl diphosphate [28]) [28] 0.099 (dimethylallyl diphosphate, A57L mutant enzyme [30]) [30] 0.108 (isopentenyl diphosphate, cosubstrate dimethylallyl diphosphate [30]) [30] 0.121 (farnesyl diphosphate) [30] 0.122 (isopentenyl diphosphate, cosubstrate geranyl diphosphate [28]) [28] 0.127 (dimethylallyl diphosphate) [28] Ki-Value (mM) 0.0012 ((E,E,E)-geranylgeranyl diphosphate) [18] 0.0012 ((Z,E,E)-geranylgeranyl diphosphate) [18]

610

2.5.1.29


pH-Optimum 6.9-7.2 [24] 7 [2, 4] 7.7 [16] 8-9 ( recombinant enzyme [29]) [29] 8.2 [7] 8.5 [12] Temperature optimum ( C) 37 ( assay at [4,9]) [4, 9] 60 ( assay at [7,8]) [7, 8] 65 [7] Temperature range ( C) 30-72 ( approx. 30% of maximal activity at 30 C, approx. 80% of maximal activity at 72 C [7]) [7]

4 Enzyme Structure Molecular weight 39000 ( native PAGE [7]) [7] 60000 ( gel filtration, enzyme consists of 2 identical subunits of 30000 Da with a tendency to dissociate [11]; recombinant truncated enzyme, gel filtration [23]) [11, 23] 66000 ( gel filtration [28]) [28] 70000 ( gel filtration [15,16]) [15, 16] 72000 ( gel filtration [24]) [24] 74000 ( gel filtration [5]; gel filtration [3]) [3, 5] 76000 ( gel filtration [24]) [24] 78000 ( gel filtration [7]) [7] 85000 ( gel filtration [12]) [12] 150000 ( Superose 12, gel filtration [18]) [18] 190000 ( Superdex 200, gel filtration [18]) [18] 300000 ( gel filtration [4]) [1, 4] Subunits ? ( x * 37500, tetramer or pentamer, SDS-PAGE [18]) [18] dimer ( 2 * 30000, the native enzyme consists of 2 identical subunits with a tendency to dissociate, SDS-PAGE [11]; 2 * 37000, SDS-PAGE [3]; 2 * 38000, SDS-PAGE [5]; 2 * 39000, SDSPAGE [7]; 2 * 31500, deduced from nucleotide sequence [23]; 2 * 38000, SDS-PAGE [24]; 2 * 32000, recombinant enzyme, SDS-PAGE [28]) [3, 5, 7, 11, 23, 24, 28]

611


2.5.1.29

5 Isolation/Preparation/Mutation/Application Source/tissue brain [9] cell suspension culture [23, 24] flower [29] fruit [14] latex ( low expression level [29]) [29] leaf [29] liver [1, 4, 6] petiole ( low expression level [29]) [29] seed ( activity increases markedly as germination proceeds [13]) [2, 13] seedling [24] Localization chromoplast ( stroma [3]) [3] etioplast [5] Additional information ( enzyme seems to be membrane associated [24]) [24] Purification (recombinant enzyme, urea, DEAE-52 [10]) [10] (polyethylene glycol, DE-52 cellulose, DEAE Trisacryl-M, Reactive blue 4-agarose, Reactive red 120-agarose [11]) [11] (partial [12]) [12] (poly(ethylene glycol) 6000, DEAE-Sephacel, aminophenethyl diphosphate affinity column [3]) [3] (partial [1]; ammonium sulfate, DEAE Sephadex a-50, hydroxylapatite [4]) [1, 4] (DEAE-Sephadex [14]) [2, 13, 14] (anion exchange chromatography, aminophenylethyl diphosphate affinity column [5]) [5] [7] (ammonium sulfate, DEAE-Sephadex, hydroxylapatite, Sephadex G100, enzyme fraction may be a mixture of EC 2.5.1.1 and EC 2.5.1.29 or a single enzyme with two independent catalytic sites, see EC 2.5.1.1 [15]) [15, 16] (Mono Q, isoelectric focusing, Superose 12, partial purification [9]) [9] (ammonium sulfate, farnesylmethyl affinity gel [18]) [18] (recombinant enzyme [22]) [22] (recombinant enzyme [23]) [23] (ammonium sulfate, phenyl Sepharose 6, Mono Q, Tosohaas-G3000, Mono P [24]) [24] (recombinant his-tagged enzyme, Ni2+ -column [29]) [29] Cloning (expression in Escherichia coli [10]) [10]

