The Enzymes. Volume XIII: Oxidation-Reduction. Part C: Dehydrogenases (II), Oxidases (II), Hydrogen Peroxide Cleavage. Third Edition

The Enzymes VOLUME XI11 OXIDATION-REDUCTION Part C DEHYDROGENASES (II) OXIDASES (II) HYDROGEN PEROXIDE CLEAVAGE Third ...

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The Enzymes VOLUME XI11

OXIDATION-REDUCTION Part C DEHYDROGENASES (II) OXIDASES (II) HYDROGEN PEROXIDE CLEAVAGE Third Edition

CONTRIBUTORS WINSLOW S. CAUGHEY

GREGORY R. SCHONBAUM

BRITTON CHANCE

DIANA L. STIGGALL

L. ERNSTER

JOHN A. VOLPE

J. IEUAN HARRIS

YOUSSEF HATEFI

WILLIAM J. WALLACE MICHAEL WATERS

J. B. HOEK

CHARLES H. WILLIAMS, JR.

J. RYDSTROM

TAKASHI YONETANI SHINYA YOSHIKAWA

ADVISORY BOARD BRITTON CHANCE BO MALMSTROM LARS ERNSTER VINCENT MASSEY

THE ENZYMES Edited by PAUL D. BOYER Molecular Biology Institute and Department of Chemistry University of California Los Angeles, California

Volume XI11 OXIDATIOWREDUCTION Part C DEHYDROGENASES (II) OXIDASES (II) HYDROGEN PEROXIDE CLEAVAGE

THIRD EDITION

ACADEMIC PRESS New York San Francisco London 1976 A Subeidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 6 1976, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN A N Y FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR A N Y INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York. New' York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l

Library of Congress Cataloging in Publication Data

Main entry under title: The Enzymes. Includes bibliographical references. CONTENTS: v. 1. Structure and control.-v. 2. netics and mechanism.-v. 3. Hydrolysis: peptide bonds. (etc.] 1. Enzymes. I. Boyer,PaulD.,ed. 1. Enzymes. QU135 B791eJ [DNLM: QP601.E523 574.1 '925 75-117107 ISBN 0-12-122713-8

PRINTED IN THE UNITED STATES OF AMERICA

Ki-

Contents . . . . . . . . . . . . . . . Preface . . . . . . . . . . . . . . .. . . Contents of Other Volumes . . . . . . . . . . . . .

List of Contributors

1.

vii ix

xi

Glyceraldehyde-3-phosphate Dehydrogenase

J. IEUANHARRISAND MICHAEL WATERS

.

I. Introduction . 11. Molecular Properties 111. Catalytic Properties

2.

. . . . . . . . . . . . . 1 . . . . . . . . . . . . , 3 . . . . . . . . . . . . . as

Nicotinamide Nucleotide Transhydrogenases

J. RYDSTRBM, J. B. HOEK,AND L. ERNSTER

.

.

. . .

.

. . . . . . . .

I. Definitions . . . . , . . . . . 11. BBSpecific Transhydrogenases . . . . . . . 111. ABSpecific Transhydrogenases . . . . . , . IV. Physiological Roles of Nicotinamide Nucleotide Transhydrogenases

3.

. .

51 52 62

79

Flavin-Containing Dehydrogenases

CHARLES H. WILLIAMS, JR.

.

I. Introduction . . . . . . . 11. Pyridine Nucleotide-Disulfide Oxidoreductases 111. Lipoamide Dehydrogenase . . . . IV. Glutathione Reductaae . . . . . V. Thioredoxin Reductase . . . . . VI. Microsomal Electron Transport VII. NADH-Cytochrome b. Reductase . . . VIII. NADPH-Cytochrome P-450 Reductase . .

.

.

4.

. . . . . . . . . . .

. . . . . . . .

. . . . .

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

. . . . . . .

90 92 106 129 142 148

154 185

Metal-Containing Flavoprotein Dehydrogenases

YOUSSEF HATEFIAND DIANA L. STIGGALL I. Introduction . . . 11. NADH Dehydrogenases

. . . . . . . . . . . . . . . . . . . . . . . . V

175

177

vi

CONTENTS

. .

. . . . . . . . . . . . 222 . . . . . 256 . . 280 . . . . . . . . . . . . 263 . . 273 . . 279 . . 286 . . . . . . . . . . . . . . . . 295

I11 Succinate Dehydrogenases IV . ~-Glycerol-3-phosphate Dehydrogenase (EC 1.1.995) V Choline Dehydrogenase (EC 1.1.99.1) . . . . . . . VI . Lactate Dehydrogenases VII . Nitrite Reductases (EC 1.6.6.4) . . . . . . . . . VIII . Adenylyl Sulfate Reductases (EC 1.8.99.2) . . . . . . IX . Sulfite Reductases (H&:NADPH Oxidoreductases) (EC 1.8.12) X . Addendum

5

.

Cytochrome c Oxidare

WINSLOW S. CAUGHEY. WILLIAMJ . WALLACE. JOHNA . VOLPE.AND SHINYA YOSHIKAWA

I . Introduction . . . . . I1 Isolation and Characterization I11. Chemical and Physical Properties IV Mechanisms

. .

.

6

. . . . . . . . . .

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

299

305 313

337

Cytochrome c Peroxidare

TAKASHI YONETANI I. Introduction . . . . . . . I1. Preparation and Molecular Properties I11. Structural Aspects IV . Enzymic Activity V. Reaction Mechanism VI . Interaction with Cytochrome c VII . General Comments . . . . .

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

.

7

. . . . .

. . . . . . .

. . . . . . .

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. . . . . .

. . . . .

. . . . .

. 363 . 366 . 369 . 388 . 409

345 347 348 352 353 356 300

Catalase

GREGORY R . SCHONBAUM AND BRITTON CHANCE I . Introduction . . . . . . I1. General Enzyme Properties . . I11. The Nature of the Active Site . IV . Catalase-Mediated Redox Reactions Author Index

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

. . . .

. . . . . . . . . . . . . . . . 435 Topical Subject Index for Volumes I-XIII . . . . . . . . . . 459

Subject Index

List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

WINSLOW S. CAUGHEY (299), Department of Biochemistry, Colorado State University, Fort Collins, Colorado BRITTON CHANCE (3631, Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

L. ERNSTER (51),Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, Stockholm, Sweden J. IEUAN HARRIS (1), Medical Research Council Laboratory of Molecular Biology, University Postgraduate Medical School, Cambridge, England YOUSSEF HATEFI (175),Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California

J. B. HOEK (51),Department of Biochemistry, University of Nairobi, Nairobi, Kenya J . RYDSTROM (51),Department of Biochemistry, Arrhenius Laboratory, University of Stockholm, Stockholm, Sweden GREGORY R. SCHONBAUM (363), Department of Biochemistry, St. Jude Children’s Research Hospital, and University of Tennessee Center for the Health Sciences, Memphis, Tennessee DIANA L. STIGGALL (175), Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California JOHN A. VOLPE (299), Department of Biochemistry, Colorado State University, Fort Collins, Colorado WILLIAM J. WALLACE (299), Department of Biochemistry, Colorado State University, Fort Collins, Colorado MICHAEL WATERS (1),Department of Biochemistry, Monash University, Clayton, Victoria, Australia vii

viii

LIST OF CONTRIBUTORS

CHARLES H. WILLIAMS, JR. (89), Veterans Administration Hospital, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan TAKASHI YONETANI (346), Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania SHINYA YOSHIKAWA (299), Department of Biochemistry, Colorado State University, Fort Collins, Colorado

Preface This is the final volume of the Third Edition of “The Enzymes.” It completes the coverage of oxidation-reduction enzymes. As with previous volumes, the quality and quantity of information a t the molecular level in this volume are impressive. The first portion of the volume includes the remaining chapters on the nicotinamide nucleotidelinked dehydrogenases, namely, the transhydrogenases and the very important glyceraldehyde-3-phosphate dehydrogenase. The second portion completes the treatment of the great family of flavin-containing enzymes, with chapters on the flavoprotein dehydrogenases and the metalloflavoprotein dehydrogenases. The last portion includes chapters on catalase and peroxidase that use hydrogen peroxide, and on cytochrome oxidase, the enzyme responsible for most of the oxygen consumption by animals. The Third Edition has proved considerably longer and contains much more information than was thought likely when the edition was launched. The privilege of editing the treatise has given me a deep respect for the collective accomplishments of the many scientists whose continued efforts have made such a treatise possible. The quality and abundance of information found in this edition are a tribute to the individual research worker, often little recognized, and to the society that has made such a work possible. I know of no finer recognition of man’s potentiality and creativity than has been my fortune to experience in editing this multivolume treatise. Again, it is a pleasure to acknowledge the indebtedness of the users of these volumes to the Advisory Board that helped plan each volume, to the contributors for their unusually high level of excellence, to the staff of Academic Press for their high professional standards, and to Lyda Boyer, whose editorial and other assistance made many tasks lighter. PAULD. BOYER ix

This Page Intentionally Left Blank

Contents of Other Volumes Volume I: Structure and Control

X-Ray Crystallography and Enzyme Structure David E isenberg Chemical Modification by Active-Site-Directed Reagents Elliott Shaw Chemical Modification as a Probe of Structure and Function Louis A. Cohen Multienzyme Complexes Lester J. Reed and David J . Cox Genetic Probes of Enzyme Structure Milton J . Scklesinger Evolution o f Enzymes Emil L. Smith The Molecular Basis for Enzyme Regulation D. E. Koshland, Jr. Mechanisms of Enzyme Regulation in Metabolism E. R. Stadtman Enzymes as Control Elements in Metabolic Regulation Daniel E. Atkinson Author Index-Subject

Index xi

xii

CONTENTS OF OTHER VOLUMES

Volume II: Kinetics and Mechanism

Steady State Kinetics W . W . Cleland Rapid Reactions and Transient States Gordon B. Hammes and Paul R . Schimmel Stereospecificity of Enzymic Reactions G. Popjhk Proximity Effects and Enzyme Catalysis Thomas C . Bruice Enzymology of Proton Abstraction and Transfer Reactions Irwin A. Rose Kinetic Isotope Effects in Enzymic Reactions J . H . Richards Schiff Base Intermediates in Enzyme Catalysis Esmond E. Snell and Samuel J. DiMari Some Physical Probes of Enzyme Structure in Solution Serge N . Timasheff Metals in Enzyme Catalysis Albert S. Mildvan Author Index-Subj ect Index

Volume 111: Hydrolysis: Peptide Bonds

Carboxypeptidase A Jean A. Hartsuck and William N . Lipscomb Carboxypeptidase B J . E . Folk Leucine Aminopeptidase and Other N-Terminal Exopeptidases Robert J . DeLange and Emil L. Smith Pepsin Joseph S. Fruton


Chymotrypsinogen : X-Ray Structure J . Kraut The Structure of Chymotrypsin I?.M . Blow Chymotrypsin-Chemical George P . Hess

Properties and Catalysis

Trypsin B. Keil Thrombin and Prothrombin Staflan Magnusson Pancreatic Elastase B. S. Hartley and D. M . Shotton Protein Proteinase Inhibitors-Molecular Aspects Michael Laskowski, Jr., and Robert W . Sealock Cathepsins and Kinin-Forming and -Destroying Enzymes Lowell M . Greenbaum Papain, X-Ray Structure J . Drenth, J. N . Jansonius, R. Koekoek, and B. G. Wolthers Papain and Other Plant Sulfhydryl Proteolytic Enzymes A . N . Glazer and Emil L. Smith Subtilisin: X-Ray Structure J . Kraut Subtilisins : Primary Structure, Chemical and Physical Properties Francis S. Markland, Jr., and Emil L. Smith Streptococcal Proteinase Teh-Yung Liu and S. D.Elliott The Collagenases Sam Seifter and Elvin Harper Clostripain William M . Mitchell and William F. Harrington

xiii

xiv


Other Bacterial, Mold, and Yeast Proteases Hiroshi Matsubara and Joseph Feder Author Index-Subject

Index

Volume IV: Hydrolysis: Other C N Bonds, Phosphate Esters

Ureases F. J . Reithel Penicillinase and Other p-Lactamases Nathan Citri Purine, Purine Nucleoside, Purine Nucleotide Aminohydrolases C . L. Zielke and C. H . Suelter Glutaminase and 7-Glutamyltransferases Standish C . Hartman L-Asparaginase

John C. Wriston, Jr. Enzymology of Pyrrolidone Carboxylic Acid Marian Orlowski and Alton Meister Staphylococcal Nuclease X-Ray Structure F. Albert Cotton and Edward E . Hazen, Jr. Staphylococcal Nuclease, Chemical Properties and Catalysis Christian B. Anfinsen, Pedro Cuatrecasas, and Hiroshi Taniuchi Microbial Ribonucleases with Special Reference to RNases TI, T,,N1, and Uz Tsuneko Uchida and Fuji0 Egami Bacterial Deoxyribonucleases I. R. Lehman Spleen Acid Deoxyribonuclease Giorgio Bernardi Deoxyribonuclease I M . Laskowski, Sr.


Venom Exonuclease M . Laskowski, Sr. Spleen Acid Exonuclease Albert0 Bernardi and Giorgio Bernardi Nucleotide Phosphomonoesterases George I . Drummond and Masanobu Yamamoto Nucleoside Cyclic Phosphate Diesterases George I . Drummond and Masanobu Yamamoto

E. coli Alkaline Phosphatase Ted W . Reid and Irwin B. Wilson Mammalian Alkaline Phosphatases H . N . Fernley Acid Phosphatases Vincent P. Hollander Inorganic Pyrophosphatase of Escherichia coli John Josse and Simon C. K. Wong Yeast and Other Inorganic Pyrophosphatases Larry G. Butler Glucose-6-Phosphatase, Hydrolytic and Synthetic Activities Robert C. Nordlie

Fructose-1,6-Diphosphatases 8.Pontremoli and B . L. Horecker Bovine Pancreatic Ribonuclease Frederic M . Richards and Harold W . Wyckoff Author Index-Subj ect Index

Volume V: Hydrolysis (Sulfate Esters, Carboxyl Esters, Glycosides) , Hydration

The Hydrolysis of Sulfate Esters A. B. Roy

xv

xvi


Arylsulf atases R. G. Nicholls and A. B. Roy Carboxylic Ester Hydrolases Klaus Krkch Phospholipases Donald J . Hanahan Acetylcholinesterase Harry C. Froede and Irwin B . Wilson Plant and Animal Amylases John A. Thoma, Joseph E . Spradlin, and Stephen Dygert Glycogen and Starch Debranching Enzymes E. Y . C. Lee and W . J. Whelan Bacterial and Mold Amylases Toshio Takagi, Hirolco Toda, and Toshizo Isemura Cellulases D. R. Whitaker Yeast and Neurospora Invertases J . Oliver Lampen Hy aluronidases Karl Meyer Neuraminidases Alfred Gottschallc and A. S. Bhargava Phage Lysozyme and Other Lytic Enzymes Akira Tszlgita Aconitase Jenny Pickworth Glusker p-Hydroxydecanoyl Thioester Dehydrase Konrad Bloch Dehydration in Nucleotide-Linked Deoxysugar Synthesis L. Glaser and H.Zarkowslcy


xvii

Dehydrations Requiring Vitamin B,, Coenzyme Robert H. Abeles Enolase Finn Wold Fumarase and Crotonase Robert L. Hill and John W . Teipel 6-Phosphogluconic and Related Dehydrases W . A. Wood Carbonic Anhydrase S. Lindslcog, L. E. Henderson, K . K . Kannan, A. Liljas, P. 0. Nyman, and B. Strandberg Author IndexSubject Index

Volume VI: Carboxylation and Decarboxylation ( Nonoxidative), lromerization

Pyruvate Carboxylase Michael C.Scrutton and Murray R. Young Acyl-CoA Carboxylases Alfred W . Alberts and P. Roy Vagelos Transcarboxylase Harland G.Wood Formation of Oxalacetate by CO, Fixation on Phosphoenolpyruvate Merton F. Utter and Harold M . Kolenbrander

Ribulose-l,5-DiphosphateCarboxylase Marvin I. Siegel, Marcia WGhnick, and M . Daniel Lane Ferredoxin-Linked Carboxylation Reactions Bob B. Buchanan Amino Acid Decarboxylases Elizabeth A. Boeker and Esmond E. Snell Actoacetate Decarboxylase Irwin Fridovich

xviii


Aldose-Ketose Isomerases Ernst A . Noltmann Epimerases Luis Glaser Cis-Trans Isomerization Stanley Seltzer Phosphomutases W. J. Ray, Jr., and E. J . Peck, Jr. Amino Acid Racemases and Epimerases E lija h Adams Coenzyme Bl,-Dependent Mutases Causing Carbon Chain Rearrangements H . A . Barker Blz Coenzyme-Dependent Amino Group Migrations Thressa C . Stadtman Isopentenylpyrophosphate Isomerase P . W . Holloway Isomerization in the Visual Cycle Joram Heller A6-3-KetosteroidIsomerase Paul Talalay and Ann M . Bemon Author Index-Subject Index

Volume VII: Elimination and Addition, Aldol Cleavage and condensation, Other C C Cleavage, Phorphorolysir, Hydrolysis (Fats, Glycoriderl

Tryptophan Synthetase Charles Yanojsky and Irving P . Crawjord Pyridoxal-Linked Elimination and Replacement Reactions Leodis Davis and David E. Metzler The Enzymatic Elimination of Ammonia Kenneth R . Hanson and Evelyn A . Havir

CONTENTS OF OTHW VOLUMES

Argininosuccinases and Adenylosuccinases Sarah Ratner Epoxidases William B. Jakoby and Thorsten A. Fjellstedt Aldolases B. L. Horecker, Orestes Tsolas, and C. Y.Lai Transaldolase Orestes Tsolas and B. L. Horecker

2-Keto-3-deoxy-6-phosphogluconicand Related Aldolaseo W. A. Wood Other Deoxy Sugar Aldolases David Sidney Feingold and Patricia Ann Hoflee 8-Aminolevulinic Acid Dehydratase David Shemin 8-Aminolevulinic Acid Synthetase Peter M . Jordan and David Shemin Citrate Cleavage and Related Enzymes Leonard B. Spector Thiolase Ulrich Gehring and Feodor Lynen Acyl-CoA Ligases Malcolm J. P. Higgim, Jack A. Kornblatt, and Harry Rudney a-Glucan Phosphorylases-Chemical and Physical Basis of Catalysis and Regulation Donald J . Graves and Jerry H . Wang Purine Nucleoside Phosphorylase R. E. Parks, Jr., and R. P. Agarwal Disaccharide Phosphorylases John J . Mieyal and Robert H. Abeles Polynucleotide Phosphorylase T . Godejroy-Colburn and M . Grunberg-Manago

xix

xx


The Lipases P. Desnuelle p-Galactosidase Kurt Wallenfels and Rudolf Wed Vertebrate Lysozymes Taiji Imoto, L. N. Johnson, A . C. T. North, D. C. Phillips, and J . A . Rupley Author Index-Subject Index

Volume VIII: Group Transfer, Part A: Nucleotidyl Transfer, Nucleoridyl Transfer, Acyl Transfer, Phosphoryl Transfer

Adenylyl Transfer Reactions E. R. Stadtman Uridine Diphosphoryl Glucose Pyrophosphorylase Richard L. T u r n p h t and R. Gaurth Hansen Adenosine Diphosphoryl Glucose Pyrophosphorylase Jack Preiss The Adenosyltransferases S. Harvey Mudd Acyl Group Transfer (Acyl Carrier Protein) P. Roy Vagelos Chemical Basis of Biological Phosphoryl Transfer S. J. Benkovic and K . J . Schray Phosphofructokinase David P. Bloxham and Henry A . L a d y Adenylate Kinase L. Noda Nucleoside Diphosphokinases R. E. Parks, Jr., and R. P. Agarwal


xxi

3-Phosphoglycerate Kinase R. K. Scope Pyruvate Kinase F. J. Kayne Creatine Kinase (Adenosine 5’-Triphosphate-Creatine Phosphotransferase) D.c. w a t t s Arginine Kinase and Other Invertebrate Guanidino Kinases J . F. Morrison Glycerol and Glycerate Kinases Jeremy W. Thorner and Henry Paulus Microbial Aspartokinases Paolo Truffa-Bachi Protein Kinases Donal A . Walsh and Edwin G. Krebs Author Index-Subject

Index

Volume IX: Group Transfer, Part B: Phosphoryl Transfer, One-Carbon Group Transfer, Glycosyl Transfer, Amino Group Transfer, Other Transferaser

The Hexokinases Sidney P. Colowick Nucleoside and Nucleotide Kinases Elizabeth P. Anderson Carbamate Kinase L. Raijman and M . E . Jones N5-Methyltetrahydrofolate-HomocysteineMethyltransferases Robert T . Taylor and Herbert Weissbach

Enzymic Methylation of Natural Polynucleotides Sylvia J. Kerr and Ernest Borelc Folate Coenzyme-Mediated Transfer of One-Carbon Groups Jeanne I. Rader and F. M . Huennekens

xxii


Aspartate Transcarbamylases Gary R. Jacobson and George R. Stark Glycogen Synthesis from UDPG W . Stalmam and H , G. Hers Lactose Synthetase Kurt E. Ebner Amino Group Transfer Alexander E. Braumtein Coenzyme A Transferases W . P. Jencks Amidinotransferages James B. Walker

N-Acetylglutamate-5-Phosphotransferase Giza De'nes Author I n d e x a u b j e c t Index

Volume X: Protein Synthesis, DNA Synthesis and Repair, RNA Synthesis, Energy-Linked ATPases, Synthetases

Polypeptide Chain Initiation Severo Ochoa and Rajarshi Mazumder Protein Synthesis-Peptide Chain Elongation Jean Lucus-Lenard and Laszlo Beres Polypeptide Chain Termination W . P. Tate and C. T . Caskey Bacterial DNA Polymerases Thomas Kornberg and Arthur Kornberg Terminal Deoxynucleatidyl Transferase F . J . Bollum Eucaryotic DNA Polymerases Lawrence A. Loeb

RNA Tumor Virus DNA Polymerases Howard M . Temin and Satoshi Mizutani


DNA Joining Enzymes (Ligases) I. R. Lehman Eucaryotic RNA Polymerases Pierre Chambon Bacterial DNA-Dependent RNA Polymerase Michael J . Chamberlin Mitochondria1 and Chloroplast ATPases Harvey S.Penefsky Bacterial Membrane ATPase Adolph Abrams and Jeffrey B. Smith Sarcoplasmic Membrane ATPases Wilhelm Hasselbach Fatty Acyl-CoA Synthetases John C. Londesborough and Leslie T . Webster, Jr. Aminoacyl-tRNA Synthetases Dieter Sol1 and Paul R. Schimmel C T P Synthetase and Related Enzymes D. E. Koshland, Jr., and A. Levitzki Asparagine Synthesis Alton Meister Succinyl-CoA Synthetase William A. Bridger PhosphoribosylpyrophosphateSynthetase and Related Pyrophosphokinases Robert L. Switzer Phosphoenolpyruvate Synthetase and Pyruvate, Phosphate Dikinase R. A. Cooper and H . L.Komberg Sulfation Linked to ATP Cleavage Harr y D . Peck, J r . Glutathione Synthesis A1ton Meis ter Glutamine Synthetase of Mammals Alton Meister

xxiii

xxiv


The Glutamine Synthetase of Escherichia coli: Structure and Control E . R. Stadtman and A. Ginsburg Author Index-Subject

Index

Volume XI: Oxidation-Reduction, Transfer (1)

Part A: Dehydrogenases (II , Electron

Kinetics and Mechanism of Nicotinamide-Nucleotide-Linked Dehydrogenases Keith Dalziel Evolutionary and Structural Relationships among Dehydrogenases Michael G. Rossmann, Anders Liljas, Carl-Ivar Brandin, and Leonard J . Banaszak Alcohol Dehydrogenases Carl-Ivar Brand&, Hans Jornvall, Hans Eklund, and Bo Furugren Lactate Dehydrogenase J. John Holbrook, Anders Liljas, Steven J. Steindel, and Michael G. Rossmann Glutamate Dehydrogenases Emil L. Smith, Brian M . Awten, Kenneth M . Blumenthal, and Joseph F. Nyc Malate Dehydrogenases Leonard J . Banaszak and Ralph A . Bradshaw Cytochromes c Richard E . Dickerson and Russell Timkovich Type b Cytochromes Bunji Hagihara, Nobuhiro Sato, and Tateo Yamanaka Author Index-Subject Index Volume XII: Oxidatiorr-Reduction, Part B: Electron Transfer ( I l l , Oxygenases, Oxidares (I1

Iron-Sulfur Proteins Graham Palmer


Flavodoxins and Electron-Transferring Flavoproteins Stephen G. Mayhew and Martha L. Ludwig Oxygenases : Dioxygenases Osamu Hayaishi, Mitsuhiro Nozaki, and Mitchel T. Abbott Flavin and Pteridine Monooxygenases Vincent Massey and Peter Hemmerich Iron- and Copper-Containing Monooxygenases V . Ullrich and W . Duppel Molybdenum Iron-Sulfur Hydroxylases and Related Enzymes R. C. Bray Flavoprotein Oxidases Harold J . Bright and David J . T. Porter Copper-Containing Oxidases and Superoxide Dismutase B. G. Malmstrom, L.-E. Andrdasson, and B. Reinhammar Author Index-Subj ect Index

xxv


Dehydrogenase J. IEUAN HARRIS

MICHAEL WATERS

I. Introduction . . . . . . . . . . 11. Molecular Properties . . . . . . . A. Isolation . . . . . . . . B. Enryme Structure . . . . . . 111. Catalytic Properties . . . . . . . A. Studies of Pyridine Nucleotide Binding B. Mechanism of Action of GAPDH . . C. Metabolic Role of GAPDH . . . .

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1

3 3 5 28 28 38

45

1. Introduction ( 1 )

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes reversibly the oxidation and phosphorylation of D-glyceraldehyde 3-phosphate (G-3P) to 1,3-diphosphoglycerate (DPGA) according to the following reaction scheme:

ocoPo~*-

CHO HAOH AHaOPOa2-

+ HPO,*- + NAD+

~

HAOH

+ H+ + NADH

LHaOPO2-

It is thus a key enzyme in the glycolytic conversion of glucose to pyruvic 1. Abbreviations used are as follows : GAPDH, GlyceraldehydeSphosphate dehydrogenase ; G 3 P , glyceraldehgde 3-phosphate ; DPGA, 1,3-diphosphoglyceric acid; LDH, lactate dehydrogenase; MDH, mdic dehydrogenase ; and ADH, alcohol dehydrogenase . 1

2

J. IEUAN HARRIS AND MICHAEL WATERS

acid which represents an important pathway of carbohydrate metabolism in most organisms. That the oxidation of G-3P was associated with the coupled phosphorylation of adenine nucleotides was originally established by Meyerhof ( l a ) and by Needham and Pillai ( 2 ) . Meanwhile the eventual isolation of the participating enzyme was prompted by the earlier observations of Lundsgaard (3)and of Green et al. ( 4 ) on the inhibition of glycolysis and of alcoholic fermentation by halogenacetic acids, and by the subsequent work of Rapkine ( 6 ) ,associating this inhibition with sulfhydryl groups of GAPDH. The precise nature of the enzymic reaction was elucidated by Warburg and Christian (6) when they succeeded in preparing GAPDH in pure crystalline form from yeast. Subsequently, isolation of the crystalline enzyme from rabbit skeletal muscle was described by Dixon and Caputto (7) and by Cori et al. (8),and in retroTABLE I

SOURCESOF PUREGAPDH’s Source

Ref.

Rabbit muscle Yeast Cat, dog, pig muscle Rabbit, ox, human, chicken, turkey, pheasant, halibut, sturgeon, lobster muscle E . coli B . stearothermophilus T . aquaticus B . cereus Coelacanth muscle Cold-adapted Antarctic fish muscle Insects Rat muscle Kangaroo muscle Pea seed Photosynthetic plants

7, 8 10

14 16

16 17,18 19

20 dl 22 93

24 25

26 d7,28

la. 0. Meyerhof, Naturwissenschaften 25, 443 (1937). 2. D. M. Needham and P. Pillai, Nature (London) 140,65 (1937). 3. E. Lundsgaard, Bwchern. Z. 217, 162 (1930). 4. D. E. Green, D. M. Needham, and J. D. Dewan, BJ 31, 2327 (1937). 5. L. Rapkine, BJ 32, 1729 (1938). 6. 0. Warburg and W. Christian, Biochem. Z.303,40 (1939). 7. R. Caputto and M. Dixon, Nature (London) 156, 630 (1945). 8. G. T. Cori, M. W. Slein, and C. F. Cori, JBC 159,565 (1945).

1. GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

3

spect there can be little doubt that K. Bailey’s “albumin” from rabbit muscle (9)“exhibiting a pronounced sheen upon agitation’’ was in fact GAPDH. Glyceraldehyde-3-phosphate dehydrogenase occurs widely and abundantly throughout nature. It comprises about 20% of the total soluble protein in yeast (10) and up to 10% of the soluble protein from muscle (8),and the relative ease of its preparation from a wide variety of different species has contributed to its popularity among enzymologists, protein chemists, and X-ray crystallographers (cf. 11). Moreover, study of the active enzyme-NAD complex has been facilitated by the fact that uniquely among NAD-linked enzymes crystalline muscle GAPDH contains firm bound NAD. Detailed reviews of these early studies have been given by Velick and Furfine ( l a ) and by Colowick et al. (IS). II. Molecular Properties

A. ISOLATION Pure crystalline GAPDH has been isolated from a number of different sources (cf. Table I) (7,8,10,14-28). Methods of purification have relied heavily upon its solubility as the enzyme-NAD complex in high concen9. K.Bailey, Nature (London) 145, 934 (1940). 10. E. G. Krebs, G. W. Rafter, and J. M. Junge, JBC 200, 479 (1953). 11. J. I. Harris, in “Structure and Function of Oxidation-Reduction Enzymes” (A. Akeson and A. Ehrenberg, eds.), p. 639.Pergam n, Oxford, 1972. 12. S. F.Velick and C. Furfine, “The Enzymes,” pol. 7, p. 243,1963. 13. S.P.Colowick, J. Van Eys, and J. H. Park, Compr. Biochem. 14,l (1966). 14. P. Elodi and E. SzorGnyi, Acta Phgsiol. 9, 339 (1956). 15. W.S.Allison and N. 0. Kaplan, JBC 239,2140 (1964). 16. G. D’Alessio and J. Josse, JBC 246, 4319 (1971). 17. R. E. Amelunxen, BBA 122, 175 (1966). 18. K. Suzuki and J. I. Harris, FEBS (Fed. Eur. Biochem. Soc.) Lett. 13, 217 (1971). 19. J. D. Hocking and J. I. Harris, FEBS (Fed. Eur. Biochem. Soc.) Lett. 34, 280 (1973). 20. K. Suzuki and K. Imahori, J . Biochem. (Tokyo) 73,97 (1973). 21. E. Kolb and J. I. Harris, BJ 130, 26P (1971). 22. F. C.Greene and R. E. Feeney, BBA 220,430 (1970). 23. C. W.Carlson and R .W. Rrosemer, Biochemistry 10, 2113 (1971). 24. N. K.Nagradova and M. K. Guseva, Biokhimiya 36, 496 (1971). 25. R. J. Simpson and B. E. Davidson, Aust. J. Biol. Sci. 24, 263 (1971). 26. R. G.Duggleby and D. T. Dennis, JBC 249, 162 (1974). 27. W.Hood and N. G. Carr, BBA 146, 309 (1967). 28. B. A. Melandri, P. Pupillo, and A. Baccarini-Melandri, BBA 220, 178 (1970).

4


trations (up to 70% saturation) of ammonium sulfate so that the pure muscle enzyme can be obtained from a low salt extract of blended muscle by direct crystallization from 65 to 70% ammonium sulfate. Methods for preparing enzyme from bacterial sources such as Escherichia coli (16) and B. stearothermophilus (18) have been improved by the use of chromatography on ion exchangers, while more recently Hocking and Harris (19) have prepared pure enzyme from the thermophiles B. stearotherrnophilw and Thermus aquaticus by means of affinity chromatography on immobilized NAD'. This method of preparation utilizes the strong affinity of the enzyme for suitably immobilized NAD' (it remains bound to NAD-Sephsrose in 0.7 M NaCl and is then eluted from the resin with a pulse of 10 mM NAD') which allows it to be obtained pure and in high yield from relatively crude bacterial extracts as shown in Fig. 1.

FIG.1. Purification of (A) T.aquaticua and (B) B. stearothermophilua GAPDH; SDS-gel electrophoresis (a) before and (b) after NAD-Sepharose (cf. 37).

1. GLYCERALDEHYDE-%PHOSPHATE DEHYDROGENASE

5

B. ENZYME STRUCTURE 1. Primary Structure

A study of the enzyme by chemical methods involving the specific labeling of catalytically active cysteine residues ($9, 30) and the characterization of peptide fragments produced by enzymic cleavage (31) led Harris and Perham to conclude that GAPDH from a given source was composed of subunits comprising approximately 330 amino acid residues corresponding to a molecular weight of 36,000. These results, considered in conjunction with the physicochemical data of Harrington and Karr (32),showed that the active enzyme with a molecular weight of 146,000 was a tetramer and that it was in all probability composed of chemically identical subunits (31). Proof that the subunits are of identical primary structure was obtained by Harris and co-workers when complete amino acid sequences were established for enzyme from lobster muscle (33), pig muscle ( 3 4 ) , and yeast (36).Comparison of the three sequences (Table 11) shows that they are strictly homologous. Moreover, 60% of the residues occur in identical sequence in the three species showing that the sequence of GAPDH has been conserved to a much greater extent than the sequence of other comparable enzymes such as, for example, alcohol dehydrogenase ( 3 6 ) .Hocking and Harris (37) have subsequently determined the sequence of GAPDH from the thermophilic bacterium T . aquaticus, and comparison of this sequence with that of the lobster muscle enzyme shows a sequence identity of 50% which is again significantly higher than was found in comparison of bacterial and liver alcohol dehydrogenase (38) or bacterial and muscle triosephosphate isomerase

-

(399) 29. J. I. Harris, B. P. Meriwether, and J. H. Park, Nature (London) 198, 154 (1963). 30. R. N. Perham and J. I. Harris, JMB 7,316 (1963). 31. J. I. Harris and R. N. Perham, JMB 13,876 (1965). 32. W. F. Harrington and G . M. Karr, JMB 13, 885 (1965). 33. B. E. Davidson, M. Sajg6, H. F. Noller, and J. I. Harris, Nature (London) 218, 1181 (1967). 34. J. I. Harris and R. N. Perham, Nature (London) 219, 1025 (1988). 35. G. M. T. Jones and J. I. Harris, FEHS (Fed. Eur. Biochem. Soc.) Lett. 22, 185 (1972). 36. H. Jornvall, Proc. Nat. Acad. Sci. U . S. 70,2295 (1973). 37. J. D. Hocking and J. I. Harris, Ezperientia (1976) (in press); J. D. Hocking Ph.D. Dissertation, University of Cambridge, 1974. 38. J. Bridgen, E. Kolb, and J. I. Harris, FEBS (Fed. Eur. Biochem. Soc.) Lett. 33, 1 (1973). 39. S. Artavanis, Ph.D. Dissertation, University of Cambridge, 1974.

TABLE I1 COMPARISON OF THE AMINO ACIDSEQUENCE OF GAPDH FROM PIOMUSCLE, LOBSTEBMUSCLE,AND YEAST^.^ 10 Asn-Gly -Phe-Gly -Arg - Ile -Gly -Arg-Leu-Val Yeast

Val-Arg-Val-Ala- Ile

Leu-Ser -&g40

Asn-Asp-Pro-Phe Gly -Ala -Gln -Val Pro-Asx-Val -Glx -Val

(Ala

&:

50

- Ile

Asx,Asx,Pro,Phe, Ile

60

Tyr-Asp-Ser -Thr-His -Gly

t

Val-Val-Glu Ser -Thr-Gly -Val -Phe Ile -Val-Glu Ala- Ile - Asp

130

120 Ala-Pro-Met-Phe-Val

Q,

Y

150

160

*C

Ser - Lys-Asp-Met-Thr-Val

Val-Ser -Asn-Ala-Ser-CYS-Thr-Thr-Asn-Cys-Leu-Ala-Pro

Leu 170

180

Glu -Gly -Leu-Met-Thr-Thr-Val -His A l a - Ile Thr-Ala -Thr-Gln-LYSAla -Val (Met-Thr, Thr, Val, His) Ser -Le 200

Thr-Val -Asp-Gly -Pro-Ger

210

220

Ser -Thr-Gly -Ah-Ala-Lys-Ala-Val-Gly -Lys-Val

230

Gly -Lys-Leu-Thr-Gly - M e t - A h -

240

Phe-Arg-Val-Pro-Thr

250

Val -Ser -Val-Val -Asp-Leu-Thr Pro-Asp Val - A s x

Glu -Thr-Thr 260

270

Leu-Gly-Tyr-Thr-GluGLx -a

TABLE I1 (Continued)

Asx Ala

Ser

Leu-Gly -Asp-Ser -His

Ser

310

300

Ser-Trp-Tyr-Asp-Asn-Glu Aax-Asx-Glx Tyr

Thr

Val Asp-Leu Met-Val H i s Met-Ala-Ser-Lys-Glu

4 ~~

From (56). b Sequences not experimentally determined for the yeast chain are given within brackets and in a provisional order that maximizes sequence homology between the yeast and muscle enzymes. c C;s-149 forms part of the active site. a

1. GLYCEBALDEHYDE-%PHOSPHATE

DEHYDROGENASE

9

The amino acid sequence results clearly imply a unique sequence for each of the enzymes examined, and there is no decisive evidence for the existence among GAPDH’s of tissue-specific isozymes that differ in primary sequence despite reports of the occurrence of multiple electrophoretic forms in several different organisms (40, 41). I n no case was it demonstrated that these multiple forms are the products of different genes, and it is entirely possible that electrophoretically different tetramers may have arisen by amide loss [as in the case of muscle aldolases (42)] or through differential binding of NAD (41).

2. X - R a y Structure of Hobenzyme The elucidation of the subunit structure and of the amino acid sequences of the subunits of different GAPDH’s provided the necessary framework for the interpretation of chemical modification studies as well as of X-ray crystallographic studies of the tertiary and quaternary structure of the active enzymeINAD complex. The first X-ray diffraction data for GAPDH were obtained by Watson and Banaszak (43) with crystals of enzyme from lobster muscle. These crystals, which displayed the yellow color that is characteristic of the holoenzyme, were orthorhombic (P2,2,2, space group) with the tetramer as the asymmetric unit. Essentially similar results have also been obtained with enzyme crystals from human muscle (4,46) and from B. stearothermophilus (cf. 18). A more detailed study of the lobster muscle enzyme by Rossmann and co-workers (46-48) led to the computation of the first interpretable high resolution (3 A) structure for GAPDH. The first map (with the tetramer as asymmetric unit) was interpreted by averaging the four chemically equivalent but crystallographically different subunits and, with the aid of the amino acid sequence (33),it then became possible to trace the polypeptide chain within the individual subunits. A coordinate system of P , Q , and R axes 40. H.G. Lebherz and W. J. Rutter, Science 157, 1198 (1967). 41. S. F. Velick in “Pyridine Nucleotide-Dependent Dehydrogenases” (H. Sund, ed.), p. 57. Springer-Verlag, Berlin and New York, 1970. 42. C.F. Midelfort and A. H. Mehler, Proc. Nut. Acad. Sci. U.S . 69, 1816 (1972). 43. H.C.Watson and L. J. Banaszak, Nature (London) 204,918 (1974). 44. A. I. Gorjunov, N. S. Andreeva, T. Baranowski, and M. Wohy, J M B 69, 421 (1972). 45. H.C.Watson, E. Due& and W. D. Mercer, Nature (London) 240, l$O (1972). 46. M. Buehner, G.C. Ford, D. Moras, K. W. Olsen, and M. G. Roasmann, Proc. Nut. Acad. Sci. U.S . 70, 3052 (1973). 47. M. Buehner, G. C. Ford, D. Moras, K. W. Olsen, and M. G. Rossmann, J M B 82, 563 (1974). 48. M. Buehner, G. C. Ford, D. Moras, K. W. Olsen, and M. G. Rossmann, J M B 90, 25 (1974).

10


Q

P

Q

P

FIO.2. Diagrammatic comparison of the association of subunits in GAPDH (left) and LDH (right) (48).

FIo. 3. Stereoviews of the Ca atom backbone in lobster muscle GAPDH: (a) one subunit viewed to illustrate the NAD+-binding and catalytic domains; (b) the NADtbinding domain viewed in the same orientation as in (a); (c) the catalytic

1.