612

2.5.1.29


(expression in Saccharomyces cerevisiae [19]; overexpression of maltose-binding protein-fusion protein or glutathion S-transferase-fusion protein in Escherichia coli [22]) [19] (expression in Saccharomyces cerevisiae [23]) [23] [25] [26] (expression in Escherichia coli [27]) [27] (expression of preprotein and truncated versions in Escherichia coli [28]) [28] (expression in Escherichia coli [29]) [29] (overexpression in Escherichia coli [30]) [30] Engineering A57L ( mutated enzyme produces mainly farnesyl diphosphate [30]) [30] D170E/M171P/I172A/S173M ( no activity with dimethylallyl diphosphate, low activity with geranyl diphosphate [20]) [20] D170E/M171P/I172A/S173R ( low activity with dimethylallyl diphosphate, approx. 60% of wild-type activity with geranyl diphosphate [20]) [20] D170E/M171P/I172R/S173V ( no activity with dimethylallyl diphosphate, low activity with geranyl diphosphate [20]) [20] L50S ( similar activity as wild-type [30]) [30] M171P ( low activity with dimethylallyl diphosphate, approx. 50% of wild-type activity with geranyl diphosphate [20]) [20] M171P/I172R ( no activity with dimethylallyl diphosphate, low activity with geranyl diphosphate [20]) [20] M171P/S173V ( very low activity with dimethylallyl diphosphate, approx. 50% of wild-type activity with geranyl diphosphate [20]) [20] S4F ( very weak activity [30]) [30] V8A ( similar activity as wild-type [30]) [30] Additional information ( the sequence between position 77 and position 86 of geranylgeranyl diphosphate synthase has been replaced with the corresponding sequences of farnesyl diphosphate synthase from human, rat, Arabidopsis thaliana and Saccharomyces cerevisiae [21]) [21]

6 Stability Temperature stability 65 ( in presence of 800 mM KCl, 30 min, stable [7]) [7] 72 ( in the presence of 3500 mM KCl [7]) [7] Storage stability , -20 C, at least 1 month, no loss of activity [4] , -20 C, at least 1 week, no loss of activity [2, 14, 16]

613


2.5.1.29

References [1] Sagami, H.; Ishi, K.; Ogura, K.: Occurence and unusual properties of geranylgeranyl pyrophosphate synthetase of pig liver. Biochem. Int., 3, 669-675 (1981) [2] Ogura, K.; Nishino, T.; Shinka, T.; Seto, S.: Methods Enzymol., 110, 167-171 (1985) [3] Dogbo, O.; Camara, B.: Purification of isopentenyl pyrophosphate isomerase and geranylgeranyl pyrophosphate synthase from Capsicum chromoplasts by affinity chromatography. Biochim. Biophys. Acta, 920, 140-148 (1987) [4] Sagami, H.; Ishi, K.; Ogura, K.: Geranylgeranylpyrophosphate synthetase of pig liver. Methods Enzymol., 110, 184-188 (1985) [5] Laferriere, A.; Beyer, P.: Purification of geranylgeranyl diphosphate synthase from Sinapis alba etioplasts. Biochim. Biophys. Acta, 1077, 167172 (1991) [6] Sagami, H.; Korenaga, T.; Ogura, K.; Steiger, A.; Pyun, H.-J.; Coates, R.M.: Studies on geranylgeranyl diphosphate synthase from rat liver: specific inhibition by 3-azageranylgeranyl diphosphate. Arch. Biochem. Biophys., 297, 314-320 (1992) [7] Tachibana, A.; Tanaka, T.; Taniguchi, M.; Oi, S.: Purification and characterization of geranylgeranyl diphosphate synthase from methanobacterium thermoformicicum Sf-4. Biosci. Biotechnol. Biochem., 57, 1129-1133 (1993) [8] Tachibana, A.; Tanaka, T.; Taniguchi, M.; Oi, S.: Potassium-stimulating mechanism of geranylgeranyl diphosphate synthase of Methanobacterium thermoformicicum SF-4. J. Biochem., 114, 389-392 (1993) [9] Sagami, H.; Korenaga, T.; Ogura, K.: Geranylgeranyl diphosphate synthase catalyzing the single condensation between isopentenyl diphosphate and farnesyl diphosphate. J. Biochem., 114, 118-121 (1993) [10] Wiedemann, M.; Misawa, N.; Sandmann, G.: Purification and enzymatic characterization of the geranylgeranyl pyrophosphate synthase from Erwinia uredovora after expression in Escherichia coli. Arch. Biochem. Biophys., 306, 152-157 (1993) [11] Lütke Brinkhaus, F.; Rilling, H.C.: Purification of geranylgeranyl diphosphate synthase from Phycomyces blakesleanus. Arch. Biochem. Biophys., 266, 607-612 (1988) [12] Takahashi, I.; Ogura, K.: Prenyltransferases of Bacillus subtilis: undecaprenyl pyrophosphate synthetase and geranylgeranyl pyrophosphate synthetase. J. Biochem., 92, 1527-1537 (1982) [13] Shinka, T.; Ogura, K.; Seto, S.: Farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthases during Cucurbito pepo germination. Phytochemistry, 13, 2103-2106 (1974) [14] Ogura, K.; Shinka, T.; Seto, S.: The purification and properties of geranylgeranyl pyrophosphate synthetase from pumpkin fruit. J. Biochem., 72, 1101-1108 (1972)

614

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[15] Sagami, H.; Ogura, K.: Geranylgeranyl pyrophosphate synthetase lacking geranyl-transferring activity from Micrococcus luteus. J. Biochem., 89, 1573-1580 (1981) [16] Sagami, H.; Ogura, K.: Geranylpyrophosphate synthetase-geranylgeranylpyrophosphate synthetase from Micrococcus luteus. Methods Enzymol., 110, 188-192 (1985) [17] Shinka, T.; Ogura, K.; Seto, S.: Comparative specificity of geranylgeranyl pyrophosphate synthetase of Micrococcus lysodeikticus and pumpkin. J. biochem., 78, 1177-1181 (1975) [18] Sagami, H.; Morita, Y.; Ogura, K.: Purification and properties of geranylgeranyl-diphosphate synthase from bovine brain. J. Biol. Chem., 269, 2056120566 (1994) [19] Ohnuma, S.-i.; Hirooka, K.; Hemmi, H.; Ishida, C.; Ohto, C.; Nishino, T.: Conversion of product specificity of archaebacterial geranylgeranyl-diphosphate synthase. Identification of essential amino acid residues for chain length determination of prenyltransferase reaction. J. Biol. Chem., 271, 18831-18837 (1996) [20] Ohnuma, S.-i.; Hemmi, H.; Koyama, T.; Ogura, K.; Nishino, T.: Recognition of allylic substrates in Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase: analysis using mutated enzymes and artificial allylic substrates. J. Biochem., 123, 1036-1040 (1998) [21] Ohnuma, S.-I.; Hirooka, K.; Ohto, C.; Nishino, T.: Conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase. Two amino acids before the first aspartate-rich motif solely determine eukaryotic farnesyl diphosphate synthase activity. J. Biol. Chem., 272, 5192-5198 (1997) [22] Ohto, C.; Nakane, H.; Hemmi, H.; Ohnuma, S.-I.; Obata, S.; Nishino, T.: Overexpression of an archaeal geranylgeranyl diphosphate synthase in Escherichia coli cells. Biosci. Biotechnol. Biochem., 62, 1243-1246 (1998) [23] Hefner, J.; Ketchum, R.E.B.; Croteau, R.: Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for Taxol production. Arch. Biochem. Biophys., 360, 62-74 (1998) [24] Laskaris, G.; van der Heijden, R.; Verpoorte, R.: Purification and partial characterization of geranylgeranyl diphosphate synthase, from Taxus baccata cell cultures. An enzyme that regulates taxane biosynthesis. Plant Sci., 153, 97-105 (2000) [25] Bouvier, F.; Suire, C.; d'Harlingue, A.; Backhaus, R.A.; Camara, B.: Molecular cloning of geranyl diphosphate synthase and compartmentation of monoterpene synthesis in plant cells. Plant J., 24, 241-252 (2000) [26] Szabo, C.M.; Matsumura, Y.; Fukura, S.; Martin, M.B.; Sanders, J.M.; Sengupta, S.; Cieslak, J.A.; Loftus, T.C.; Lea, C.R.; Lee, H.-J.; Koohang, A.; Coates, R.M.; Sagami, H.; Oldfield, E.: Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a potential route to new bone antiresorption and antiparasitic agents. J. Med. Chem., 45, 21852196 (2002)