GLYCERALDEHYDE-%PHOSPHATE DEHYDROGENASE

11

similar to that used previously for lactate dehydrogenase (LDH) (49) and malate dehydrogenase (MDH) (60) has been used (Fig. 2) to define the GAPDH structure in order to draw attention to the striking structural similarities that exist between the three dehydrogenases (46, 61). The major feature of the structure is that, although exhibiting apparent 222 symmetry, the tetramer consists functionally of a dimer of dimers related across the Q axis (cf. 46). The only true twofold axis is the Q axis whereas the other axes exhibit pseudosymmetry within the limits

domain viewed in the same orientation as in (a) ; and (d) complete tetramer viewed down the P axis demonstrating the dumbbell silhouette with the four active sites close to the center of the molecule (48, 6.5). 49. M. J. Adams, A. McPherson, Jr., M. G. Rossmann, R. W. Schevitz, and A. J. Wonacott, J M B 51, 31 (1970). 50. E. Hill, D. Tsernoglou, L. Webb, and L. J. Banaszak, J M B 72,577 (1972). 51. M. G. Rossmann, A. Liljas, C.-I. Branden, and J. J. Banaszak, Chapter 2, Volume XI.

12


of resolution obtained. The region of major interaction between subunits is across the P axis; Q-axis-related contacts are relatively few and not highly conserved, while R-axis-related contacts are again more numerous and highly conserved. The conformation of C a backbone atoms in the GAPDH subunit is shown in Fig. 3a. The subunit is envisaged as consisting of two domains (Figs. 3b and 3c), each with a specific function. The first, comprising residues 1-149, is mainly involved in NAD+ binding while the second domain, comprising residues 149-334, provides residues for substrate binding, specificity, and catalysis. The “catalytic” domain also contains most of the residues that are involved in intersubunit contacts. a. The NAD-Binding Domain. The fold of the NAD+-binding domain in GAPDH is shown diagrammatically in Fig. 4. It consists of a sixstranded parallel /3 sheet flanked by helices and is similar to analogous nucleotide binding structures in LDH, MDH, and ADH (cf. 6 1 ) . The NAD+ in each of the four subunits is bound close to the molecular waist (cf. Figs. 2 and Fig. 3d) of the tetramer and close enough to interact via a section of antiparallel sheet (comprising residues 179-200) that extends across the R axis into the adjacent subunit. This intersubunit interaction was thought to link Lys-183 in one subunit to the pyrophosphate

Fra. 4. Diagrammatic representation of the NAD+-binding domain showing the six-stranded parallel p sheet flanked by helices (48,63).

1.

GLYCERALDEHYDE-3-PHOSPHATE

DEHYDROGENASE

13

moiety of NAD+ in the adjacent subunit (46, 48), compatible with earlier chemical evidence implicating Lys-183 in coenzyme binding ( 5 2 ) . A revised structure (@, 53) for this part of the molecule shows, however, that Lys-183 does not interact directly with either NAD+ or substrate. Nevertheless, it remains possible that interactions between other residues in the S-shaped loop (such as, for example, Pro-188 and Trp-193 with NAD+ in the adjacent subunit could be responsible for the cooperativity of NAD+ binding (cf. Section III,A,l) and the NAD+-promoted tetramerization of the dimeric moiety. I n this respect, and as shown in Fig. 2, GAPDH differs from LDH where each molecule of NAD’ is bound entirely within each subunit with little possibility for direct interaction between binding sites within the tetramer. The conformation of the NAD+ in GAPDH is nevertheless similar to that found in LDH. Thus it is bound in an open extended configuration in each of the four subunits but with the important difference of a 180° rotation about the C-1 to N-1 glycosidic bond linking the nicotinamide ring to the ribose. This change ensures that the “B” face of the ring is exposed to the substrate for hydride ion transfer giving GAPDH its B specificity. The B or syn configuration is stabilized by hydrogen bonds formed between the carboxyamide group and the invariant Asn-313 and with the nicotinamide phosphate. It should be noted that the alternative “A” configuration of the ring that occurs in MDH, LDH, and ADH is prohibited in GAPDH due to steric hindrance involving the main chain residues Ala-120 and Pro-121 and the carboxyamide group of the nicotinamide ring. The main chain hydrogen bonding scheme in the NAD+-binding domain is shown in Fig. 5 and the topography of the NAD-binding site itself is shown diagrammatically in Fig. 6. The adenine ring binds between Phe-34 and Phe-99; a t the side of the adenine binding pocket there are hydrophobic residues Pro-33, Met-77, and Pro-79, while the inside of the pocket is more hydrophilic in character owing to the presence of Asn-6 and Asn-31. Aspartate-32 forms a hydrogen bond to the 02’ atom on the adenosine ribose while Gly-7 approaches it closely from one side. The phosphates interact with the part of the chain comprising Gly-9, Arg-10, and Leu-11; Gly-97 and Ala-120 provide a hydrophobic environment for the nicotinamide ribose while, as mentioned previously, the carbonyl group of the nicotinamide forms a hydrogen bond to Asn-313. It should be noted that the residues found to be interacting with NAD’ are highly conserved in different 52. J. H. Park, D. C. Shaw, E. Mathew, and B. P. Meriwether, JBC 245, 2946 (1970). 53. D. Moras, I7.5) and phosphorolysis of the acyl enzyme a t low pH. This is because the rate of phosphorolysis is highly p H dependent, possibly increasing more than 2 X 104-fold from pH 5.4 to 8.6, while the rate of NADH release is independent of pH over this range, with the result that the two converge around pH 7.5. The pH dependence of phosphorolysis (190) may reflect a requirement for the phosphate trianion (PO,3-). At high enzyme concentrations (> 0.1 mg/ml) , the conversion of the predominant gem-diol form of G-3p to its reactive aldehyde form becomes rate limiting (146, 199). This in vitro interconversion does not apply in vivo since the reactive aldehyde form of G-3P is the product of both the aldolase and triosephosphate isomerase reactions, and the hydration of the aldehyde is a slow process. I n the reverse reaction, the rate determining step is a process associated with NADH binding, probably a conformational change, at high pH, and release of G-3P a t low pH (189, 1 9 9 ~ )At . high ionic strengths, acylation becomes rate limiting (83)* With the natural substrate it is now generally agreed that all four sites of the muscle enzyme tetramer are simultaneously active both in the forward and reverse reactions (160, 161, 189) despite earlier claims (169) that only the fourth site turns over. Smith and Velick (194) have undertaken an extensive steady-state kinetic analysis of forward and reverse reactions catalyzed by the liver and muscle enzymes, under pseudophysiological conditions, in a n effort 198. I. Krimsky and E. Racker, Biochemistry 2, 512 (1963). 199. D. R. Trentham, C. H. McMurray, and C. I. Pogson, BJ 114, 19 (1969). 199a. An alternative proposal now favored by Trentham which removes the necessity to postulate an NADH-induced conformational change is that aldehyde release is rate limiting under all conditions of low salt. The precursor for aldehyde release is the NAD+-aldehyde enzyme. A t high pH this complex is in rapid equilibrium with the NADH-acyl enzyme, which is the major species and therefore the predominant steady-atate intermediate. At low pH, however, the rapid equilibrium can favor the aldehyde-apoenzyme complex suggesting that NAD' dissociation from the NAD+-aldehyde enzyme is favored at low pH.

42

J. IEUAN HARRIS AND'MICHAEL WATERS

to understand the factors allowing gluconeogenesis through GAPDH in liver. Although the conclusions with regard to the forward reaction are complicated by product inhibition caused by DPGA, the results obtained for the reverse reductive dephosphorylation reaction provide an explanation for the possible metabolic significance of negative cooperativity in muscle and in liver. At the low DPGA concentrations encountered in vivo, NAD' acts cooperatively to convert the DPGA saturation curve from a sigmoidal to a hyperbolic form, thus sensitizing the enzyme to the lower concentrations of the acyl phosphate. At the same time, NAD+ acts to abolish substrate inhibition by NADH toward nonacylated enzyme sites. At higher concentrations of acyl phosphate, NAD' acts as a weak competitive inhibitor toward NADH, in contrast to the strong competitive inhibition by NADH toward NAD' seen in the forward reaction. The latter observations can be explained by the decrease in binding affinity for NAD' that occurs on acylation (GO),which Smith and Velick (194) suggested is largely the result of an isomerization process (cf. 143).Presumably, because the enzyme is almost totally acylated and because of the high NAD' concentrations, cooperative effectcl are not seen in the forward reaction in vitro; i.e., the enzyme exists in a single conformation. While there is no doubt that bound NAD+ enhances acylation (117) as well as deacylation (7O),it is difficult to reconcile the above-mentioned cooperative effects with the observation (165)that NAD' occupation of one site in the rabbit tetramer did not affect the catalytic activity of the other three sites toward aldehyde substrates. Evidence against a slow isomerization of the T + R variety, which might be expected to affect thiol reactivity in other subunits, is the fact that the sturgeon apoenzyme is almost instantaneously active in acylation, following the addition of NAD' (116). The nature of the NAD+ enhancement of acylation and deacylation is of considerable interest since it has a bearing on the mechanism of enzyme catalysis in general, and also because it explains the NAD' requirement of a number of the minor activities of GPD (see Section III,B,2). Studies with the simple alkylating agents iodoacetate and iodoacetamide have shown that NAD' promotes alkylation of Cys-149 by negatively charged iodoacetate, but inhibits alkylation by the uncharged iodoacetamide molecule (117,196).Since NADH and the adenine nucleotides do not facilitate alkylation by iodoacetate or arsenolysis of acetyl phosphate (200-202), it has been suggested that the positively charged pyridinium ring facilitates attack by negatively charged alkylating or 200. S. H. Francis, B. P. Meriwether, and J. H. Park, JBC 246, 5427 (1971). J. H. Park, JBC 248, 5433 (1971).

201. S. H. Francis, B. P. Meriwether, and 202. A. Feneslau, JBC 245, 1239 (1970).

1. GLYCERALDEHYDE-3-PHOSPHATE

DEHYDROGENASE

43

deacylating agents through ion pair formation. This "ion pair" concept is supported by the inhibitory effect of high ionic strength on both alkylation by iodoacetate and acylation by acetyl phosphate, which presumably occurs because the positive charge is masked by interaction with anions (83, 122). Cseke and Boross (122,203) have shown that the PKa of Racker band absorbance and of thiol anion carboxymethylation is lowered from about 8 in the apoenzyme to around 5.5 (204) in the presence of NAD'. This has been taken as evidence that NAD' has lowered the pK, of Cys-149 (117,203), especially since the pK, of carboxymethylation and of Racker band absorbance vary together, depending on the nature of the solvent anion. If the nucleophilicity of the thiol anion is insensitive to its basicity, as the evidence suggests, then this lowered thiol group dissociation induced by NAD' would explain the thiol group reactivity a t lower pH (< 7.0). It cannot explain the fact that the reactivity is only exhibited toward iodoacetate and not iodoacetamide, since reactivity of the thiol group should be identical toward the two alkylating agents. Consequently, it is still necessary to postulate ion pair formation in the presence of NAD'. Whatever the nature of this effect it is manifest a t low ratios of NAD+:enzyme (< 1 mole/mole) which suggests migration of NAD' from alkylated sites to nonalkylated sites induced by the lowered affinity of NAD' for alkylated subunits (117, 202). While the scheme outlined above accounts for the NAD+-dependent activation of thiol-149, it does not account for the fact that Cys-149 is highly activated compared to simple aliphatic thiols even in the absence of NAD'. It therefore becomes necessary to postulate the presence of a basic group to activate the thiol group by hydrogen bonding to its proton in a manner similar to that found with papain and thiol-subtilisin, where histidine is the basic group (83, 205, 206'). The effect of NAD' on the pK, of the thiol could then be explained by postulating that NAD' binding alters the conformational alignment of the thiol and basic residues in order to draw a proton further away from the thiol group. The inhibitory effect of anions and the anion-induced variation in the pK, of the thiol base proton would then be the result of interference in both base thiol and pyridinium thiol interactions (122).It has been suggested that a strongly basic group adjacent to the thiol base pair is responsible for anion binding and that the bound anion creates an electron-rich region which in turn increases the pK, of the thiol base pair (122, 20od). The 203. E. Cseke and L. Boross, Acta Biochim. Biophys. 2, 39 (1967). 204. M.T.A. Behme and E. H. Cordes, JBC 242,5500 (1967). 205. G. Loae, Phil. Trans. Roy. Snc. London, Ser. B 257, 237 (1970). 206. L. Polghr, FEBS (Fed. Eur. Biochem. Soc.) L e t t . 38, 187 (1974).

44


H
O.lmM

12

100 100 100

100 100 100

100 100

None None

None None

12 44

None None None

>0.25mM

From Hatefi and Stempel (40). Per mole of flavin, this activity is considerably higher in complex I than in the soluble, low molecular weight dehydrogenase. c A t 0.15 mM NADH; V ~ ~ ~ ( C= N685. ’6 d This activity results from the presence in complex I of 0 . 5 1 % complex I11 contamination.

0

Y

9 z

tl

ii ;s

i% e3

198

YOUSSEF HATEFI AND DIANA L. STIGGALL

given. According to these investigators, a considerable amount of flavin can be removed from the enzyme by treatment with Florisil or Bio-Gel. The depleted enzyme retains its ferricyanide reductase activity, but loses considerable activity for reduction of dichloroindophenol and cytochrome c. The latter is partially restored by addition of large amounts of FMN. The authors concluded from these data that reducing equivalents from NADH first go to the iron-sulfur moiety of the enzyme, then to flavin. Ferricyanide accepts electrons from the iron-sulfur moiety, but indophenol, quinones, and cytochrome c are reduced a t the flavin site. I n agreement with this conclusion they have shown that chromatography of the enzyme on DEAE-cellulose at pH 6.8 results in nearly complete removal of flavin and labile sulfide, and about two-thirds of the iron. This preparation had no reductase activity with any of the acceptors, even when assayed in the presence of added FMN. The above mechanistic conclusions are not generally accepted, however, because (a) the preparation of Kumar et al. has very low reductase activities (probably related to its low content of iron and labile sulfide) , (b) the cytochrome c reductase activity restored by addition of FMN is only about 1% of the maximal cytochrome c reductase activity of the more active preparations of the enzyme (do),and ( c ) no attempt was made to reconstitute the ironsulfur moiety of the DEAE-cellulose-treated enzyme by treatment with sulfide and ferrous ions to see whether ferricyanide reductase activity can be restored. It has been stated by Yang (74)that the partial loss of ferricyanide reductase activity (and 450 nm absorption) of the preparation of Kumar et al. upon aging in air a t room temperature could be restored to a considerable extent by treatment of the enzyme with 2mercaptoethanol, FeCl,, and Na,S followed by filtration through a column of Sephadex G-25. However, the validity of this type of reconstitution experiment rests on several important controls which were not presented. 4. Relevance of the Low and High Molecular Weight Preparations

to Mitochondria1 NADH Dehydrogenase The ubiquinone reductase activity of their low molecular weight dehydrogenase led Pharo et al. (64, 75) to conclude that the enzyme represented the mitochondria1 NADH-ubiquinone reductase. However, it has been shown that the quinone reductase activity of the low molecular weight dehydrogenase is different from that of intact respiratory particles or complex I in many important respects, including kinetic constants, re74. C. S. Yang, in “Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.), p. 664. Univ. Park Press, Baltimore, Maryland, 1971. 75. D. R. Sanadi, R. L. Pharo, and L. A. Sordahl, in “Non-Heme Iron Proteins” (A. San Pietro, ed.), p. 429. Antioch Press, Yellow Springs, Ohio, 1966.

4.

METAL-CONTAINING FLAVOPROTEIN DEHYDROGENASES

199

mM DPNH

FIG. 11. Effect of NADH concentration on the ferricyanide reductase activities of complex I and the soluble, low molecular weight dehydrogenase. From Hatefi and Stempel (40).

sponse to inhibition by Amytal, rotenone, and piericidin A, and the apparent involvement of several EPR-active iron-sulfur centers. As compared to respiratory particles or complex I, the soluble enzyme exhibits low ferricyanide reductase activity per mole of flavin and very high reductase activities with respect to menadione, 2,6-dichloroindophenol, or cytochrome c as electron acceptor. The latter activity, in contrast to that found in submitochondrial particles or complex 1-111, is insensitive to inhibition by rotenone, piericidin A, or antimycin A, and 4 s marked by a very high K , for cytochrome c (600 p.M versus 12 p M in the case of complex 1-111) (Tables VII and VIII; also see 40). In addition, the ferricyanide reductase activity of complex I is sharply inhibited at NADH concentrations above 0.1 mM whereas the activity of the soluble dehydrogenase is not (Fig. 11). Conversely, the former activity is not inhibited by mercurials, but the latter is. It has also been shown that the ubiquinone reductase activity of particles is 70% inhibited by 50 m M guanidineHCl, whereas the same activity catalyzed by the low molecular weight dehydrogenase is 75% activated (68, 76). The cytochrome c reductase activity of the soluble enzyme was first discovered by Mahler and co-workers (5‘7); hence, the designation “Mahler’s DPNH-cytochrome c reductase.” However, these investigators had recognized the unphysiological nature of this activity, and Mahler and Glenn (7’7) pointed 76. Y. Hatefi, PTOC.Nat. Acad. Sci. U . S. 60,733 (1968). 77. H. R. Mahler and J. L. Glenn, in “Inorganic Nitrogen Metabolism” (W. D. McElroy and B. Glass, eds.), p. 575. Johns Hopkins Press, Baltimore, Maryland, 1956.

200


out that the cytochrome c reductase activity of their dehydrogenase might be because cytochrome c behaves as a single electron acceptor similar to, but not identical with, the physiological electron acceptor in the respiratory chain. This early prediction is interesting since a single electron accepting iron-sulfur structure is very likely the natural acceptor for the membrane-bound dehydrogenase. It should also be mentioned that interaction with cytochrome c is not a peculiar property of the above enzyme. A number of flavoproteins containing FMN or FAD as prosthetic group and utilizing NADH or NADPH as electron donor are known which interact with cytochrome c (29, 46). Examples of this unphysiological phenomenon are in the case of Old Yellow Enzyme (78) and Straub’s diaphorase (lipoyl dehydrogenase) (79). It is clear from the above section that the differences between the enzymic properties of the soluble, low molecular weight NADH dehydrogenase and the particulate system represented by complex I are very large, even though the former is obviously a component of the latter enzyme system. Singer and his colleagues believe that the low molecular weight enzyme is a “peptide fragment” of the high molecular weight NADH dehydrogenase (16, 19). While in essence the parent-progeny relationship is obvious, the vigorously espoused views concerning peptide fragment and the equivalence of the high molecular weight preparation to mitochondrial NADH dehydrogenase are no longer acceptable. It has been pointed out that the small molecular weight dehydrogenase is not likely to be a peptide fragment because the procedures leading to its isolation are not likely to cleave peptide bonds (69, 80). Furthermore, as will be seen below, complex I and the corresponding section of respiratory particles catalyze the dehydrogenation of NADPH without the intermediation of NAD and the transhydrogenase reaction. Studies on complcx I with NADH and NADPH as substrates have shown that flavin and iron-sulfur center 1 are reduced by NADH, but apparently not by NADPH. Therefore, there appears to exist in complex I a segment, containing flavin and a portion of the total iron and labile sulfide, which is specific for dehydrogenation of NADH. It is highly probable that the small molecular weight NADH dehydrogenase represents this segment of complex I, except that conversion from membrane-bound to soluble state has modified its enzymic properties, some of which (e.g., wide acceptor specificity) might be simply the result of better access of acceptors to the soluble enzyme. Much has been written by Singer and his colleagucs in defense of thc 78. A. Akeaon, A. Ehrenberg, and H. Theorell, “The Enzymes,” 2nd ed., Voi. 7, p. 477,1963.

79. V. Massey, BBA 30, 205 (1958). 80. Y. Hate6 and W. G. Hanstein, Biochemistry 12,3515 (1973).

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thesis that their preparation is the true NADH dehydrogenase of the respiratory chain (14, 15, 19, 2 3 ) . They have done extensive studies on their preparation and have compared its composition, enzymic, and E P R properties to complex I and the low molecular weight dehydrogenase preparations. They have pointed out quite correctly that theirs is the first preparation of a NADH dehydrogenase which displays a high ferricyanide reductase activity comparable on the basis of NADH dehydrogenase flavin to the activity of complex I and intact respiratory chain preparations. By comparison, the low molecular weight NADH dehydrogenases have very low ferricyanide reductase activity. However, as compared to complex I, the dehydrogenase of Singer and co-workers appears to have several important differences. The comparison to complex I as a reliable point of reference is valid since (a) the above dehydrogenase resembles complex T in its size, ferricyanide reductase activity, and content of nonheme iron, and ( b ) complex I appears to be the smallest segment yet isolated which displays all the catalytic and inhibitor-response properties of the NADH-ubiquinone reductase portion of intact respiratory chains, including the important ability of conserving oxidative energy and reacting with mitochondria1 coupling factors to synthesize ATP ( 3 5 ) .The differences are as follows: a. The NADH dehydrogenase of Singer and co-workers is incapable of reducing ubiquinone, which is the physiological electron acceptor for complex I. This inability has been referred to the fact that the dehydrogenase is essentially devoid of lipids. That phospholipids are necessary for ubiquinone reduction by complex I has been demonstrated by Ragan and Racker (36 ) as discussed above. However, the latter investigators were able to restore ubiquinone reductase activity by adding phospholipids to deficient complex I preparations. I n the absence of such activity restoration experiments, the view that the preparation of Singer e t al. has not sustained irreversible damage during isolation would remain an assumption. According to Ragan and Racker ( 3 6 ) ,special reducing conditions are needed during the removal of lipids from complex I in order to preserve the ability of the complex to exhibit ubiquinone reductasc activity upon readdition of lipids. It remains to be seen whether the dehydrogenase of Singer et al. can be isolated under similar reducing conditions with preservation of ubiquinone reductase activity when assayed in the presence of added lipids. b. As stated above the ratio of flavin to iron to labile sulfide is 1:16-18:16-18 for complex I. This ratio is stated to be 1:17-18:27-28 for the NADH dehydrogenase. The molar excess of labile sulfide as compared to iron is surprising and contrary to literature data for all species of iron-sulfur proteins known. However, this high labile sulfide value

202


might have resulted from the low extinction coefficient (21,000 liters mole-' cm-l) used by the authors (81) for labile sulfide determination by the method of Fog0 and Popowsky (82). A more correct molar extinction coefficient is between 27,500 and 30,000, which-when applied to the labile sulfide value published for NADH dehydrogenase-would lower it to about 20, a value in much better agreement with the iron content of the preparation. c. According to Watari et al. (83) and Singer and Cremona ( I d ) , the K , for NADH of their preparation is 108 p M . This value is more than 15-fold greater than the K , of complex I for NADH (7 p i l l ) determined similarly in the NADH-ferricyanide reductase assays (Table VIII) . This difference is rather serious because the high K,,, value is characteristic of the low molecular weight NADH dehydrogenase derived from complex I. The K , for NADH of the low molecular weight enzyme, also determined in the ferricyanide reductase assay, is about 65 (Table VII), and as detailed above it is generally agreed that the isolated low molecular weight dehydrogenase shows major differences in catalytic properties as compared to its membrane-bound counterpart. Thus, with respect to its K,,, for substrate, the NADH dehydrogenase of Singer and co-workers is similar to the modified, low molecular weight enzyme, and differs from complex I and other submitochondrial particles. This difference might be associated with structural modifications responsible for the inability of the high molecular weight NADH dehydrogenase to interact with ubiquinone under appropriate conditions. Although it was not recognized as a reaction involving a separate mechanism, the published data of Singer's laboratory show clearly that their preparation also has NADPH dehydrogenase activity (84). Consequently, the high molecular weight NADH dehydrogenase preparations appear to be segments of the respiratory chain related to complex I. These preparations appear to have preserved the ferricyanide reductase activity of the system but irreversibly lost the physiological ubiquinone reductase activity. The argument as to which preparation-the high or the low molecular weight enzyme-represents the respiratory chain NADH dehydrogenase is perhaps irrelevant in view of our present knowledge. Both are clearly derived from complex I. However, the low molecular weight preparation has grossly modified enzymic properties (see also Section II,B), and the high molecular weight preparation appears to have retained the NADH and NADPH dehydrogenase activities of com81. 82. 83. 84.

C. J. Lusty, J. M. Machinist, and T. P. Singer, JBC 240, 1804 (1965). J. K. Fog0 and M. Popowsky, Anal. Chem. 21,732 (1949). H. Watari, E. B. Kearney, and T. P. Singer, JBC 238, 4063 (1963). C. Rossi, T. Cremona, J. M. Machinist, and T. P. Singer, JBC 240, 2634 (1965).

4.

METAL-CONTAINING

FLAVOPROTEIN DEHYDROGENASES

203

plex I, but only with respect to ferricyanide as electron acceptor. These considerations lead, therefore, to the conclusion that proper purification of NADH dehydrogenase beyond the stage of complex I has yet to be achieved.

5. Inhibitors of N A D H Dehydrogenase Thiol reagents, barbiturates, rotenoids, and piericidin A inhibit NADH dehydrogenation and ubiquinone reduction in appropriate preparations. According to Singer and his colleagues (19,23, 85-87) there are five types of --SH groups in various preparations of NADH dehydrogenase. Type I is the -SH group found in small molecular weight NADH dehydrogenase, and is apparently involved in mercurial inhibition of its reductase activities. According to Kumar et al. ( 6 3 ) , NADH treatment increases the mercurial sensitivity of the enzyme with respect to ferricyanide and cytochrome c, but not dichloroindophenol, reduction. Hatefi et al. (68) have shown that (a) incubation of the enzymes with 1-2 pM pCMS results in activation by as much as 20-300/0, while higher concentrations inhibit, and (b) contrary to thc results of others (37) N-ethylmaleimide does not cause inhibition even after partial inactivation by heat a t pH 4.8 or in the presence of 3 M urea. In contrast to the low molecular weight enzyme, the ferricyanide reductase activity of the high molecular weight dehydrogenase or complex I is not inhibited by mercurials, suggesting that type I -SH groups are not accessible to mercurials in these preparations. The type I1 -SH group appears to be peculiar to the high molecular weight dehydrogenase of Singer et al. At Oo it reacts rapidly and reversibly with -SH reagents without inhibiting ferricyanide reductase activity, but incubation a t 15O-3Oo results in gradual and irreversible inactivation. The temperature effect suggests structural destabilization and recalls the temperature dependence of the resolution of complex I by chaotropic agents. Davis and Hatefi (69) have shown that in the presence of moderate concentrations of NaC104 the resolution of complex I does not occur at temperatures below 15O (Fig. 5 ) . Type I11 -SH groups are found in particles and high molecular weight preparations. This type of -SH group, which was discovered by Tyler et al. (48), reacts with mercurials and results in inhibition of electron transport only after the preparation is pretreated with NADH. The conditioning by NADH is reversible inasmuch as addition of ferricyanide to the NADH-treated enzyme reverts it to the mercurial insensitive state. This type of -SH 85. T. Cremona and E. B. Kearney, JBC 240,3645 (1965). 86. H. Mersmann, J. Luthy, and T. P. Singer, RBRC 25,43 (1966). 87. M. Gutman, H. Mersmnnn, J . Luthy, and T. P. Singer, Biochemistry 9, 2678 (1970).

204

YOUSSEF H A T E F I AND DIANA L. STIGGALL

group is considered to be located very near the substrate binding site, but possibly not directly involved in electron transport activity, since relative to the turnover number of the enzyme both the NADH conditioning and the ferricyanide reversal are slow processes (23).Type IV -SH groups are also found in particles and high molecular weight preparations. According to Singer and co-workers (19, 23), they react readily with low levels of mercurials, and mercaptide formation with this type of thiol group is considered to affect the ferricyanide reductase reaction by increasing both the K,,, and the V,,, for ferricyanide by severalfold. Type V -SH groups are detectable only in complex I and parent particles because they affect ubiquinone reduction and piericidin binding. Mercaptide formation with this type of -SH group requires relatively high concentrations (30-80 p M ) of mercurials and results in inhibition of electron transport from NADH to ubiquinone, but not to ferricyanide and other acceptors reacting on the substrate side of ubiquinone (Table VIII) . I n submitochondrial particles, mercaptide formation with type V -SH groups also results in the loss of one of two specific binding sites for rotenone and piericidin A, and a sigmoidal to hyperbolic change in the piericidin inhibition titration curves (23, 88). The ubiquinone reductase activity of complex I is inhibited by barbiturates (e.g., Amytal and Seconal), Demerol, rotenone, or piericidin A. These compounds appear to inhibit electron transfer from the iron-sulfur centers of complex I to ubiquinone. Absorption and fluorescence spectroscopic studies on submitochondrial particles had suggested to Chance et al. (89) the existence of two consecutive flavoproteins between NADH and ubiquinone. These authors placed the site of rotenone and Amytal inhibition between the two flavoproteins. Hatefi (76) showed that in complex I there is only one type of flavoprotein, and that the additional bleaching by substrate a t the wavelength pair 475 minus 510 nm used by Chance et al. results from reduction of the iron-sulfur components of complex I. Therefore, the site of Amytal and rotenone inhibition could be between the flavoprotein and an iron-sulfur moiety of complex I. While the conclusion regarding the absence of two consecutive flavoproteins in the complex I region of the respiratory chain was correct and confirmed (go), the interpretation regarding the site of inhibition of rotenone and Amytal was not. It is now generally agreed that the flavin and all the EPR-active iron-sulfur moieties of complex I are located on the substrate side of the inhibition site of Amytal, rotenone, and piericidin A. 88. M. Gutman, T. P. Singer, and J. E. Casida, JBC 245, 1992 (1970). 89. B. Chance, L. Ernster, P. B. Garland, C.-P. Lee, P. A. Light, T. Ohnishi, C. I. Ragan, and D. Wong, Proc. N u t . Acad. Sci. U.S. 57, 1498 (1967). 90. C. I. Ragan and P. B. Garland, BJ 10, 399 (1969).

4. METAL-CONTAINING FLAVOPROTEIN DEHYDROGENASES

205

Palmer et al. (91) have suggested that in addition to the above site, rotenone and piericidin A also inhibit electron transport immediately on the substrate side of cytochrome cl. This view has not been accepted by others. Teeter et al. (9.2) have shown that secondary effects of rotenone and piericidin can be observed a t other regions of the respiratory chain when high concentrations of the inhibitors are used, as by necessity did Palmer et al. in their EPR experiments. The studies of Horgan, Singer, and their colleagues (19, 22, 23, 88, 93, 94) with radioactive piericidin A and rotenone have led these authors to the following conclusions :

(a) There are specific and unspecific binding sites for both rotenone and piericidin A, the latter being reversible by washing of the particles with bovine serum albumin. ( b ) Rotenone (and several other rotenoids), piericidin A, and Amytal bind noncovalently and inhibit a t the same specific binding site in phosphorylating and nonphosphorylating preparations. (c) Piericidin binds more tightly than rotenone, and titration data indicate that 2 moles of piericidin bind with comparable affinity per mole of NADH dehydrogenase in submitochondrial particles. (d) Titration curves relating the degree of NADH oxidase inhibition to inhibitor concentration are sigmoidal, thus indicating that the two binding sites are not equivalent in terms of their contribution to inhibition of electron transport. (e) Unlike submitochondrial particles, the number of binding sites per mole of NADH dehydrogenase in complex I and complex 1-111 is close to unity. Other aspects of rotenone and piericidin inhibition studied by Singer and co-workers are related more to submitochondrial particles than to complex I. These studies have been compiled in reviews (.22, 23) by these investigators and will not be detailed here. As stated above, the low molecular weight NADH dehydrogenase of Pharo et al. (64) was considered incorrectly to be the NADH-ubiquinone reductase of the respiratory chain. This was in part because the ubiquinone reductase activity of the preparation could be partially inhibited by Amytal and by very low concentations of rotenone. It was demonstrated by others that these effects were different from the inhibitions 91. G.Palmer, D.J. Horgan, H. Tisdale, T. P. Singer, and H. Beinert, JBC 243, 844 (1968). 92. M. E. Teeter, M. L. Baginsky, and Y. Hatefi, BBA 172, 331 (1969). 93. D. J. Horgan, T. P. Singer, and J. E. Casida, JBC 243, 834 (1968). 94. D.J. Horgan, H. Ohno, T. P. Singer, and J. E. Casida, JBC 243, 5967 (1968).

206


obtained with complex I or submitochondrial particles (19, 22, $2). The results of Hatefi et al. (42) show the following differences: (a) Substantial inhibition of NADH-ubiquinone-1 reductase activity of the soluble, low molecular weight enzyme requires more than 100-fold as much rotenone as is necessary for a comparable degree of inhibition of complex I. (b) Barbiturates inhibit the ubiquinone reductase activity of the former enzyme only when higher ubiquinone isoprenologs are used as electron acceptor. Unlike the reaction catalyzed by complex I, barbiturates do not inhibit the ubiquinone-1 reductase activity .of the soluble dehydrogenase. (c) Piericidin A, which is the most potent inhibitor known for ubiquinone reduction by complex I and submitochondrial particles, is essentially ineffective on ubiquinone reduction by the soluble enzyme. Among several iron chelators used, only o-phenanthroline inhibited the soluble dehydrogenase (42). It was shown by Hatefi et al. (42) that incubation of the enzyme with o-phenanthroline results in the loss of labile sulfide, while pretreatment with bathophenanthroline sulfonate, Tiron (1,2-dihydroxybenzene 3,5-disulfonate) or ethylenediamine tetraacetate protects the enzyme against the loss of labile sulfide and inhibition of activity upon subsequent incubation with o-phenanthroline. The unique destructive ability of o-phenanthroline has been demonstrated by these investigators for several iron-sulfur proteins (95,96). While in contrast t o complex I the ferricyanide reductase activity of the low molecular weight dehydrogenase is not inhibited at high NADH concentrations (>0.2 mM) , its quinone reductase activities are. Indeed, there seems to be a correlation between NADH inhibition, the apparent Km of the enzyme for NADH, and whether the acceptor is a single-electron (ferricyanide, cytochrome c) or a two-electron (ubiquinones, menadione, and dichloroindophenol) recipient (40). With single-electron acceptors, the apparent K m for NADH is about 65 pM and the reaction is relatively insensitive to the concentration of NADH. However, with two electron acceptors, the apparent K , for NADH is twice as much (133 UJM) and the reaction is sharply inhibited a t NADH concentrations greater than 0.25 mM. Hatefi and Stempel (40) have suggested that these phenomena might be a consequence of the dissimilar affinity for NADH of the various oxidation-reduction states of the enzyme (i.e., oxidized, half-reduced, fully reduced) transiently produced during electron transfer to one-electron versus two-electron acceptors. Millimolar concentrations 95. Y. Hatefi and W. G. Hanstein, ABB 138,73 (1970). 96. R. M. Kaschnitz and Y. Hatefi, ABB 171,292 (1975).

4.


207

of NAD partially inhibit NADH dehydrogenase ( 4 0 ) . A similar inhibition is caused by AMP, ADP, and ATP, but not by adenosine ( 4 2 ) . Guanidinium hydrochloride and alkylguanidinium salts have been implicated as inhibitors of electron transport and oxidative phosphorylation a t site 1 (97, 98). Hatefi et al. (42) have shown that the ubiquinone reductase activity of complex I is inhibited by guanidinium hydrochloride (10-50 mM), but the ferricyanide reductase activity of complex I and all the reductase activities of the low molecular weight dehydrogenase are activated. Guanidinium ion was more potent than the alkylated derivatives] and the activation effect analyzed for menadione reduction indicated a decrease in K , for NADH and an increase in V,,,, both of which were dependent on the concentration (10-100 m M range) of guanidinium ion.

6. N A D P H Oxidation and N A D P H to N A D Tramhydrogenation b y Complex I The ability of submitochondrial particles to catalyze transhydrogenation from NADPH to NAD has been known for many years. The reverse reaction, i.e., from NADH to NADP, is slow but can be accelerated when energy is supplied to the system (e.g., ATP). The energy requirement of the reverse reaction and the different equilibria of the transhydrogenase reaction in the absence and presence of an energy supply are ~ U Z zling thermodynamic problems. The mitochondrial transhydrogenase reaction has been under vigorous investigation (99-102), a recent review is available (103), and the topic is covered in Chapter 2. Until 1973, it was generally agreed that the mitochondrial respiratory chain is incapable of oxidizing NADPH directly (103-105).NADPH oxidation was considered to occur only through the transhydrogenase reaction and with the obligatory intermediation of NAD (103-105). In 1973, 97. J. B. Chappell, JBC 238,410 (1963). 98. B. Chance and G. Hollunger, JBC 238,432 (1963). 99. J. Rydstrom, A. Teixeira da Cruz. and L. Ernster, Eur. J. Biochem. 17, 56 (1970). 100. A. Teixeira da Cruz, J. Rydstrom, and L. Ernster, Eur. J. Biochem. 23, 203 (1971). 101. J. Rydstrom, A. Teixeira da Cruz, and L. Ernster, Eur. J. Biochem. 23, 212 (1971). 102. R.R.Fisher and N. 0. Kaplan, Biochemistry 12, 1182 (1973). 103. N.0.Kaplsn, Harvey Lect. 66, 105 (1972). 104. L. Ernster, C.-P. Lee, and U. B. Torndal, in “The Energy Level and Metabolic Control in Mitochondria” (S. Papa et al., eds.), p. 439. Adriatrica Editrice, Bari, 1969. 105. F. A. Hommes, in “Energy-Linked Functions of Mitochondria” (B. Chance, ed.), p. 39. Academic Press, New York, 1963.

208


(CI

FIQ.12. (A) First-derivative E P R spectra of complex I treated with NADH or NADPH. Conditions: complex I, 45 mg/ml; temperature 14°K; microwave frequency 9.225 GHz; power, 2 mW, modulation amplitude, 6.3 G ; gain, 50. g = 2 was at 3295 G. Where indicated 1.5 mM NADH or NADPH was added. Small letters of the alphabet in this, (B), (C), and Fig. 13 denote the same signals as in Fig. 4. (B) The EPR spectrum of NADH-treated complex I shown in (A) a t gain of 200 and 0.3 mW power. (C) The E P R spectrum of NADPH-treated complex I shown in (A) at gain of 200 and 0.3 mW power. From Hatefi and Hanstein (80).

Hatefi (106-108) and Hatefi and Hanstein (80) demonstrated, however, that NADPH is oxidized directly by the respiratory chain a t a site close to, but apparently not identical with, the site of NADH oxidation. It was found that NADPH oxidation by respiratory particles is very slow a t neutral pH (about 50 nmoles/min/mg protein) , but quite appreciable a t pH values between 5 and 6 ( 2 2 5 0 nmoles/min/mg protein). Indeed, the rate difference between pH 9 and pH 6 was found to be about 35-40-fold, 106. Y.Hatefi, BBRC 50, 978 (1973). 107. Y. Hatefi, i n “Dynamics of Energy Transducing Membranes” (L. Ernster R. W. Estabrook, and E. C. Slater, eds.), p. 125. Elsevier, Amsterdam, 1974. 108. Y.Hatefi, Fed. Proc., Fed. Amer. SOC. Exp. Biol. 32,595 (1973).

4.

m

I 0

r

t

-1

209


G=100

I-

n

s

\/

H-

FIQ. 13. Computer-derived difference of NADH-treated minus NADPH-treated complex I shown in Fig. 12. From Hatefi and Hanstein (80).

an indication of the fact that others working a t neutral or more alkaline pH values had missed (or dismissed as resulting from the presence of traces of NAD) the direct oxidation of NADPH by the respiratory chain. Both NADPH dehydrogenase (assayed with ferricyanide as acceptor) and NADPH-to-NAD transhydrogenase activities of respiratory particles were found to fractionate mainly into complex I. Electron paramagnetic resonance studies on complex I a t neutral pH showed that NADPH reduced iron-sulfur center 2 and partially the overlapping iron-sulfur centers 3 4. Iron-sulfur center 1 was not detectably reduced by NADPH, nor was the flavin of complex I as evidenced from the difference absorption spectra of NADPH-treated minus NADH-treated complex I. In agreement with previous findings described above, all iron-sulfur centers were reduced by NADH. These results are depicted in Figs. 12-14. Sub-

+

-0.0

400

500

600

Wavelength Inm)

FIG.14. Absorption spectrum of NADPH-treated minus NADH-treated complex I. Conditions: complex I, 6 mg protein/ml of 0.66 M sucrose containing 50 mM Tris-HC1 ( p H KO), 1 mM histidine, and 0.25% (v/v) Triton X-100. The sample cuvette was treated with 200 pM NADPH, and the reference cuvette with 100 p M NADH. Dashed line, untreated complex I in both cuvettes. From Hatefi and Hanstein (80).

210


5

6

7

a

9

PH

FIQ.15. pH dependence of NADPH oxidase, NADPH to 3-acetylpyridine adenine dinucleotide (AP-DPN) transhydrogenase, and NADH oxidase activities of submitochondrial particles (ETP), Conditions: oxidase activities were measured in the presence of 2 mM NADH or NADPH, 0.25 121 sucrose, 100 mM sodium phosphate for pH values 6-9, and 100 mM sodium acetate for pH values 5.0 and 5.5. ETP concentration was 2.16 mg/ml for the NADPH oxidase, and 0.216 mg/ml for the NADH oxidase assays. The transhydrogenasc reaction was measured by the AmincoChance spectrophotometer at 400 minus 450 nm. The extinction coefficient used for reduced 3-acetylpyridine adenine dinucleotide a t 400 nm was 2300 liters mole-' cm-'. Media were the same as in the oxidase assays. Dotted lines indicate uncertainty about the pH 5 rates because of possible acidity damage to ETP. The ordinate refers to nanomoles of NADPH or NADH oxidized min-' x mg-' of ETP protein at 30". From Hatefi and Hanstein (80).

sequent studies of Hatefi and Bearden (109) indicated that as compared to NADH reduced complex I, the low field signal due to overlapping centers 3 4 was not only smaller but also a t a position approximately 3 G upfield (corresponding to Ag = 0.002) when it was generated with NADPH as reductant. These results indicated, therefore, that the partial center 3 4 reduction by NADPH might have resulted mainly or entirely from center 3 (see Table IV). Thus, i t appears that a t neutral pH NADPH can reduce those components of complex I (iron-sulfur centers 2 and 3) whose reduction potentials appear to be close to zero, but not those whose reduction potentials are between -300 and -200 mV (flavin and iron-sulfur centers 1 and 4, see Section II,A,7). As stated above, submitochondrial particles and complex I exhibit both NADPH dehydrogenase and NADPH-to-NAD transhydrogenase activities. These activities have similar pH dependencies (Fig. 15) and are

+

+

109. Y . Hatefi and A. J. Bearden, unpublished.

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211

both specific with respect to abstraction of the 4-B hydrogen of NADPH (10'7). The NADH dehydrogenase of the respiratory chain is also 4-B specific, whereas transhydrogenation from NADH to NADP is 4-A specific in agreement with the 4-B specificity of the reverse reaction with regard to NADPH. The similarities between NADPH dehydrogenase and NADPH-to-NAD transhydrogenase have suggested that both reactions might be catalyzed by the same enzyme. They have also created suspicion regarding the noninvolvement of NAD in NADPH dehydrogenation in spite of the differences detailed above in the reduction of respiratory components by NADH and NADPH and the demonstration of the absence of detectable NAD in the experiments involving complex I (submitochondrial particles contain about 0.2 nmole NAD per mg protein) (80). Two lines of evidence have furnished unambiguous evidence, however, that NADPH oxidation by submitochondrial particles and complex I can occur under conditions that the transhydrogenase reaction is completely inhibited. The differences in the reduction of respiratory components with NADH or NADPH as substrate is reflected in the degree of bleaching afforded by these substrates a t 475 minus 510 nm in complex I and rotenoneor piericidin-treated submitochondrial particles. Thus, as seen in Fig. 16 (left-hand trace), addition of NADH doubles the bleaching a t 475 minus 510 nm obtained by addition of NADPH to piericidin-treated particles. The middle trace shows a similar effect when NAD is added instead of NADH. This results from the presence of transhydrogenase activity, which yields NADH from NAD and excess NADPH. I n the right-hand

475.510 nm

#l

i 1 min+

0 CI

>

FIG.16. Effect of palmitoyl-coenzyme A on reduction of chromophores a t 475 minus 510 nm in ETP via NADPH to NAD transhydrogenation. Conditions: ETP, 2.2 mg protein/ml; NADPH, 60 p M ; NADH, 60 p n l ; NAD, 140 pM; sodium succinate, 1.75 m M ; piericidin A , 5.3 pM ; antimycin A, 1 p M ; 2-thenoyltrifluoroacetone (TTFA), 1 m M ; palmitoyl-CoA (P-CoA), 200 pM. From Hatefi and Hanstein (80).

212


trace the preparation has been treated with appropriate amounts of palmitoyl coenzyme-A (110) to inhibit the transhydrogenase reaction. The bleaching by NADPH is seen, the NAD effect resulting from the transhydrogenase reaction is largely, abolished, but subsequent NADH addition is still effective. These results clearly show that NADPH reduction of components as measured a t 475 minus 510 nm can occur under conditions that transhydrogenation to NAD is inhibited. That the latter reaction was substantially inhibited in these experiments is clear because in the presence of a piericidin block even a slow production of NADH would have resulted in bleaching just as NADPH did under conditions of low electron flux (pH 7.5 and NADPH concentration equivalent to 0.1 K,,,). Similar results were reported for complex I (80). I n addition, it has been shown by Djavadi-Ohaniance and Hatefi (111) that trypsin treatment of submitochondrial particles can distinguish among NADH oxidation, NADPH oxidation, and NADPH-to-NAD transhydrogenation. The exceptional sensitivity of the latter reaction to trypsin was demonstrated earlier by Ernster and his colleagues (119). Taking advantage of this sensitivity, the former investigators have shown that treatment of submitochondrial particles a t Oo with appropriate amounts of trypsin can completely destroy the NADPH-to-NAD transhydrogenase activity without affecting appreciably either the NADH or the NADPH oxidase activity (Fig. 17). Incubation of the particles a t 30° in the presence of trypsin then led to gradual loss of NADPH oxidase activity without affecting NADH oxidase activity. These results clearly demonstrate that the three reactions shown in Fig. 17 are independently affected by trypsin: transhydrogenase activity is rapidly destroyed a t Oo, NADPH oxidase is gradually destroyed a t 30°, while NADPH oxidation is unaffected by trypsin under these conditions. An important question that is raised by these experiments is the relationship between the NADPH dehydrogenase and the NADPH-to-NAD transhydrogenase activities of submitochondrial particles. On the one hand, they are clearly distinguishable on the basis of their sensitivities to trypsin and palmitoyl coenzyme-A. On the other hand, they exhibit common features with regard to their stereospecificities for abstraction of the 4-B hydrogen of NADPH, their extremely large response to pH change, and their copurification into complex I. It is entirely possible that the two activities might be catalyzed by the same enzyme because (a) many nicotinamide110. J. Rydstrorn, A. V. Panov, G. Paradies, and L. Ernster, BBRC 45, 1389 (1971). 111. L. Djavadi-Ohaniance and Y . Hatefi, JBC, in press. 112. K.Juntti, U. B. Torndal, and L. Ernster, in “Electron Transport and Energy Conservation” (J. M. Tager et a l , eds.), p. 257. Adriatica Editrice, Bari, 1970.

4.

m


5

(a)

15

25

40

213

55

(b)

FIG. 17. Effect of trypsin on the NADH oxidase, NADPH oxidase and the NADPH-to-NAD transhydrogenase activities of submitochondrial particles. The particles suspended in 0.25 M sucrose and 100 mM sodium phosphate, pH 7.0, were treated with 0.1 mg trypsin per mg particle protein and incubated a t (a) 0" or (b) 30". At the intervals shown samples were removed and assayed at pH 6.0 and 7.0 for the activities shown. Transhydrogenase activity was measured either directly by reduction of 3-acetylpyridine adenine dinucleotide at 375 nm in the presence of cyanide-treated particles or by the increase in the rate of NADPH oxidation by submitochondrial particles after the addition of NAD. (A) NADH + O,, ( 0 ) NADPH + 02,and (0) NADPH + NAD. From Djavadi-Ohaniance and Hatefi (111).

adenine dinucleotide dehydrogenases, including the mitochondrial NADH dehydrogenase, exhibit transhydrogenase activity in the presence of a suitable analog, and (b) the exceptional trypsin sensitivity of the transhydrogenase reaction might be concerned mainly with the NAD ( H ) binding site. For example, it is possible that similar to Rhodospirillum rubrum (113) the mitochondrial transhydrogenase system also involves a soluble protein cofactor. This protein factor or its association with the membranes might be susceptible to the action of trypsin. A more attractive possibility is suggested by the recent work of Vallee and his colleagues (114). They have found that in a number of enzyme-active sites arginyl residues serve as the positively charged recognition sites for negatively charged substrates, and have identified arginyl residues a t the NADH binding site of a number of alcohol dehydrogenases from various sources. A peptide bond involving the carboxyl group of arginine, if present a t the substrate binding site of the mitochondrial transhydrogenase, could be particularly susceptible to attack by trypsin. 113. R. R. Fisher and R. J. Guillory, JBC 244, 1078 (1969). 114. L. G. Lange, 111, J. F. Riordan, and B. L. Vallee, Biochemistry 13, 4361 (1974) .

214


7. Energy Conservation and Coupling by Complex I It has long been known that the first site of energy conservation in the respiratory chain is located between NADH and ubiquinone-cytochrome b. Schatz and Racker (44) demonstrated ATP synthesis by submitochondria1 particles a t the expense of NADH oxidation by externally added ubiquinone-1, and more recently Ragan and Racker (36)reconstituted oxidative phosphorylation a t site 1 in a system composed of complex I and appropriate membrane proteins, coupling factors, and phospholipids. The important experiments of Ragan and Racker showed that in principle the isolated and purified complexes are capable of energy conservation and coupling. The studies of Hatefi, Galante, and You (106, 107, 115) have shown that comparable ATP yields (P/O = 2.62.8) are obtained as a result of oxidation of NADH or NADPH by submitochondrial particles. Since the components of complex I reduced by both NADH and NADPH are iron-sulfur centers 2 and 3 and ubiquinone, the above experiments implicate these electron carriers. in site 1 energy conservation. Gutman and his colleagues (116) have shown that in piericidin-treated submitochondrial particles iron-sulfur center 2 remains reduced after exhaustion of added NADH through the piericidin leak. Iron-sulfur center 2 could be reoxidized by addition of ATP. This reaction was sensitive to uncouplers, and appeared to result from energy-linked reverse electron transfer to NAD. On the basis of these observations, Gutman et al. have concluded that coupling site 1 is located on the oxygen side of iron-sulfur center 1 and the substrate side of both iron-sulfur center 2 and the site of roteaone-piericidin block. These conclusions are in general agreement with the results of Hatefi et al. (80,106-108) regarding the NADPH reducible iron-sulfur centers and P/O > 2 obtained during NADPH oxidation. It is also interesting to note that energy-linked transhydrogenation can be induced by ATP or as a result of succinate oxidation in rotenonetreated particles (117). Further, Van de Stadt et al. (118) and Skulachev (119) have presented data regarding energy production in rotenoneblocked particles as a result of transhydrogenation from NADPH to NAD. Therefore, it appears that energy communication with the transhydrogenase reaction also occurs a t or near site 1. This would indeed 115. Y. Hatefi, Y. Galante, and K. S. You, unpublished. 116. M. Gutman, T. P. Singer, and H. Beinert, Biochemistry 11, 556 (1972). 117. C.-P. Lee, G. F. Azzone, and L. Ernster, Nature (London) 201, 152 (1964). 118. R. J. Van de Stadt, F. J. R. M. Nieuwenhuis, and K. Van Dam, BBA 234, 173 (1971). 119. P. Skulachev, Curr. T o p . Bioenerg. 4, 127 (1971).

4.

METAL-CONTAINING

215

FLAVOPROTEIN DEHYDROGENASES (SDH Fe/S) +3omV

Rotenone NADH t) (Center lo.lb)(Center3,4)-(Center

Halfreduction potentiol

-305mV

-245rnV

-20rnV

2) -(Center

1

5 ) +-+(R~eske’s

+4OrnV

Fe/S)-

O2

+ 280rnV

FIG. 18. Thermodynamic profile of iron-sulfur centers in pigeon heart mitochondria : SDH, succinate dehydrogenase ; Rieske’s Fe/S, iron-sulfur protein of complex 111. From Ohnishi (121).

be plausible if as suggested by their similar enzymic features NADPH oxidation and nicotinamide-adenine dinucleotide transhydrogenation shared through a common enzyme the same linkage to the respiratory chain. Ohnishi et al. (120, 121) have suggested that coupling site 1 involves not only iron-sulfur center 2 but also half of iron-sulfur center 1 (designated center l a ) . Their conclusion is based on an apparent change in the reduction potential of these centers induced by ATP as estimated from the measurement of E P R signals in the presence of redox mediators. Similar criteria were used by Wilson and Dutton (122, 123) to identify cytochromes b, and a3 as energy transducing components a t coupling sites 2 and 3, respectively. However, these experiments have been criticized by Caswell (124) and Lambowitz e t al. (125).They feel that complications resulting from improper equilibration of the redox mediators with the electron carriers under study and ATP-induced reverse electron transfer to and from these components have been the underlying basis of the measured redox potential changes brought about by A T P addition. According t o Ohnishi (121) , the half-reduction potentials of the various iron-sulfur centers of the respiratory chain a t pH 7.2 are as shown in Fig. 18. The value for center 1 is essentially in agreement with the results of Orme-Johnson et al. (46, 54) who found that reduced acetylpyridine adenine dinucleotide (Eo’ = -248 mV) can only reduce this center by 50%, while its oxidized form can effectively oxidize iron-sulfur center 120. T. Ohnishi, D. F. Wilson, and B. Chance, BBRC 49, 1087 (1972). 121. T. Ohnishi, BBA 301, 105 (1973). 122. D. F. Wilson and P. L. Dutton, BBRC 39,59 (1970). 123. D. F. Wilson and P. L. Dutton, A B B 136, 583 (1970). 124. A. H. Caswell, A B B 144,445 (1971). 125. A. M. Lambowitz, W. D. Bonner, Jr., and M. K. F. Wikstrom, Proc. N u t . Acud. Sci. U . S. 71, 1183 (1974).

216


-.I

NADPHNADP

FeS4

FIQ. 19. Proposed electron transfer pathways for oxidation and reduction of NADH/NAD and NADPH/NADP, and energy coupling site 1 in complex I. Where applicable broken arrows indicate energy-linked electron or hydride ion transfer. FeS, iron-sulfur center.

1 in dehydrogenase preparations. These investigators feel, however, that the reduction potential of center 3 is greater than or equal to that of center 2, since upon titration of complex I with graded amounts of NADH or dithionite reduced centers 2 and 3 appear long before centers 4 and 1 are reduced. Thus, it seems that complex I contains two iron-sulfur centers (1 and 4) with reduction potentials close to that of NADH (Eo’= -315 mV), and two centers (2 and 3) with potentials close to that of ubiquinone (Eo’N +65 mV) . Accordingly, the largest single-step energy drop in the NADH pathway is between iron-sulfur centers 4 and 2 + 3 (AE = -225 mV; AGO‘ for 2e = -10,400 cal), and in the NADPH pathway is, so far as known, between NADPH and iron-sulfur centers 2 3 (AE = -295 mV; AGO’ for 2e = -13,600 cal). I n theory, therefore, the energy liberated a t each of these two steps appears to be compatible with the amount required for ATP synthesis a t site 1 with either NADH or NADPH as substrate. The above considerations are summarized in Fig. 19.

+

B. NADH DEHYDROGENASES OF YEAST The NADH dehydrogenase of yeast is of considerable interest because in Saccharomyces cerevisiae and Saccharomgces carlsbergensis coupling site 1 is absent, whereas in Candida utilis its existence depends on the growth phase of the cells and can be altered by adaptations to culture conditions and by catabolite repression. In 1961, Vitols and Linnane (126) reported that mitochondria isolated from S. cerevisiae showed identical P/O values for oxidation of succinate and NAD-linked substrates. These results were the first indication of the absence of coupling site 1 in S. cerevisiae. Mackler et al. (127) and 126. E. Vitols and A. W. Linnane, J. Biophys. Biochem. Cytol. 9, 701 (1961). 127. B. Mackler, P. J. Collipp, H. M. Duncan, N. A. Rao, and F. M. Huennekens, JBC 237, 2968 (1962).

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Mahler and co-workers (128) isolated respiratory particles from bakers’ yeast, and showed that NADH oxidation by these particles was insensitive to inhibition by Amytal, Seconal, and rotenone. The former authors also demonstrated that, unlike mammalian respiratory particles, the flavin of S. cerevisiae particles was exclusively FAD of which approximately 50% was acid-extractable. Extraction of the remainder required prior digestion of the particles with trypsin. Duncan and Mackler (129) isolated a low molecular weight NADH dehydrogenase from these particles by the acid-ethanol procedure (see above). The preparation contained per mg protein 10.6 nmoles of FAD, 6.3 ng atoms of iron, and no labile sulfide. The molecular weight was determined by equilibrium sedimentation to be approximately 55,000. Thus, it appeared that during isolation the enzyme had lost about 40% of its flavin, assuming one mole of FAD per mole of enzyme. The possibility of iron and labile sulfide loss is also suggested by the use of acid pH, prolonged dialysis, and DEAE-cellulose chromatography for isolation and purification of the enzyme. The dehydrogenase could react with ferricyanide, dichloroindophenol, and cytochrome c as electron acceptors, and in all instances added FAD increased the activity and p-mercuriphenyl sulfonate inhibited it. Thus, in terms of size, acceptor specificity, and mercurial sensitivity, the NADH dehydrogenase from S. cerevisiae respiratory particles appears to be comparable to a similar preparation from mammalian mitochondria. The major difference is that the flavin of the latter is FMN. According to Kim and Beattie (130),the appearance of NADH dehydrogenase activity in mitochondria of glucose derepressed S. cerevisiae is blocked by cycloheximide, but not by chloramphenicol, suggesting that NADH dehydrogenase does not contain products of mitochondria1 protein synthesis. The NADH dehydrogenase system of C . utilis is very similar to that of mammalian mitochondria. Both systems are inhibited by rotenone and piericidin A, conserve energy a t coupling site 1, contain multiple forms of EPR-active iron-sulfur center, have F M N as their flavin prosthetic group, react best with ferricyanide as electron acceptor, and are inhibited a t high substrate concentrations (131-133).Biggs et al. (132) were unsuccessful in dissociating a high molecular weight type enzyme from C. utilis 128. H. R. Mahler, B. Mackler, S. Grandchamp, and P. P. Slonimski, Biochemistry 3, 668 (1964). 129. H. M. Duncan and B. Mackler, Biochemistry 5, 45 (1966). 130. I. C.Kim and D. S. Beattie, Eur. J . Biochem. 36,509 (1973).

131. P.A. Light, C. I. Ragan, R. A. Clegg, and P. B. Garland, FEBS (Fed. Eur. Biochem. Soc.) Lett. 1, 4 (1968). 132. D. R. Biggs, H. Nakamura, E. B. Kearney, E. Rocca, and T. P. Singer, ABB 137, 12 (1970). 133. S. 0. C. Tottmar and C. I. Ragan, BJ 124,853 (1971).

218

YOUSSEF HATEFI AND DIANA L. STIGGALL 0.06 r Wavelength (nm)

h

350

400

450

500

550

600

650

700

Wnvalongth (nm)

FIG.20. Absorption spectra of the purified NADH dehydrogenase of C. utilis at 0.8 mg/ml. Trace (a), oxidized enzyme; trace (b) after addition of 0.1 mM NADH; inset, (b) minus (a). From Tottmar and Ragan (138).

particles by digestion with phospholipase A. However, Tottmar and Ragan (133) have isolated such a preparation with the use of deoxycholate and Triton X-100. The preparation contains per mg protein 0.5-0.6 nmole of flavin ( F M N ) , 15-17 ng-atoms of iron, and 15-17 nmoles of labile sulfide. It reacts efficiently with ferricyanide as electron acceptor, but poorly with menadione, cytochrome c, dichloroindophenol, and ubiquinone-1. The K , and K i of the enzyme with respect to NADH are, respectively, 83 pM and 0.2 m M ; the K , for ferricyanide is 1.0 mM. The preparation exhibits a g = 1.94 E P R signal, which is characteristic of complex I and the high molecular weight dehydrogenases from heart mitochondria. Its absorption spectrum (Fig. 20) is qualitatively comparable to that of the mammalian high molecular weight dehydrogenase, and similar to complex I it can oxidize NADPH a t a slow rate (133) (see Table V I ) . The absence of rotenone-piericidin sensitivity and coupling site 1 in S. cerevkiae and S. carlsbergensis, and their presence in C. utilis have suggested a possible connection between the two phenomena. Light et al. (131) demonstrated that in C. utilis iron-limited growth conditions result in a decrease of mitochondria1 cytochromes and nonheme iron, loss

4.

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219

of site 1 phosphorylation, loss of sensitivity of NADH oxidation to inhibition by rotenone and piericidin A, and loss of piericidin A binding capacity. These changes were not reflected, however, in the respiratory activity of the mitochondria. Site 1 phosphorylation and rotenone-piericidin sensitivity were recovered when the iron-deficient cells were incubated with FeS04 in the absence of an added carbon source. Others have reported that under various growth conditions, energy conservation a t site 1 reappears upon aeration of deficient cells concomitant with (134) or without (135) the appearance of piericidin sensitivity. These developments have been reviewed by Ohnishi (f.21). More recently, a careful study of the problem has been done by Grossman et al. (136) in which they have followed NADH dehydrogenase activity, appearance and loss of piericidin sensitivity, and the nature of various E P R signals and cytochromes during the exponential and stationary phases of C . utilis growth as well as after catabolite repression. They found that the respiratory particles of cells harvested during the exponential growth phase have very low piericidin sensitivity and NADHferriayanide reductase activity, but high NADH-juglone (5-hydroxy-1,4naphthoquinone) reductase activity (Fig. 21). Successive washing of the particles with low osmolarity buffer resulted in the loss of NADH dehydrogenase activit,y. The EPR spectra of antimycin-treated particles reduced with NADH or dithionite showed a n absence of signals resulting from iron-sulfur centers 1 and 2, but centers 3 4 were present. Signals were also present a t gll = 2.01 and gL = 1.92, which appeared t o result from an iron-sulfur center. The temperature sensitivity of this new species was similar t o that of center 1. Particles of cells in the stationary phase showed an increase in P/O ratio, suggesting the appearance of site 1 coupling, presence of piericidin sensitivity, rise in ferricyanide reductase activity, loss of juglone reductase activity (Fig. 21), appearance of EPR signals resulting from centers 1 and 2, and loss of the new EPR signal seen in exponential phase particles. Further, the dehydrogenase of the stationary phase was stable to washing of the particles. Catabolite repression of late stationary phase cells by addition of ethanol resulted in the loss of the above characteristics and reacquisition of exponential phase properties with regard to enzymic activity, piericidin sensitivity, and the EPR signals associated with NADH dehydrogenase. These changes were prevented by cy cloheximide.

+

134. T. Ohnishi, P. Panebianco, and B. Chance, BBRC 49,99 (1972). 135. R. A. Clegg and P. B. Garland, BJ 124, 135 (1971). 136. S. Grossman, J. G. Cobley, T. P. Singer, and H. Beinert, JBC 249, 3819 (1974).

220


10

20

30 40 50 Hours FIG.21. Characteristics of NADH oxidation by submitochondrial particles from C. utilis during transformation from exponential to stationary phase. Candida utilis was grown in 1.5% (v/v) ethanol in a fermentor at 30". Cells were harvested a t the times shown for isolation of mitochondria and preparation of submitochondrial particles. NADH oxidase activity is expressed as microatoms of oxygen per min per mg protein at 30". NADH dehydrogenase activity is expressed as micromoles of NADH oxidized per min per mg particle protein a t 25" at V,, with respect to Fe(CN).'-; sensitivity to piericidin A (0.5 nmole/mg protein) is expressed as percent inhibition of NADH oxidase; and turbidity is given as absorbance at 650 nm in 1 cm light path. The pH was maintained during growth by automatic addition of 6 N KOH (pH-stat) at 5.0 until 25 hr, after which no further acid development occurred but the pH rose to between 5.0 and 6.2. From Grossman et al. (136).

The authors feel that during transition from the exponential to the stationary phase, a different type of NADH dehydrogenase is synthesized (136). However, a very interesting possibility suggests itself when one compares the characteristics of NADH dehydrogenase in the stationary phase and exponential phase (or catabolite repressed) particles, respectively, with the properties of complex I and chaotrope-destabilized complex I (see above). Stationary phase particles and complex I are capable of energy conservation a t site 1, are piericidin-sensitive, have high ferricyanide reductase and low naphthoquinone reductase activities (juglone was used with stationary phase particles and menadione with. complex I ) , and exhibit EPR signals resulting from centers 1 (responsible for g = 1.94 signal) and 2. In both cases, the membrane-bound dehydrogenase is stable. In contrast, destabilized complex I and particles from

4.

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221

exponential phase or catabolite-repressed C . utilis cells exhibit low ferricyanide reductase and high naphthoquinone reductase activities,' loss of piericidin sensitivity and g = 1.94 E P R signal, and appearance of new EPR signals (46, 136). In the fractions isolated from destabilized complex I, the modified EPR signal is seen in the soluble, low molecular weight dehydrogenase ( 4 6 ) , which presumably originates from the temperature-insensitive center 1 signal of the unperturbed complex. Further, the dehydrogenase activities of both destabilized complex I and exponential phase or repressed particles are unstable. These analogies suggest, therefore, that in C . utilis cells at exponential growth phase or subjected to catabolite repression a t late stationary phase there exist species of NADH dehydrogenase with features akin to the mammalian low molecular weight enzyme derived from destabilized particles. Assuming that the low molecular weight dehydrogenase of mammalian mitochondria is a bona fide component rather than a degradation product, then its different properties when membrane bound might be a consequence of integration into the membrane and interaction with other respiratory components. By analogy, it is conceivable that during the exponential growth phase a species comparable to the low molecular weight dehydrogenase is present and in the process of being assembled into the respiratory chain, while after catabolite repression the membrane becomes degraded and this species of dehydrogenase exhibits once again the properties of the unbound enzyme.

C. NADH DEHYDROGENASE OF Azotobacter winelandii Low molecular weight NADH dehydrogenases have been isolated by DerVartanian (137) from A. winelandii grown under normal and ironlimited conditions. Both preparations are reported to have a molecular weight of 56,500, and to contain FRIN. The enzyme from cells grown under iron-limited conditions contained 1 g-atom of molybdenum, 2 g-atoms of iron, and 2 moles of labile sulfide per mole of FMN, while in the enzyme from cells grown under normal conditions the iron and labile sulfide content was twice as much. The two preparations exhibited different EPR signals but comparable NADH-menadione and NADHferricyanide reductase activities. It is stated that, unlike the mammalian low molecular weight enzyme, the purification of the Azotobacter low molecular weight NADH dehydrogenase is not accompanied by changes in catalytic properties. 137. D. V. DerVartanian, Z. Naturforsch. 27, 1082 (1972).

222


111. Succinate Dehydrogenarer

A. MAMMALIAN SUCCINATE DEHYDROGENASE (EC 1.3.99.1) Succinate dehydrogenase is the only enzyme of the citric acid cycle which is bound to the inner membrane of mitochondria. It is also one of three flavoproteins known in which flavin is covalently linked to the protein. The other two are monoamine oxidase of the outer membrane of liver mitochondria (138) and Chromatiurn cytochrome c-552 (139). Succinate dehydrogenase was solubilized from beef heart mitochondria in 1954 (140) and purified in 1970 (141-143). In the intervening years modified or new procedures for isolation and purification of the enzyme were reported by Bernath and Singer (144), Basford et al. (145),Wang et al. (l46‘),Keilin and King (147, 148),Veeger et al. (149), and Cerletti et al. (160). The preparations of various laboratories differed in their content of covalently bound flavin (hence in degree of purity), nonheme iron, and labile sulfide, and exhibited different enzymic activities, the most important of which was the ability of the enzyme to transfer electrons to the respiratory chain. Consequently, unresolvable controversies developed and strong positions were taken regarding the molecular weight, composition, activities, and regulatory properties of succinate 138. E. B. Kearney, J. I. Salach, W. H. Walker, R. Seng, W. Kenney, E. Zeszotek, and T. P. Singer, Eur. J. Biochem. 24, 321 (1971). 139. W. C. Kenney, D. Edmondson, R. Seng, and T. P. Singer, BBRC 52, 434 ( 1973).

140. T. P. Singer and E. B. Kearney, BBA 15, 151 (1954). 141. Y. Hatefi, K. A. Davis, W. G. Hanstein, and M. A. Ghalambor, A B B 137, 286 (1970). 142. W. G. Hanstein, K. A. Davis, and Y. Hatefi, in “Energy Transduction in

Respiration and Photosynthesis” (E. Quagliariello, S. Papa, and C. S. Rossi, eds.), p. 495. Adriatica Editrice, Bari, 1971. 143. K. A. Davis and Y. Hatefi, Biochemistry 10, 2509 (1971). 144. P. Bernath and T. P. Singer, “Methods in Enzymology,” Vol. 5, p. 597, 1962. 145. R. E. Basford, H. D. Tisdale, and D. E. Green, BBA 24, 290 (1957). 146. T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sci. Sinicn 5, 73 (1956). 147. D. Keilin and T. E. King, Nature (London) 181, 1520 (1958). 148. T. E. King, JBC 238, 4037 (1963). 149. C. Veeger, D. V. DerVartanian, and W. P. Zeylemaker, “Methods in Enzymology,” Vol. 13, p. 81, 1969. 150. P. Cerletti, G. Zanetti, G. Testolin, C. Rossi, F. Rossi, and G. Osenga, in “Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.), p. 629. Univ. Park Press, Baltimore, Maryland, 1971.

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dehydrogenase. These issues have been discussed in a number of reviews by Singer and his colleagues from their laboratory’s standpoint. Their most recent are references 23 and 25. 1. Molecular Properties Singer and co-workers extracted succinate dehydrogenase from mitochondrial acetone powder a t alkaline pH, and applied purification procedures involving adsorption on calcium phosphate gel and ammonium sulfate fractionation (140, 144). Summarizing their results, Singer ( 1 5 ) concluded in 1966 that succinate dehydrogenase had the following molecular properties: “The minimum molecular weight of homogeneous preparations from nonheme iron content is 49,000 and from flavin content 200,000. Physical measurements support a molecular weight of approximately 200,000. This value is also in accord with gel exclusion studies on Sephadex G-200. Thus the enzyme contains 1 mole of flavin and 4 g-atoms of nonheme iron per mole. . . . The sedimentation velocity of the beef heart enzyme at 10-15 mg protein/ml is 6.5 S . . . .” This preparation could oxidize succinate in the presence of ferricyanide or phenazine methosulfate (PMS) as electron acceptor but was unable to transfer electo be unable to interact with the respiratory chain. Wang et al. (146) reported in 1956 the isolation of succinate dehydrogenase from heart muscle preparations treated with succinate and cyanide. The enzyme was extracted with 20% aqueous ethanol a t p H 9.0. The final product, after adsorption on calcium phosphate gel and ammonium sulfate fractionation, was stated to be electrophoretically homogeneous and to contain 1 mole of flavin and 4 g-atoms of iron per 140,000160,000 g of protein. While apparently purer than the preparations of Singer’s group, the cyanide-treated enzyme was also shown subsequently to be unable to interact with the respiratory chain. In 1958, Keilin and King (147) reported that succinate dehydrogenase isolated essentially by the method of Wang et al. (146), but without the use of cyanide, had an important property. Unlike other preparations, the Keilin-King enzyme was capable of electron transfer to the respiratory chain (148). It was subsequently shown by King (148) that the presence of succinate during extraction of the enzyme was essential for its ability to interact with the respiratory chain. Preparations of the enzyme contained 2.4-3.6 nmoles of flavin per mg protein and high levels of nonheme iron and labile sulfide. An average of several preparations showed a ratio of iron to sulfide to flavin of 8.5:8.1: 1 (151). By compari151.

T.E. King, BBRC 16,511 (1964)

224


son to the preparation of Singer and co-workers and Wang et al., the Keilin-King enzyme contained twice as much iron per mole of flavin but less flavin per unit weight of protein. These data suggested the presence in the latter preparation of an additional iron-sulfur protein linking succinate dehydrogenase to the respiratory chain. One difficulty with regard to this possibility was that omission of succinate during enzyme isolation resulted in a preparation with similar composition, absorption spectrum, and dye reductase properties, but with complete lack of the ability to interact with the respiratory chain. Further, Veeger et al. (149) showed that modifications of the Keilin-King procedure yielded a preparation of succinate dehydrogenase which retained the ability to interact with the respiratory chain, but contained 1 mole of flavin, 8 g-atoms of iron, and 4-8 moles of labile sulfide per 200,00CL250,000 g of protein. Thus, it appeared that the ability to reconstitute with the respiratory chain is an important property of succinate dehydrogenase, which was lost in the earlier low-iron preparations. I n 1959, Ziegler and Doeg (152-164) reported the isolation of a particulate preparation from beef heart mitochondria which was capable of electron transfer from succinate to ubiquinone. This preparation was subsequently recognized by Hatefi et al. (27, 29) to be one of the four electron transfer complexes of the respiratory chain and is now generally referred to as complex 11. Preparations of complex I1 contain approximately 5 nmoles of covalently bound flavin per mg protein, and 7-8 g-atoms of iron and 7-8 moles of labile sulfide per mole of flavin. I n addition, it was shown by Ziegler and Doeg that complex I1 contained cytochrome b at a molar concentration comparable to flavin. These data cast strong doubt on the molecular weight of 200,000 for succinate dehydrogenase since, on the basis of its flavin content, complex I1 had a similar molecular weight while containing in addition cytochrome b and possibly other proteins. In agreement with this, Baginsky and Hatefi (155,156) showed in 1969 that a preparation of succinate dehydrogenase with a flavin content of 6-7 nmoles/mg protein could be isolated from complex 11. I n spite of these indications, it was generally assumed, however, that succinate dehydrogenase had been purified and its molecular weight was 200,000. The enzyme was finally purified in 1970 by selective resolution of complex I1 with chaotropic agents (141-143). It was shown to have a flavin 152. D. M. Ziegler and K. A. Doeg, BBRC 1, 344 (1959). 153. D. M. Ziegler and K. A. Doeg, ABB 97,41 (1962). 154. D. M. Ziegler, in “Biological Structure and Function” (T. W. Goodwin and 0. Lindberg, eds.), Vol. 2, p. 253. Academic Press, New York, 1961. 155. M. L. Baginsky and Y. Hatefi, BBRC 32,945 (1968). 156. M. L. Baginsky and Y. Hatefi, JBC 244,5313 (1969).

4.


225

content of 10.3 +- 4% nmoles per mg protein, a molecular weight of about 100,000, two unlike subunits, an iron:labile su1fide:flavin ratio of 7-8:7-8:1, very high dye reductase activity, and the ability to interact with the respiratory chain. Certain features of the chaotrope-induced resolution of complex I1 will be described because (a) the procedure is novel and interesting and ( b ) insofar as the authors have been able to ascertain this is the only published procedure which yields pure succinate dehydrogenase (156~). The molecular and enzymic properties of the above preparations are summarized in Table IX. a. Resolution of Complex I I with Chotropic Ions. The purification of succinate dehydrogenase is a simple process, which involves selective extraction of the enzyme from complex I1 in the presence of a chaotropic agent followed by precipitation with ammonium sulfate (72, 143). Before the addition of ammonium sulfate to the resolved complex, it is necessary to separate the soluble enzyme from the remainder of complex 11, which is particulate, by centrifugation. Otherwise, the addition of ammonium sulfate will reverse the resolution process, as will be seen below. Figure 22 shows the effect of several chaotropic agents on the resolution of complex 11. The ordinate is percent succinate-ubiquinone (solid lines) or SUCcinate-PMS (dotted line) reductase activity, and the abscissa is time. 156a. Subsequent to the announcement of the above findings by Hatefi’s group (141, I @ ) , two laboratories (167, 165) have claimed the purification of succinate dehydrogenase by modifications of earlier procedures. Righetti and Cerletti (167) have stated that using an earlier method (150) they have obtained pure succinate

dehydrogenase “containing 7-8 nmoles of peptide bound flavin.” Purification details for this preparation, which according to its stated flavin content can be at best 80% pure, have, not been given. The earlier procedure (150), according to which the above enzyme was made, showed in the best fraction a flavin content of 5.5-5.7 nmoles/mg protein. The other preparation claimed to be pure (23, 26) is that of Coles et 01. (15,s).This preparation was made by the original procedure of Singer and co-workers (144) with subsequent purification by chromatography on Sephadex G-200 columns (details not given). However, Coles et al. (165) stated that “Although the peak fractions from such columns appeared homogeneous in sucrose gradients, their histidyl flavin content was no higher (4 to 5 nmoles/mg) than previously reported ( 5 nmole/mg).” This agreed with the earlier results of Singer’s (16) quoted above that their molecular weight of 200,000 was “in accord with gel exclusion studies on Sephadex G-200.” Coles et al. further stated that “correction for impurities” observed on polyacrylamide gels stained with Coomassie Blue “raised the histidyl flavin content to 8 to 9 nmoles/mg. . . .” Such calculated values referred to elsewhere should not be misunderstood, however, to represent the actual flavin content of the preparation, which was not more than 5 nmoles/mg protein, thus indicating a t best 50% purity. 157. P. Righetti and P. Cerletti, FEBS (Fed. Eur. Biochem. Soc.) LetL.13, 181 (1971). 158. C. J. Coles, H . D. Tisdale, W. C. Kenney, and T. P. Singer, Physiol. Chem. Phys. 4, 301 (1972).

TABLE IX

MOLECULAR AND ENZYMIC PROPERTIES OF REPRESENTATIVE PREPARATIONS OF SIJCCINATE DEHYDROOENASE

Preparation Bernath-Singera wang et al! King" Veeger et a1.d Complex 118 Davis-Hatefie

Molecular weight

Flavin (nmole/mg protein)

Flavin :iron : labile sulfide ratio

200,000

4.2-5.0

1:4:?

-6,000

1.3

1:4:? 1:8.5:8.1 1:8:4-8

2,00O-3,500 -6,000

0.58 0.3

1:7-8:7-8 1:7-8 :7-8

10,000-11,000 10,000-11,000

140,OOO-160,000

329,000

200,000-250,000 200, OOO

97,000

6-7 3.04 4-5 5.0 10.3

Turnover number at

v,PMS

-3,900

KZa (mM)

0.3

0.3

Reconstitution activity Absent Absent Present

0

Present Present Present

X

From Singer (16)and Bernath and Singer (I&). The latest preparation of Coles el al. (168)haa similar properties. Turnover number calculated from data in Wang et al. (146). ~Flavinand flavin:iron:labile sulfide are average values from (161).Turnover number and molecular weight calculated from highest activity in King (171)and average flavin content, respectively. d Turnover number calculated from V , activity at 25" in DerVertanian et al. ($18). From Hate6 et d. (%), and references therein. a

20°K, and activation-deactivation effects. Therefore, it appears that these phenomena do not require the intactness of both iron-sulfur centers. The presence of PMS reductase activity in the 2-iron preparations further suggests the possible presence in these preparations of a stable 2 iron-2 sulfur center or of a fraction with higher iron-sulfur content which is responsible for the g = 1.94 signal and PMS reduction. The two subunits resolved by Davis and Hatefi (143, 166) by the use of chaotropes and freeze-thawing were inactive, separately and in combination, for succinate oxidation or fumarate reduction. However, the possibilities have not been fully explored. Nor has this sort of resolution been performed on the cyanide-treated enzyme to see whether one or the other subunit can be preferentially modified. Further work in these areas might 218. D. V. DerVartanian, C. Veeger, W. H. OrmeJohnson, and H. Beinert, BBA 191, 22 (1969).

254


provide important clues to the reaction mechanism of succinate dehydrogenase and the role of the subunits.

B. SUCCINATE DEHYDROGENASE IN MICROORGANISMS All aerobic organisms, including yeast, appear to have a membranebound succinate dehydrogenase containing iron and covalently bound flavin (15, 16, 25). In contrast, the enzyme in anaerobic organisms is found in the cytoplasm and appears to be more effective as a fumarate reductase, a modification which is in accord with the physiological requirements of the organism. I n facultative anaerobes such as E. coli and S. cerevisiae, both the membrane-bound succinate dehydrogenase and the cytoplasmic fumarate reductase are found, their synthesis and concentration depending on the growth conditions. The succinate dehydrogenase of yeast mitochondria was isolated by Singer et al. (15, 219) in 1957, and stated to have a molecular weight of 200,000 and an iron:flavin ratio of 4:1, similar to the mammalian enzyme. These studies antedated, however, the purification of mammalian succinate dehydrogenase by Davis and Hatefi. Therefore, the exact molecular weight and composition of the yeast enzyme will have to be reexamined in light of present information. Hatefi et al. (220) have isolated the succinate dehydrogenase of Rhodospirillum rubrum by extraction of chromatophores with NaC10,. The enzyme has two subnits of molecular weights of approximately 60,000 and 25,000 (Fig. 34) (22Oa). Both contain iron-sulfur chromophores (221), and the larger subunit carries the covalently bound flavin. I n the intact enzyme, the ratio of flavin:iron:labile sulfide is approximately 1:8 :8. The enzymic properties of this prokaryotic enzyme are also very similar to the mammalian succinate dehydrogenase. Ferricyanide and PMS are reduced a t comparable V,,,,, rates, and the former inhibits above 1 mM. K , values with respect to succinate, PMS, and ferricyanide are 0.23, 0.11, and 0.3 mM, respectively (220). At 77OK, the succinate-reduced enzyme exhibits a free radical signal a t g = 2.00 and an iron-sulfur type of signal a t g = 1.93 (220).The absorption spectrum of the R . rubrum enzyme is very similar to that of mammalian mitochondria. A very interesting observation is that the R . rubrum succinate dehydrogenase can cross-interact with alkali-inactivated mammalian respiratory 219. T. P. Singer, V. Massey, and E. B. Kearney, ABB 69, 405 (1957). 220. Y . Hatefi, K. A. Davis, H. Baltscheffsky, M. Baltscheffsky, and B. C. Johansson, ABB 152, 613 (1972).