615


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[27] Burke, C.; Croteau, R.: Geranyl diphosphate synthase from Abies grandis: cDNA isolation, functional expression, and characterization. Arch. Biochem. Biophys., 405, 130-136 (2002) [28] Burke, C.; Croteau, R.: Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate. J. Biol. Chem., 277, 3141-3149 (2002) [29] Takaya, A.; Zhang, Y.-W.; Asawatreratanakul, K.; Wititsuwannakul, D.; Wititsuwannakul, R.; Takahashi, S.; Koyama, T.: Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis. Biochim. Biophys. Acta, 1625, 214220 (2003) [30] Kawasaki, T.; Hamano, Y.; Kuzuyama, T.; Itoh, N.; Seto, H.; Dairi, T.: Interconversion of the product specificity of type I eubacterial farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase through one amino acid substitution. J. Biochem., 133, 83-91 (2003)

616

trans-Hexaprenyltranstransferase

2.5.1.30

1 Nomenclature EC number 2.5.1.30 Systematic name all-trans-hexaprenyl-diphosphate:isopentenyl-diphosphate stransferase

hexaprenyltran-

Recommended name trans-hexaprenyltranstransferase Synonyms HepPP synthase [10] HepPS [9] all-trans-heptaprenyl-diphosphate synthase heptaprenyl diphosphate synthase heptaprenyl pyrophosphate synthase heptaprenyl pyrophosphate synthetase synthase, heptaprenyl pyrophosphate CAS registry number 74506-59-5

2 Source Organism no activity in Escherichia coli [4] Bacillus subtilis (ISW1214 [10]; PCI-219 [1-3]) [1-3, 6, 7, 9, 10] Bacillus subtilis (operon gerC [4,5]) [4, 5] Bacillus stearothermophilus (HepPS gene cluster [8]) [6, 8]

3 Reaction and Specificity Catalyzed reaction all-trans-hexaprenyl diphosphate + isopentenyl diphosphate = diphosphate + all-trans-heptaprenyl diphosphate ( Ile76 is essential for termination of chain elongation, mechnism [8]; catalytic mechanism [5]) Reaction type alkenyl group transfer condensation [2,4-9]

617


2.5.1.30

Natural substrates and products S (E,E)-farnesyl diphosphate + isopentenyl diphosphate ( part of the biosynthesis of the side chain of menaquinone-7 [4,5]; successive condensation of 4 molecules of isopentenyl diphosphate with farnesyl diphosphate as primer [2,4,5,8]) [2-10] P all-trans-heptaprenyl diphosphate + diphosphate Substrates and products S (E,E)-farnesyl diphosphate + 3-ethylbut-3-enyl diphosphate (Reversibility: ir [9,10]) [9, 10] P (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate + diphosphate [9, 10] S (E,E)-farnesyl diphosphate + 3-propylbut-3-enyl diphosphate (Reversibility: ir [10]) [10] P (all-E)-3-propyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate + diphosphate [10] S (E,E)-farnesyl diphosphate + but-3-enyl diphosphate ( but-3-enyl diphosphate condenses only once with farnesyl phosphate [9,10]) (Reversibility: ir [9,10]) [9, 10] P (E)-norgeranylgeranyl diphosphate + diphosphate [9, 10] S (E,E)-farnesyl diphosphate + isopentenyl diphosphate ( isoleucine 76 is essential for termination of chain elongation [8]; recombinant enzyme, consisting of individually expressed components I and II [5]) (Reversibility: ir [9,10]; ? [1-8]) [1-10] P all-trans-heptaprenyl diphosphate + diphosphate [1, 2, 4-10] S (E,E,E)-geranylgeranyl diphosphate + isopentenyl diphosphate ( recombinant enzyme, consisting of individually expressed components I and II [5]) (Reversibility: ? [1,2,8]) [1, 2, 8] P all-trans-heptaprenyl diphosphate + diphosphate [1, 2, 8] S Additional information ( no activity with 3-butylbut-3-enyl diphosphate and norgeranylgeranyl diphosphate [10]; no activity with norfarnesyl diphosphate [9,10]; product chain length with primers (E,E)-farnesyl diphosphate and (E,E,E)-geranylgeranyl diphosphate, wildtype and mutants, overview [8]; recombinant enzyme, consisting of individually expressed components I and II, no activity with short-chain allylic diphosphates, dimethylallyl diphosphate and geranyl diphosphate [5]; no activity with isopentenyl diphosphate + dimethylallyl diphosphate or geranyl diphosphate [1,2]; elimination of 2-pro-R hydrogen of isopentenyl diphosphate without accumulation of any prenyl diphosphate shorter than C-35 [1]) [1, 2, 5, 8-10] P ? Metals, ions Mg2+ ( 1 mM, required, recombinant enzyme, consisting of individually expressed components I and II [5]; required [1,2,5,10]) [1, 2, 5, 8, 10]

618

2.5.1.30


Mn2+ ( can replace Mg2+ at 2 mM, recombinant enzyme, consisting of individually expressed components I and II [5]; cannot replace Mg2+ [1]; one-third as effective as Mg2+ [2]) [2, 5] Additional information ( not susceptible to monovalent cations or detergents [2]) [2] Specific activity (U/mg) 0.000002 ( wild-type enzyme and mutants A79Y and S80F [8]) [8] 0.000003 ( mutant I76G [8]) [8] Additional information [1] Km-Value (mM) 0.0071 0.0083 0.0085 0.0128 0.0133 0.0167

(farnesyl diphosphate) [5] (geranylgeranyl diphosphate) [1, 2] (geranylgeranyl diphosphate) [5] (isopentenyl diphosphate) [1, 2] (farnesyl diphosphate) [1, 2] (isopentenyl diphosphate) [5]

pH-Optimum 6.5-7 [1, 2] 7.5-9 ( broad optimum, recombinant enzyme, consisting of individually expressed components I and II [5]) [5] 8.5 ( assay at [8]) [8] pH-Range 5.5-8 ( about 50% of activity maximum at pH 5.5 and pH 8.0 [1]) [1] Temperature optimum ( C) 37 ( assay at [1,2,10]) [1, 2, 10] 55 ( assay at [8]) [8] Additional information ( temperature optimum of different hybrids of the 2 components from Bacillus subtilis and Bacillus stearothermophilus [6]) [6]

4 Enzyme Structure Molecular weight 45000 ( gel filtration [1]) [1] 60000 ( complexed recombinant components I and II, gel filtration [6]) [6] 66000 ( recombinant enzyme, components I and II in presence of Mg2+ and farnesyl diphosphate, gel filtration [5]) [5] Subunits dimer ( 1 * 29000, component I + 1 * 36000, component II, recombinant, gel filtration, in absence of substrates, Mg2+ or phosphate [5]; x * 30000, component I + x * 30000, component II, gel filtration [2, 3, 6]) [2, 3, 5, 6] 619


2.5.1.30

Additional information ( three-dimensional structure [8]; component II shows a MW of 36 kDa in SDS-PAGE [6]; formation of a ternary complex of the 2 subunits, and essentially farnesyl diphosphate and Mg2+ representing the catalytically active state of the enzyme [5]; enzyme consists of 2 essential protein components: I and II, either of the components itself has no catalytic activity, active when combined which each other [2-8,10]; resolution of I and II by DEAE-Sephadex chromatography and MW-estimation by gel filtration [3]) [2-8, 10]