220a. K. A. Davis, I. P. Crawford, and Y . Hatefi, in preparation.

4.


255

FIG.34. Electrophoresis of Rhodospirillum rubrum succinate dehydrogenase on SDS-polyacrylamide gel. The protein bands were visualized with Coomassie blue. From Davis et al. ( 2 2 0 ~ ) .

particles to reconstitute succinoxidase activity (Fig. 35). This activity is inhibited by TTFA. According to Hatefi et al. ( 2 2 0 ) , affinity of the R. rubrum enzyme for reconstitution with the mammalian respiratory chain is also similar to that of the mammalian succinate dehydrogenase. Rhodospirillurn rubrum succinate dehydrogenase can also reconstitute with alkali-inactivated R. rubrum chromatophores, but cross-interaction of the latter with the mammalian enzyme, though it occurs, is not equally efficient ( 2 2 1 ) . Tisdale et al. (222) have shown that several isoeymes of fumarate reductase occur in brewer’s yeast, ranging in molecular weight from 34,000 to 112,000. The predominant species had a molecular weight of 62,000221. Y. Hatefi, unpublished. 222. H. Tisdale, J. Hauber, G. Pragcr, P. Turini, and T. P. Singer, Eur. J. Biochem. 4, 472 (1968).

256


42rnin

FIQ.35. Reconstitution of succinoxidase activity of alkali-inactivated bovine heart ETP with bovine and R . rubrum succinate dehydrogenases (SD). Left-hand trace : 114 pg alkali-treated ETP (alk-ETP) and 37 pg bovine SD per ml. Right-hand trace: 172 pg alk-ETP and 46 pg R. rubrum SD per ml. I n both cases alk-ETP and SD a t 10 times the concentrations indicated above were premixed with 10 m M succinate and preincubated for 3 min a t 30" before addition t o the assay mixture. Alk-ETP, bovine SD, or R . rubrum SD d o n e resulted in no oxygen uptake. Where indicated, 5.9 m M TTFA and 0.15 mM PMS were used. Assay temperature 30". S.A., specific activity. From Hatefi et nl. (220).

63,000. The enzyme contains noncovalently bound FAD, nonheme iron, possibly copper, but no labile sulfide. Electron paramagnetic resonance studies showed signals resulting from copper and high-spin ferric ions a t g = 4.3.

IV. ~-Glycerol-3-phosphateDehydrogenase (EC 1.1.99.5 1

The oxidation of L-glycerol 3-phosphatc to dihydroxyacetone phosphate is catalyzed by two different enzymes. One is the cytoplasmic NAD-linked a-glycerophosphate dehydrogenase, and the other is the niitochondrial enzyme, which appears to contain flavin and iron. The latter enzyme was first studied by Green in 1936 (223). It was shown to be associated with respiratory particles, and widely distributed in animal tissues. The highest concentration of the enzyme was found in the brain. Lardy and co-workers (264) studied the enzyme in deoxycholatesolubilized particles obtained from skeletal muscle, confirmed the finding 223. D.E. Green, BJ 30, 629 (1936). 224. T. Tung, I,. Anderson, and H. A. Lardy, ABB 40,191 (1952).

4.


I

400

500

257

600

Wavelength nm

FIG.36. Absorption spectrum of partially purified a-glycerophosphate dehydrogenase, protein concentration 13 mg/ml. (0) Spectrum of oxidized enzyme. The difference spectra (shown in the insert) were recorded after the addition of a few granules of sodium hydrosulfite ( 0 )or 5 pmoles substrate (0) in a total volume of 0.2 ml. I n the difference spectra, a decrease in optical density indicates bleaching. From Ringler (226).

of Green with regard to the specificity of the enzyme for L-a-glycerophosphate as substrate, and demonstrated that the reaction product was dihydroxyacetone phosphate. a-Glycerophosphate dehydrogenase has been solubilized by treatment of pig brain mitochondria with phospholipase A (225). Only partial purification of the enzyme has been achieved. The best preparations of Ringler and Singer (225-227) were shown to contain 1 mole of flavin per 2.1 X 10” g of protein and 1 g-atom of nonheme iron per 3.5 X lo5 g of protein. I t has been claimed that the flavin is FAD (227). The absorption spectrum of the above preparation is shown in Fig. 36. Phenazine methosulfate, ferricyanide, 2,6-dichloroindophenol, and methylene blue have been used as electron acceptors. With PMS a s electron acceptor, the best preparations of Ringler and Singer (226) from pig brain were shown to oxidize a-glycerophosphate a t 38O and p H 7.6 a t a rate of 3.4 pmoles/min x mg protein. Under these conditions K, for a-glycerophosphate was shown to be 9.5 mM, the same as the particle-bound enzyme (223). Dihydroxyacetone phosphate is a competitive inhibitor of the mammalian enzyme; K , = 0.18 m M a t 3 8 O and p H 7.6. Attempts a t reversing the action of a-glycerophosphate dehydrogenase in the presence of dihydroxyacetone phosphate plus reduced FMN, leucobenzylviologen, 225. R. L. Ringler, JBC 236, 1192 (1961). 226. R . L. Ringler and T. P. Singer, “Methods in Enzymology,” Vol. 5, p. 432, 1963. 227. T. P. Singer, “The Enzymes,” 2nd ed., Vol. 7, p. 345,1963.

258


or leucomethylviologen as electron donor have not been successful (226). a-Glycerophosphate dehydrogenase is believed to be located in the outer phase of the mitochondria1 inner membrane (228). The enzyme appears to donate electrons to the respiratory chain beyond the level a t which Amytal inhibits NADH oxidation (229). It has been shown with the use of pentane-extracted mitochondria that electron transfer from the enzyme to the respiratory chain, but not to dyes, requires the presence of ubiquinone, and is inhibited by antimycin A (230). Therefore, i t appears that a-glycerophosphate dehydrogenase interacts with the mitochondrial electron transport system a t the level of ubiquinone, which is also the point of convergence of complexes I, 11, and I11 (29, 33, 231). I n agreement with the above findings, Ringler and Singer (177) have made the important observation that in antimycin-treated brain mitochondria the oxidation of a-glycerophosphate to dihydroxyacetone phosphate can be linked by way of succinate dehydrogenase to the reduction of fumarate. Further, Szarkowska and Drabikowska (232) have demonstrated the reduction of exogenous ubiquinone-6 by the particle-bound and the phospholipase-solubilized a-glycerophosphate dehydrogenase from pig brain. Both systems were inhibited by 3-phosphoglycerate. The best rate reported by these investigators for the soluble enzyme is 0.24 pmole QG reduced/min x mg protein at 37O and pH 7.2, which is only 7% of the PMS reductase activity of similar preparations. This low activity might in part have resulted from assay difficulties, which are usually encountered when water-insoluble homologs of ubiquinone are used as electron acceptor. It is also possible that (a) by analogy to the early preparations of succinate dehydrogenase (see Section 111), the preparation of a-glycerophosphate dehydrogenase used in these studies was damaged with respect to ubiquinone reduction, and (b) the reduction of ubiquinone by the dehydrogenase occurs by way of an unknown electron carrier in which the enzyme preparation used was deficient. Mitochondria from the flight muscle of house flies, Musca dornestica, have been shown to oxidize a-glycerophosphate a t exceptionally high rates (233, 234). This activity was shown to be inhibited by EDTA. It is believed that in these and other mitochondria the combined action of the soluble and the particle-bound a-glycerophosphate dehydrogenases 228. 229. 230. 231. 232. 233. 234.

M. Klingenberg, Eur. J. Biochem. 13, 247 (1970). B. Chance and B. Sacktor, ABB 76, 509 (1958). J. I. Salach and A. J. Bednarz, ABR 157, 133 (1973). Y. Hatefi, Clin. Chem. 11, 198 (1965). L. Szarkowska and A. K. Drabikowska, LifeSci. 7,519 (1963). R. W. Estabrook and B. Sacktor, JBC 233, 1014 (1958). B. Sacktor and D. G. Cochran, ABB 74,266 (1958).

4.

METAL-CONTAINING FLAVOPROTEIN DEHYDROGENASES ouler

membranes

259

inner

Cytosol Glycerol- 1 -P

Dihydroxyacetone-P

It

FIG.37. The a-glycerophosphate cycle for the oxidation of extramitochondrial NADH by the mitochondrial respiratory chain. From Klingenberg (928).

provides the principal route for the transfer of reducing equivalents from cxtramitochondrial NADH to the mitochondrial electron transport SYStem (228, 229, 233-237). This process has been termed the “a-glycerophosphate cycle” (Fig. 37). Lee and Lardy (238) and others (239-242) have found that in the rat the a-glycerophosphate dehydrogenase activity of mitochondria from liver, kidney, adipose tissue, and heart were increased severalfold upon feeding of desiccated thyroid glands. The activity increase appeared to be organ-specific and particularly marked in liver, which showed a 20-fold increase after 10 days. The activity of the enzyme in brain, lung, spleen, stomach, small intestine, and testis was not appreciably increased. Thyroidectomy resulted in the decrease or disappearance of particle-bound a-glycerophosphate dehydrogenase activity in several organs, and a single injection of triiodothyronine restored the activity within 48 hr. The increased activity of a-glycerophosphate dehydrogenase induced by the thyroid hormone appears to result from synthesis of new enzyme (239).The possible role of this striking, organ-specific effect of the thyroid hormone has been discussed in relation to increased carbohydrate degradation in response to the increased oxidation of extramitochondrial NADH by the a-glycerophosphate cycle (238), as well as in relation to its effect on phospholipid synthesis ( 243) . 235. R. W. Estabrook and B. Sacktor, ABB 76, 532 (1958). 236. B. Sacktor, L. Packer, and R. W. Estabrook, .4BB 80, 68 (1959). 237. B. Sacktor and A. Dick, JBC 237,3259 (1962). 238. Y.-P. Lee and H. A. Lardy, JBC 240, 1427 (1965). 239. Y.-P. Lee, A. E. Takernori, and H. Lardy, JBC 234, 3051 (1959). 240. H. A. Lardy, Y.-P. Lee, and A. Takemori, Ann. N . Y. Acad. Sci. 86, 506 (1960). 241. 0. Z. Sellinger and K.-L. Lee, BBA 91, 183 (1964). 242. G. H. Isaacs, B. Sacktor, and T. A. Murphy, BBA 177, 196 (1969). 243. W. R. Frisell and J. R. Cronin, ir, “Electron and Coupled Energy Transfer in Biological Systems” (T. E. King and M. Klingenberg, eds.), Vol. 1, Part A, p. 177. Dekker, New York, 1971.

260


Flavin-containing a-glycerophosphate dehydrogenases have been found also in Streptococcus faecalis (244) and Propionibacterium arabinosum (245). The enzyme from 8. faecalis is reported to contain FAD, have a K , for a-glycerophosphate of 4 mM, and a pH optimum of 5.8. I n addition t o dyes, this enzyme can interact directly with molecular oxygen to form H,O,. The preparation from P . arabinosum is particle-bound, has a K , for a-glycerophosphate of 26 p M , and is claimed to contain flavin and nonheme iron.

V. Choline Dehydrogenase (EC 1.1.99.1 1

The oxidation of choline to betaine is catalyzed by two enzymes. First, choline is oxidized to betaine aldehyde by a n enzyme which is found in mitochondria in membrane-bound form. This enzyme is believed to be a flavoprotein containing nonheme iron. Betaine aldehyde is then oxidized to betaine by a soluble enzyme, which is NAD-linked. Betaine aldehyde dehydrogenase appears to be present both in mitochondria and the soluble fraction of liver (243,246‘). The existence of choline dehydrogenase was first demonstrated by Mann and Quastel in 1937 (247, 248) in extracts of rat liver and kidney. These authors also obtained evidence that the first oxidation product of choline was betaine aldehyde. Others showed subsequently that choline oxidase activity resided in the mitochondria1 fraction of rat liver and is linked to the respiratory chain (249, 250). Detergents (251, 252), solvent treatment of fragmented mitochondria (253), and venom phospholipase (254-256‘) have been used for extraction and solubilization of choline dehydrogenase. Among these, the best method reported to date appears to be the digestion of acetone-powdered mitochondria with venom phospholipase. Choline dehydrogenase, partially purified from phospholipase extracts of rat liver mitochondria, contains 1 mole of flavin and 4 g-atoms of nonheme iron per 850,000 g protein. The flavin is claimed to be acid244. 245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256.

N. J. Jacobs and P. J. VanDemark, ABB 88,250 (1960). N. Sone, J . Biochem. (Tokyo) 74,297 (1973). J. L. Glenn and M. Vanko, ABB 82, 145 (1959). P. J. G. Mann and J. H. Quastel, BJ 31, 869 (1937). P. J. G. Mgnn, H. E. Woodward, and J. H. Quastel, BJ 32, 1025 (1938). C. J. Kensler and H. Langemann, JBC 192, 551 (1951). J. N. Williams, Jr., JBC 194, 139 (1952). J. N. Williams, Jr. and A. Sreenivasan, JBC 203, 899 (1953). M. Korgenovsky and B. V. Auda, BBA 29,463 (1958). K. Ebisuzaki and J. N. Williams, Jr., BJ 60, 644 (1955). G . Rendina and T. P. Singer, BBA 30,441 (1958). G. Rendina and T. P. Singer, JBC 234, 1605 (1959). T. Kimura and T. P. Singer, “Methods in Enzymology,” Vol. 5, p. 562, 1962.

4.


261

extractable FAD, and the enzyme preparation is reported to contain trace amounts of a b-type cytochrome. For assay of the activity of membrane-bound enzyme, molecular oxygen, cytochrome c, ferricyanide, PMS, and dichloroindophenol can be used (254).The soluble enzyme reacts only with the latter two electron acceptors (227, 255, 256), thus indicating that other acceptors interact indirectly by way of the niitochondrial electron transport system. With PMS as electron acceptor, the K , for choline a t 38O and p H 7.6 is about 7 mM (227, 255). The best preparations of Kimura and Singer (256) oxidize choline a t a rate of 5.3 pmoles/min x mg protein a t 3 8 O . I n addition to choline, the particulate enzyme has been reported to oxidize arsenocholine (248) and other choline analogs (25‘7).Choline dehydrogenase is very sensitive to thiol inhibitors, and choline has been reported to protect against inhibition by p-mercuribenzoate (258).The oxidation of choline is competitively inhibited by betaine aldehyde ( K i = 2 mM) (256).Nitrogen mustard has also been reported to be a strong competitive inhibitor ( 2 5 9 ) , but others have reported no inhibition of choline dehydrogenase by nitrogen mustard ( 2 6 0 ) . Information regarding the involvement of flavin and iron in enzyme catalysis is not available. Rothschild et al. (258) have reported that dialysis of rat liver particles resulted in the loss of choline-cytochrome c reductase activity, which could be restored by addition of FAD but not FMN. However, these results have not been substantiated by others (255). Singer has stated that the difference spectrum of the enzyme “shows bleaching by substrate in both the flavin and the iron regions” (227).This spectrum has not been published. Relation to the Electron Transport System

It was shown by Strength et al. (261) that the oxidation of choline by a particulate preparation from rat liver was considerably enhanced upon addition of NAD. Others showed that choline oxidation by isolated rat liver mitochondria was completely inhibited by Amytal when oxygen, cytochrome c, ferricyanide, or methylene blue was the electron acceptor (262-664). Choline dehydrogenase activity of particles and soluble prepa257. I. C. Wells, JBC 207, 575 (1954). 258. H. A. Rothschild, 0. Cori, and E. S. G. Barrbn, JBC 208, 41 (1954). 259. E. S. G. Bnrron, G. R. Bartlett, and Z. B. Miller, J . E x p . M e d . 87, 489 (1948). 260. A . Sivak, A. J. Mahoney, Jr., and W. I. Rogers, Biochem. Pharmacol. 16, 1919 (1967). 261. D. R. Strength, J . R. Christensen, and L. J. Daniel, JBC 203, 63 (1953). 262. L. Packer, R. W. Estabrook, T. P. Singer, and T. Kimura, JBC 235,535 (1960). 263. L. Ernster, 0. Jalling, H. Low, and 0. Lindberg, Exp. Cell R e s . Suppl., 3, 124 (1955). 264. 0. Rendina and T. P. Singer, F e d . Proc., F e d . A m e r . SOC.Exp. Biol. 18, 308 (1959).

262


rations was reported to be insensitive to Amytal when assayed with P M S as electron acceptor (227, 262). Bianchi and Azzone (265) confirmed the findings of Strength et al. and showed that choline oxidation by intact rat liver mitochondria, but not by swollen mitochondria, was partially inhibited by rotenone. They further demonstrated that addition of choline to liver mitochondria in the presence of A D P resulted in the reduction of intramitochondrial nicotinamide nucleotides, and that under anaerobic conditions choline oxidation could be linked to the reduction of oxaloacetate to malate in the absence of an energy supply. These and other results led to the conclusion that reducing equivalents from choline dehydrogenase to the respiratory chain of intact mitochondria passed in part through the rotenone- and Amytal-sensitive site 1 of phosphorylation in the NADH oxidase pathway, and in part through another Amytal-sensitive, but rotenone-insensitive . point to ubiquinone and cytochrome b (243).

Kimura e t al. (266) showed that, unlike a-glycerophosphate, the oxidation of choline to betaine aldehyde in anaerobic mitochondria could not be linked to fumarate reduction. They also reported t h a t the choline oxidase activity of rat liver mitochondria was partially resistant to inhibition by antimycin A and quinoline oxide. They concluded, therefore, t h a t the mitochondria1 choline and succinate oxidase pathways were separate. The two chains were interlinked only between cytochrome cI and oxygen, and the choline chain involved an autoxidixable cytochrome b. These complications and the NAD stimulation of choline oxidation were resolved to a considerable extent by the work of Feinberg et al. (267) and Estabrook and his colleagues (268). The former group showed that the NAD stimulation was abolished when semicarbazide was present during choline oxidation. Under these conditions, semicarbazide interacted with, and prevented the NAD-linked oxidation of, betaine aldehyde which was formed as a result of choline oxidation. I n the absence of semicarbazide, choline oxidation was stimulated by NAD. These and other workers concluded that the rotenone inhibition of choline oxidation, which occurred in intact mitochondria, even in the presence of semicarbazide, involved an interference with the rate of entry of choline into intact mitochondria. Estabrook and co-workers (268) showed very clearly ( a ) that the antimycin-resistant oxygen uptake by mitochondria in the presence of choline resulted from inhibitor-resistant oxidation of endogenous substrates ; (b) that rotenone has little effect on choline oxidation by submitochondrial 265. 266. 267. 268.

G. Bianchi and G. F. Azzone, JBC 239, 3947 (1964). T. Kimura, T. P. Singer, and C. J. Lusty, BBA 44, 284 (1960). R. H. Feinberg, P. R. Turkki, and P. E. Witkowski, JBC 242, 4614 (1967). D. D. Tyler, J. Gonze, and R . W. Estabrook, ABB 115, 373 (1966).

4.


263

particles, whereas under the same conditions Amytal exerts a strong inhibitory effect; (c) that contrary to the previous report of others, the oxidation of choline by submitochondrial particles in the presence of PMS as electron acceptor was inhibited by Amytal; (d) that the concentration of Amytal needed for 50% inhibition of NADH (or 3-hydroxybutyrate) and choline oxidation by submitochondrial particles were 0.2 and 0.7 mM, respectively; and (e) that on the basis of spectra recorded a t 77OK, the nature and the degree of cytochromes reduced in r a t liver mitochondria were the same when the system was allowed to reach anaerobiosis as a result of succinate oxidation in the presence of rotenone or choline oxidation in the presence of rotenone and malonate. It has been shown that the oxidation of choline by isolated r a t liver mitochondria is biphasic (269). The initial phase of choline oxidation is slow and coupled to the uptake of inorganic phosphate. The ensuing phase is 3-5 times faster and not coupled t o phosphorylation. The slow phase can be extended in the presence of Mg2+and A D P or ATP. These compounds are considered t o control the permeability of mitochondria to choline ( 2 7 0 ) .Calcium ions and conditions which result in mitochondrial swelling and membrane disruption have been shown to increase choline oxidation (266, 271).

VI. lactate Dehydrogenases

Three types of lactate dehydrogenase are found in yeast, which may be considered as metal-containing flavoproteins. These are L-lactate :cytochrome c reductase or cytochrome b,, D-lactatc dehydrogenase, which is found in anaerobic yeast, and D-1actate:cytochrome c reductase, which is associated with the mitochondria of aerobic cells. A.

L (+)-LACTATE: CYTOCHROME c OXIDOREDUCTASE

(CYTOCHROME b,) ( E C 1.1.2.3) This enzyme [also known a t L (+)-lactate dehydrogenase] was first extracted from bakers’ yeast by Bernheim in 1928 (272). Bach et al. (273) showed in 1942 that lactate dehydrogenase copurified with a species of cytochronie b, which contained protoheme as prosthetic group. The 269. 270. 271. 272. 273.

T. Kagawa, D. R. Wilken, and H. A. Lardy, JBC 240, 1836 (1965). D. R. Wilken, T. Kagawa, and H. A. Lardy, JBC 240, 1843 (1965). G. R. Williams, JBC 235, 1192 (1960). F. Bernlieini, BJ 22, 1179 (1928). S. J. Bach, M. Dixon, and L. G. Zerfas, Nature (London) 149, 48 (1942).

264


cytochrome was designated cytochrome b, by Keilin and co-workers (274). The enzyme was crystallized by Appleby and Morton in 1954 (275, 276) and shown to contain FhlN in amounts equimolar to heme. This also marked the first crystallization of a cytochrome. These studies were confirmed by others (277) and extended to show that lactate dehydrogenase was specific for L (+) -lactate, and inhibitable by p-mercuribenzoate and Atebrin. Appleby and Morton (278) showed subsequently that the crystalline enzyme contained 5-6% DNA, but that the polynucleotide was not essential for activity. The crystalline preparation containing DNA is known as type I cytochrome b,, and the preparation from which DNA has been removed is known as type I1 cytochrome b,. These early studies have been reviewed (279-282). 1. Physical Properties

Cytochrome b, is found as a soluble protein in the autolysates of Saccharonayces cerevisiae. The crystalline preparations of Appleby and Morton (278) were shown to sediment as a single peak in the ultracentrifuge. Minimum molecular weight based on amino acid analysis and a heme extinction coefficient of 232 mM-l cm-’ was calculated to be 53,000 (283). The heme extinction coefficient was then corrected to 183 mM-l cm-’, and the minimum molecular weight per mole of heme recalculated to be 58,600 ( 2 8 4 ) . It was concluded that cytochrome b, is a tetrameric structure. This conclusion agreed with the results of X-ray diffraction studies on type I and type I1 crystals, which indicated molecular weights of 235,000 2 10,000 and 234,000 & 8,000, respectively, for these two preparations of cytochrome b, (285).The oxidized and reduced spectral bands of cytochrome b, are given in Table XIV. Subsequent studies showed that reduction and carboxymethylation of crystalline cytochrome b, yielded two unlike subunits of approximately 274. S. J. Bach, M. Dixon, and D. Keilin, Nature (London) 149, 21 (1942). 275. C. A. Appleby and R. K. Morton, Nature (London) 173, 749 (1954). 276. C. A. Appleby and R. K. Morton, BJ 71, 492 (1954). 277. E. Boeri, E. Cutolo, M. Luzzati, and L. Tosi, ABB 56, 487 (1955). 278. C. A. Appleby and R. K. Morton, BJ 75,258 (1960). 279. T. P. Singer, “The Enzymes,” 2nd ed., Vol. 7, p. 345, 1963. 280. A. P. Nygaard, “The Enzymes,” 2nd ed. Vol. 7, p. 557, 1963. 281. T. P. Singer, C. Gregolin, and T. Cremona, in “Control Mechanisms in Respiration and Fermentation” (B. Wright, ed.) , p. 47. Ronald Press, New York, 1963. 282. A. P. Nygaard, in “Control Mechanisms in Respiration and Fermentation” (B. Wright, ed.), p. 27. Ronald Press, New York, 1963. 283. C. Jacq and F. Lederer, E w . J . Biochem. 12, 154 (1970). 284. P. Pajot and 0. Groudinsky, Eur. J. Biochem. 12, 158 (1970). 285. C. Monteilhet and J. L. Risler, Eur. J. Biochem. 12, 165 (1970).

4.

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TABLE XIV SPECTRAL PROPERTIES OF CYTOCHROME b t (TYPE II)O Oxidized form Band a

B r

s

uv

Reduced form

x

B

x

€

(nm)

(mM-1 cm-1)

(nm)

(mM-' cm-l)

560 530 413 360-365 275

9.2 11.3 129.5 34.4 83.5

557 528 424 328 269

30.9 15.6 183 39

88

From Pajot and Groudinsky (284).

21,000 and 36,000 daltons (286).These subunits had different amino acid compositions, and other results suggested that the heme binding site is on the heavy chain. At this time, Baudras (287) showed that L ( + ) -lactate: cytochrome c reductase isolated from the yeast Hansenula anomala was very similar to the Saccharamyces enzyme in molecular weight and heme and flavin content, but was considerably more stable and six times more active. Moreover, unlike the Saccharamyces enzyme, the activity of Hansenula cytochrome b, was inhibited in the presence of excess substrate. The Hansenula cytochrome b, appeared to be composed of four subunits of approximately 61,000 ? 5,000 daltons each (288). Baudras and Spyridakis (689) suggested, therefore, that the 21,000- and 36,000dalton subunits of the Saccharomyces enzyme were the result of artifactual splitting during isolation and crystallization of the type I cytochrome b,. The differences between the Hansenula and the Saccharomyces preparations of cytochrome b, were resolved by Jacq and Lederer (290) who showed that, when prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, the Saccharomyces enzyme does not crystallize as before, and shows a subunit size comparable to that of the Hansenula cytochrome b,. The enzyme so prepared had considerably improved stability and enzymic properties, and was inhibited at high lactate concentrations. It was concluded that the uncleaved, physiological form of Saccharomyces cytochrome b, has a molecular weight of 230,000, and is composed of four identical subunits, each associated with one FMN 286. 287. 288. 289. 290.

F. Lederer and A.-M. Simon, Eur. J . Biochem. 20, 469 (1971). A. Baudras, Biochimie 53, 929 (1971). F. Labeyrie and A. Baudras, Eur. J. Biochem. 25,33 (1972). A. Baudras and A. Spyridakis, Biochimie 53, 943 (1971). C. Jacq and F. Lederer, Eur. J. Biochem. 25, 41 (1972).

266


TABLE XV PARAMETERS OF INTACT A N D CLEAVED CYTOCHROME bp MOLECULAR Parameter N-Terminal residues

C-Terminal residues

Intact

Cleaved a-Subunit

iLys

a-Subunit

I

Glu

Ala

Val

LYE Ala ASP

/%Subunit ( Ala Minimum molecular weight per heme

58,100+7% (amino acids)

53,000 i~ 3% (amino acids) 58,600 f 2% (dry weight)

Molecular weight of peptide chains

57,500

=-Subunit 33,000-36,000 8-Subunit 21,000

Molecular weight

220,000 f 10% (gel filtration)

220,000 f 10% (gel filtration) 234,600 f 4% (crystallography) 240,000 f 4% (ultracentrifugation)

From Jacq and Lederer (291).

and one heme ($991).The amino acid composition of the enzyme prepared in the presence of phenylmethylsulfonyl fluoride has been determined, and it has been shown that alanine and glutamic acids are the C- and Nterminal residues, respectively (Table XV) (2991).These results indicated that, by comparison, the early crystalline preparations involved nearly 10% loss of peptide material, and circular dichroism spectra a t the Soret region of cytochrome b, showed a modification of the heme environment in the cleaved enzyme (2991). 2. Cytochrorne b, Core

Tryptic hydrolysis of cytochrome b, yields a polypeptide fragment which carries the heme and has a molecular weight of approximately 291. C. Jacq and F. Lederer, Eur. J. Biochem. 41,311 (1974).

4.

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267

11,000 (292). This material, designated cytochrome b, core, resembles the whole enzyme in its reduction potential, light absorption, and EPR spectra. Moreover, cytochrome b, core was shown to resemble soluble preparations of liver microsomal cytochrome b, in several respects, including cytochrome absorption spectrum, extinction coefficient, reduction potential, E P R signals at alkaline pH, and proton NMR spectra of the oxidized and reduced preparations (293, 294). Recently, Guiard et al. (295) have shown that the amino acid sequence of cytochrome b, core is very similar to that of microsomal cytochrome b,. They have also indicated that the amino acid sequence of cytochrome b, core is compatible with the peptide chain folding recently determined by others for cytochrome b,, and thus affords a similar heme environment as well. Cytochrome b, is reduced in microsomes by the enzyme cytochrome b, reductase, which is a flavoprotein. I n cytochrome b,, both the flavin and the heme are found in association with the same polypeptide chain. Thus, Guiard et al. (295) considered the possibility of a common ancestral origin for cytochromes b, and b,. They suggested that a pair of genes coding for cytochrome b, and cytochrome b, reductase might have fused in the course of evolution leading to cytochrome b,. 3. Enzymic Properties

Cytochrome b, is stereospecific for L ( + ) -lactate. It also oxidizes other a-hydroxymonocarboxylic acids a t slow rates (280, 298). As electron acceptors ferricyanide, methylene blue, 2,6-dichloroindophenol, 1,a-naphthoquinone 4-sulfonate, and cytochrome c have been used. This wide acceptor specificity is characteristic of a number of flavoproteins, which are generally capable of reducing quinoid structures and ferric compounds (297). However, as will be seen below, cytochrome c is considered to be the physiological electron acceptor for the yeast L-lactate dehydrogenase. Much of the available enzymic work on cytochrome b, has been performed on type I and type I1 enzymes which, as mentioned above, appear to have suffered limited proteolysis and peptide cleavage of the subunits 292. F. Labeyrie, 0. Groudinsky, Y. Jacquot-Armand, and L. Naslin, BBA 128, 492 (1966). 293. H. Watari, 0. Groudinsky, and F. Labeyrie, BBA 131, 592 (1967). 294. R. Keller, 0. Groudinsky, and K. Wiithrich, BBA 328, 233 (1973). 295. B. Guiard, 0. Groudinsky, and F. Lederer, Proc. Nat. Acad. Sci. U . S., 71, 2539 (1974). 296. R. H. Symons and L. A . Burgoyne, “Methods in Enzymology,” Vol. 9, p. 314, 1966. 297. M. Dixon, BBA 226,269 (1971).

268


during purification. These preparations are very unstable and their enzymic properties as compared to crude yeast extracts reflect the structural damage they have sustained during purification (290, 291 ) . Comparative data regarding molar activities, K , values for substrate and cytochrome c, and inhibition by high levels of substrate have been published for the intact and cleaved Saccharomyces enzymes as well as for the intact cytochrome b, isolated from Hansenula anomalu (287, 289-291). It is generally agreed that the rate-limiting step is the transfer of reducing equivalents from substrate to the enzyme, that the initial reaction rate is first order with respect to substrate concentration, that flavin is the first electron acceptor (298-300), and that the transfer of electrons from flavin to the heme occurs intramolecularly (300). Anaerobic titration with L-lactate has indicated that the enzyme accepts three electrons (301). It has also been shown by EPR studies that upon reduction of the enzyme with L-lactate, a flavin semiquinone is formed to the extent of about 20% of the flavin content of the enzyme (301). However, it is not known whether the flavin semiquinone is a kinetic intermediate during enzyme catalysis. Ferricyanide appears to accept electrons from both the flavin and the heme (299-302), and it is believed that heme is required for cytochrome c reduction. Forestier and Baudras (30.2)have reported that, by treatment with guanidinium chloride, preparations of cytochrome b, could be rendered partially deficient in flavin and heme. Thus, enzyme preparations were obtained which contained 65-75% flavin and variable amounts of heme from about 12 to 100%. The low heme preparations showed considerably greater loss of cytochrome c reductase than ferricyanide reductase activity. When preparations with increasing content of heme relative to flavin were tested, both the ferricyanide and the cytochrome c reductase activities increased as a linear function of heme to flavin ratio (up to heme: flavin = 1) , but the increase in the heme content had a much greater effect on the cytochrome c reductase activity of the enzyme. The apoenzyme of cytochrome b , has been prepared. However, reconstitution with FMN, heme, and F M N plus heme in all cases resulted in extremely 298. M. Iwatsubo, A. Baudras, A. di Franco, C. Capeillere, and F. Labeyrie, in “Flavins and Flavoproteins,” 2nd Int. Symp. (K. Yagi, ed.), p. 41. Univ. Park Press, Baltimore, Maryland, 1968. 299. A. Baudras, C. Capeillere-Blandin, M. Iwatsubo, and F. Labeyrie, in “Strbcture and Function of Oxidation Reduction Enzymes” (A. Akeson and A. Ehrenberg, eds.), p. 273. Pergamon, Oxford, 1972. 300. R. K. Morton and J. M. Sturtevant, JBC 239, 1614 (1964). 301. K. Hiromi and J. M. Sturtevant, JBC 240, 4662 (1965). 302. J.-P. Forestier and A . Baudras, in “Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.), p. 599. Univ. Park Press, Baltimore, Maryland, 1971.

4.


269

low activities (SOZ-SO4). Now that intact preparations of cytochrome b, are available, further efforts on these lines might yield clearer results regarding the roles of flavin and heme in the reduction of cytochrome c and artificial acceptors. The kinetics of cytochrome c reduction by L-lactate dehydrogenase are somewhat complicated because the enzyme binds cytochrome c strongly M ) (299, 300, 305). The cleaved enzyme binds one mole of (K, = cytochrome c per mole, but the intact preparations of Haiisenula bind 4 moles of cytochrome c per mole of enzyme, i.e., one mole of cytochrome c per subunit (287). The cytochrome b,-cytochrome c adduct of Saccharoinyces can be crystallized. Examination of the crystals have suggested that the crystal lattice of cytochrome b, can accommodate cytochrome c without an apparent change in the crystal structure (299, 305). The association of cytochrome c with L-lactate dehydrogenase does not depend on the oxidation-reduction state of either cytochrome, and similar to L-lactate protects the enzyme against denaturation by 3 M urea (299, 305). The interaction of cytochrome c with L-lactate dehydrogenase is considered to be specific. I n addition to the above results, i t has been shown that lysozyme, which is similar to cytochrome c in size and charge, does not compete for the binding of cytochrome c to the enzyme (299, 305). The role of L-lactate dehydrogenase in the physiology of aerobic yeast is not clear. It has been shown that its presence in yeast depends on the availability of oxygen (306), and that in the presence of antimycin A, which inhibits electron transfer to cytochrome c from NADH-linked substrates, L-or D-lactate can partially support the growth of Saccharomyces cerevisiae (307). Under these conditions, cyanide inhibited the growth. Therefore, i t has been concluded that L- and D-lactate-cytochrome c reductases can feed electrons to the respiratory chain at the level of cytochrome c and provide energy through the third site of oxidative phosphorylation (30’7).

B. D(-)-LACTATE:CYTOCHROME c OXIDOREDUCTASE (EC 1.1.2.4) This enzyme is tightly associated with the mitochondria of aerobic yeast. Similar to L-lactate: cytochrome c reductase, it is produced in yeast R. K. Morton and K. Sheplcy, Biochem. Z . 338, 122 (1963). M. Mevel-Ninio, P. Pajot, and F. Labeyrie, Biochimie 53, 35 (1971). A. Baudras, M. Krupas, and F. Labeyrie, Eur. J. Biochem. 20, 58 (1971). F. Labeyrie and M. Somlo, “Homologous Enzymes and Biochemical Evolution Colloquium” (Nguyen van Thoai*and J. Roche, eds.), p. 93. Gordon & Breach, New York, 1968. 307. P. Pajot and M. Claisse, Proc. Znt. Congr. Biochem., 9,1973 p . 239 (1973). 303. 304. 305. 306.

270

YOUSSEF HATEFI AND DIANA

L.

STIGGALL

during oxygen adaptation. It was suggested that both the D- .and the L-lactate cytochrome c reductases arise during oxygen adaptation from the D-2-hydroxyacid dehydrogenase of anaerobic yeast. However, this hypothesis has not found experimental support (308-310). 1. Physical Properties D-Lactate: cytochrome c reductasc has been extensively purified from the respiratory particles of bakers’ yeast by two different methods (308, 311-313). One method involves the treatment of particles with acetone and n-butanol, and the other involves treatment with Triton X-100, phospholipase A, and bacterial protcase. The latter method appears to result in greater purification, and higher yield, activity, and stability of the enzyme (312, 313). According to Gregolin and Singer (312), purified preparations of D-lactate: cytochrome c reductase contain 1 mole of FAD per 50,000 5,000 g protein, and 1 g-atom of Zn2+ per 22,000-27,000 g protein. They have concluded that the flavin content and the sedimentation constant of S = 6.8 suggest that the enzyme has a molecular weight of about 100,000 and contains 2 moles of FAD and 6 6 g-atoms of Zn2+ per mole. These conclusions are subject to change, however, because the diffusion constant and the partial specific volume of the enzyme are not known, and partial loss of flavin during purification of the enzyme cannot be ruled out.

*

2. Enzymic Properties D-Lactate :cytochrome c reductase can oxidize D-2-hydroxymonocarboxylic acids, but only D-lactate and D-2-hydroxybutyrate are oxidized at appreciable rates. The enzyme exhibits a similar high specificity for electron acceptors. It reacts with cytochrome c and phenazine methosulfate as electron acceptors, but not with ferricyanide, methylene blue, 2,6dichloroindophenol, and menadione (308, 312, 313). With Dlactate as substrate and a t V,,, with respect to acceptor, phenazine methosulfate is reduced a t 30° eight times as fast as cytochrome c (308). The K , values a t 30° and pH 7.5 are D-lactate, 0.29 mM; ~-2-hydroxybutyrate, 1.4 mM; phenazine methosulfate, 4.5 mM; and cytochrome c, 5.4 p M . The turnover number of the enzyme, isolated with the use of Triton 308. C. Gregolin and T. P. Singer, BBRC 4, 189 (1961). 309. A. P.Nygaard, JBC 236, 1585 (1961). 310. T. P. Singer, E. B. Kearney, C. Gregolin, E. Boeri, and M. Rippa, BBA 54, 52 (1961). 311. A. P.Nygaard, JBC 236, 920 (1961). 312. C.Gregolin and T. P. Singer, BBA 67, 201 (1963). 313. T. P. Singer and T. Cremona, ‘‘Methods in Enzymology,” Vol. 9, p. 302, 1966.

4.

27 1


TABLE XVI I N H I B I T O R S O F D ( -)-LACTATk::

CYTOCHROME

C REDUCTASEa

Concentration (M)

Inhibitor p-Mercuriphenyl sulfonate p-Mercuriphenyl sulfonate H202

Oxalate Oxalate Oxalate EDTA EDTA o-Phenanthroline* o-Phenan throline"

x x I x 6 x 1x 5 x 4 x 1x 3.5 x 3.5 x 5 5

10-7

10-3 10-3 10-8 10-6 10-3 10-3 10-2 10-3 10-3

Inhibition (%) 60 60 0 22 50 92 25 51 95 90

~

From Gregolin and Singer (319). * Overnight dialysis a t p H 6.5 against the indicated concentration of inhibitor. Incubated for 15 min a t 30'. a

X-100 and phospholipase A, is reported to be 90,000 moles lactate/ min x mole flavin at 30° and pH 7.5. The reaction of the enzyme does not appear to be reversible (312). D-Lactate :cytochrome c reductase is inhibited by p-mercuriphenyl sulfonate salts, metal chelators, and dicarboxylic acids such as oxalate and oxaloacetate (Table XVI) (312, 314, 315). According to Nygaard (314), salts (cations) inhibit a t the acceptor site, and dicarboxylic acids a t the substrate site. Cremona and Singer (315) have studied the inhibitions by metal chelators and by oxalate. They recognized two types of inhibition. One type of inhibition is that which is caused by E D T A or oxalate. This kind of inhibition is reversed immediately upon dilution of the enzyme-inhibitor mixture. The second is that which results from addition of o-phenanthrolinc. Enzyme preparations treated with o-phenanthroline bind 2 moles of the chelator per mole of Zn2+.This complex is stable and inactive, and does not result in the release of Zn2+.The inactive o-phenanthroline-enzyme complex can be reactivated by dialysis, addition of divalent metal ions such as Zn2+, Co2+,Mn2+, and Fez+, or by incubation at elevated temperaturcs ( 5 4 5 O ) (312, 3 1 5 5 1 7 ) . It has been shown that heat treatment does not involve the release of o-phenanthroline. The authors suggested that thermal reactivation of the o-phenan314. A. P. Nygaard, JBC 236, 2128 (1961). 315. T. Cremona and T. P. Singer, JBC 239, 1466 (1964). 316. A. Ghiretti-Magaldi, T. Cremona, T. P. Singer, and P. Bernath, BBRC 5, 334 (1961). 317. T. Crernona and T. P. Singer, BBA 57, 412 (1962).