5 Isolation/Preparation/Mutation/Application Purification (recombinant His-tagged protein from Escherichia coli [10]; recombinant mutants of component I, from Escherichia coli [7]; partial [1,2]; partial, component I and II, recombinant from Escherichia coli JM109 [6]) [1, 2, 7, 10] (component I and II, recombinant from Escherichia coli JM109 [5]) [5] (partial, component I and II, recombinant from Escherichia coli JM109 [6]) [6] Cloning (overexpression of His-tagged enzyme in Escherichia coli [10]; overexpression of component I mutants in Escherichia coli JM109 [7]; expression of component I and II individually in Escherichia coli K12 strain JM109 [6]) [6, 7, 10] (overexpression of gerC1 and gerC3 in Escherichia coli JM109 [5]; 2 cistrons of the gerC operon, orfs gerC1 and gerC3, encode the 2 subunits of the enzyme, cloning and functional expression in Escherichia coli [4]) [4, 5] (expression of wild-type and mutant enzymes in Escherichia coli JM109 [8]; expression of component I and II individually in Escherichia coli K12 strain JM109 [6]) [6, 8] Engineering A79Y ( site-directed mutagenesis, mutant enzyme yields mainly C20 products instead of C35 product from the wild-type enzyme [8]) [8] D27H ( natural mutant of gerC1, no effect on catalytic activity, but slightly altered stability of component I [4]) [4] D73A ( site-directed mutagenesis, slightly increased activity, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] D97A ( site-directed mutagenesis, yields shorter chain prenyl diphosphates as products, increased activity, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] E128V ( site-directed mutagenesis, slightly increased activity, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] 620

2.5.1.30


E91D ( natural mutant of gerC3, no effect on catalytic activity, but slightly altered stability of component II [4]) [4] I76G ( site-directed mutagenesis, mutant can catalyse condensations of isopentenyl diphosphate beyond the native chain length of C35 [8]) [8] K130I ( site-directed mutagenesis, only very slightly decreased Km values for isopentenyl diphosphate [7]) [7] K245E ( natural mutant of gerC3, no effect on catalytic activity, but slightly altered stability of component II [4]) [4] L102S ( site-directed mutagenesis, slightly increased activity, only slightly altered Km values for isopentenyl diphosphate in complex with wildtype component II [7]) [7] L92V ( natural mutant of gerC3, no effect on catalytic activity, but slightly altered stability of component II [4]) [4] L94S ( site-directed mutagenesis, residue is located in component I B-region, highly reduced activity, 7fold increased Km for farnesyl diphosphate in complex with wild-type component II [7]) [7] N127A ( site-directed mutagenesis, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] N26K ( natural mutant of gerC1, no effect on catalytic activity, but slightly altered stability of component I [4]) [4] Q294E ( natural mutant of gerC3, no effect on catalytic activity, but slightly altered stability of component II [4]) [4] S100G ( site-directed mutagenesis, reduced activity, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] S80F ( site-directed mutagenesis, mutant enzyme yields mainly C20 products instead of C35 product from the wild-type enzyme [8]) [8] T76V ( site-directed mutagenesis, slightly reduced activity, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] V93G ( site-directed mutagenesis, residue is located in component I B-region, highly reduced activity, 10fold increased Km for farnesyl diphosphate in complex with wild-type component II [7]) [7] Y103S ( site-directed mutagenesis, final products with C40 prenyl chain length, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] Y104S ( site-directed mutagenesis, residue is located in component I B-region, reduced activity, 3fold increased Km for farnesyl diphosphate in complex with wild-type component II [7]) [7] l107S ( site-directed mutagenesis, slightly reduced activity, only very slightly altered Km values for the substrates farnesyl diphosphate and isopentenyl diphosphate in complex with wild-type component II [7]) [7] Additional information ( no activity of hybrids formed between a component of the enzyme with the component A or B of hexaprenyl diphosphate synthase from Micrococcus luteus [6]; construction of several 621


2.5.1.30

hybrids between 2 of the components of the medium-chain length prenyl diphosphate synthases of Bacillus subtilis and Bacillus stearothermophilus, the only active hybrid is the combination of component I from Bacillus subtilis with component II from Bacillus stearothermophilus [6]) [6]

6 Stability Temperature stability 50 ( 5 min, almost complete inactivation of component II, 30 min, no loss of component I activity [2,3]) [2, 3] Additional information ( heat stability of different hybrids of the 2 components from Bacillus subtilis and Bacillus stearothermophilus [6]) [6] Storage stability , 0 C, complete loss of activity after 1 week [1, 2]