272

POUSSEF HATEFI AND DIANA L. STIGGALL

throline-enzyme complex is the result of a change in the conformation of the enzyme molccule. Other studies have suggested to these authors that Zn?+is involved in the binding of substrate to the enzyme (312). It has been shown that by treatment with ammonium sulfate a t acid pH, flavin can be partially removed from the enzyme (318). Addition of FAD, but not FMN, reactivated the enzyme. Zinc is not removed under these conditions, and its addition is not required for reactivation. The metal appears to be very tightly bound to the enzyme (312) ; its removal without protein denaturation has not been achieved.

c. D-2-HYDROXYACID DEHYDROGENASE (EC 1.1.99.6) It was discovered in 1958 that anaerohically grown yeast contains a form of lactate dehydrogenase which is different from the D- and L-lactate:cytochrome c reductases of aerobic yeast (306, 319). The enzyme has been partially purified (320, 321), and shown to contain flavin (320-322). Gel filtration studies have suggested a molecular weight of about 100,000 (320, 321). Preparations of the enzyme oxidize several D-2hydroxyacids to the respective keto acids in a reversible manner (320). For the forward reaction, ferricyanide, 2,6-dichloroindophenol, menadione, and methylene blue have been used as electron acceptors, and for the reverse reaction leucomethyl viologen and FMNH, are effective electron donors (320).A number of L-2-hydroxyacids and 2-keto acids have been shown to be competitive inhibitors. Oxalate, cyanide, o-phenanthroline, and EDTA are also potent inhibitors (320, 321, 323, 324). The inhibition by metal chelators develops slowly and is reversed by addition of Zn2+,Co2+,Mn2+,or Fez+ (320, 323-326). Substrates prevent the inhibition by chelators a t concentrations considerably lower than their respective K, values (327). It has been suggested that EDTA inactivation involves the removal of a metal, most probably Zn2+,from the substrate binding site of the enzyme (325, 326, 328, 329). However, others have 318. C. Gregolin and T. P. Singer, BBA 57,410 (1962). 319. P. P. Slonimski and W. Tysarowski, C . R. Acad. Sci. 246, 1111 (1958). 320. T. Cremona, JBC 239, 1457 (1964). 321. J. Rytka and W. Tysarowski, Acta Biochim. Pol. 12,229 (1965). 322. M. Iwatsubo, BBA 77, 568 (1963). 323. E. Boeri, T. Cremona, and T. P. Singer, BBRC 2,298 (1960). 324. A. Curdel, L. Naslin, and F. Labeyrie, C. R. Acad. Sci. 249, 1959 (1959). 325. A Curdel and F. Labeyrie, BBRC 4,175 (1961). 326. A. Curdel, C. R . Acad. Sci. 254, 4092 (1962). 327. F. Labeyrie and E. Stachiewicz, BBA 52, 136 (1961). 328. E. Stachiewicz, F. Labeyrie, A. Curdel, and P. P. Slonimski, BBA 50, 45 (1961). 329. M. Iwatsubo and A. Curdel, BBRC 6, 385 (1961).

4.


273

shown that, similar to D-lactate :cytochrome c reductase, EDTA-treated D-2-hydroxyacid dehydrogenase can be reactivated by dialysis or by incubation a t elevated temperatures in the absence of added metals (320, 330, 331). The latter authors believe that chelator treatment results in the formation of an inactive enzyme-chelator complex without the removal of metal. This complex can be reactivated by addition of metal ions or dialysis, which presumably will result in the removal of the chelator, or by heat treatment, which converts the inactive complex to an active form. A similar mechanism has been invoked for the inhibition of D-lactate :cytochrome c reductase by o-phenanthroline. Thus, the presence and possible role of Zn2+and the nature of the flavin prosthetic group of D-Zhydroxyacid dehydrogenase have yet to be unambiguously demonstrated. Howeger, it might be added that the enzyme can be inactivated by treatment with ammonium sulfate a t acid pH, and reactivated by FAD, but not by F M N (329). Further, the flavin in EDTA-inactivated preparations is not reduced by D-lactate, but addition of Zn? results in rapid bleaching at 450 nm (330). These results have been considered as evidence that the flavin prosthetic group is FAD, and that the metal is necessary for the reduction of flavin by substrate. Soluble D-lactate dehydrogenases with enzymic properties similar to those of the D-2-hydroxyacid dehydrogenase of anaerobic yeast have been isolated from rabbit kidney mitochondria (322-334) and from a species of Mycobacterium ( 3 3 5 ) .It is not clear whether these enzymes are metalcontaining flavoproteins.

VII. Nitrite Reductases (EC 1.6.6.4)

Nitrate reduction and assimilation is a fundamental biological process in plants and various microorganisms. In this process nitrate is reduced ultimately to ammonia. Thus, as shown in Eq. ( 5 ) , the reduction of nitrate to ammonia requires eight electron or hydrogen equivalents. HN03

+8H+

NH.7

+ 3H20

(5)

The first reduction product of nitrate is nitrite. This reaction is catalyzed 330. A. Ghiretti-Magaldi, T. Cremona, T. P. Singer, and P. Bernath, BBRC 5, 334 (1961). 331. T. Cremona and T. P. Singer, Nature (London) 194,836 (1962). 332. P. K. Tubbs, BBRC 3, 513 (1960). 333. P. K. Tubbs and G. D. Greville, BJ 81, 104 (1961). 334. P. K. Tubbs, BJ 82,36 (1962). 335. T. Szumilo and M. Szymona, Physiol. Chem. Phys. 4, 407 (1972).

274


by the molybdenum- and FAD-containing enzyme, nitrate reductase, which is discussed in Volume XII, Chapter 6, p. 402. Enzyme systems which catalyze nitrite reduction have been observed in bacteria (336-343), fungi (344-351), green algae (348, 352-355) , and higher plants (344, 356-362). While the assimilatory nitrite reductases convert nitrite to NH,, the denitrifying organisms reduce it to nitric oxide (338, 340, 363) or nitrogen gas (336). Examples of denitrifying nitrite reductases are the enzymes of Pseudomoms denitnficans ( S d O ) , and P . aeruginosa (338, 364-366), which convert nitrite to nitric oxide, and of P. stutzeri (336), which reduces nitrite to NO and N,. The nitrite reductase of P. denitrificuns has been partially purified. The enzyme reduces 336. C. W. Chung and V. A. Najjar, JBC 218,617 (1956). 337. D. Spencer, H. Takahashi, and A. Nason, J. Bacterial. 73, 553 (1957). 338. G. C. Walker and D. J. D. Nicholas, BBA 49, 350 (1961). 339. R. A. Lazzarini and D. E. Atkinson, JBC 236, 3330 (1961). 340. B. C. Radcliffe and D. J. D. Nicholas, BBA 153,545 (1968). 341. 0. Prakash and J. C. Sadana, ABB 148,614 (1972). 342. J. M. Vega, M. G. Guerrero, E. Leadbetter, and M. Losada, BJ 133, 701 (1973). 343. C. D. Cox, Jr. and W. J. Payne, Can. J. Microbial. 19, 861 (1973). 344. A. Nason, R. G. Abraham, and B. C. Averback, BBA 15, 159 (1954). 345. J. Rivas, M. G. Guerrero, A. Paneque, and M. Losada, Plant Sci. Lett. 1, 105 (1973). 346. D. J. D. Nicholas, A. Medina, and 0. T. G. Jones, BBA 37,468 (1968). 347. K. Yamafuji, Y. Osajima, H. Omura, and 8. Hatano, Enzymologia 21, 245 (1960). 348. E. Kessler, Annu. Rev. Plant. Physiol. 15, 57 (1964). 349. K. A. Cook and G. J. Sorger, BBA 177,412 (1969). 350. R. H. Garrett, BBA 264, 481 (1972). 351. M. A. Lafferty and R. H. Garrett, Abstr. 7Jrd. Annu. Meet Amer. Sac. Microbiol. p. 194 (1973). 352. E. Kessler and F. C. Czygan, Experientia 19, 89 (1963). 353. M. G. Guerrero, J. Rivas, A. Paneque, and M. Losada, BBRC 45, 82 (1971). 354. W. G. Zumft, BBA 276, 363 (1972). 355. A. Hattori and I. Uesugi, Plant Cell Physiol. 9, 689 (1968). 356. G. G. Roussos and A. Nason, JBC 235,2997 (1960). 357. R. H. Hageman, C. F. Cresswell, and E. J. Hewitt, Nature (London) 193, 247 (1962). 358. K. W. Joy and R. H. Hageman, BJ 100, 263 (1966). 359. K. Asada, G . Tamura, and R. S. Bandurski, JBC 244, 4904 (1969). 360. J. Cardenas, J. L. Barea, J. Rivas, and C. G. Moreno, FEBS (Fed. Eur. Biochem. Sac.) Lett. 23, 131 (1972). 361. M. J . Dalling, N. E. Tolbert, and R. H. Hageman, BBA 283, 505 (1972). 362. M. J. Dalling, N. E. Tolbert, and R. H. Hageman, BBA 283, 513 (1972). 363. A. Nason, Bacterial. Rev. 26, 16 (1962). 364. T. Yamanaka, A. Ota, and K. Okunuki, BBA 44,397 (1960). 365. T. Yamanaka and K. Okunuki, BBA 67,379 (1963). 366. T. Yamanaka, Nature (London) 204, 253 (1964).

4.


275

nitrite to nitric oxide in the presence of NADH or NADPH and FMN, FAD, or riboflavin. It can also use artificial electron donors, such as reduced benzyl viologen or leucomethylene blue, in the absence of flavins. Inhibitor studies have suggested the involvement of metals and active thiol. Walker and Nicholas (338)have reported the isolation and 600-fold purification of an enzyme from P. aeruginosa, which reduces nitrite to nitric oxide. The preparation contained 1.5 nmoles of FAD per mg protein, a c-type cytochrome and an absorption band a t 630-635 nm, suggestive of copper. As electron donors, reduced FMN, FAD, riboflavin, pyocyanine, and methylene blue were effective, but not NADH, NADPH, or reduced cytochrome c. The preparation required phosphate or sulfate for maximal activity. The cytochrome and the 630-635-nm band were reduced under anaerobic conditions with a suitable electron donor and readily oxidized by nitrite. The K , for NaNO, is reported to be 3.1 X lo-” M . The presence of an active thiol in the enzyme is indicated by p-mercuribenzoate inhibition and glutathione reactivation. Yamanaka and co-workers (364-366) have crystallized a cytochrome oxidase from P . aeruginosa which oxidizes Pseudomonus ferrocytochrome 0551. It is also capable of nitrite reduction with a turnover number of 4000 moles nitrite reduced under anaerobic conditions to nitric oxide per minute a t 37O. It is an adaptive enzyme, nitrate being essential for its biosynthesis. The enzyme has a molecular weight of 120,000, with two subunits of equivalent molecular weight, 2 heme c and 2 heme d groups per mole (Fig. 38) (36%). Nitrite reductase activity is 94% inhibited M KCN, but only by CO. The lack of CO inhibition appears by 8 X to be related to the fact that the enzyme has a greater affinity for nitrite than for carbon monoxide. Nitrite reduction in assimilatory nitrate-reducing Neurospora crassa, Tomlopsis nitratophila, Azotobacter vinelandii, and Azotobacter chroococcum appears to be catalyzed by enzyme systems which require flavin and metals. The enzyme from N . crassa has been partially purified, and its molecular weight has been estimated to be 300,000 (344, 346, 351, 367). The enzyme reduces both nitrite and hydroxylamine to ammonia and utilizes NADH or NADPH as electron donor. It is reported to be a FAD-dependent enzyme and to contain iron, copper, and active thiol (346, 367). Three moles of NADH are oxidized per mole of nitrite reduced to ammonia. It has been suggested that the reduction of nitrite occurs in three steps, each involving two electrons. Thus, hyponitrite and hydroxylamine have been proposed as successive intermediates in the re366a. J. C. Gudat, J. Singh, and D. C. Wharton, BBA 292, 376 (1973); D. C. Wharton, private communication. 367. A. Medina and D. J. D. Nicholas, BBA 25, 138 (1957).

276


Wavelength (nm)

FIG.38. The absorption spectra of crystalline Pseudomonas cytochrome oxidase. The crystals were dissolved in 0,2 M phosphate buffer (pH 7.0). (---) Oxidized, (-) reduced with sodium dithionite. From Yamanaka and Okunuki (366).

duction of nitrite to NH, (36'7). The nitrite reductase of N . crassa is inducible by nitrate or nitrite and repressed by ammonia (350). The nitrite reductase of Torulopsis nitratophila is specific for NADPH and FAD, and can utilize reduced benzyl or methyl viologen as electron donor, but not reduced flavins (345). With NADPH as electron donor, nitrite reduction is inhibited by cyanide and mercurials. Michaelis constants for FAD and nitrite have been reported to be 45 n M and 19 p M , respectively. Unlike the Neurospora enzyme, the nitrite reductase of T. nitratophila could not reduce hydroxylamine in the presence of NADPH and FAD. That hydroxylamine might not be an obligatory intermediate, or occur as a free intermediate, in the reduction of nitrite to ammonia is suggested by the properties of nitrite reductases of Azotobacter chroococcum and Escherichia coli. The former is an adaptive enzyme, the formation of which requires nitrate or nitrite in the culture (342).It is FAD-dependent and presumably contains metals and p-mercuribenaoate inhibitable

4.

METAL-CONTAINING


277

thiols. It reduces nitrite to ammonia in the presence of NADH as electron donor and does not appear to produce hydroxylamine as an intermediate. Cyanide competitively inhibits the reduction of nitrite with a K i= 32 nM. The K , values for nitrite and NADH are 5.5 and 15 p M , respectively. The enzyme is inhibited upon preincubation with NADH. Nitrite protects against NADH inhibition and reverses it. Sucrose density gradient centrifugation has suggested a molecular weight of 67,000 for the A . chroococcum enzyme. The E. coli enzyme can reduce nitrite and hydroxylamine to ammonia a t the expense of NADPH (339). However, with the use of 15N-nitrite it was shown that hydroxylamine was not an intermediate in the reduction of nitrite. No cofactor requirements were shown for the E . coli enzyme, but similar to other flavin and metal requiring nitrite reductases it was inhibited by cyanide and mercurials. The nitrite reductase of Azotobacter vinelandii ( A . agile) was extracted in soluble form by Nason and his colleagues (337). The preparation reduced nitrite and hydroxylamine in the presence of reduced nicotinamide-adenine dinucleotides and required flavin for maximal activity. FAD was shown to be specific for nitrite reduction, whereas both FAD and FMN were active for hydroxylamine reduction. The hydroxylamine reductase activity of the preparation was enhanced in the presence of Mn2+.Ammonia was shown to be the product of nitrite reduction, but the product of hydroxylamine reduction was not identified. Another nitrite and hydroxylamine reductase, which had a MnZ+requirement, was also isolated and partially purified in Nason’s laboratory from soybean TABLE XVII THEPHYSICAL PROPERTIES OF PURIFIED NITRITEREDUCTASE FROM Achromobacter fscherio Sedimentation constant,, slo,w Diffusion coefficient, D ~ o . ~ Molecular weight (Archibald procedure)b Molecular weight (calculated from diffusion and sedimentation constants)b Heme content (nmoles/mg protein) Minimum molecular weight (from heme content) Iron content Minimum molecular weight (from iron content) Isoelectric point

5.2 s 5.56 X lo-’ om2 sec-l 95,000 +_ 4,000 84,000 19 52,500 0.102-0.105% 54,000 Around p H 4,. 5

From Prakash and Sadana (341). In the calculation, a value for the partial specific volume of 0.73 ml/g for nitrite reductase is assumed.

278


I

t

0.9

4 20

I!

i!

Wavelength (nm)

FIQ.39. The absorption spectra of Achromobacter fischeri nitrite reductase. Spectra were recorded in 0.05 1cI phosphate buffer, p H 7.5, at 0.41 mg enzyme protcin/ml. (-) Oxidized, (- * -) reduced with dithionite, (---I NO,- (or hydroxylamine) added to the dithionite reduced enzyme. From Prakash and Sadana (341).

leaves (356).However, the enzyme preparation did not require flavin, but had an absolute requirement for an unidentified, heat-stable factor, which had an absorption peak a t 312 and 315 nm, respectively, in 0.1 N HC1 and 0.1 M pyrophosphate, pH 7.0. The peak shifted to 358 nm in 0.1 N NaOH. The nitrite reductase system of Achromobacter fischeri appears to be composed of two separable enzymes (341). The first enzyme is a flavin reductase and utilizes NADH or NADPH to reduce FMN or FAD. The second interacts with the flavin reductase and converts nitrite and hydroxylamine to ammonia. The nitrite reductase enzyme has a molecular weight of 95,000 f 4,000 (Table XVII), contains two heme c per mole, and is inhibited by p-mercuribenzoate, cyanide, and carbon monoxide.

4. METAL-CONTAINING


279

The latter inhibition is reversed by light. Urea inactivation-reactivation studies showed parallel loss and recovery of nitrite and hyroxylamine reductase activities, and nitrite was shown to inhibit hydroxylamine reduction. These results have suggested that the enzyme has a common binding site for nitrite and hydroxylamine. The absorption spectra of the A . fischeri enzyme (oxidized, reduced, and reduced plus nitrite or hydroxylamine) are shown in Fig. 39.

VIII. Adenylyl Sulfate Reductases (EC 1.8.99.2)

Two major pathways are known for the reduction of sulfate. One is the assimilatory pathway, which reduces sulfate to the extent necessary for satisfying the nutritional requirements of the organism. I n this pathway, which has been extensively studied in yeast by Robbins and Lipmann (368) and Bandurski and his colleagues (369, 370), sulfate is first activated in the presence of ATP by the enzyme ATP-sulfurylase t o form adenosine 5'-phosphosulfate (APS). Then in a second reaction, APS is phosphorylated in the 3' position by ATP to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) ATP APS

+ Solz+ ATP

+ +

APS PP PAPS ADP

(6) (7) In the presence of appropriate enzymes, the sulfate group of PAPS can be donated to various acceptors, such as carbohydrates, steroids and phenols, or become reduced to sulfite for assimilatory purposes. Figure 40 shows a unified scheme for sulfate and sulfite assimilation by algae as proposed by Abrams and Schiff (371). The second pathway by which sulfate is reduced is the dissimilatory pathway in which sulfate is the terminal electron acceptor and leads to the formation of large quantities of H,S. During the dissimilatory reduction of sulfate, APS is formed as in Eq. ( 6 ) . Then APS is reduced directly to sulfite and AMP by the enzyme APS-reductase. Table XVIII shows the data of Peck (372) on the pathway of sulfate reduction in various microorganisms. Adenylyl sulfate (APS) reductase is a flavoprotein, which contains iron and possibly acid-labile sulfide. It catalyzes the reduction of APS in the -+

+

368. P. W. Rohbins and F. Lipmann, JACS 78, 6409 (1956). 369. L. G. Wilson, T. Asahi, and R. S. Bandurski, JBC 236, 1822 (1961). 370. K. Torii and R. S. Bandurski, BBA 136, 286 (1967). 371. W. R. Abrams and J. A. Schiff, Arch. Mikrobiol. 94, 1 (1973). 372. H. D. Peck, Jr., J. Bacteriol. 82, 933 (1961).

SULFATE ESTERS

h3

TRANSFERASES

m

*

0

I

I

n

I

I

-o-s-o-2 ;

;

SUFATE OUTSIDE

1 1 1

[Cor-s-]

-0-2-0-

0

I

SUFATE INSIDE

.

SULFURYLASE

!

P-PI

I

I

-

FERREDOXIN OXIDIZED

AMP

'R-S-

I I

X

I

t

--+-I

E -0-s - 0SULFITE OUTSIDE

FIG.40. A proposed unified scheme of sulfate assimilation in algae. Adenylyl sulfate (APS) transfers the sulfo group via APS-sulfotransferase to form Car-SSO; (Car = carrier), which is reduced further by thiosulfonate reductase to Car-SS- which yields the thiol group of cysteine. In addition, if sulfite is released from Car-S-SOa- (i.e., by thiol or from mutated sites) or if it enters the cell from outside, i t can be reduced via a separate sulfite reductase. From Abrams and Schiff (371).

r

4.

281


TABLE XVIII PATHWAY OF SULFATE REDUCTION I N VARIOUS TYPES OF MICROORGANISMS~ APS reductaseb Met,hyl viologenc

Organism

PAPS reductaseb Methyl viologen

NADPH

0.4

2.4

3.7

0.0

3.4

2.8

0 0.6 0 0 0 0 0 0 0 0

0 5.6 0.1 3.3 4.0 12.2 0.1 8.7 0.3 0

1.1 7.3 8.9 0.6 21.4 0 0 0 0

0 0.4 0

0 0.1

1. Assimilatory sulfate reducers

Escherichia coli (grown aerobically) E. coli (grown anaerobically) Yeast Aerobacter aerogenes Proteus mirabilis P . vulgaris Pseudomonas hydrophila Aeromonas punctata Clostridium kluyveri C. pasteurianum Rhodopseudomonas spheroides R . palustris 2. Dissimilatory sulfate reducers Desulfovibrio desulfuricans Clostridium nigrificans Vibrio cholinicus 3. Sulfur oxidizers Thiobacillus thioparus T . thiooxidans T . denitri’cans Chromatium sp.

1,750 310 907

0

640 162 1,260 0.3

From Peck (372). Specific activity is expressed aa nanomoles of acid-volatile sulfur formed per hr per mg protein. No activity was observed with NADPH. a

presence of an appropriate electron donor [reduced methyl viologen, or reduced cytochrome c3 in Desuljovibrio vulgaris (373, 37Sa) ] to sulfite and AMP [Eq. ( S ) ] . APS

+ 2e

S0a2-

+ AMP

(8) It can also catalyze the reverse reaction when ferricyanide or cytochrome c is used as electron acceptor (374, 375). The phosphosulfate bond of 373. H. D. Peck, Jr., Proc. N a t . Acad. Sci. U . S. 45,701 (1959). F!

373a. D. V. DerVartanian and J. LeGall, B B A 346,79 (1974). 374. H. D. Peck, Jr., B B A 49, 621 (1961). 375. R. M. Lyric and I. Suzuki, Can. J. Biochem. 48, 344 (1970).

282


APS is energy-rich (AGO = 18-19 kcal/mole). Therefore, the reversal of reaction (8) is rather interesting, because it can capture oxidation energy and convert it to a biologically utilizable form. For example, the enzyme ADP-sulfurylase can catalyze the synthesis of ADP from APS and inorganic phosphate as shown in Eq. (9) (376, 377). AD P-sulfurylase

APS

+ Pi,

'

ADP

+ SOa2-

(9)

APS reductase is found in dissimilatory sulfate reducing bacteria, such as Desulfovibrio and Desulfotomaculum, in certain Thiobacilli, in Thiocapsa roseopersicina, and in the alga Chlorella pyrenoidosa. Table XIX, compiled by Schiff (378), gives the properties of various APS reductases from plants and microorganisms. I n Thiobacilli and Desulfovibrio, APS reductase constitutes as much as 1-5% of the cell protein, which suggests the important role of this enzyme in the metabolism of these organisms (375). The APS reductase of Desulfovibrio vulgaris has been extensively studied by Peck and his co-workers. The enzyme is reported to have a molecular weight of 220,000, and to contain 1 mole of FAD and 6-8 g-atoms of nonheme iron per mole (379). The oxidized and reduced absorption spectra of the enzyme are shown in Fig. 41. Spectrophotometric studies have shown that in the absence of AMP the enzyme is partially bleached between 350 and 500 nm upon addition of sulfite. The rate of bleaching achieved with sulfite was shown by stopped-flow kinetic measurements to be comparable to the turnover number of the enzyme when sulfite oxidation was assayed in the presence of ferricyanide as electron acceptor. These findings, plus the increased absorption of the sulfitetreated enzyme a t 320 nm, have suggested to Peck and co-workers that sulfite oxidation involves the interaction of sulfite with the enzyme to form a flavin-sulfite adduct in position N-5 of the isoalloxazine ring (379). The authors pointed out that these results are analogous to the data of Massey and co-workers (380, 381) on the effect of sulfite on various flavoproteins. The latter authors found similar spectral changes when sulfite was added to glucose oxidase, D- and L-amino acid oxidases, oxynitrilase, lactate oxidase, and glycollate oxidase. They concluded that the flavoproteins which are capable of interacting with oxygen (APS re376. H. D. Peck, Jr., JBC 237, 198 (1962). 377. H. D. Peck, Jr., T. E. Deacon, and J. T. Davidson, BBA 96, 429 (1965). 378. J. A. Schiff and R. C. Hodson, Annu. Rev. Plant Physiol. 24, 381 (1973). 379. G. B. Michaels, J. T. Davidson, and H. D. Peck, Jr., BBRC 39,321 (1970). 380. V. Massey, F. Miillcr, R. Feldberg, M . Schurnan, P. A . Sullivan, L. G. Howell, S. G. Mayhew, R. G. Matthews, and G. P. Foust, JBC 244, 3999 (1969). 381. F. Miiller and V. Massey, JBC 244, 4007 (1969).

TABLE XIX PROPERTIES OF ADENYLYL SULF.ATEREDUCTASES FROM PLANTS A N D MICROORGANISMS~

Organism Bacteria Desulfovibrio vulgaris T hiobacillus thioparus

Enzyme

Electron donor or acceptor

PH optimum

K,

MW

Remarks Contains 1 mole FAD, 6-8 g-atoms nonheme iron

APS reductase

Fe(CN)?

7.4

S032-,2 m M

220,000

APS reductase

Fe(CN)F

7.4

170,000

APS reductase

Cytochrome c

9.5

APS reductase

Fe(CN)2-

7.2

APS reductase

Fe(CN)63-

8.0

APS reductase

Cytochrome c

9.0

S032-, 2.5 m M AMP, 0.1 m M S032-, 0.017 m M AMP, 0.0025 m M SO3$-, 1.5 m M AMP, 0.041 m M S032-, 1.5 m M AMP, 0.073 m M S032-, 0.093 m M AMP, 0.059 m M

Fungi Saccharom yces cerevisiae

PAPS reductase

NADPH

7.5 (tris)

Algae Chlorella p yrenaidosa

APS reductase

Thiol

Thiobacillus denitri’cans Thiocapsa roseopersicina

a

From Schiff and Hodson (378).

-

170,000 -

180,000 180,000

Contains 1 mole FAD, 8-10 g-atoms nonheme iron Contains 1 mole FAD, 6-11 g-atoms nonheme iron Contains 1 mole FAD, 4 gatoms nonheme iron, 2 gatoms heme iron. Purified 60-80-fold ; homogeneous upon ultracentrifugation Partially purified into 3 fractions A, B, C. Some activity with APS. Fraction A purified 60-fold, fraction C to apparent homogeneity in ultracentrifugation

-

330,000

Partially purified. PAPS is active in the presence of a 3’-nucleotidase

284


o’6 0.5

1 -

0.4

-

w. C

5 03-

::

n

a

0.2-01 0.1

-

300

3K)

460

450

500

550

600

nrn

FIa. 41. Absorption spectrum of purified APS reductase from Desulfovibrio vulA : difference spectrum obtained from tracing of “oxidized” and “reduced” enzyme. Insert B: spectrum obtained after boiling APS reductase and removing protein by centrifugation. From Peck et al. (377). garis. The enzyme concentration was 2.5 mg/ml. Insert

ductase reacts slowly with oxygen) can form a flavin-sulfite adduct, and that the N-5 position of the isoalloxazine ring is very likely involved. Addition of AMP to the sulfite-treated APS reductase resulted in further bleaching between 350 and 500 nm. Peck et al. (382, 383) have shown by EPR spectroscopy near liquid helium temperature that addition of either sulfite or AMP alone does not result in the formation of an iron signal a t g = 1.94. However, when AMP and sulfite are added together, a g = 1.94 signal is produced, which is approximately 80% of that obtained when the enzyme is reduced with dithionite. Thus, the authors suggested that APS reductase catalyzes an intramolecular electron transfer during sulfite oxidation as shown in Fig. 42 from Peck et al. ( 382).

Whereas Peck and his co-workers have not reported the presence of acid-labile sulfide in the APS reductase of D.vulgaris, Lyric and Suzuki (376) have shown that the enzyme from Thiobacillus thioparus contains 4-5 moles of labile sulfide per mole. The T . thioparus enzyme appears to have a molecular weight of 170,000, and contains, in addition to labile sulfide, 1 mole of FAD and 8-10 g-atoms of iron per mole. That the en382. H. D. Peck, Jr., R. Bramlett, and D. V. DerVartanian, 2. Nuturforsch. B 27, 1084 (1972). 383. R. N. Bramlett and H. D. Peck, Fed. Proc., Fed. Amer. SOC.E z p . Biol. 32, 668 (1973).

4.

METAL-COKTAINING FLAVOPROTEIN DEHYDROGENASES

X

=

285

nonheme iron centers

FIG.42. A proposed mechanism for APS reductase. From Peck e t al. (388).

zyme of Peck et al. very likely contains labile sulfide is suggested both by its absorption spectrum and by its characteristic iron-sulfur signal centered a t g = 1.94. Another APS reductase of interest is that which has been isolated by Triiper and Roger (384) from Thiocapsa roseopersicina. The enzyme is reported to have a molecular weight of 180,000 and to contain 1 mole of flavin (presumably FAD), 4 g-atoms of nonheme iron, 6 moles of labile sulfide, and 2 c-type hemes per mole. The spectral properties of the enzyme are shown in Fig. 43. It utilizes cytochrome c and ferricyanide as 0.7

1

iL17nm

Wavelength (nm)

FIG.43. Absorption spectra of the purified APS reductase from Thiocapsa roseopersicina: ox, oxidized enzyme; red, enzyme reduced with 1 mg sodium dithionite per ml. From Triiper and Rogers ( 3 8 4 ) . 384. H. G. Truper and L. A. Rogers, J. Bacterial. 108, 1112 (1971).

286


electron acceptors, and the reaction to cytochrome c is especially sensitive to thiol inhibitors. The heme groups of the enzyme are suggested to be involved in electron transfer from sulfite to added cytochrome c. However, it has not been shown that these heme groups can be reduced by treatment of the enzyme with substrate ( 3 8 4 ~ ) .

IX. Sulfite Reductases (H,S:NADPH Oxidoreductases) (EC 1.8.1.2)

As pointed out in the preceding section, sulfate assimilation in yeast has been shown to involve the activation of sulfate by ATP successively to adenosine 5’-phosphosulfate and once again to 3’-phosphoadenosine 5’-phosphosulfate. The latter is then reduced in the presence of NADPH to sulfite and 3’,5’-diphosphoadenosine (372). Enzymes catalyzing the 6-electron reduction of sulfite to sulfide have been observed in bacteria (339,385-397), yeast (398-401), fungi (402-404), and higher plants (359, 405). These enzymes may be divided into two classes depending on 384a. Dr. H. G. Truper has informed us that the APS reductase of Chlorobium limicola, recently purified in his laboratory, does not contain any heme groups, but is otherwise similar to the APS reductases of sulfate reducing bacteria and Thiobacilli. 385. M. Ishimoto, J. Koyama, and Y. Nagai, J . Biochem. (Tokyo) 42, 41 (1955). 386. J. Mager, BBA 41,553 (1960). 387. J. Dreyfuss and K. J. Monty, JBC 238, 3781 (1963). 388. J. M. Akagi, BBRC 21, 72 (1965). 389. J. LeGall and N. Dragoni, BBRC 23, 145 (1966). 390. L. M. Siegel and H. Kamin, in “Flavins and Flavoproteins,” 2nd Int. Symp. (K. Yagi, ed.), p. 15.Univ. Park Press, Baltimore, Maryland. 1968. 391. N. Gilboa-Garber and J. Mager, BBA 220,602 (1970). 392. P. A. Trudinger, J . Bncteriol. 104, 158 (1970). 393. W.D. Hoeksema and D. E. Schoenhard. J . Bacteriol. 108, 154 (1971). 394. L. M.Siegel, H. Kamin, D. C. Rueger, R. P. Presswood, and Q. H. Gibson, in “Flavins and Flavoproteins,” 3rd Int. Symp. (H. Kamin, ed.), p. 523. Univ. Park Press, Baltimore, Maryland, 1971. 395. K. Kobayashi, E.Takahashi, and M. Ishimoto, J . Biochem. (Tokyo) 72, 879 (1972). 396. J.-P. Lee, J. LeGall, and H. D. Peck, Jr., J . Bacteiiol. 115, 529 (1973). 397. L. M. Siegel, M. J. Murphy, and H. Kamin, JBC 248, 251 (1973). 398. T. Wainwright, BJ 83, 39P (1962). 399. N. Naiki, Plant Cell Physiol. 6,179 (1965). 400. A. Yoshimoto and R. Sato, BBA 153, 555 (1968). 401. K. Prabhakararao and D. J. D. Nicholas, BBA 180,253 (1969). 402. A. Yoshimoto, T. Nakamura, and R . Sato, J . Biochem. (Tokyo) 50,553 (1961). 403. A. Yoshimoto, T . Nakamura, and R. Sato, J . Biochem. (Tokyo) 62, 756 (1967). 404. L. M. Siegel, F. J. Leinweber, and K. J. Monty, JBC 240, 2705 (1965). 405. G. Tamura, J . Biochem. (Tokyo) 57,207 (1965).

4. METAL-CONTAINING


287

COOH I

FIQ.44. Postulated structural formula for the siroheme prosthetic group. From Murphy et al. (418).

whether or not they can use reduced nicotinamide adenine dinucleotide (specifically NADPH) for the reduction of sulfite. The NADPH-sulfite reductases appear to contain flavin, nonheme iron, acid-labile sulfide, and a novel heme (extractable by acid acetone) of the isobacteriochlorin type with characteristic a-absorption peak a t 582-589 nm. This heme, in which two adjacent pyrrole rings are reduced, has been named “siroheme” (Fig. 44). The sulfite reductases, which cannot utilize NADPH as reductant, are generally of smaller molecular weight, do not require flavin, but exhibit the cytochrome-like absorption peaks comparable to those of the siroheme-containing enzymes. Sulfite reduction by this group of enzymes is usually studied in the presence of appropriate dyes (e.g., reduced methyl viologen) as electron donors. Enzymic and genetic studies have suggested that NADPH-sulfite reductases are composed of a flavoprotein (NADPH dehydrogenase) , and a hemoprotein (sulfite reductase) which can utilize reduced methyl viologen as electron donor. A. NADPH-SULFITEREDUCTASES NADPH-sulfite reductases are found in E . coli (386, 390, 391, 397, 406-416)) Salmonella typhimurium (587, 394, 417-419)) yeast (598-401, 406. F. J. Leinweber and K. J. Monty, BBA 63, 171 (1961). 407. L. M. Siegel and H. Kamin, “Methods in Enzymology,” Vol. 17B, p. 539, 1971. 408. L. M. Siegel, E. J. Faeder, and H. Kamin, 2.Naturjorsch. B 27,1087 (1972).

288


420-424), and Neurospora crassa (404, 425, 426). The E. coli enzyme

has been purified and extensively studied by Kamin, Siegel, and their colleagues (390, 397, 407-416). The enzyme has a molecular weight of 670,000, and contains 4 moles of FAD, 4 moles of FMN, 20-21 g-atoms of iron, 14-15 moles of acid-labile sulfide, and 3 4 moles of heme per 670,000 g protein (390, 397). The absorption spectrum of E. c d i NADPH-sulfite reductase is shown in Fig. 45. The oxidized enzyme (trace A ) has absorption maxima a t 278, 386, 455, 587, and 714 nm. The 455-nm peak results largely from flavin and is bleached upon treatment of the enzyme with NADPH (trace B) or dithionite (trace C ) . Electron paramagnetic resonance studies have shown a signal centered a t g = 6, which is characteristic of high-spin ferric heme, and only under special conditions a signal a t g = 1.94, characteristic of an iron-sulfur center, has been observed (413). The enzyme catalyzes electron transfer from NADPH to sulfite, nitrite, hydroxylamine, cytochrome c, ferricyanide, dichloroindophenol, menadione, FMN, FAD, and molecular oxygen. It is also capable of transhydrogenation from NADPH to acetylpyridine adenine dinucleotide phosphate, and electron transfer from reduced methyl viologen (MVH) to sulfite, nitrite, hydroxylamine, or NADP. All the NADPH-dependent reductions, except the reduction of acetylpyridine adenine dinucleotide phosphate, are inhibited by p-mercuriphenyl sulfonate, but not the reduction of sulfite, nitrite, and hydroxylamine by MVH. The reduction of the latter compounds by NADPH or ~

~~~

409. M. J. Murphy, L. M. Siegel, H. Kamin, D. V. DerVartanian, J.-P. Lee, J. LeGall, and H. D. Peck, Jr., BBRC 54, 82 (1973). 410. M. J. Murphy and L. M. Siegel, JBC 248, 6911 (1973). 411. M. J. Murphy, L. M. Siegel, and H. Kamin, JBC 248, 2801 (1973). 412. M. J. Murphy, L. M. Siegel, S. Tove, and H. Kamin, Proc. Nut. Acad. Sci. U. S. 71, 612 (1974). 413. L. M. Siegel, P. S. Davis, and H. Kamin, JBC 249, 1572 (1974). 414. L. M. Siegel and P. S. Davis, JBC 249, 1587 (1974). 415. E. J. Faeder, P. 9. Davis, and L. M. Siegel, JBC 249, 1599 (1974). 416. M. J. Murphy, L. M. Siegel, and H. Kamin, JBC 249, 1610 (1974). 417. J. Dreyfuss and K. J. Monty, JBC 238, 1019 (1963). 418. L. M. Siegel, E. M. Click, and K. J. Monty, BBRC 17, 125 (1964). 419. L. M. Siegel and K. J. Monty, BBRC 17,201 (1964). 420. T.Wainwright, BJ 103, 56p (1967). 421. A. Yoshimoto and R. Sato, BBA 153,576 (1968). 422. K.Prabhakararao and D. J. D. Nicholas, BBA 218, 122 (1970). 423. A. Yoshimoto and R. Sato, BBA 220, 190 (1970). 424. A. Yoshimoto, N. Naiki, and R. Sato, “Methods in Enzymology,” Vol. 17B, p. 520, 1971. 425. F. J. Leinweber, L. M. Siegel, and K. J. Monty, JBC 240, 2699 (1965). 426. F.J. Leinweber and K. J. Monty, JBC 240, 782 (1965).

4.


so

I 400

I 450

I

MO

I 5

~

I

I

)600

650

289

mo

Wavelength (nm)

FIG.45. Absorption spectra of E . coli sulfite reductase in the presence of reducing agents. All experiments contained enzyme at a final concentration of 1.54 p M in the sample cell. Spectra were recorded versus a buffer blank as soon as possible after addition of components. A, enzyme in buffer; B, enzyme plus 0.3 mM NADPH (0.1 ml of 23.1 fiM enzyme was added to 1.4 ml of a solution of NADPH which had been bubbled with N, for 30 min); and C, enzyme plus sodium dithionite. From Siege1 et al. (397).

MVH is inhibited by CO, cyanide, arsenite, and sulfide. Carbon monoxide, cyanide, and arsenite react only with the reduced enzyme. Spectral modifications of the heme and other results have indicated that the heme is the site of action of these inhibitors as well as the site a t which sulfite, nitrite, and hydroxylamine are reduced. The Michaelis constants of the enzyme for sulfite and NADPH are both about 4-5 ,AM. Treatment of E . coli sulfite reductase with p-mercuriphenyl sulfonate results in the specific release of F M N from the enzyme (390). FMNdepleted sulfite reductase can be prepared also by photodestruction of FMN. The enzyme-FMN dissociation constant is 10 n M a t 2 5 O , and light irradiation can deplete the enzyme of F M N by destroying the released flavin. These treatments do not lead to removal or destruction of other components of the enzyme. The FMN-depleted enzyme is no longer capable of NADPH-dependent reduction of sulfite, nitrite, hydroxylamine,

290


and diaphorase-type acceptors such as ferricyanide, cytochrome c , and menadione. However, it is fully capable of the reduction of acetylpyridine adenine dinucleotide phosphate by NADPH, and the reduction of sulfite, hydroxylamine, and nitrite by MVH. Further, the remaining flavin (essentially FAD), but not the heme, is still reducible by NADPH as rapidly as the most rapidly reduced flavin of the native enzyme (k = 190 sec-l) . These results and kinetic studies (416) have indicated that FAD is probably the first acceptor of reducing equivalents from NADPH, that FMN is the link between FAD and heme as well as the site of reduction of diaphorase-type acceptors, and finally that the heme is the last component of the enzyme t o be reduced. The FMN-depleted sulfite reductase can be reactivated by added FMN, FAD, and a number of other flavins (413, 415).

By treatment with 5 M urea and chromatography on DEAE-cellulose, it has been possible to dissociate the E. coli NADPH-sulfite reductase into a flavoprotein and a hemoprotein fraction. The flavoprotein fraction has been shown to be an octamer of a single polypeptide of molecular weight 58,000-60,000 and to contain F M N and FAD in equimolar amounts, but no heme, nonheme iron, or labile sulfide. The hemoprotein fraction is a tetramer of a polypeptide of molecular weight 54,000-57,000, and contains heme, nonheme iron, and labile sulfide, but no flavin. Thus NADPH-sulfite reductase is considered to be an enzyme of asp4 subunit composition. The amino acid composition of the whole enzyme and the flavoprotein and hemoprotein fractions have been determined (414). The hemoprotein fraction has no NADPH-dependent activities, but reduces sulfite in the presence of MVH. The flavoprotein fraction catalyzes electron transfer from NADPH to diaphorase-type acceptors and to acetylpyridine adenine dinucleotide phosphate. It does not reduce sulfite, nitrite, or hydroxylarnine with either NADPH or MVH as electron donor. The molecular weight of the flavoprotein is estimated to be 470,000 (two-thirds of the whole enzyme). A similar flavoprotein with a molecular weight of 460,000has been isolated from a S. typhimurium mutant, which requires cysteine for growth. Other genetic data on the S. typhimurium enzyme (994), which appears to be essentially identical to the E. coli sulfite reductase, are in agreement with the above results. Thus mutants lacking the flavoprotein or the hemoprotein component of the enzyme and containing only the appropriate partial activities have been obtained and the respective partial enzymes isolated. The absorption spectra of sulfite reductase preparations from the wild type and from these mutants are shown in Fig. 46, and the proposed structure for the two components of the wild-type enzyme is shown in Fig. 47.Reconstitution of NADPH-sulfite reductase by recombination of the flavoprotein

4.


291

FIG.46. Comparison of the absorption spectra of wild-type and mutant (cys G-439 and cys 1-68) sulfite reductases from Salmonella typhimurium. Spectra of S. typhimurium sulfite reductase, cys G-439 NADPH-cytochrome c reductase, and cys 1-68 NADPH-cytochrome c reductase, each dissolved in 0.05 M potassium phosphate buffer, pH 7.7, containing 0.1 mM EDTA, were read against a blank containing only buffer. The spectrum of each enzyme is presented in terms of its millimolar extinction coefficients, assuming 8 moles of flavin per mole of enzyme. Light broken line, calculated difference spectrum between those of wild-type and cys G enzymes when both enzyme solutions contain equal concentrations of flavin. From Siegel et al. (394).

and hemoprotein fractions separated by urea treatment has been achieved (414). Similarly, appropriate partial enzymes isolated from S. typhimuSum mutants have been recombined in vitro to reconstitute NADPHsulfite reductase activity.

cyt c

T

MV

FIG.47. Schematic diagram of the proposed structure and function of the S. typhimurium NADPH-sulfite reductase and its component “subenzymes.” From Siegel et al. (394).

292


co

pCMPS

- -

NADPH. AcPyADP’ , NADP’

FAD

FMN

1

MVH

/

\ *

CN AsO;

,’

Heme

- SO,’-. NOz-. NH,OH

Diaphorase Acceptors. 0 2

FIG.48. Proposed minimum linear scheme of electron flow within the sulfite reductase molecule. The dotted arrow between FMN and heme indicates that the mechanism of electron flow from flavin to heme is not clear. From Siege1 et al. (413).

The above results are summarized in the scheme shown in Fig. 48. Thus, the NADPH-sulfite reductase of enterobacteria appears to be composed of an octameric flavoprotein and a tetrameric hemoprotein, which also contains iron and labile sulfide. The flavoprotein contains 4 moles of FAD and 4 moles of F M N per mole, and appears to bind 1 mole of NADP per mole of FAD. Electron transfer occurs from NADPH to FAD to FMN, and the two flavin sequence is considered to be a device for “stepping down” a two-electron donor, NADPH, to a one-electron acceptor, the heme (413). This is in agreement with the findings that flavin free radical seems to appear after full reduction of the flavins, and that the rate of FH. formation is too slow for the radical to serve as electron donor in the diaphorase reactions (390). The flavoprotein segment catalyzes electron transfer to the hemoprotein, to diaphorase-type acceptors, and to acetylpyridine adenine dinucleotide phosphate. The latter reduction does not require the presence of FMN. The hemoprotein accepts electrons from the flavoprotein or from appropriate dyes and in turn reduces sulfite, nitrite, and hydroxylamine, apparently by direct electron transfer through the heme. The role of iron and labile sulfide is not clear. They might be involved in electronic communication between F M N and the heme. It is also possible that electrons from MVH enter the system a t the level of the iron and labile sulfide. The iron and labile sulfide are likely associated in the form of clusters found in iron-sulfur proteins. However, unlike most iron-sulfur proteins, these clusters appear to be resistant to destruction by mercurials (397). Another interesting point is that it has been suggested that both the heme and the iron-sulfur moieties of NADPH-sulfite reductase have reduction potentials considerably more negative than that of the electron donor, NADPH (415). The NADPH-sulfite reductase of S. cerevisiae (398-401, 420-424) has properties similar to the reductase from enterobacteria. The enzyme has been purified to near homogeneity by Yoshimoto and Sato (400). It contains 1 mole each of FAD and FMW and 5 g-atoms of iron per 350,000

4.


293

g protein, and a hemelike chromophore with absorption peaks a t 386 and 587 nm. The oxidized enzyme is greenish yellow, and its spectrum (Fig. 49) is very similar to that of the E . coli sulfite reductase. The yeast enzyme catalyzes the reduction of sulfite, nitrite, and hydroxylamine by NADPH or MVH, and the reduction of diaphorase-type acceptors (Le., quinones and ferric compounds) by NADPH. The NADPH-dependent activities are inhibited by NADP, 2’-AMP, and p-mercuribenzoate. The Michaelis constants for NADPH, sulfite, nitrite, and hydroxylamine are 18-21 p M , 14 p M , 1 mM, and 4.5 mM, respectively. The NADPH-treated enzyme is inhibited by carbon monoxide or cyanide (390,401). The latter treatment results in the formation of a reddish violet color with peaks a t 397 and 411 nm. Cyanide and carbon monoxide are considered to react with the heme moiety and inhibit the enzyme a t the site of reduction of sulfite, nitrite, and hydroxylamine. Yoshimoto and Sat0 (421) have isolated sulfite reductases from four mutants of S. cerevisiae incapable of sulfite assimilation. These enzymes were inactive for sulfite reduction by NADPH, but could utilize MVH as electron donor. All the mutant enzymes contained the chromophore responsible for the 386- and 587-nm peaks, nonheme iron, and labile sulfide. Three of these mutant enzymes contained F M N ; no flavin was detected in the fourth. The sedimentation coefficients of these preparations

1 2a

!i

e

:: 9 1.0

1

300

4 00

500

600

Wavelength (nm)

FIG.49. Absorption spectra of purified NADPH-sulfite reductase from ’Saccharomyces cerevisiae. Curve A : 3.38 mg of enzyme protein in 2.0 ml of 0.3 M potassium phosphate (pH 7.3) containing 1 mM EDTA. Curve B: a mixture containing 3.38 mg of enzyme protein, 0.2 pmole of NADP, 10 pmoles of glucose 6-phosphate, 8 units of glucose-6-phosphate dehydrogenase, 0.3 M potassium phosphate buffer (pH 7.3), and 1 mM EDTA in n final volume of 2.0 ml was incubated anaerobically for 60 min. The reference cell contained all the components except sulfite reductase. From Yoshimoto and Sat0 (400).

294


were 5.1 S for the enzyme lacking FMN, 6.6 S for the three enzymes containing FMN, and 14.8 S for the wild-type enzyme. The authors have concluded, therefore, that the yeast sulfite reductase is composed of a t least three components, one each carrying FAD, FMN, and the heme. The FAD-containing component is the site of NADPH oxidation, and the heme-containing component the site of sulfite (also nitrite and hydroxylamine) reduction, Thus, the mutant enzymes lacking the former component can reduce sulfite only in the presence of an artificial electron donor such as MVH which could reduce both F M N and the heme (Fig. 50). These conclusions regarding the yeast sulfite reductase are essentially in agreement with our current knowledge of the mechanism of sulfite reduction by NADPH and MVH in the E. coli enzyme. Since the reduction of sulfite to sulfide is a six-electron reaction, twoelectron reduction steps may be written as

so2- + (SO,2-)

(sol-)

(15) However, in both the yeast and the E. coli systems, the stoichiometries for NADPH:S2- and S03*-:S2- are 3 : l and 1:1, respectively. These results and the inability to detect 2-electron- and 4-electron-reduced intermediates in these systems have suggested that such intermediates, if present at all, must be firmly held on the surface of the enzyme. It has further been suggested that the presence of multiple flavins and hemes in the enzyme might be a device for achieving a rapid six-electron reduction of sulfite without the release of intermediates (414). This situation is analogous to the four-electron reduction of 0, to 2H,O by cytochrome oxidase and the six-electron reduction of nitrite to ammonia by various assimilatory nitrite reductases. However, unlike cytochrome oxidase,

-

NADPH-reacting site

,4,8

FAD (1)

i

FMN

(n)

+

--f

52-

M$=reacting site

j 587 Chromophore

;

(rn)

Wild-type

Strain 6 , l l and 20

5,,

I

587 Chromphore

cm,

Strain 21

FIG.50. Schematic illustration of a tentative relationship between NADPH-sulfite reductase from the wild-type strain of Saccharomyces cerevisiae and two categories of MVH-sulfite reductases from various mutant strains. From Yoshimoto and Sat0 (421).

4.


295

which does not seem to reduce H,O,, several nitrite and sulfite reductases can reduce hydroxylamine to ammonia. This fact indicates that these sulfite and nitrite reductases are capable of catalyzing a two-electron reduction reaction. Indeed, sulfur compounds of oxidation states between sulfite and sulfide have been observed during sulfite reduction by MVHsulfite reductases (395).

B. REDUCED METHYLVIOLOGEN-SULFITE REDUCTASES The methyl viologen-sulfite reductases have been isolated from Aspergillus nidulans (4OZ, .4OoS), Desulfotomaculum nigrificans (392),Desulfovibrio gigas (389, 427), Desulfovibrio vulgaris (396), and from higher plants, such as spinach (359) and Allium odorum (405). These sulfite reductases are incapable of utilizing NADPH or NADH as electron donor. With the possible exception of the sulfite reductase of A . nidulans, they also appear to lack flavin. They all exhibit, however, absorption maxima characteristic of siroheme. Indeed, it has been shown by Murphy and his colleagues (410, 412) that a number of sulfite and nitrite reductases appear to contain the siroheme-type tetrahydroporphyrin. In addition to heme, the sulfite reductase preparation of D.nigrificans also contains nonheme iron, labile sulfide, and zinc. I n general, the methyl viologen-sulfite reductases appear to have lower molecular weights than the NADPH-sulfite reductases. For the enzymes from D . nigrificans, spinach leaves, and D. vulgaris, the reported molecular weights are, respectively, 145,000, 84,000, and 26,800. The physiological electron donor for the MVH-sulfite reductases is not known. However, similar to the ferredoxin-nitrite reductases, certain MVH-sulfite reductases have been shown to use ferredoxin as electron donor (388, 389; see also 373a).

X. Addendum

This additional material is intended to bring to the readers’ attention the recent major developments. For easy identification, the following comments are marked by the same section designations to which they pertain in the text of the chapter.

II,A,3 SDS-Acrylamide gel electrophoresis of the soluble NADH dehydrogenase derived from complex I has shown that this enzyme is composed of two subunits with molecular weights of approximately 28,000 and 56,000 (G. Dooijewaard and E. C. Slater, private communication). 427. H. D. Peck, BBRC 22, 112 (1966).

296


II,A,6 Hatefi et al. (428) have shown that incubation of submitochondrial particles or complex I with 2,3-butanedione, in the presence of borate buffer a t pH 9.0, inhibits the NADPH to NAD transhydrogenase activity with little or no effect on the NADH and NADPH dehydrogenase activities. Presence in the incubation mixture of NAD, NADP, and more effectively NAD NADP, prevented the inhibition of transhydrogenase activity by butanedione. Since butanedione specifically reacts with protein arginyl residues, these findings agreed with the sensitivity of the transhydrogenase activity to trypsin (see Fig. 17) and suggested that the nucleotide-binding site of the transhydrogenase enzyme contains a susceptible arginyl residue. II,A,7 Ohnishi (429) has published revised Em values for the ironsulfur centers of complex I. These values for iron-sulfur centers 1, 2, 3 and 4 a t pH 7.2 are center 1, component a, -380 -+ 20 mV; center 1, component b, -240 -C 20 mV; center 2, -20 20 mV; center 3, -240 20 mV; center 4, -410 f 20 mV. Additional centers ( 5 and 6 with Em value of -260 f 20 mV) are also claimed by Ohnishi to exist in the NADH-ubiquinone segment of the respiratory chain. III,A According to Ohnishi and collaborators (430, 4 3 l ) , succinate dehydrogenase preparations which are capable of electron transfer to the respiratory chain contain 3 iron-sulfur centers, designated centers S-1, S-2 and 5-3. The Em values of these centers a t pH 7.4 have been given as follows: center S-1, O r + 10 mV; center 5-2, -4OOk 15 mV; center 15 mV. Centers S-1 and S-2 are thought to contain one Fe,S2 S-3, +SO cluster each. Center 5-3 is considered to contain one Fe,S, core, and to be the oxygen-sensitive center necessary for electron transfer from succinate dehydrogenase to the respiratory chain. The findings of Ohnishi and co-workers reported here and above have not yet been confirmed by other groups. VI,A A communication from L. Lederer has pointed out that the N-terminus of the intact cytochrome b, chain is Asn, not Glu, (cf. Table XV). Also a note has appeared from the same laboratory (432) on additional similarities between cytochrome b, and liver microsomal cytochrome b, (cf. Section VI,A,2).

+

*

*

*

428. Y. Hatefi, L. Djavadi-Ohaniance, and Y. Galante, in “Electron-Transfer Chains and Oxidative Phosphorylation” (E. Quagliariello, et al., eds.) . NorthHolland Publ., Amsterdam (in press). 429. T. Ohnishi, BBA 387, 475 (1975). 430. T. Ohnishi, D. B. Winter, J. Lim: and T. E. King, BBRC 61, 1017 (1974). 431. T. Ohnishi, J. S. Leigh, D. B. Winter, J. Lim, and T. E. King, BBRC 61, 1026 (1974). 432. B. Guiard, F. Lederer, and C. Jacq, Nature (London) 255, 422 (1975).

4.

METAL-CONTAINING


297

VII According to Husain and Sadana (433), the earlier preparation of Achromobacter fischeri nitrite reductase with a molecular weight of 95,000 2 40,000 (341) was found to be polydisperse. A monodisperse preparation subsequently studied (433) had a molecular weight of 80,000, and was shown to be composed of two subunits of approximate molecular weights of 39,000 with methionine as the sole N-terminal residue. The subunits are stated to be linked together by disulfide bridges. I n a private communication, Henry Kamin has indicated to us that (a) J. Vega, R. H. Garrett and L. Siege1 have demonstrated recently that the nitrite reductase of Neurospora has siroheme as its prosthetic group, and have given us permission to cite this new finding. Kamin has further suggested that we emphasize the fact that enzymic properties and patterns of repression and derepression clearly show that the E. coli nitrite reductase is distinct from the sulfite reductase of this organism, which is also capable of nitrite reduction (Section IX,A). VIII Recent data of Bramlett and Peck (434) indicate that, as predicted in the above review, the adenylyl sulfate reductase of Desulfovibrio vulgaris does contain acid-labile sulfide. The enzyme with a molecular weight of 220,000 has been shown to contain 1 mole of FAD, 12 g-atoms of iron and 12 moles of labile sulfide per mole. I n addition, SDS-gel electrophoresis has revealed the presence of subunits with molecular weights of 20,000 and 72,000. X A new flavoprotein, containing iron and labile sulfide, has been discovered in the mitochondria1 inner membrane independently by Ruzicka and Beinert (435) and Hatefi et al. (436).The protein contains acid-extractable FAD, and 4 g-atoms of iron and 4 moles of labile sulfide per mole of flavin. The molecular properties and the enzymic function of this iron-sulfur flavoprotein are not clear.

ACKNOWLEDGMENTS The authors are grateful to the investigators whose work has been reviewed for kindly providing them with reprints and preprints in advance of publication. They also wish to thank Mrs. C. Schaeggl for typing the manuscript. The work of this laboratory reported in Sections I1 and I11 was supported by USPHS grants AM08126 and CA13609 to Y. H.

M. Husain and J. Sadana, Rur. J. Biochem. 42, 283 (1974). R. N. Bramlett and H. D. Peck, Jr., JBC 250, 2979 (1975). F. J. Ruzirka and H. Beinert, BBRC 66, 622 (1975). Y. Hatefi, Y. M. Galante, D. L. Stiggall, and L. Djavadi-Ohaniance, in “The Structural Basis of Membrane Function” ( Y . Hatefi and L. Djavadi-Ohaniance, edu.), p. 169. Academic Press, New York, 1976. 433. 434. 435. 436.


Cytochrome c Oxidase WINSLOW S. CAUGHEY WILLIAM J . WALLACE JOHN A . VOLPE SHINYA YOSHIKAWA 1. Introduction . . . . . . . . . . . . . . . . 299 A . The Role of Cytochrome c Oxidase in Biological Systems . 299

B . History . . . . . . . . . . . . . . . C . The Chemical and Physical Properties of Cytochrome c Oxidase . . . . . . . . . . . . . . D . The Chemistry of Oxygen Reduction . . . . . . I1. Isolation and Characterization . . . . . . . . . . A . Preparation . . . . . . . . . . . . . B . Metal Components . . . . . . . . . . . C . Protein . . . . . . . . . . . . . . . D . Lipids . . . . . . . . . . . . . . . I11. Chemical and Physical Properties . . . . . . . . . A . Models . . . . . . . . . . . . . . . B. Electronic Spectroscopy . . . . . . . . . . C . Ligand Binding Studies . . . . . . . . . . D . Potentiometry . . . . . . . . . . . . . E . Electron Paramagnetic Resonance Studies . . . . F . Interaction of Cytochrome c Oxidase with Cytochrome c G . Kinetic Studies . . . . . . . . . . . . IV . Mechanisms . . . . . . . . . . . . . . . .

.

300

. 301

. . . .

. . .

. . . .

. . . .

302 305 305 307 309 312 313 314 315 319 325 329 334 335 337

I. Introduction

c OXIDASEIN BIOLOGICAL SYSTEMS A. THEROLEOF CYTOCHROME

Cytochrome c oxidase. the terminal oxidase in the respiratory metabolism of all aerobic organisms. plants. animals. yeasts. algae. and some bacteria. is responsible for catalyzing the reduction of dioxygen to water . 299

300

W. S. CAUGHEY, W. J. WALLACE, J. A. VOLPE, AND S. YOSHIKAWA

The electrons are provided by reduced cytochrome c in the following overall reaction: 02

+ 4 cyt c*+ + 4 H+ -+

2 H20

+ 4 cyt c3+

(1) The free energy developed in oxygen reduction is used to promote oxidative phosphorylation and, in consequence, becomes available, as ATP, to satisfy the energy requirements of the cell. It is not surprising then that this oxidase is found in high concentrations in tissues where the energy requirements are high. Especially high levels have been observed in heart muscle ( I ) , flight muscles of birds (2) and insects (S),red skeletal muscles ( 4 ) , liver mitochondria ( 5 ) , brain gray matter (6),corpora lutea of sheep (Y), the parasitic worm Ascaris (8),and sugar cane roots ( 9 ) .It is estimated that 90% of the energy for heart muscle contraction (1) and 96% of the energy for bird flight muscle contraction (2) is provided through aerobic metabolism via cytochrome c oxidase. Chronic muscular disease gives rise to oxidase depletion (10). A further indication of its importance in the energetics of biological systems is the suggcstion by Malmstrom (11) that 90% of biological oxygen consumption is directed through the oxidase. The locus of this activity is in the inner membrane of mitochondria in eukaryotes and in the plasma membrane of prokaryotes.

B. HISTORY In 1886, MacMunn ( 1 2 ) discovcred the respiratory pigment, myohematin, which was widely distributed in plant and animal tissues. This important observation attracted little attention a t the time of its publication and became cffectively lost in the literature (IS). I n 1925, Keilin (14) 1. D. R. Challoner, N a l w e (London) 217, 78 (1968). 2. A. Tucker, Science 154, 150 (1966). Ser. B 98, 312 (1925). 3. D. Keilin, Proc. R o y . SOC., 4. M. S. Gordon, Science 159, 87 (1968). 5. D. L. Drabkin, Physiol. R e v . 31, 345 (1954). 6. S. Manocha and G. H. Bourne, E x p . Brain Res. 2, 230 (1966). 7. L. Arvy and P. Mauleon, C . R . SOC.Biol. 158, 453 (1964). 8. M. H. Smith, N a l w e (London) 223, 1129 (1969). P . R . 50, 131 (1966). 9. A. G. Alexander, J. Ag?. Unit!. 10. F. W. Booth and J. R. Kelso, Can. J. Physiol. Phurmucol. 51, 679 (1973); V. P. Andrcev, Dokl. Akad. Nauk Beloniss. SSR 17, 470 (1973). 11. B. G. Malmstrom, Quart. Reu. Biophys. 6,389 (1973). 12. C. A. MacMunn, Phil. Trans. R o y . Soc. London 177, 267 (1886). 13. D. Keilin. in “The History of Cell Respiration and Cytochromes” (J. Keilin, ed.), p. 95. Cambridge Univ, Press, London and New York, 1966. 14. D. Keilin, Proc. R o y . SOC.,Ser. B 98, 312 (1925).

5.

CYTOCHROME C OXIDASE

301

rediscovered the MacMunn pigment, proved it to be a mixture‘ of three spectroscopically identifiable components which he named cytochromes a, b, and c, and showed them to be links in the respiratory chain that connected activated substrates to activated dioxygen. Cytochromes a and c showed a special relationship to each other; cytochrome a was the sole physiological oxidizing agent for cytochrome c. Hence, the name cytochrome c oxidase ( 1 5 ) . The ligand binding and autoxidizability studies seemed compatible with the presence of two components, cytochromes a and as, of which only as was considered to be autoxidizable and able to combine with carbon monoxide or cyanide. Keilin and Hartree (16) identified this a3 component spectroscopically with the Atinungsjerment which Warburg had shown on the basis of the photochemical action spectrum of its CO complex to be a heme protein ( 1 7 ) .Further advances in the understanding of this important enzyme were not to come for another 25 years until renewed interest and improved isolation techniques paved the way for further progress. Lemberg (18) and Lemberg and Barrett (19) have summarized the development of this understanding. Less extensive reviews have been provided recently by Wharton (20) (emphasizing the role of copper) , by Nicholls and Chance (21) (emphasizing kinetic measurements) , and by Malmstrom (11) (emphasizing physicochemical measurements). AND PHYSICAL PROPERTIES OF CYTOCHROME c C. THECHEMICAL OXIDASE

The focus of this article will be upon those aspects of the structure and function of cytochrome c oxidase that contribute particularly to an understanding of the chemical events that lead to the reduction of dioxygen to water. This important function is, however, only one aspect of its physiological role. The functioning enzyme is provided with electrons from the electron transport chain by cytochrome c, uses these electrons to reduce dioxygen bound at the active site, communicates the energy released in this reduction to the site of oxidative phosphorylation, 15. 16. 17. 18. 19. 1972. 20. 21.

D. Keilin and E. F. Hartree, Proc. Roy. Soc., Ser. B 121, 173 (1936). D. Keilin and E. F. Hartree, Nature (London) 141, 870 (1938). 0. Warburg and €3. Negelein, Biochem. 2.214,64 (1929). R. Lemberg, Physiol. Rev. 49, 48 (1969). R. Lernberg and J. Barrett, “The Cytochromes.” Academic Press, New York,

D. C. Wharton, Metal Zons Biol. Syst. 3, 157 (1974). P. Nicholls and B. Chance, in “Molecular Mechanisms of Oxygen Activation” (0. Hayaishi, ed.), p. 479. Academic Press, New York, 1974.

302

W. S. CAUGHEY, W. J . WALLACE, J . A. VOLPE, AND S. YOSHIKAWA

and is strictly controlled in these functions by respiratory control processes. The structure of the oxidase and its placement in the cell reflect the multiplicity of roles it is required to play. The active site of the enzyme, which contains iron, as heme A, and copper, is the locus for oxygen binding and reduction; thus, it is necessary to provide pathways to get electrons in and energy out of the active site. The multisubunit lipoprotein in which the active site is embedded is itself embedded in the inner mitochondria1 membrane in such a way that it, along with NADH dehydrogenase and possibly cytochrome b, spans the membrane ( 2 2 ) . Thus, a t each site of energy conservation there appears to be an electron carrier which spans the entire membrane. Perhaps this transmembranous configuration provides an extended surface for interaction with electron carriers and may also provide for an important interaction with ATPase. Such an interaction is supported by the observation of Wilson et al. (23,24) that binding of ATP to ATPase influences the redox potentials and ligand binding characteristics of oxidase. Thus, cytochrome c oxidase appears very carefully tuned to a variety of specific tasks. The components are so adjusted that a low energy pathway is available to entering electrons, an efficient nonthermal energy transport pathway is available for energy conservation, and a set of electron donors has been assembled into an array that will permit facile reduction of dioxygen via an efficient low energy pathway that is unprecedented in simple systems. Nonenzymic reduction of dioxygen to water is often slow and usually involves a complex series of steps (25-27). The enzymic reaction is fast and appears to be accomplished in either a single step or in a series of concerted steps; no evidence for intermediate reduction products (i.e., superoxide or peroxide) has been found.

D. THECHEMISTRY OF OXYGEN REDUCTION The chemical inertness of dioxygen a t first seems surprising because the transformation to water is so strongly thermodynamically favorable ( 4 3 0 kcal) (Fig. 1 ) (28, 2 9 ) . However, on the basis of the standard redox potentials, the simplest reduction step, the one-electron step to 22. E. Racker, Hosp. Pract. p. 87 (1974). 23. J. G. Lindsay and D. F. Wilson, Biochemistry 11, 4613 (1972). 24. D. F. Wilson and K. Fairs, A B B 163, 491 (1974). 25. C. T. Mathews and R. G . Robins, Australas. Znst. Mining Met., Proc. C31 242, 47 (1972). 26. D. V. Stynes, H. C. Stynes, J. A. Ibers, and B. R. James, JACS 95, 1142 (1973). 27. I. A. Cohen and W. S. Caughey, Biochemistry 7,636 (1968). 28. M. S. Tsao and W. K. Wilmarth, Advan. Chem. Ser. 36, 113 (1962). 29. K. Sehested, 0. L. Rasmussen, and H. Fricke, J . Phys. Chem. 72,626 (1968).

5.

303


m

Q-%O$B%

d*%V

B A2OV

+0.82V

FIG.1. Standard oxidation-reduction potentials for the steps involved in the conversion of dioxygen to water at 25” and pH 7. superoxide, is thermodynamically highly disfavored (SO). Hence, reactions involving dioxygen must either have enormous driving energies to go through the superoxide or have access to a two-electron step to peroxide. Although this conclusion depends upon reasoning based upon standard potentials (SO) (all concentrations 1 M and pH 7), i t seems valid since oxygen reduction by a low energy pathway is found to proceed via the two-electron reduction to peroxide as the first recognizable product (31) .

The other property of dioxygen that contributes to the slowness of its reactions is its electronic structure (3.2). I n common with most stable molecules dioxygen has an even number of electrons. Uncommonly, though, the molecule is paramagnetic with two unpaired electrons in the two highest occupied molecular orbitals. Since both peroxide and oxide are completely spin paired, reactions involving dioxygen must involve spin reversal and are therefore spin forbidden and slow. The forbiddenness can be removed if dioxygen can interact with a paramagnetic center to participate in exchange coupling. The transition metal ions frequently have unpaired electrons and turn out to be excellent catalysts for dioxygen reduction. Despite the long history of transition metal ion induced reduction of dioxygen surprisingly little is known about the mechanism of the reaction ( 2 5 ) .Perhaps the most studied of these metal ion oxygenation reactions is that between the nitrogen ligand complexes of cobalt (11) and dioxygen. The traditional method for preparing cobalt (111) ammine complexes was to assemble the desired ligands on cobalt(I1) and oxygenate the solution. Upon long oxygenation a cobalt (111) complex was formed ( 3 3 ) .It is now known that the first step in this reaction is the formation of an unstable Co”02 complex (34) and upon standing, a second cobalt(I1) ion is added to produce the p-peroxo bridged complex [reactions (2) and (3)1. I n the 30. P. M. Wood, FEBS (Fed. Ew. Biochem. Soc.) Lett. 44, 22 (1974) ; P. George, in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), p. 3. Wiley, New York, 1965. 31. R. G. Wilkins, Adwnn. Chem. Ser. 100, 111 (1971). 32. H. Taube, J . Gen. Physiol. 49, 29 (1965). 33. A. Werner, 2. Anorg. Chem. 3, 267 (1893). 34. J. Simplicio and R. G. Wilkins, JACS 89,6092 (1967).

304

W. S. CAUGHEY, W. J. WALLACE, J.

A. VOLPE,

AND S. YOSHIKAWA

presence of a second bridging ligand, such as amido or hydroxo, the p-peroxo complex is stabilized and can be isolated (36) [reaction ( 4 ) ] .

con-0,

+ con

-

com-0,

L

corn- 0,0-corn

(3)

0-co"'

coF0NL\ -o,Coul

L = NR, or OR

(4)

I n the absence of the second bridging ligand, further oxidation occurs by a series of, as yet, not completely understood steps (36) and the cobalt (111) product results [reaction ( 5 )1. coin -0,

0-co'"

-

2 Com-OH

(5)

I n a similar way chromium(I1) (37) and copper(1) (38)react with dioxygen by way of a p-peroxo intermediate. And, of special relevance to cytochrome c oxidase, the reactions of hemes (27, 39, 40) as well as simple aquo ferrous iron (26, 41) with dioxygen seem to proceed through two-electron reduction of bridged intermediates. Thus, dipyridine hemes react cleanly with dioxygen to form py-FeII-py-

(7)

+ py-Fen-py-Fem-O,-Fem-py

(8)

py-FenI-O~Fem-pyt++Fdn-O-Fenl Fem-O, FeIV-0.

(6)

+ py

-py-Fen-O,

py-Fen py-FeI1-O0,

+ 0,

py-Fen

0 Fen'

+

-2

+

2 py

+

py

(9)

Fe"-O.

py-Ferl-Feln-O-Fem

(11)

35. M. Mori and J. A. Weil, JACS 89, 3732 (1967). 36. L. G. Stadtherr, R. Prados, and R. B. Martin, Znorg. Chem. 12,1814 (1973). 37. T. B. Joyner and W. K. Wilmarth, JACS 83, 516 (1961). 38. C. DeMarco, S. Dupre, C. Crifo, G. Rotilio, and D. Cavallini, ABB 144, 496 (1971). 39. I. A. Cohen and W. S. Caughey, in "Hemes and Hemoproteins" (B. Chance, R. W. Estabrook, and T. Yonetani, eds.), p. 577. Academic Press, New York, 1966. 40. W. S. Caughey, J. L. Davies, W. H. Fuchsman, and S. McCoy, in "Structure and Function of Cytochromes" (K. Okunuki, M. D. Kamen, and I. Sekuzu, eds.), p. 20. Univ. of Tokyo Press, Tokyo, 1968. 41. P. George, JCS p. 4349 (1954).

5.

305


p-oxobishemins (42) as shown by reactions (6) through ( 9 ) . Kinetic data fully support reactions ( 6 ) through (8) but have not yet provided information on the steps from the bridged oxygen species (presumably p-peroxobishemin) to p-0x0 dimer. When the solvent medium is able to provide protons, solvolysis to produce H,O, would likely follow formation of the p-peroxobishemins complex and sequence ( 6 ) , ( 7 ) , and (8). However, where solvolysis cannot occur, as in aprotic solvents, formation of the ferry1 (FexVO)intermediate as suggested by reactions (10) and (11) is reasonable (40). But in no case does our certain knowledge about the mechanism of the reduction reaction extend beyond the bridged dimer. Apparently the peroxide formed in the initial reaction is a kinetically inert (fully spin-paired) molecule that does not readily accept additional electrons despite the favorable thermodynamics for reduction to water (Fig. 1). The most commonly suggested mechanism (43) for the subsequent reduction steps in protic media is shown by reactions (12), (13), and (14). Mi--O\ MI- 0, 0-H MI--+

H+

0-MI

+

M;+

+

H,O, -MIOH

MI--,

0- H

+

M:

+ M,OH+

M,-o+

+

0,

+

H+

(13)

(14)

It is anticipated that despite the specially favored environment provided for oxygen reduction by the protein the fundamental principles of chemistry in simple systems will apply to the enzyme. Thus, any proposed mechanism for the enzymic reduction of dioxygen will have to accommodate two electron steps leading sequentially to peroxide and water and provide a means to overcome the characteristic stability of the peroxide intermediate. II. Isolation and Characterization

A. PREPARATION In general cytochrome c oxidase has been isolated from mitochondria or mitochondria1 fragments by initial extraction of proteins with a sur42. N. Sadasivan, H. I. Eberspaecher, W. H. Fuchsman, and W. S. Caughey, Biochemistry 8,534 (1969). 43. M. L. Kremer, Trans. Faraday Soc. 59, 2535 (1963); E. Zidoni and M. L. Kremer, ABB 161, 658 (1974).

SPECIFIC

ACTIVITIESO F CYTOCHROME

C

TABLE I OXIDASE PREPAR.%TIONS

FROM

DIFFERENTISOL.4TION

Conditions of assay

Preparation

Buffer

PH

Yonetani Griffiths and Wharton Okunuki et al. Horie and Morrison Sun and Jacobs Wainio Fowler et al. Kuboyama et al. I-olpe and Caughey

0.050 M phosphate

5.9 7.0 5.95

a

Data not provided.

0

0.075 M phosphate

0 . 1 0 M phosphate 0.070 M phosphate 0.10 M phosphate

pl

Specific activity Initial concn. cytoTemp. chrome c*+ 25 38 25

D

6.0 6.0 6.0 5.7 5.9

4

PROCEDURES

25 25 25 23 22

15 18 15 24 15

pg

protein/ml 6.6 0.55 1.81 1.22

( I

22 20 15

0.5 0.5 0.33

s-l/mg protein/3 ml 4.50 2.70 5.20 4.70 0.08 6.7 14.3 16.0 15.4

Ref.

44 45 46

47 48 43 60 51 5.9

5.


307

face-active agent followed by removal of contaminating detergent and protein (Table I ) , (44-52). Procedures differ in the nature and amount of detergent used and retained. Recently, a preparation with high s o h bility in detergent-free media was reported ( 5 2 ) . The preparations obtained vary widely in activity (Table I ) , and this suggests that the detailed manipulative history is critical to the “nativeness” of the isolated enzyme. Nevertheless, there is good reason to believe that the oxidases isolated by many procedures are similar, but not identical, in response to chemical and physical probes (18).