References [1] Takahashi, I.; Ogura, K.; Seto, S.: Heptaprenyl pyrophosphate synthetase from Bacillus subtilis. J. Biol. Chem., 255, 4539-4543 (1980) [2] Sagami, I.; Fujii, H.; Koyama, T.; Ogura, K.: Heptaprenylpyrophosphate synthetase from Bacillus subtilis. Methods Enzymol., 110, 199-205 (1985) [3] Fujii, H.; Koyama, T.; Ogura, K.: Essential protein factors for polyprenyl pyrophosphate synthetases. Separation of heptaprenyl pyrophosphate synthetase into two components. FEBS Lett., 161, 257-260 (1983) [4] Zhang, Y.-W.; Koyama, T.; Ogura, K.: Two cistrons of the gerC operon of Bacillus subtilis encode the two subunits of heptaprenyl diphosphate synthase. J. Bacteriol., 179, 1417-1419 (1997) [5] Zhang, Y.W.; Koyama, T.; Marecak, D.M.; Prestwich, G.D.; Maki, Y.; Ogura, K.: Two subunits of heptaprenyl diphosphate synthase of Bacillus subtilis form a catalytically active complex. Biochemistry, 37, 13411-13420 (1998) [6] Koike-Takeshita, A.; Koyama, T.; Ogura, K.: Intersubunit structure within heterodimers of medium-chain prenyl diphosphate synthases. Formation of a hybrid-type heptaprenyl diphosphate synthase. J. Biochem., 124, 790797 (1998) [7] Zhang, Y.W.; Li, X.Y.; Sugawara, H.; Koyama, T.: Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase. Biochemistry, 38, 14638-14643 (1999) [8] Hirooka, K.; Ohnuma, S.-I.; Koike-Takeshita, A.; Koyama, T.; Nishino, T.: Mechanism of product chain length determination for heptaprenyl diphosphate synthase from Bacillus stearothermophilus. Eur. J. Biochem., 267, 4520-4528 (2000) [9] Nagaki, M.; Kimura, K.; Kimura, H.; Maki, Y.; Goto, E.; Nishino, T.; Koyama, T.: Artificial substrates of medium-chain elongating enzymes, hexaprenyl-

622

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and heptaprenyl diphosphate synthases. Bioorg. Med. Chem. Lett., 11, 2157-2159 (2001) [10] Nagaki, M.; Kuwahara, K.; Kimura, K.; Kawakami, J.; Maki, Y.; Ito, S.; Morita, N.; Nishino, T.; Koyama, T.: Substrate specificities of medium-prenylchain elongating enzymes, hexaprenyl- and heptaprenyl diphosphate synthases. J. Mol. Catal. B, 22, 97-103 (2003)

623

Class 2 Transferases VI

Class 2 Transferases: EC 2.7.11.17-2.8 (Springer Handbook of Enzymes)

Class 2 Transferases V (Springer Handbook of Enzymes)

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Генрих VI (Часть 2)

Class 2 Transferases III: EC 2.3.1.60 - 2.3.3.15 (Springer Handbook of Enzymes)

PzKpfw. VI Tiger vol.2

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Paulys Realencyclopädie der classischen Altertumswissenschaft: neue Bearbeitung, Bd.6 2 : Euxantios - Fornaces: Bd VI, Hbd VI,2

Kilo Class

Class Mothers

Class Struggle

Class Warfare

After Class

The Class

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Class 2 Transferases VI

Class 2 Transferases: EC 2.7.11.17-2.8 (Springer Handbook of Enzymes)

Class 2 Transferases V (Springer Handbook of Enzymes)

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Bomb Queen VI #2

Генрих VI (Часть 2)

Class 2 Transferases III: EC 2.3.1.60 - 2.3.3.15 (Springer Handbook of Enzymes)

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Ombres, tome VI : Le Crâne 2

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FASTES VI

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Capital & Class, June 2011, 35 (2)

Happy House: Class Book Level 2

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Volume VI

Писмо VI

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Paulys Realencyclopadie der classischen Altertumswissenschaft: neue Bearbeitung, Bd.6A 2 : Timon - Tribus: Bd VI A, Hbd VI A,2

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