B. METALCOMPONENTS

It is now widely agreed that both copper and iron are essential components (52-56). The metal content (11 nmoles/mg protein) and the iron to copper ratio (1.0) are well established for the bovine enzyme, whereas in yeast the reported metal contents are higher and more variable (5-15 nmoles of iron per milligram of protein) and the copper to iron ratio is greater than unity (-1.5) (Table 11) (44-46, 48-52, 57-59). The iron is present as the unusual heme, heme A, with an apparently unique structure (Fig. 2) (60). The coordination environment of copper is far less clear, but the easy reducibility of copper seems to require a ligand envi44. T. Yonetani, JBC 236, 1680 (1961). 45. D. E. Griffiths and D. C. Wharton, JBC 236, 1850 (1961). 46. K. Okunuki, I. Sekuzu, T. Yonetani, and S. Takemori, J . Biochem. (Tokyo) 45, 847 (1958). 47. S. Horie and M. Morrison, JBC 238, 1855 (1963). 48. F. F. Sun and E. E. Jacobs, BBA 143,639 (1967). 49. W. W. Wainio, JBC 239, 1402 (1964). 50. L. R . Fowler, S. W. Richardson, and Y. Hatefi, BBA 64, 170 (1962). 51. M. Kuboyama, F. C. Yong, and T. E. King, JBC 247, 6375 (1972). 52. J. A. Volpe and W. S. Caughey, BBRC 61,502 (1974). 53. D. E. Griffiths and D. C. Wharton, JBC 236,1857 (1961). 54. H. Beinert, in “Biochemistry of Copper” (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), p. 213. Academic Press, New York, 1966. 55. E. C. Slater, B. F. Van Gelder, and K. Minnaert, in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 667. Wiley, New York, 1965. 56. W. W. Wainio, in “Oxidasas and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 622. Wiley, New York, 1965. 57. M. S.Rubin and A. Tzagoloff, JBC 248, 4269 (1973. 58. T. L. Mason, R. 0. Poyton, D. C. Wharton, and G. Schatz, JBC 248, 1346 ( 1973). 59. P. G. Shakespeare and H. R. Mahler, JBC 246,7649 (1971). 60. W. S. Caughey, G. A. Smythe, D. H. O’Keeffe, J. Maskasky, and M. L. Smith, JBC 250, 7602 (1975).

308


TABLE I1 COMPOSITION OF CYTOCHROMF: c OXIDASE PREPARATIONS

Preparation

Species

Yonetani Griffiths and Wharton Okunuki et al. Wainio Fowler et al. Sun,and Jacobs Kuboyama et al. Volpe and Caughey Rubin and Tzagoloff Mason et al. Shakespeare and Mahler

Bovine Bovine

0

Bovine Bovine Bovine Bovine Bovine Bovine

Copper (nmole/mg protein)

Iron (nmole/mg protein)

a

7.2 8.2-9.4

9.2-10.6 a

11.0

10.0 11.5 8.4-8.7 8.2 11.1 10.9

Yeast

21.3

15.0

Yeast Yeast

15.8 6.2-11.6

9.4 5.5-7.2

(I

9.4 n

11.8

Phospholipid (%) Ref. 10 24 (I

9 a

22 20 20 3.8

44 46 46 49 60 48 61

68

67

2

68

a

69

Data not provided.

ronment that stabilizes Cu(1) relative to Cu(I1). Thus, sulfur might serve as a ligating atom (as in a disulfide) (61) as may an interaction with the T system of the long side chain of the heme (6‘2). The latter possibility is attractive because it provides a role for the uiiusual side chain by affording a mechanism for electronic coupling between iron and copper (Fig. 3 ) . Whatever the nature of the binding forces, the copper must be well sequestered by the protein because it is not readily moved by the usual complexing agents [EDTA, BCS (62a), and CN-] (53),and there is no evidence that any ligand or any inhibitor binds directly to copper a t the active site (53, 63-65). In addition, it is now clear that there are two quite distinct kinds of copper. One kind is observed by electron paramagnetic resonance (EPR) to be rapidly reduced. The other 61. P. Hemmerich, in ‘LBiocl~emistry of Copper” (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), p. 15.Academic Press, New York, 1966. 62. W. S. Caughey, Adunn. Chem. Ser. 100,248 (1971). 62a. BCS = bathocuproin sulfonate or 2,9-dimethy1-4,7-diphenyl-l,lO-phenanthroline sodium disulfonate. 63. Q.H.Gibson and C. Greenwood, JBC 240,2694 (1965). 64. K.J. H.Van Buuren, P. F. Zuurendonk, B. F. Van Gelder, and A. 0. Muijsers, BBA 256, 243 (1972). 65. J. A. Volpe, M. C. O’Toole, and W. S. Caughey, BBRC 62, 48 (1975).

5.

309


H2

F\

Y3

7H2 04\OH

04bi

FIG.2. The structure of heme A.

1

FIG.3. Schematic representation of a possible conformation of the 2-alkyl group of heme A.

kind is not detected directly spectroscopically, but is reduced slowly ( 6 6 ) . Similar observations suggest that, despite the fact that the only heme is heme A, two kinds of iron are present ( 6 6 ) .These observations must mean that the minimal functional unit contains two iron atoms and two copper atoms.

C. PROTEIN The 11 nmoles of iron per milligram of protein of bovine preparations corresponds t o an empirical molecular weight of about -90,000. When 20% lipid is added a total of 108,000 is obtained. If the functional unit contains two iron and two copper atoms, the minimum molecular weight then becomes -200,000 with the additional possibility that multiples of 66. C. R. Hartzell, R. E. Hansen, and H. Beinert, Proc. N u t . Acad. Sci. U S . 70, 2477 (1973).

310

W . S. CAUGHEY, W. J . WALLACE, J. A. VOLPE, AND 9. YOSHIKAWA

TABLE I11 AMINO ACID COMPOSITION OF CYTOCHROME c OXIDASE Number of residues Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine" Phenylalanine NH8 Tryptophan Ethanolamine Total residues Molecular weightc Molecular weightd a

Kuboyama el al."

Matsubara rt alSh

28 20 21 52 51 53 52 48 53 55 7 45 13 40 79 29 43 63 27

39 30 31 60 53 A4 60 46 59 62 7 51 35 43 87 33 47 59 30

716 80,054 89,400

827 93,802

From Kuboyama et al. (61).

* From Matsubara rt al. (YO). c

d

Based on amino acid composition; the prosthetic groups have not been included. Based on heme content.

this minimum unit could also be observed. Direct physical measurements of the molecular weight have led to values of 72,000 for an inactive preparation (67) and about 200,000 (68, 69) and 430,000 (51) for active preparations. An empirical molecular weight for protein alone of about 90,000 (51,70) was suggested by amino acid analysis of two preparations 67. R S. Criddle and R. M. Bock, BBRC 1,138 (1959). 68. A. Tzagoloff, P. C. Yang, D. C. Wharton, and J. S. Reiske, BBA 96, 1 (1965); W. W. Wainio, T. Laskawska-Klita, J. Rosmm, and D. Grebner, J. Bioenerg. 4, 455 (1973). 69. B. Love, S. H. P. Chan, and E. Stotz, JBC 245,6664 (1970). 70. H. Matsubara, Y. Orii, and K. Okunuki, BBA 97,61 (1965).

5.


311

(Table 111).Extraneous protein and variable amounts of lipid can result in differences in determined molecular weight values. However, a molecular weight of about 200,000 is widely accepted for functioning oxidase with 20% lipid. Nevertheless, treatment with alkali (69) or with sodium dodecyl sulfate (71) has been reported to result in a molecular weight of about 100,000 for a monomer (72) which exhibited activity. Such a catalytically active monomer has been difficult to reconcile with different roles for each of two coppers and of two hemes. For this reason, the dimer is still broadly considered the normal form of the native oxidase. Subdivision below the monomer level occurs in the presence of sodium dodecyl sulfate and thiols (mercaptoethanol). The oxidase is thus identified as a multisubunit protein. Both yeast (57, 73) and Neurosporu crussu (74) oxidases were shown to be composed of seven subunits. Bovine heart oxidase, on the other hand, has been reported to have between two (75, 76) and six (57, 76) subunits. The subunits from yeast have molecular weights in the range of I, 40,000; 11, 33,000; 111, 22,000; IV, 14,000; V, 12,700; VI, 12,700; and VII, 4,600 (57, 7 7 ) . The situation for bovine heart is less clear but the six subunits are reported to have molecular weights around I, 40,000; 11, 25,000; 111, 19,000; IV, 14,000; V, 10,000; and VI, 8,000 ( 7 6 ) .When fewer than six subunits are found, their molecular weights invariably correspond to some of the six reported (75-78). The subunit sizes differ for yeast and bovine heart ( 5 7 ) .That the protein compositions differ is also reflected in the failure of antibodies against subunits I1 and VI of yeast oxidase to cross-react with bovine heart oxidase ( 7 9 ) . In both yeast (80) and Neurosporu crassa (74) biosynthetic studies have shown that the four small polypeptides are of cytoplasmic origin while the three large polypeptides are of mitochondria1 origin. Similar studies are unavailable for bovine heart. The polypeptide associations of the copper and heme are still far from clear, but hints that the metals 71. Y. Orii, Y. Matsumura, and K. Okunuki, in “Oxidases and Related Redox Systems” (T. E. King, H. S. Mason, and M. Morrison, eds.), p. 666. Univ. Park Press, Baltimore, Maryland, 1973. 72. S. H. P. Chan, B. Love, and E. Stotz, JBC 245,6669 (1970). 73. R. 0. Poyton and G. Schatz, JRC 250,752 (1975). 74. W. Sebald, W. Machleidt, and J. Otto, Eur. J. Biochem. 38, 311 (1973). 75. H. Komai and R. A. Capaldi, FEBS ( F e d . Eur. Biochem. SOC.) Lett. 30, 273 (1973). 76. T. Yamamoto and Y. Orii, J. Biochem. ( T o k y o ) 75, 1081 (1974). 77. R. A . Capaldi and H. Hayashi, FEBS (Fed. Eur. Biochem. SOC.) Lett. 26, 261 (1972). 78. J. J. Keirns, C. S. Yang, and M . V. Gilmour, BBRC 45,835 (1971). 79. R. 0. Poyton and G. Schatz, JBC 250, 762 (1975). 80. T. L. Mason and G. Schatz, JBC 248, 1355 (1973).

312

W. S. CAUGHEY, W. J. WALLACE, J. A. VOLPE, AND S . YOSHIKAWA

are coupled with the low molecular weight subunits have appeared (76, 78, 81). The picture that emerges, albeit imperfectly, from these studies is of a multisubunit protein synthesized partly in the cytoplasm and partly in the mitochondria (@). The subunits are then assembled in the mitochondria1 membrane, where the functioning enzyme resides, with the times of incorporation of copper, heme, and lipid quite uncertain.

D. LIPIDS The role of the lipid component remains in doubt but is becoming somewhat clearer. The phospholipid content is reported for several preparations (Table IV) (83-86).Somewhat more diphosphatidylglycerol and TABLE I V COMPOSITION OF CYTOCHROME C OXIDASK MITOCHONDRIA FROM BOVINEHEART

PHOSPHOLIPID

AND

Percent of total lipid Phosphatidylcholine

Phosphatidylethanolamine

10 5

37 38

31 30

16 18

83

9 11

26 27 46 32

25 21 26 30

30 31 11 30 73

83 86 86

PhosphatidylPreparation investigators inositol Mitochondria Fleischer et al. Awasthi et al. Cytochrome c oxidase Fleischer et al. Brierley and Merola Yu el al. Awasthi et al. Awasthi et al.@

Diphosphatidylglycerol Ref.

84

84 84

"Lipid-free" cytochrome c oxidase was obtained by treatment of mitochondria with Triton X-100 and Triton X-114. The remaining lipids (27%) were not identified but were possible breakdown products of diphosphatidylglycerol. 0

81. G.Schatz, G.S. P. Groot, T. Mason, W. Rouslin, D. C. Wharton, and J. Saltzgaber, Fed. Proc., Fed. Amer. SOC.Exp. Biol. 31,21 (1972). 82. W. L. Chen and F. C. Charalampous, BBA 294,329 (1973). 83. S. Fleischer, H.Klouwen, G. Brierley, E. Carpenter, and T. Moran, JBC 236, 2936 (1961). 84. Y . C. Awasthi, T. F. Chuang, T. W. Keenan, and F. L. Crane, BBA 226, 42 (1971). 85. G.P. Brierley and A. J. Merola, BBA 64,205 (1962) 86. C. Yu, L.Yu, and T. E. King, JBC 250,1383 (1975).

5.


313

somewhat less phosphatidylethanolamine are found in the oxidase than in whole mitochondria, but the amount and kind of phopholipid in an enzyme preparation is dependent upon the treatment it has received ( 8 4 ) . Conventional purification procedures preferentially remove phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These lipids are extracted readily with acetone (84, 8 7 ) , methanol chloroform ( 8 8 ) , or nonionic detergents such as Triton X-100 or X-114 (89, 90) to leave behind a small amount of lipid (-1.6-1.7 pg phosphorus per milligram of protein) that is largely cardiolipin (Table IV) ( 8 4 ) . This residual cardiolipin (about 2 moles/mole of oxidase) (87) is both difficult t o extract (only chloroform-methanol-ammonia proved efficacious) and insensitive to digestion by phospholipase A under conditions where the phospholipids of mitochondria are readily hydrolyzed ( 8 4 ) . As the other phospholipids are removed, the enzyme becomes progressively less active (87), but the activity is restored by adding back purified lipids, mitochondrial lipid, or detergent (such as Emasol) (84, 85, 8 7 ) . However, if the residual cardiolipin is removed, restoration of activity is minimal ( 8 4 ) . Such studies suggest a t least three ways in which phospholipid interacts with and affects the activity of the enzyme: (1) in the incorporation of the oxidase into membranes, with a concomitant increase in the accessibility of the active site (91) ; (2) in the formation of a complex between cytochrome c and the oxidase (84); and ( 3 ) in the stabilization of active conformations ( 9 2 ) . It is evident that particular attention must be paid to both lipid and detergent contents before many of the differences in properties among preparations can be rationalized.

111. Chemical and Physical Properties

The chemical and physical properties of cytochrome c oxidase have been widely studied in intact mitochondria, in mitochondria1 particles, and as the isolated enzyme. Aside from variations in activity mentioned above and the recently observed effect of detergent on intensities of electronic spectra (52), the properties have proved remarkably insensitive 87. W. L. Zahler and S. Fleischer, J . Bioenerg. 2, 209 (1971). 88. Y. C. Awasthi, T. F. Chuang, T. W. Keenan, and F. L. Crane, BBRC 39, 822 (1970). 89. E. E. Jacobs, F. H. Kirkpatrick, Jr., E. C. Andrews, W. Cunningham, and F. L. Crane, BBRC 25, 96 (1966). 90. F. F. Sun, K. S. Preebindowski, F. L. Crane, and E. E. Jacobs, BBA 153, 804 (1968). 91. T . F. Chuang, Y . C. Awasthi, and F. L. Crane, J . Bioenerg. 5, 27 (1973). 92. T. F. Chuang and F. L. Crane, J. Bidenerg. 4, 563 (1973).

314


to its physical state. These studies have encompassed a wide range of techniques, and in this section it is intended to review the information available from them with particular emphasis upon the evaluation and interpretation of those data which may be of importance in unraveling the chemkal events that lead to the reduction of dioxygen to water. A. MODELS In the development of ideas about the fundamental mechanisms of dioxygen reduction one expects to be able to carry over into the protein system the basic physical and inorganic chemistry of copper and heme iron. Consequently, it is important to have a clear understanding of the response of copper complexes, heme A, and, perhaps, copper-heme A complexes to those chemical and physical probes that might be used with the oxidase. Although copper (I) and copper (11) complexes have been subjected to extensive investigation and their properties are quite well understood, few probes are effective in following copper in the oxidase and thus so little is known about the copper environment in the enzyme that the transposition of knowledge from simple systems to the enzyme system is difficult. The main probe is EPR spectroscopy where a portion, and only a portion, of the copper(I1) is visible. There is a probable requirement for copper to retain substantially the same coordination environment in the oxidized and reduced forms under rapid turnover conditions and for different environments for the two copper atoms. Fortunately, there are more opportunities to probe the iron and its associated porphyrin. Several physical properties can be examined and the structure-property relationships have been quite extensively worked out with some hemes, namely, heme B (protoheme) and other 2,4-disubstituted deuterohemes (60, 62, 93). However, the heme studies to date are not as relevant to the oxidase as future studies can be for two reasons. One is that heme A, although its structure has recently been elucidated and a few properties examined (60), has still not been as thoroughly studied as heme B. This is important, since heme A, in those few properties that have been studied differs significantly from heme B. A second factor is the paucity of data on electron exchange interactions between any heme system and another heme or a copper or another donor (or acceptor). Several p-oxobishemins, including p-oxobishemin A, have been thoroughly characterized (60, 94) , p-hydrazine-bishemes have been preRared 93. W. S. Caughey, C. H. Barlow, D. H. O’Keeffe, and M. C. O’Toole, Ann. N . Y . Acad. Sci. 206,296 (1973). 94. D. H. O’Keeffe, C. H. Barlow, G. A. Smythe, W. H. Fuchsman, T. H. Moss, H. R. Lilienthal, and W. S. Caughey, Bioinorg. Chem. (in press).

5.


315

(93),and copper-heme A interactions have been indicated (95); but, nevertheless, in sum, relatively little is known about interactions between hemes, coppers, and other donor-acceptor systems of the types likely to be present in cytoohrome c oxidase. Then, the application of the ideas of inorganic chemistry to the oxidase must be largely inferential but nevertheless can lead to useful generalizations about mechanistic pathways that might be accessible to dioxygen en route to water. Furthermore, model studies cannot be expected to hold all the answers t o the oxidase function because the protein is able to provide environments for electron transfer and an active site geometry that may not be readily duplicated in conventional chemical systems. Simple metal atom chemistry very rarely provides assemblies of metal ions in an ordered yet flexible environment where cooperative interactions can facilitate, in a single step, reactions that require a number of steps by the separate ions.

B. ELECTRONIC SPECTROSCOPY 1. Absorption Spectra

Electronic spectra provide a simple and convenient way to monitor changes induced in the oxidase by various chemical treatments. Indeed, spectral observations were a t the core of the pioneering observations of MacMunn ( l a ) , Keilin (96),and Warburg (97); and more recently many investigators have examined the spectra of isolated oxidase, mitochondrial particles, and electron transport particles. The spectra of the fully oxidized [oxidase ( I V ) ] (97a) and the fully reduced [oxidase ( 0 ) ] oxidase have been well characterized (52) (Table V). In Table VI are spectral parameters for ligand complexes of various oxidation states (98-105). Although the spectra of most of these complexes have been 95. R. A. Bayne, G. A. Smythe, and W. S. Caughey, in “Probes of Structure and Function of Macromolecules and Membranes” (B. Chance, T. Yonetani, and A. S. Mildvan, eds.), Vol. 2, p. 613. Academic Press, New York, 1971. 96. D. Keilin, Proc. Roy. SOC.,Ser. B 98, 312 (1925). 97. 0. Warburg, Biochem. 2.152,479 (1924). 97a. Hereafter, the oxidation state (number of electrons removed) of cytochrome oxidase will be represented by a roman numeral, 0 to IV, in parentheses. 98. W. H. Vanneste, Biochemistry 5, 838 (1966). 99. C. Greenwood, M. T. Wilson, and M. Brunori, BJ 137,205 (1974). 100. R. Lemberg and J. Stanbury, BBA 143,37 (1967). 101. A. 0. Muijsers, K. J. H. Van Buuren, and B. F. Van Gelder, B B A 333, 430 (1974). 102. R. Wever, A. 0. Muijsers, B. F. Van Gelder, E. P. Bakker, and K. J. H. Van Buuren, BBA 325, 1 (1973). 103. Y. Orii and K. Okunuki, J. Bioehem. ( T o k y o ) 55,37 (1964).

316

W. S. CAUGHEY, W. J. WALLACE, J . A. VOLPE, AND S. YOSHIKAWA

TABLE V ELECTRONIC ABSORPTION SPECTRAL DATAFOR CYTOCHROME c OXIDASEWITH A N D WITHOUT DETERGENT' Fully oxidized: oxidase(1V)b 830 1.2 1.1

Xmax,nm

emx with detergente e , ~without

detergentd

660 2.4 2.0

598 8.7 6.6

545 8.2 6.3

515 8.3 6.3

418 79 59

560 7.7 6.3

517 7.2 5.3

443 100 78

Fully reduced: oxidase(0)~ 603 19.3 14.5

Xmax,nm

with detergent" without detergentd

C ~ M

em^

Volpe and Caughey (68). As isolated. "Detergent, Tween 20. Oxidase(1V) dissolved in 0.1 M phosphate buffer 0.75% Tween 20, p H 7.4. d Oxidase(1V) dissolved in 0.01 M phosphate p H 7.4. Reduced with slight excess of sodium dithionite. a

observed many times (18) and may, on this basis, be considered well established, there remain some troublesome difficulties in their detailed interpretation (11). Assignment of frequency and intensity values to band maxima for heme a, heme a,, and copper components (98) has been accepted rather widely (18). However, the EPR evidence (66) for strong interaction among the metal components in terms of facile electron exchange and magnetic coupling indicates the likelihood that changes induced a t one component (e.g., oxidation or ligand binding) will affect the electronic spectra (and other properties) of the other components. Thus, it is risky indeed to ascribe individuality to the hemes or coppers on the basis of monotonic dependence of the spectra upon the states of the individual TABLE VI WAVELENGTHS OF ABSORPTIONMAXIMAFOR VISIBLEAND SORET OF CYTOCHROME c OXIDASE' SPECTRAOF COMPLEXES Oxidase-ligand complexb Oxidase(0) .CO Oxidase(II1). CO Oxidase(0) . O p Oxidase(II1) .02 Oxidase(1V) .FOxidase(1V). N8Oxidase(1V) .CN-

Ref.

Xrnnx.nrn

603

638 660

603 605 598 598 598

590(sh) 590

551 547 550 550 545 545 545

517

440(sh)

431. 429 428 428 421 421 425

98 99

100 99 101 108 10s

Preparative methods different but all contained detergent. The precise nature of ligand binding has not been established in these complexes.

5.


317

metal centers (11, 104, and conclusions drawn from experimental observations which depend upon this separation for quantitation and analysis should be viewed with caution. These comments apply both to attempts to synthesize spectra for cytochromes u and u3 on the assumption that the properties of one heme are independent of the oxidation state of, and ligands bound to, the other metals (98),and to the assignment of the 830-nm band to copper (105). Copper may be an active contributor to the 830-nm band since changes in EPR signals resulting from copper (11) have been noted to follow the intensity of the 830-nm band, but present evidence does not show that copper is the unique contributor, or even a contributor a t all, to the 830-nm band intensity (106-108). A comparison of the spectral differences between cytochrome c oxidase and heme A derivatives with those for hemoglobin or myoglobin and heme B derivatives reveals similar effects of protein environment on the heme moieties. The magnitude and direction of wavelength shifts upon going from heme species to proteins are comparable for the three proteins (Tables V, VI, and V I I ) . There is therefore little doubt that the 8-formyl group of heme A remains intact upon incorporation into the apoenzyme. The conversion from deoxy to carbon monoxy to oxy species results in similar spectral shifts (Table VII) (52, 65, 109-112). An exception is the blue shift in the 605-nm band upon reaction with CO, whereas hemoglobin (Hb) and myoglobin (Mb) experience a red shift upon binding CO. Nevertheless, the remaining spectral evidence supports similar terminal CO to Fe binding for the three proteins as do infrared C-0 stretch bands (113-116). Similar binding of 0, among the three proteins can also be assumed; infrared 0-0 stretch band data have shown this to be 104. W. S. Caughey, Annu. Rev. Biochem. 36, 611 (1967). 105. D. C. Wharton and A. Tzagoloff, JBC 239,2036 (1964). 106. W. S. Caughey and S. McCoy, in “Biochemistry of Copper” (J. Peisach, P. Aisen, and W. E. Blumberg, eds.), p. 271. Academic Press, New York, 1966. 107. L. N. Mackey, T. Kuwana, and C. R. Hartzell, FEBS (Fed. Eur. Biochem. Soc.) Lett. 36, 326 (1973).

108. L. E. AndrCasson, B. G. Malmstrom, C. Stromberg, and T. Vanngard, FEBS (Fed. Eur. Biochem. SOC.) Lett. 28,297 (1972). 109. R. Banerjee, Y. Alpert, A. F. Leterrier, and R. J. P. Williams, Biochemistry 8,2862 (1969). 110. Y. Sugita and Y. Yoneyama, JBC 246,389 (1971). 111. A. 0. Muijsers, R. H. Tiesjema, and B. F. Van Gelder, BBA 234, 481 (1971). 112. K. D. Hardrnan, E. H. Eylar, D. K. Ray, L. J. Banaszak, and F. R. N. Curd, JBC 241, 432 (1966). 113. J. 0. Alben and W. S. Caughey, Biochemistry 7, 175 (1968). 114. S. McCoy and W. S. Caughey, in “Probes of Structure and Function of

Macromolecules and Membranes” (B. Chance, T. Yonetani, and A. S. Mildvan, eds.), Vol. 2, p. 295. Academic Press, New York, 1971. 115. W. S. Caughey, R . A. Bayne, and S. McCoy, JCS,D p. 950 (1970). 116. W. S. Caughey, Ann. N . Y . Acnd. Sci. 174, 148 (1970).

TABLE VII DATAFOR OXYAND CARBONYL COMPLEXES OF HEMOGLOBIN, VISIBLEA N D SORETSPECTRAL MYOGLOBIN, A N D CYTOCHROME c OXIDASE Complex 53(13.3) 569( 15.0) 576(15.2)

Oxidase(0)b Oxidase(0)COb Oxidase(0)OZc

603(14.5) 603( 14.0) 603(12)

Mba MbCO MbO,

556(12) 579(12.2) m(14.6)

a

539(14.9) 542(14.3) s590(11.5)

560(6.3) 551 (7.4) 550(10) 540( 14.0) 543(13.7)

Here, Hb stands for hemoglobin and Mb for myoglobin. determined in the absence of detergent. srn~ determined in the presence of 1.0 % cholate.

m Ref.

Xmax.nm(ernM-')

Hba HbCO HbOr

?

517(5.3) 517(6.3)

444 (50)

430(145) 420(208) 415(135)

109 109

?

109,110

4

443(77) 431(62) 428(84)

51 66 111

434( 114) 424( 187) 418(128)

111 111 111

F-1 ?

e,,,~

m

5.


319

true for HbO, (117)and MbOz (118) but not for oxidase(O).O, as yet. The marked difference in the spectra of the oxidase(1V) and p-OXObishemin A derivatives indicates a simple p-oxobishemin A structure is not present. However, interactions of copper with a p-oxobishemin moiety could result in the spectrum found for oxidase(1V) and explain also the generally low sensitivities of the visible spectra of oxidase to changes in ligation and oxidation state. 2. Circular Dichroic Spectra

The circular dichroic (CD) spectra of many cytochrome c oxidase derivatives have been observed (119-123) with reproducible results. Here also the insensitivity of the C D spectra to ligand substitution makes them difficult to interpret. Thus, the general shapes of the C D spectra for cytochromes a and a3 generated by the algebraic addition of spectra obtained from a number of derivatives were sufficiently independent of the specific ligands on the complexes used to generate the spectra that it was concluded that the hemes were acting independently (121, 123). However, detailed examination of the spectra revealed sufficient ligand-dependent differences in band positions and intensities to suggest cooperative interactions of the hemes (12U,122). Thus, directly opposed interpretations of the same data were presented. On the one hand, the traditional concept of identifiable cytochromes a and a3 is maintained, while on the other, the increasingly supportable notion of cooperativity between the hemes is suggested. Here also it has proved difficult to distinguish between the hemes (cytochromes a and a,) in oxidase(0) and oxidase(1V). But these hemes can be distinguished upon reaction with ligands or upon changing the oxidation state. Current evidence favors heme-heme interaction which results in changes induced a t one heme influencing the behavior and properties of the other. C. LIGAND BINDING STUDIES In addition to studies that have involved the determination of the thermodynamic states (potentiometric) and the observable valence states 117. C. H. Barlow, J. C. Maxwell, W. J. Wallace, and W. S. Caughey, BBRC 55, 91 (1973). 118. J. C. Maxwell, J. A. Volpe, C. H. Barlow, and W. S. Caughey, BBRC 58, 166 (1974). 119. D. W. Urry, W. W. Wainio, and D. Grebner, BBRC 27, 625 (1967). 120. D. W. Urry and B. F. Van Gelder, in “Structure and Function of Cytochromes” (K. Okunuki, M. D. Kamen, and I. Sukuru, eds.), p. 210. Univ. of Tokyo Press, Tokyo, 1968. 121. Y. P. Myer, JBC 248, 1241 (1971). 122. R. H. Tiesjema and B. F. Van Gelder, BBA 347,202 (1974). 123. F. C. Yong and T. E. King, BBRC 40, 1445 (1970).

320


(EPR)of the electron acceptors in cytochrome c oxidase, there have been other chemical studies directed toward understanding the changes that occur a t the catalytically active center as it goes from oxidized to reduced and back to oxidized again. These studies, as with the potentiometric and EPR studies, must be interpreted with some caution because they must of necessity be conducted under nonturnover conditions. I n these circumstances the steps that emerge from the analysis of the data may not correspond to any transitions that occur in the functioning enzyme. Nevertheless, such work has provided valuable insight into the interrelationships between the components of the active site. The binding of ligands and the consequent alteration of physical and chemical properties have been used to probe for the identification and differentiation of the cytochromes a and as (18).The observational techniques employed have varied from UV-visible and CD spectroscopy (discussed above) through potentiometry (124, 125) and EPR spectroscopy (126)( to be examined later), but there remains a measure of uncertainty in the interpretation of the results. The stoichiometry and stereochemistry of complexes have both been inferred from the changes in electronic spectra that accompany ligand binding and/or changes in the oxidation state (98, 127). However, while such spectral changes are convenient monitors of ligand interaction, they do not measure directly either the nature or the extent of ligand binding. Consequently, the complexes under study are frequently ill-defined despite the extensive examination that they may have undergone. 1. Azide, Fluoride, and Cyanide Fluoride (101, 128), azide (102, 128-150), and cyanide (103,128, 129, 151-156) have been observed spectrophotometrically to bind reversibly to the oxidase. The absorbance changes are small in all cases and the kinetics complex-at least two forms of complex are kinetically observ124. J. S. Leigh, Jr., D. F. Wilson, C. S. Owen, and T. E. King, ABB 160, 476 ( 1974).

125. Y. Fujihara, T. Kuwana, and C. R. Hartzell, BBRC 61, 538 (1974). 126. C. R. Hartzell and H. Beinert, BBA (in press). 127. T. Yonetani, JBC 235, 845 (1960). 128. S. Yoshikawa and Y. Orii, J . Bioch.em. ( T o k y o ) 71, 859 (1972). 129. S. Yoshikawa and Y. Orii, J . Biochem. ( T o k y o ) 71,873 (1972). 130. R. Wever, A. 0. Muijsers, and B. F. Van Gelder, BBA 325,8 (1973). 131. P. W. Camerino and T. E. King, JBC 241,970 (1960). 132. K. J. H. Van Buuren, P. F. Zuurendonk, B. F. Van Gelder, and A. 0. Muijsers, BBA 256, 243 (1972). 133. K. J. H. Van Buuren, P. Nicholls, and B. F. Van Gelder, BBA 256, 258 (1972). 134. S. Yoshikawa and Y. Orii, J . Biochem. ( T o k y o ) 73, 637 (1973). 135. S. Yoshikawa and Y. Orii, J . Biochem. ( T o k y o ) 76, 271 (1974).

5.


321

able in each case. Where the stoichiometry of the ligand binding reaction was tested (cyanide and azide), only a single (133) 1:l complex (102, 132) was formed. Fluoride was suggested, on the basis of its inhibitory behavior, to bind 2 moles/mole of oxidase although EPR spectra suggest little interaction with the heme iron (128). The site(s) of ligand binding has, then, not been clear but the suggestion has been made (136, 137) that azide inhibits a t a site common to both electron transport and energy conservation. In support of this contention, Wilson (137)has shown that 5-13 (5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylamide) releases azide inhibition of ATPase and electron transfer and that 1.35 molecules of s-13 per respiratory chain are needed to release inhibition of respiration. I n the presence of azide, the redox potential is altered, (138) and the EPR visible iron undergoes a high- to low-spin transition (139).Infrared observations of oxidase(1V) in the presence of azide revealed that even upon long standing the vNS- is not shifted from its free solution value ( V = 2047 em-', Av% = 28 cm-') (140). However, oxidase(I1) treated with azide followed by reoxidation t o oxidase (IV) exhibited a frequency (2038 cm-', h v , = 14 em-') consistent with iron-bound azide (140, 141). Clearly, azide was bound to the iron in the latter case but not in the former. It is important to discriminate among the possible ligand bonding configurations, but such differences in binding could not be established on the basis of electronic spectra alone. Infrared difference spectroscopy has enjoyed considerable success in the elucidation of the nature of binding of a variety of ligands to hemes and heme proteins (116, 1411, and since the method provides a direct measure of the character of the ligand bonds it promises to be a powerful tool in probing oxidase ligands, even within intact tissue (142), despite the large size of the enzyme.

2. Carbon Monoxide Carbon monoxide binds readily to iron (11), but not iron (111), porphyrins to form complexes that are quite distinctive in terms of the spectral properties both of the heme and of the bound CO. Thus, CO has been widely used as a probe of the active site of heme proteins (113, 136. D. F. Wilson and B. Chance, BBRC 23,751 (1966). 137. D. F. Wilson, Biochemistry 8, 2475 (1969). 138. D. F. Wilson, J. G. Lindsay, and E. S. Brocklehurst, BBA 256, 277 (1972). 139. B. F. Van Gelder and H. Beinert, BBA 189, 1 (1969). 140. W. S. Caughey, C. H. Barlow, J. C. Maxwell, J. A. Volpe, and W. J. Wallace, Ann. N . Y . Acad. Sci. 244, 1 (1975). 141. S. McCoy and W. S. Caughey, Biochemistry 9,2387 (1970). 142. J. C. Maxwell, C. H. Barlow, J. E. Spallholz, and W. S. Caughey, BBRC 61, 230 (1974).

322


114). In the past, such investigations with cytochrome c oxidase have been limited to visible UV spectroscopy ; however, more recently, infrared (66, 116), EPR (124), and potentiometric (138) techniques have been employed to investigate the reaction of CO. Carbon monoxide may bind a t iron(I1) (62, 115) or copper(1) (65,115) 0

II

either as a terminal (M-CO) or bridging (M-C-M) ligand. The presence of the C-0 stretch band a t 1963.5 cm-l for the CO complex with oxidase(0) provided firm evidence for binding a t iron(II), and not a t copper(I), in a terminal (end-on) fashion similar to HbACO (v-1951 cm-1) (115) and MbCO (v-1944 cm-1) (114). Furthermore, the extreme narrowness of the band showed a well-ordered (nonrandom) environment about the bound CO well isolated from external medium. A similar band was found for CO bound to the oxidase in intact heart muscle (142). These data are not consistent with suggestions that CO is bound cooperatively by both copper(1) and iron(I1) in CO (143). The stoichiometry of CO binding to the oxidase has been considered to be one CO per two hemes since Keilin's original interpretations of visible spectra (13). Results from many subsequent attempts (98, 144-147) to establish such a stoichiometry though variable, were interpreted as consistent with the Keilin suggestion. However, recent evidence from infrared intensities and exchange of CO from the oxidase to Hb gave clear evidence for one CO for each heme A (66). The very narrow bandwidth ( A V ~ , ~6, cm-I) shows the vc0 values for each heme are either identical or very nearly so since if more than one bond were present the frequencies would necessarily be nearly the same if such a narrow width were to result. Since vco is a very sensitive probe of cis, trans, and medium effects in heme proteins (49, 6 2 ) , the essentially identical vc0 for each heme A provides strong evidence that each heme A is of the same structure including trans ligand (histidine?) and that the environment about each bound CO ligand is the same. The CO ligands in HbACO are found at 1950 and 1952 cm-*, corresponding to the a! and p subunits, respectively, to give a v l I Z of 8 cm-' (148). It is of interest that in the presence of one iron(II1) heme, e.g., oxidase (11).CO, only one CO binds with 143. J. G. Lindsay and D. F. Wilson, FEBS (Fed. E w . Biochem. S o d Lett. 48, 45 (1974). 144. Q.H. Gibson and C. Greenwood, BJ 86,541 (1963). 145. M. Morrison and S. Horie, JBC 240, 1359 (1965). 146. Q. H. Gibson, G. Palmer, and D. C. Wharton, JBC 240, 915 (1965). 147. G. E. Mansley, J. T. Stanbury, and R. Lemberg, BBA 113, 33 (1966). 148. J. A. Volpe, J. C. Maxwell, W. J. Wallace, W. S. Caughey, and S. Charache, Fed. Proc., Fed. Amer. SOC.Exp. Biol. 34, 687 (1975).

5. CYTOCHROME

C OXIDASE

323

vc0 a t 1965 cm-’ slightly, but only slightly shifted from the value for the fully reduced oxidase CO complex (149). Little is known about the relative affinities of the two heme A sites for CO. The infrared and H b exchange evidence noted above demonstrates that two CO ligands can bind, a t least in certain enzyme preparations. However, it is reasonable to expect the first CO to be bound with greater affinity than the second; therefore, in some preparations, only one CO may bind. I n mono-CO complexes, the heme to which CO binds can be called a3. But, there is no basis for knowing which of the two liemes in the fully reduced oxidase represents the preferred binding site or even whether there is a preferred binding site. Once CO becomes , other heme may in consequence adopt differbound to one heme ( a 3 ) the ent properties and become heme a. A reasonable interpretation of infrared (65) and other (99) data is one in which with either one or two electrons added to the fully oxidized enzyme, one CO binds a t iron. And, as discussed below, potentiometric and EPR evidence that CO binding affects the properties of other metal centers has been obtained (1.24, 137).

3. Dioxygen The infrared spectra show that CO binds to the oxidase (65, 114) in much the same way it binds to H b and Mb (113, 114). Hence, it.might be expected that the enzyme would form oxidase.0, in the same way that H b and M b form HbO, and MbO,. Formation of such an “oxygenated” complex represents a quite logical initial step in the sequence of reactions that lead to the reduction of oxygen to water by oxidase. Consequently, there have been a number of attempts to identify and characterize an “oxygenated” oxidase. In 1958, Okunuki and Sekuzu discovered a new form of cytochrome c oxidase, characterized by a Soret band a t 426-428 nm and designated i t “oxygenated” oxidase (160).The presence of the band a t 428 nm has been taken as a clear indication of the formation of the oxygenated complex (100) and is, in fact, the only evidence for the formation of such a complex. And, despite some uncertainty about its spectral characteristics (151), all discussion of the oxygenated complex has been cast in terms of the variation in both position and intensity of the band at 428 nm. I n the original concept of Okunuki and Sekuzu, dioxygen was thought 149. J. G. Lindsay, ABB 163, 705 (1974). 150. K. Okunrrki, B. Hagihara, I. Sekuzu, and T. Horio, in (‘Proceedings of the International Symposium on Enzyme Chemistry, Tokyo and Kyoto, 1957” (K. Ichihara, ed.), p. 264. Academic Press, New York, 1958. 151. H. Beinert, C. R. Hartaell, and W.. H. Orme-Johnson, in “Probes of Structure

and Function of Macromolecules and Membranes” (B. Chance, T. Yonetani, and A. S.Mildvan, eds.), Vol. 2, p. 575. Academic Press, New York, 1971.

324

W. S. CAUGHEY, W. J . WALLACE, J. A. VOLPE, AND S. YOSHIKAWA

to bind reversibly to oxidase(O), but the only direct evidence for this has been the observation (158) that upon evacuation the dioxygen appears t o be removed and replaced by carbon monoxide. Greenwood et al. (99) have prepared the “oxygenated” oxidase by photolyzing ferricyanide oxidized oxidase(0) .CO in the presence of 0,. The complex is formed readily and is fairly stable. Decay appears to occur through a slow intermolecular transfer reaction (153-155). Clearly, the mixed valence complex oxidase(II1) -0, is not readily formulated except on the familiar HbO, bent end-on model since only one reduced heme iron is available and there is no evidence to suggest that copper contributes to ligand binding. With two hemes present, two different mono dioxygen complexes are possible, but a t present there is no experimental basis upon which to decide whether the two complexes have the same or different properties or whether one is preferred over the other. However, the presumed observation of relatively stable mixed valence oxygenated oxidase complexes suggests that it may be possible to subject intermediate steps in the reduction of dioxygen to scrutiny by more discriminating techniques (such as infrared) and that this may provide the clues that will lead to a detailed understanding of the steps involved in the important enzymic reduction. The ability to observe oxidase(0) .O, a t low temperature and oxidase(II.1) or (11)*02 a t room temperature is quite understandable in chemical terms. Many of the other observations on “oxygenated” oxidase are rather more difficult to rationalize. Both Gilmour et al. (156) and Tiesjema et al. (157) observed that oxidase(0) reacts rapidly ( 14 mK). It is possible that these two classes correspond to tetrahedrally distorted coordination (Type I) and rhombohedrally distorted coordination (Type 11). However, too little is known about the coordination environment of copper in proteins to take such assignments seriously and the Type I and Type I1 classification should be considered only as a convenient empirical observation. Nevertheless, it is interesting that the inherent copper of oxidase(1V) (911 = 2.17, gm = 2.03, All 5 3 mK) (173) falls distinctly outside the range for Type I Cu2+. Thus, there appears to be a clear distinction between copper that is integrally bound in the oxidase and adventitious copper that appears as a variable contaminant in most preparations. This difference was not clearly understood in the early literature and led to some confusion about the role played by copper (174). The copper(I1) atom&) that give rise to the characteristic oxidase(1V) E P R signal are strongly bound within the enzyme and are not readily removed by treatment with complexing agents (53, l o g ) , but upon denaturation of the protein a more normal copper(I1) E P R signal is seen (175) and the copper then becomes susceptible to removal by complexing agents (53). Griffiths and Wharton (53) were able to show by a chemical method (176) that in oxidase(1V) both copper atoms were in the +2 oxidation state and this was later confirmed by the coulometric titrations of Heineman et al. (165).It was then surprising to find that the intensity of the E P R signal corresponded to only about 40% of the total copper known to be present as copper(I1) in oxidase(1V) (173) or, as now appears likely, 80% of one copper and none of the second copper. The low signal intensity together with the lack of fine structure in the E P R signal suggests that the copper(I1) interacts with other metal centers in such a way as to quench the inherent paramagnetism of divalent copper and produce the observed lowering of the E P R signal intensity. 171. B. Malmstrom and T. Vanngard, J M B 2, 118 (1960). 172. T. Vanngard, in “Biological Applications of Electron Spin Resonance” (H. M. Swartz, J. R. Bolton, and D. C. Borg, eds.), p. 411. Wiley (Interscience), New York, 1972. 173. H. Beinert, D. E. Griffiths, D. C. Wharton, and R. H. Sands, JBC 237, 2337 (1962). 174. A. Ehrenberg and T. Yonetani, Acta Chem. Scnnd. 15, 1071 (1961). 175. H. Beinert and G.Palmer, in “Oxidases and Related Redox Systems” (T.E. King, H. S. Mason, and M. Morrison, eds.), Vol. 2, p. 567. Wiley, New York, 1965 176. G. Felsenfeld, ABB 87, 247 (1960).

5.

331


2. Iron

The E P R signal resulting from iron(II1) because of its breadth and partial submersion under the copper(I1) signal has proved much more difficult to study quantitatively. Nevertheless, it now seems well established that in oxidase(1V) iron(II1) is represented by signals a t g = 3, 2, and 1.5 with intensities that correspond to about 40% of the heme iron present (126, 139). Although the oxidation state of the iron in oxidase (IV) has never been directly determined, the electron capacity of the fully oxidized enzyme suggests that all the iron must be iron(II1). Then, the low E P R signal intensity is most conveniently interpreted in terms of metal-metal interaction between iron(II1) (low spin) and some other paramagnetic center which partially quenches the signal expected from the S = .2 net spin on the iron. Both heme-heme and heme-copper (11) interactions have been suggested (138). Further elucidation of this problem might be forthcoming on the basis of careful magnetic susceptibility and Mossbauer studies. Unfortunately, the single published accounts of magnetic susceptibility (174) and Mossbauer (17'7) studies are difficult t o interpret. Nevertheless, it is interesting to note on the most naive basis that the magnetic susceptibility results of Ehrenberg and Yonetani (17'4) correspond t o -80% of two unpaired electrons. This is just as expected on the basis of the E P R observations on intrinsic copper and iron. 3. The Effect of Valence State Changes on the EPR Spectrum

Behavior of the signals resulting from E P R visible iron and copper during reduction provides clues that may be important to the understanding of the chemistry involved in oxygen reduction. Experiments carried out on a relatively long time scale show that the first electrons to enter the system give rise to a diminution of the low-spin iron(II1) ( g 3) signal and a corresponding increase in the broad g 6 signal attributed to high-spin iron(II1) (139). The g 6 signal reaches maximum intensity (corresponding to about 40% of the heme A) when two electrons per mole of oxidase have been supplied to the system and then declines leaving a very small rhombohedra1 signal when four electrons have been supplied. The copper (11) signal remains unchanged (perhaps increasing slightly) during entry of the first pair of electrons and then diminishes t o zero during entry of the second pair (139, 178). Leigh et al. .(124) have shown that the high-spin iron (111) signal makes its appearance with a half-reduction potential of 380 mV and disappears with a half-reduc-

-

-

177. G. Lang, S. L. Lippard, and S. RosBn, BBA 336,6 (1974). 178. C. R. Hartzell and H. Beinert, BBA (in press).

-

332

W. S. CAUGHEY, W. J. WALLACE, J. A. VOLPE, AND S . YOSHIKAWA

tion potential of 210 mV. The copper(I1) signal disappears with a halfreduction potential of 250 mV. Similar observations have been made for both isolated oxidase (124, 125) and pigeon heart mitochondria and phosphorylating submitochondrial particles (168). It is apparent then, that the E P R results are in substantial agreement with the potentiometric results in showing that the iron and copper are reduced as two iron-copper pairs. At low temperature (99

95

10-8 (M-1

x kl’ sec-l)*

9.0 6.4 4.1 2.2 0.6

% Activity

0.05

100 71 46 25 7 0.6

0.01

k, (IOIa).Essentially, then, Compound I is not the primary enzyme-substrate complex (161). The formation of Compound I entails the reduction of substrate (peroxide) a t the active site (compare Schemes I and 11). The recent discovery that nearly one mole of Compound I is formed in the 1 :1 reaction between catalase ferriheme and peracetic 161. P. George, ABB 45,21 (1953).

7.

393

CATALASE 25pM CH,CO,H

1 $ 4.25pMCH,COSH

I,

8.5pM CH,CO,H

,i \

4 -

I

I

0

10 10' [CH,CO,

20

30

H] (M)

FIG.6. Titration of -15 fiM horse erythrocyte catalase-ferriheme with peracetic acid, 0.08 M phosphate, pH 7.23, 25" ( 1 0 1 ~ ) .

acid (Fig. 6) further supports this conclusion ( 1 6 1 ~also ; see 169, 166, 163). As in enzyme-hydrogen peroxide reactions, the unionized peracid is the immediate substrate (162) and, according to Jones and Middlemiss (163),one mole of acetic acid is released per mole of catalase ferriheme converted to Compound I. No information is available as to the individual steps of the reaction, and none is likely to issue from kinetic studies with higher alkyl peroxides (C 2 2) or peracids. The reasons are implicit in the data of Table VIII (1,69, 101a, 162, 164), which show that the observed rate with peracetic acid, hyconstants for Compound I formation ( k ,),, droxymethyl hydroperoxide, and ethyl hydrogen peroxide are of the same order of magnitude (2.5 0.5 X lo4 M-* sec-') in spite of differences in the following:

*

0-0

dissociation energies for EtOOH (43 kcal/mole) and peracids kcal/mole) , the basicities of the leaving groups (pK,:acetic acid, 4.76; ethanol, 18), and the ionization properties of peroxides (pK, :peracetic acid, 8.2; EtOOH, 11.8) (-35

161a. The stoichiometry of the reaction is unaccountably variable, the average of several titrations showing that approximately 0.87 & 0.08 mole of Compound I is formed per mole of peracetic acid. 162. P. Jones and D. N. Middlemiss, BJ 130,411 (1972). 163. P. Jones and D. N. Middlemiss, BJ 143,473 (1974). 164. S. Marklund, BBA 289,269 (1972).

394 GREGORY R. SCHONBAUM AND BRITTON CHANCE

e:

7.

395

CATALASE

We must assume, then, that the rate-limiting step is virtually independent of the above parameters. Nor can it be involved in the redox-dependent rearrangement of iron-protoporphyrin since the reactions with H,O, and CH,OOH are faster than those with EtOOH or CH,CO,H. A relationship between k, ap,, and van der Waals volumes of peroxides is the only discernible pattern. This strongly suggests that enzyme-peroxide association is the rate-determining step and, as such, is rather irrelevant to the elucidation of the redox transformations. The cocatalytic role of the apoprotein in facilitating Compound I generation is increasingly regarded as that of a general acid-general base (17, 75,159, 159a), although different functional groups are seen as fulfilling this role. Jones and Suggett ('?5),pointed to the possible involvement of >C(NHr),+ group from an arginine residue (BH') and a carboxylate (A-) as in Eq. (20). H ,

H

+

Hgoz

B H ,F

H,O-Fe

l

I

,

?I

H,

(20)

O/O--Fe

I

H

9_ _ _ _ 0_ _ _ _ _ _ Fe I

H

,

As already mentioned, a less detailed scheme may also be developed, using only Y H 3 His [formulas (VII) and (VIII) ]

' 7 p-".

Enzyme

3,

,'

Enz-yy

2

H, -

R

or

I

H.,,

Y'

"0

3

H (VE)

(VIII)

The central theme that the apoprotein facilitates the scission of the 0-0 bond is based on the established mechanisms of peroxide heterolysis (165).By invoking "concerted" proton transfer (s) in the transition state, such schemes illustrate that oxygen-oxygen heterolysis need not be attended by an electrostatically unfavorable charge separation. I n addition, they offer some rationale for the observed high entropy of activation in 25 cal mole-' deg-I) (166). the primary H,O,-catalase reaction (-AS* This should be the case in a rigid lattice of interactions implied in Eq. (20) and formulas (VII) and (VIII). All such suggestions are clearly most tentative since the nature of the oxidation product, Compound I, is still unresolved. The stoichiometry of the enzyme-peroxide reaction merely demands that its formal oxidaN

165. L. Bateman and K. R. Hargrave, Proc. Roy. Soc., Ser. A 224,339 (1954). 166. G. K. Strother and E. Ackerman, BBA 47,317 (1960).

396

GREGORY R. SCHONBAUM AND BRITTON CHANCE

tion state be Fe(V), i.e., two oxidation equivalents above the native enzyme, Fe (111).Such an assignment tallies well with the magnetic susceptibility for a compound with three unpaired electrons (6000-6500 X lo-’’ emu) (166) but does not uniquely define their distribution. For example, assuming that the oxidation is confined to the prosthetic group, other structures compatible with the magnetic susceptibility data can be expressed as a radical combined with Fe(1V) or as a diradical in conjunction with low-spin Fe (111).None of these structures singularly reflects the nature of Compound I but, as emphasized by Hamilton (167),all in varying degrees could contribute to its resonance form, for the term “oxidation state” has little chemical significance in compounds with a substantial covalent character, as evident in coordination compounds with delocalized ground states such as metal dithienes (168, 169) and metal-nitric oxide complexes (170). In Compound I, such polarization interactions should involve an extensive delocalization of electrons from the porphyrin toward the metal ion; an extreme case of which is a porphyrin-r-cation radical combined with Fe(1V) as shown in Eq. (21),

where X.6+denotes a radical moiety. Pertinent to this discussion are recent studies on porphyrin-r-cation radicals derived from magnesium and cobaltic oct.aethy1 porphyrins (171, 172),and zinc and magnesium tetraphenyl porphyrins (171).In all cases, the optical spectra of such radicals share features also found in Compound I (Fig. 7). These are (a) a decrease of r-r” transitions associated with the Soret band and (b) the appearance of bands between 600 and 700 nm. The chief objection to the proposal implicating “a free radical” moiety stems from the absence of a distinct E P R signature for Compound I. This is not an overriding restriction since, conceivably, an electron localized on the porphyrin will couple through exchange interactions with spin localized on the metal (173),resulting in broadening of the EPR signal beyond the limit of detection. 167. G. A. Hamilton, Advnn. Enzymol. 32,55 (1969). 168. G. N. Schrauzer, Accounts Chem. Res. 2,72 (1969). 169. J. A. McCleverty, Progr. Znorg. Chem. 10, 49 (1968). 170. J. A. Lewis, Sci. Progr. (London) 46,506 (1959). 171. J. H. Fuhrhop and D. Mauzerall, JACS 90,3875 (1968). 172. R. H. Felton, D. Dolphin, D. C. Borg, and J. Fajer, JACS 91, 196 (1969). 173. D. Dolphin, A. Forman, D. C. Borg, J. Fajer, and R. H. Felton, Proc. Nut. Acud. Sci. U.S. 68, 614 (1971).

397 120’ 100-

7 80E

-

-

‘I60-

5 40: 20OJ

400

350

450

500

550

600

I

700

650

‘ 0

A (nm)

FIG.7. Spectrum of horse erythrocyte catalase (---) and its peracetic acid deriva(Compound I) ; 0.08 M phosphate, pH 7.23, 25” ( 1 0 1 ~ ) . tive (-)

Among other factors contributing to electron delocalizations, the influence of environment, particularly that of the trans ligands, must also be taken into account. Ideally, such ligands should be polarizable but not readily oxidizable. An imidazole group a t L, could fulfill such a function and, as L,, the oxygen anion would be appropriate because of its ability to enter into u-type coordination and x-donor bonding. The nature of Compound I being still unresolved, it is not surprising that the mechanisms of its reduction have been variously expressed as: transfer of oxygen from Compound I (EO) to the reductant (HXOH) followed by the rearrangement of the “hydroxylated” intermediate (118) [Eq.

hydride transfer (17,18,75,174,175) [Eq. (2311 EO

+

XHOH

+

c .I EO

HiCQ-,,

E(H,O)

+

XO

(23)

outer sphere electron exchange (17.9) inner sphere electron transfer (17,159~4167) [Eq. (24)] EO

+

HXOH

[ -

+

E+-X

(/]

p---H

E(H,O)

+

XO

(24)

174. L. L. Ingraham, “Biochemical Mechanisms,” p. 71. Wiley, New York, 1962. 175. P. Nicholls, BJ 00, 331 (1964).

398

GREGORY R. SCHONBAUM AND BRITTON CHANCE 17mM CH,OOH

I.3pM CH,OOH

421-480nm

FIG.8. Reduction of horse erythrocyte catalase Compound I by methyl hydrogen peroxide; at pH 7, 4". Note also the ready oxidation of Compound I1 to Compound I11 by H201where knpp 3 x 1oJ M-'sec-' (164).

-

The reactions outlined in Eqs.. (22)-(24) are widely recognized in "model" redox systems. Their relevance to the catalase-mediated oxidations is the central issue-the subject of a lively current debate. I n this context, the following criteria and guidelines must be considered : 1. The HXOH donors (hydrogen peroxide, hydroxylamine, formic acid, and alcohols) and nitrous acid reduce Compound I to ferricatalase without detectable participation of Compound I1 (176). Accordingly, the reaction occurs either via two-electron equivalent reductions, or the rate of Compound I1 formation is smaller than the rate of its reduction (17'7, 178) [Eq. (25)]. Compound I + HXOH +[Compound I1 XOH] +catalase + XO (25) slow fast

If so, then Compound I1 should be reducible by radicals derived from HXOH (e.g., HOz' and NO,'). Contrary to these expectations, Nicholls (1'79) failed t o adduce evidence indicating reduction of Compound I1 by NO,'; and Compound I1 is the dominant derivative in the presence of reagents which generate superoxide. Apparently, as in the corresponding peroxidase reactions (180) catalase Compound I1 is not readily reducible by an externally generated superoxide. These observations, although not entirely conclusive, do bring into question the scheme of Eq. (25) but pertain only to compound I-HXOH interactions. With other substrates, for example, with hydroperoxides (16.4) (Fig. S), phenols (178), or oximes (101a) one-electron equivalent reductions of Compound I do occur. 2. No pH dependence is apparent in EO--(HXOH) reactions, when ionization state of the reductant is taken into account (36)(Fig. 9). All 176. B. Chance and D. Herbert, BJ 4,402 (1950). 177. D. Keilin and P. Nicholls, BBA 29, 302 (1958). 178. P. George, BJ 52, XIX (1952). 179. P. Nicholls, BBA 81, 479 (1963). 180. B. H. Bielski, D. A . Comstock, A. Haber, and P. C. Chan, BBA 350, 113 (1974).

7. CATALASE

399

4

15' 0

tb

6 PH

Fro. 9. Effect of pH upon activity of Compound I toward hydrogen donors (38).

reactions conform t o the rate law

+

(26)

d(EO)/dt = ~ ~ ' ( E O ) ( X T ) / [(Ka/H+)I ~

where XT is the total concentration of the reductant, Ka the ionization constant [Ka = (H+)(HXO-)/HXOH], and k:/[l Ka/H+] = k4 app. Thus, as in the reactions of the resting enzyme with ligands, only the un-ionized donor interacts with Compound I. Yet, following the formation of a n outer sphere complex, EO(HXOH), ionization of the substrate is a likely prerequisite t o HXOH oxidation. The point a t issue is illustrated by the relative reactivities of formic acid and formamide: the former is an excellent donor (k:Tpp 9 X 105 IT-' s-l ) (36) and the latter is inert ( 1 0 1 ~ )Similarly, . N-methyl hydroxylamine is a reductant comparable t o ethanol but 0-methyl hydroxylamine is relatively inactive ( 1 0 1 ~ ) . It is also a much weaker acid.

+

-

Donor k:',",,(M-' sec-1)

CHsCHZOH 1000

CHSNHOH 350

CH30NHz < 10

3. The reactivity pattern ( k 4 a p p : CHaCH20H> CHaNHOH > CH30NH2) would not be expected if either exchangeability of X--H hydrogen or nitrogen nucleophilicity were of importance in governing k d a p p , and the results are not peculiar to methoxy amine. I n general, amines are not effective Compound I reductants (k!c;p 12

24

Data from Chance (36), Chance and Herbert (176), and Schonbaum (10Ia).

404


TABLE XI11 THERMODYNAMIC ACTIVATION PARAMETERS FOR SOME APPARENT CATALASE COMPOUND I-MEDIATEDOXIDATIONS' Donor Methanol Trideuteromethanol Ethanol 1,1-Dideuteroethanol Ally1 alcohol 2-Propy n-l-ol 0

AHS (kcal mole-')

ASS (cal mole-' deg-1)

8.4 9.3 10.8

-17.4 -17.6 -8.9 -9.1 -10.2 -21.9

11.1

9.3 0.4

Data from White and Schonbaum (118b).

tope effects, which are principally attributable to AH* (Table XIII) obtain in the oxidation of 1-deutero alcohols (CDaOH and CHaCD20H) but not in the oxidation of deuteroformate (DC02H) (Tables XIV and XV) or deuteroethanol (CHaCH20D)(Table XVI). In particular, the large differences in k4 values and kH/kD ratios for formate and methanol oxidations cannot be simply attributed to steric constraints. Rather for formate, the formation of the Compound I-donor complex must be assumed to be rate determining. This being the case no isotope effect is expected; indeed, kFapp and k?,,, are nearly equal ( k H / k ~ 1.1 f 0.05) (Table XIV). Note also that rate constants characterizing the interaction of formic acid with the resting enzyme (kl loe M-* sec-I) (37) and with Compound I (k4 0.9 X 106 M-1 sec-1) are nearly the same. Apparently, the active site is equally accessible to substrates in different

-

.,,-

ISOTOPE

TABLE XIV EFFECTSI N BEEF LNER CATALASE-MEDIATED OXIDATIONS' Formic acid

Substrate k4 sppo

10-6

(M-1 sec-1)

k4'b

(M-1 sec-1) kdkn

-

Ethanol

Methanol

HCOzH DCOpH CHaCHzOH CH,CDzOH CHsOH CDIOH 486 8.0s

400 7.64 1.16

890

1.96

455

720

-

130

5.54

In 10 mM potassium phosphate, pH 7, 24.7'. For un-ionized form using pK.(HC02H) 3.75; pK.(DC02H) 3.78 [White and Schonbaum (118b)l. (I

7.

405

CATALASE

TABLE XV ISOTOPE

EFFECTS I N CATALASE-MEDIATED OXIDATIONS O F A N D DEUTEROETHANOLS~ ETHANOL

Rate constant (M-l sec-l) Horse erythrocyte

Substrate

1020 f 20 1020 f 20 460 f 10 2.22

Ethanol S-(-)-1JDeuteroethanol 1,l-Dideuteroethanol Pentadeuteroethanol kcE,c~,on/kcHIcDtoH a

Beef liver

&I. lysodeikticus

890 f 20

21.5 f 0 . 5 20.0 f 0 . 2 14.2 0 . 3 1.51

455 f 10 445 f 10 1.98

*

I n 10 m M potassium phosphate, p H 7, 24.7" [Schonbaum (lola)].

oxidation states of the enzyme. The simplest permissible reaction scheme is therefore EO

+ H X O H kk-il ' E O ( X H 0 H ) 2 E(H20) + XO

(36)

and

Hence, if k ) > k-l, then k 4 k l . Seemingly, such conditions are met in the Compound I-formic acid reaction. I n contrast, since in the oxidation of methanol kH/kD > 5 , scission of the C-H bond must be rate limiting, suggesting that k t < k-1, and k l a p p = ( k l k z ) / k _ , where k l a p p 830 M-l sec-'l (Table IX). N

TABLE XVI SOLVENT ISOTOPE EFFECTSI N HoRSle ERYTHROCYTE-MI.:DIATED OF ETHANOL OXIDATION Solvent

kc npp (M-I sec-l)

H20n 90 % D20-10 % H i 0 (v/v)* knlo/kDno

1020 20 960 k 30 1.06

*

I n 10 m M potassium phosphate, p H 7.0. In 10 m M potassium phosphate, pD 7.1; 24.7' [White and Schonbaum ( l l 8 b ) l .

406


Therefore, k2/k-1 = 8 X 10-4, provided that as in the reactions of catalase with methyl hydrogen peroxide (Table VIII) or with formic acid (37), kl- 106 M-' sec-1. Further, since even a t 2 mM MeOH, the kinetics of Compound I reduction by methanol obey the second-order rate law ( I & ) , it follows that k-l/lcl 2 20 m M . Hence, k-1 2 2 X lo'sec-' and kt 2 16 sec-'. The above analysis, which merely indicates the possible lower limits of k-1 Llpp and kz app, presupposes the formation of one EO(HX0H) intermediate in the course of the redox reaction. On this score definitive evidence is lacking. Even this minimum postulate may be inadequate to account for the observation that in reactions of catalase with methyl or butyl hydroperoxides less than 15% of the initial peroxide is converted into the corresponding aldehydes unlike the catalase-ethyl hydrogen peroxide system where formation of acetaldehyde from EtOOH exceeds 50% (Table V I I ) . I n the reactmionwith MeOOH, the low yield of formaldehyde could be attributed to a rapid diffusion of methanol from the active site:

%EO + CH,OH

EO(HOCH*)

(38)

Conversely, high conversion of EtOOH to CH,CHO would result from slower diffusion of ethanol from EO (HOC,H,) ; accordingly, the yield of butyraldehyde from BuOOH should be a t least equally high. Since this is not so, we can only conclude that, compared to ethanol, either the residence time of butanol a t the active site is unaccountahly shorter or that prior to oxidation of ROH, the outer sphere complex, TO (HOR b , rearranges to another intermediate ; an intermediate having a configuration in which the slowly reacting alcohols (Tables IX and X) cannot be readily accommodated. An outer sphere complex differing from EO(HOR), the possible initial product of E ( R O 0 H ) reaction, or an inner sphere complex [Eq. (24) ] could meet such criteria. However, no distinctions can be even attempted a t IUP,S(i i r We shall merely note the following: (a) Lack of isotope effect in the oxidation of C H,OD (Table XV) is inconsistent with the reaction pathway outlined in Ey. (23) ; (b) Formation of an inner sphere complex would be subject to a stringent steric control ; (c) Significant displacement of metal ion frorn the porphyrin plane,. or doming of the porphyrin macrocycle should be prerequisites to an inner sphere ligation ; (d) Addition a t iron, if any, cannot be random; otlierwise oxidations would not proceed stereospecifically (Table XI) ;

7.

407

CATALASE

(e) Oxidations proceeding by an inner sphere mechanism are formally analogous to various metal-catalyzed reactions, among others by Cr (VI) (189, 190), Pb(OAc), (191-193), or V ( V ) (194-196), and could be expressed as suggested by Taqui Khan and Martell (197) [Eqs. (38a) and (38b)l: /--

,.o -P0, H

OxE-F e y ’“OH

T__

E-Fe-

+

0,

+

OH-

(384

where OxE is a two-electron oxidized form of the active site, or as outlined in Eqs. (39)-(41) and Fig. 11.

A reminder that the above suggestions are no more than working hypotheses should hardly be necessary. 189. K. B. Wiberg, in “Oxidations in Organic Chemistry” (K. B. Wiberg, ed.), Part A, p. 69. Academic Press, New York, 1965. 190. J. K. Beattie and G. P. Haight, Jr., i n “Inorganic Reaction Mechanisms” J. 0. Edwards, ed., Vol. 17, Part 11, p. 93. Wiley (Interscience), New York, 1972. 191. K. Heusler and J. Kolvoda, Angew. Chem., Int. Ed. Engl. 3, 525 (1964). 192. Y. Pocker and B. C. Davis, JACS 95,6216 (1973). 193. R. Criegee, in “Oxidations in Organic Chemistry” (X. B. Wiberg, ed.), Part A, p. 277. Academic Press, New York, 1965. 194. K. Kustin and D. L. Toppen, Inorg. Chem. 12, 1404 (1973). 195. J. S. Littler and W. W. Waters, J . Chem. Soc., London p. 2767 (1960). 196. J. S. Littler, A. I. Mallet, and W. W. Waters, J. Chem. SOC.,London p . 2761 (1960). 197. M. M. Taqui Khan and A. E. Martell, “Homogeneous Catalysis by Metal Complexes,” Vol. I, pp 139-142. Academic Press, New York, 1974.

408


ACKNOWLEDGMENTS Research in the authors' laboratories and preparation of this review were supported by USPHS GM-12202, AA-00292 and HL-16061 (to B.C.),by the Medical Research Council of Canada (MT-12701, and by NSF-GB 41635 (to G.R.S.)

Author Index Numbers in parentheses are reference numbers and indicate that an author’s work is referred to, although his name is not cited in the text. Alm, R., 70, 88(130) Alonso, C., 65 Alpert, Y., 317, 318(109) Abacherli, E., 26 Altschul, A. M., 345, 346(1, 2), 347(1, 21, Abatwrov, L. B., 14, 31(56) 348(1, 21, 353(1) Abe, K., 367, 368, 393(69), 394(69) Amelunxen, R. E., 2(17), 3, 19(17) Abeles, R. H., 143 Anan, F., 367,368,393(69), 394(69) Abney, R., 133 Anderson, B. M., 30 Abraham, R. G., 274,275(344) Abrams, R., 345, 346(1, 21, 347(1, 21, 348 Anderson, L., 256 Andrkasson, L. E., 317, 336(108), 336(108) (1, 2), 353(1, 2) Andreev, V. P., 300 Abrams, W. R., 279, 280 Andreeva, N. S., 9 Ackerman, E., 395 Ackrell, B. A. C., 177, 245, 247, 248(26), Andreoli, T. E., 70, 76(127), 77(127) Andrews, E. C., 313 24906, 1991, 253(184) Andrews, L. J., 384 Adams, M. J., 10, 24 Anfinsen, C. B., 86, 132 Adelson, G. I., 105 Anselme, J. P., 379 Adija, D. L., 29 Antonini, E., 335, 336(192), 349 Aebi, H., 365, 367,379(63), 280(63) Appell, G., 366, 367(41), 370(41), 371(41) Agar, N. S., 142 Appleby, C. A, 264 Agatova, A. I., 25 Appleman, D., 376 Agner, K., 366, 385 Archakov, A. I., 153 Agrawal, B. B. L., 377 Arigoni, D., 252 Aikawa, T., 109 Arnold, H., 48 Akagi, J. M., 286, 295(388) Arnold, L. J., 29 Akeson, A., 200 Alben, J. O., 317, 321(113), 322(113), 323 Arnon, D. I., 54, 55(12), 66(12), 66(12), 366 (1131, 337(113) Arosio, P., 246, 247(191) Alberty, R. A., 188, 189(57), 199(57) Arrigoni, O., 237 Albracht, S. P. J., 187 Arscott, L. D., 92, 93, 94(36), 100(36, 371, Alexander, A. G., 300 103(36, 63), lOQ(61,6 3 , 105(85), 106 Alifano, A., 73 (36), 107(36), 118(63), 119(63, 85), Alimov, G. A,, 153 120(61, 63), 121(63), 123(61), 129, 135 Allen, G. A., 23,24(85), 34 (601, 136(60), 137(60), 143(36), 144 Allgyer, T. T., 110 (36) Allison, W. S., 2(15), 3, 20,21(66), 28(15), Artavanis, S., 5 30, 39 409

A

410 Arvy, L., 300 Asada, K., 274,286(359), 295(359) Asahi, T., 91, 133(7), 143(7), 279 Asakura, T., 346,347,348(27, 28,29, 30, 31,

AUTHOR INDEX

Barlow, C. H., 314, 315(93), 319, 321, 322

(1421, 323(140), 329(93), 334(94), 337 (117, 118), 338(94), 339(117,118), 365, 368, 389 Baron, J., 150 32, 37), 349(28, 30, 321, 360(27, 31) Barrett, J., 301 Asano, A., 66, 72(89), 73(89), 76(89) Barldn, E. S. G., 261 Asnis, R. E., 91 Barry, R. E., 164 Asriyants, R. A., 48 Bartlett, G. R., 261 Asyis, R. Aat., 48 Atchison, R. W., 54, 56(18), 57(18), 58 Bartsch, R. G., 371(84), 372 Basford, R. E., 222 (18) Basolo, F., 375 Atkin, C. L., 143 Atkinson, D. E., 274, 277(339), 286(339) Basu, D. K., 106, 107(98) Bateman, L., 395 Atkinson, M. R., 109 Batke, J., 25, 40 Auda, B. V., 260 Auts, S. D., 165, 166(369), 167(374), 168 Bathe, G. R., 84 Baudhuin, P., 368 (374), 169(374) Baudras, A., 265, 268(287, 289), 269(287, Averback, B. C., 274,275(344) 299, 302) Awasthi, Y. C., 312; 313(84) Bauer, V. A., 143 Assi, A., 72 Baugh, R. F., 182, 187, 188, 189, 190(43) Azzone, G. F., 67, 72(102), 214, 282 Baum, H., 70 B Bayne, R.. A., 315,317,337(115) Bearden, A. J., 210 Baccarini-Melandri, A., 2(28), 3 Beattie, D. S., 81(176), 217 Bach, S. J., 263.264 Beattie, J. K., 407 Bachmanova, G. I., 153 Beck, W. S., 143 Bader, P., 235 Baggott, J. P., 33, 34(156), 166, 167(380) Bednars, A. J., 258 Baginsky, M. L., 106, 107(112), 108(112), Beetlestone, J., 346, 372, 375(99) 112(112), 205, 224, 236(156), 243, 244 Behme, M. T. A., 43 Beinert, H., 100, 101(69), 179, 184(34), 185 (156), 246(156) (34, 46), 186(46), 187(34,46), 193(46), Bailey, K., 3 205, 214, 215(46, 541, 216(116), 219, Bakker, E. P., 315, 316(102), 320(102), 220(136), 221(46, 1361, 226(218), 235, 321(102) 244, 245, 253, 297, 307, 309, 316(66), Balastero, F., 237 320, 321, 323, 330, 331(139), 332(126, Baldesten, A., 93, 143(46), 144(46, 275) 1391, 333(139), 335(66, 126), 336(66, Balegh, M. S., 370 194) Balthasar, W., 31 Bell, J. J., 65, 83(84), 84(84) Baltscheffsky, H., 74, 254, 256(220) Benjamin, B. M, 39, 45(181) Baltscheffsky, M., 74, 254,256(220) Benohr, H. C., 130,131 Banaszak, J. J., 10, 11(51), 12(51) Berger, A., 379 Banaszak, L. J., 9, 10, 35, 317, 318(112) Berger, T. J., 68,71(115), 79(115) Bandi, L., 83 Bandurski, R. S., 91, 133(7), 143(7), 274, Berghauser, J., 24 Berglund, O., 143 279, 286(359), 295(359) Bernath, P., 222, 223(144), 225(144), 226, Banerjee, R., 317, 318(109) 271, 273 Baranowski, T., 9 Bernhard, S., 33, 34, 35(160), 36(160), 37 Barea, J. L., 274 (174, 175), 41(160), 42(165), 48(165), Barela, T. D., 15 49(165) Barker, H. A., 143

411

AUTHOR INDEX

Bernheim,. F., 263 Betheil, J. J., 31,34(137) Betz, G., 150 Beutler, E.,131,132(212),132 Beychok, S.,123, 125(168) Bianchi, G.,262 Bielski, B. H., 398 Biggs, D. R , 217 Bilimoria, M. H.,167, 168(384, 386), 170 (386) Bittmann, R.,31 Bjorkhem, I., 83 Black, S.,91,133, 141, 143(6) Blair, P.V.,237 Blakley, R. L.,143 Bloch, W., 34,42(165),48(165),49(165) Blumberg, W. E.,368, 369, 370(77, 78), 376(77), 380,381(126b), 390(126b) Bock, R. M.,310 Boger, P.,54, 62 Boeri, E.,264, 270, 272 Boers, W., 28,33 Bolotina, I. A., 25, 31(105) Bond, J. C, 24 Bondi, A., 401 Bonner, W.D., Jr., 215 Bonnichsen, R. K.,366 Booth, F.W.,300 Borg, D. C., 356,396,397(173) Boross, L., 28, 43 Borst, P.,80 Bossi, E., 365 Bourne, G.H., 300 Boxer, G.E.,47 Boyd, G.S.,83 Boyer, P.D., 39,40,74, 75, 101 Braams, R.,335 Brady, A. H.,123,125(168) Brady, W. T.,93 Bragg, P. D., 64,68(62), 72(62), 79, 80 Bramlett, R.,284,285(382),297 Branden, C. I., 10,11(51),12(51) Brandt, K.G.,94,112(52,53),113(52,53), 114(53), 135(52,53),137(53), 139(53), 141(53) Branzoli, U.,101 Bresters, T.W., 106, 107(114),114(114) Bridgen, J., 5,22 Brierley, G.,180,312,313(85) Brill, A. S.,351, 365,369, 370(74, 791,372.

(74,79,81),374(81), 376(79), 385(16), 389(16), 390,396(156) Brocklehurst, E. S., 321,322(138), 326(138 327(138),331(138), 333(138), 335(138) Brodie, A. F., 64, 65, 66(60), 68(60), 72 (60) Brodie, J. D.,237 Bronk, J. R.,62,65(34), 67(34) Brosemer, R. W., 2(23), 3 Brosnan, J. T., 82,86(186) Brown, J. P.,104,118(86),119(86), 120,121 (86) Brown, N. C., 142. 143(262) Brown, S.B., 389 Brownie, A. C., 83,84(192) Brumby, P. E.,114, 122(155), 123(155) Bruni, A., 239,244,246 Brunori, M.,315, 316(99), 323(99), 324 (99),335, 336(99, 192), 389 Bryla, J., 110 Bucher, T., 81 Buege, J. A., 165, 166(369, 3741, 167(374), 168(374),169(374) Buehner, M., 9,10(46), ll(46, 48),24(47), 29(47), 39(47, 48), 44(46, 47) Bulger, J. E.,94, 112(52, 53), 113(52, 53), 114(53),135(52,531, 137(53), 139(53), 141 (53) Burgoyne, L. A.,267 Burleigh, B. D., Jr., 92, 100(33), 103(33), 104(33,61), 105(33), 119(33), 120(61), 123(61) Burma, D. P., 106,107(98) Butler, W. L.,239,240(178) Butlin, J . D., 68,78,79 Butow, R.A.,186,203(48) Buzard, J. A.,129

C Caifa, P., 247 Caldwell, B. V.,84,88(225) Camerino, P. W.,320, 333(131) Cameron, B. F.,372,373(96) Cammer, W., 65,84(83) Canellakis, Z.N..142,143(262) Cantz, M.,365, 367,379(63), 380(63) Capaldi, R. A., 180, 311, 312(75) Capeillere, C., 268, 269(299) Caputo, A., 349

412 Caputto, R., 2,3(7) Cardenas, J., 274 Carlson, C. W.,2(23), 3 Carpenter, E.,312 Cam, M.L.,150 Carr, N. G.,2(27), 3 Carraway, K.L., 48 Carroll, W.R.,10s Casida, J. E.,177, 204, 205(22, 881, 206 (22) Casola, L., 112(150), 113, 114(150), 122 (150, 155), 123(155) Caswell, A. H.,215 Caughey, W. S., 302, 304, 305(40), 306 (52), 307, 308(52), 313(52), 314(60, 62), 315(52, 93), 316, 317(52, 651, 318 (52, 65), 319, 321(113, ll6), 322(62, 65, 113, 114, 115, 1421, 323(65, 113, 114, l40), 325(65), 328(65), 329(65, 93), 330(104), 334(60), 337(15, 113, 115, 117, 118),338(27, 40, 80,62, 651, 339(117, 1181, 340(116), 343(60, 621, 364, 389 Cavallini, D., 304 Cederbaum, A. I., 81(176), 402 Cepure, A., 101 Cerletti, P.,222, 225(150), 230(157), 231, 234, 237, 246, 247(191), 248 Challoner, D. R., 300 Chamalaun, R.A. F. M.,82 Chan, P. C., 398 Chan, S. H.P.,310,311(69) Chance, B.,31, 72, 204, 207, 215,219,258, 259(229), 301, 321, 324, 335(21), 336 (1551, 346, 348(22), 351(22), 352(6, 22),353(4,6, 221, 356(5), 363(5), 364, 365, 366, 369(1), 372, 374(37), 385 (37), 388(37, 52), 388(36), 389, 390 .(150), 391(150), 393(1), 394(1), 398 (36,1541,399(36), 400(146), 401, 403, 404(37), 406(37, 146) Chang, J. Y.,377,378 Chang, 5. H.,133 Changeaux, J. P.,31 Chantrenne, H.,347, 348(43) Chappell, J. B.,207 Charache, S., 322 Charalampous, F. C., 312 Chen, W.L.,312 Christensen, J. R.,261

AUTHOR INDEX

Christian, W., 2 Chuang, T.F.,312,313034) Chung, A. E., 53, 55, 56(10, 18), 57(10, 181, 58(10, 181, 60 Chung, C. W., 274 Cilento, G.,28 Ciotti, M.M.,30, 52, 53(1), 54(1), 56(l), 58(1, 31, 62(1), 65(31), 86(75) Claisse, M., 269 Clark, W.M.,130 Clegg, R.A.,217,219(131), 220(135) Cleland, W.W.,40, 76, 88, 139 Click, E. M.,288 Clodfelder, P.,39,45(185) Cobley, J. G.,219,220(136), 221(136) Cochran, D.G.,258,259034) Cocriamont, C., 347 Cohen, B. S.,151, 152(318, 320), 153 Cohen, I. A., 302, 304, 338(27) Cohen, P. T.,53, 54, 56(17), 57(6), 58, 59 (17), 60, 61 Cohn, M. L., 100, 103(65), lOQ(65) Coleman, R.,180, 181(41) Coles, C. J., 225, 226, 230(158), 234(15@, 248(158) Colli, W.,88 Collins, D. M., 376 Collipp, P.J., 216 Colman, R. F.,141 Colowick, S. P.,3, 28, 29(13), 38(13), 39 (131,40(13), 44, 52, 53(1), 54, 56(1), 58(1, 2, 3, 5), 62(1 ), 65(30), 67(30), 69(30), 70(30), 73(30), 76(30), 167 Comstock, D. A.,398 Conn, E. E.,92 Conney, A. H.,149 Connors, M.J., 30, 39 Conover, E.,72 Constantinides, S. M.,25 Conway, A., 31, 33, 34(143), 42(143) Cook, D.E.,153 Cook, K.A.,274 Coon, M.J., 91, 149, 150(295), 151(295), 153, 165(286), 166(370, 3711, 167(370, 371), 169 Cooper, A., 371(85), 372 Cooper, D.Y.,83, 91, 152, 166 Coratelli, P.,88 Corcoran, D., 148, 151(284a), 164 Cordes, E. H.,43

413

AUTHOR INDEX

Cori, C. F., 2, 3(8), 48 Cori, G. T., 2, 3(8), 48 Cori, O., 261 Cornforth, J. W., 252 Corrall, R. J. M., 402 Correa, W., 131 Corte, E. D., 132 Coulson, A. F. W., 347, 350, 353(34), 355 (34, 36)

Countryman, R., 376 Cox, C. D., Jr., 274 Cox, D. J., 93 Cox, G. B., 64,68, 78(73), 79, 81(73) Crane, F. L., 68, 79, 312, 313(84) Crawford, I. P., 254, 256(220) Creaghan, I. T., 110 Cremona, T., 69, 78, 177, 184, 188, 189, 190(58, 841, 202, 203, 270, 271, 272, 273 (320) Cresswell, C. F., 274 Creutz, C., 375 Criddle, R. S., 310 Criegee, R., 407 Crifo, C., 304 Cronin, J. R., 259, 260(243), 262(243) Cross, D. G., 29 Cseke, E., 28, 43 Cunningham, L. W., 39, 45(185) Cunningham, W., 313 Curdel, A., 272, 273(329) Curnyn, C., 66, 72(.87) Curti, B., 101 Cusanovich, M. A., 325 Cutolo, E., 264 Czerlinski, G., 117 Czygan, F. C., 274

D Dade, E., 46 Dahlen, J. V., 88 Daigo, K., 93, 108(43) D’Allessio, G., 2(16), 3, 4(16) Dalling, M. J., 274 Dallner, G., 166 D’Aloya, R., 63,87(53) Dalziel, K., 139 Dandliker, W. B., 24 Daniel, L. J., 261

Danielson, L., 64, 65(56, 57, 58), 67(56, 57, 58), 68(56, 57, 581, 72(56, 57, 58), 73(57, 581, 74(56, 57, 581, 77(56, 57, 58) Danielsson, H., 83 Darnall, D. W., 15 Davidson, B. E., 2(25), 3, 5,9(33), 20(33)

Davidson, D. W., 83 Davidson, J. T., 282, 284(377) Davies, J. L., 304, 305(40), 338(40) Davies, P. L., 80 Davis, B. C., 407 Davis, K. A., 179, 191, 192(71, 73), 193 (32, 691, 200(69),. 203, 222, 224(141, 142, 143), 225(141, 142, 143), 226(32), 227, 228, 229, 230(73, 141, 142, 143), 231, 232(71, 166), 233, 234, 235(143), 236, 237(166), 239,240(166), 241(166), 242(166), 243(166), 244(143), 245 (143), 253,254,246(143), 248(71,166), 254, 255, 256(220) Davis, P. S., 170, 288, 290(413, 414, 4151, 291(414), 292(413, 415), 294(414) Davison, A. J., 324 Dayhoff, M. O., 105, 371(83), 372 Deacon, T. E., 282, 284(377) Deal, W. C., Jr., 25, 30(108), 48(108) DeBernard, B., 155, 189 de Duve, C., 365, 367(23), 368 de Haan, E. J., 63, 82, 86(191), 87(53) Deisseroth, A., 365, 385(18), 389(18), 397 (18) De Kok, A., 101, 106, 107(114), 114(114), 124, 125(172), 238 De La Chica, G., 133 De Lorenzo, F., 132 De Luca, C., 65 De Luca, H. F., 83 De Marco, C., 304 Dennis, D. T., 2(26), 3, 39, 40(!26), 45 (188), 48(26) De Rosier, D., 126 DerVartanian, D. V., 124, 125(172), 187, 221, 222, 224(149), 226, 236(149), 237 (149), 238(149), 251, 252, 253(149), 281, 284, 285(282), 288, 295(373a) Deutsch, H. F., 366,382(39) Devichensky, V. M., 153 De Vijlder, J. J. M., 31, 32, 33(144, 1511, 34(144), 41(159)

414

AUTHOR INDEX

Devlin, T. M., 62, 65(33) Eichner, R. D., 29 De Wael, J., 131 Eichorn, J., 83 Dewan, J. D., 2 Eigen, M., 31 Diamond, L. S., 66 Eik-Nes, K. B., 83 Dick, A., 259 Eisele, B., 28, 33 Eisenberg, D., 371(851, 372 Dickerson, R. E., 371(85), 372 Ejima, A., 108 Diehl, H., 152 Ekstrand, V., 93, 94(40), 138(40), 139 di Franco, A,, 268 (40), 140(40) Dixon, M., 2,3(7), 109, 263, 264, 267 Djavadi-Ohaniance, L., 212, 213, 296, 297 Eldjarn, L., 130 Eley, M. H., 108 Doeg, K. A., 224,236(153), 239 Elias, H. G., 24 Dolphin, D., 356, 396, 397(173) Ellfolk, N., 347, 348(40, 41, 42) Dontsov, A. E., 74,75(148) Elliott, J., 46 Dorfman, R. I., 83 Elliott, P., 143 Dorsey, J. A., 88 Dounce, A. L., 365, 366, 367, 385(18), 389 Elodi, P., 2(14), 3, 23, 25, 26, 31 Engel, P. C., 91,100 (18), 397(18) Englard, S., 31, 34(137) Drabikowska, A. K., 258 Eriksson, A., 133 Drabkin, D. L., 300 Eriksson, L. E .G., 235 Dragoni, N., 286, 295(389) Eriksson, S. A,, 132 Dreyfuas, J., 286, 287(387), 288 Dro t t, H R., 347, 348(32), 349(32), 360 Erman, J. E., 347, 350, 353(34), 355(34), 372, 376(88) DuBus, R., 91 Ernest, M. J., 130 Due& E., 9, lO(45) Duggleby, R. G., 2(26), 3, 39, 40(26), 45 Ernster, L., 64, 65(56, 57, 58), 67(56, 57, 58), 68(56, 57, 581, 68(66), 69(68, 69, (188), 4806) 106), 70(59, 67), 72(56, 57, 58, 67), Duncan, H. M., 216 73(57, 58, 59, 102, 103, 106, 135, 136, Duncan, I. W., 93, 94(40), 138(40), 139 137), 74(56, 57, 58, 106, 136, 1371, 75 (40), 140(40) (67, 68, 69), 76(59, 67, 68,69), 77(56, Dunstan, W. R., 399 57, 58, 59, 67, 68, 69, 106, 135, 136, Duppel, W., 149, 150(295), 151(295) 137), 78, 82, 86(191), 87(74, 1841, 88 Dupre, S., 340 (129, 130), 166, 168, 204, 207, 212, 214, Durchschlag, H., 32 246, 249(188), 261 Dus, K., 371(84), 372 Escamilla, E., 133 Dutton, P. L., 215, 325 Estabrook, R. W., 63, 65, 66(37), 81(37), E 83, 84(83), 86(37), 91, 151, 152(318, 320, 321), 153, 168, 186, 203(48), 258, Eberhard, C. A., 106, 107(97) 259(233), 261, 262(262), 387 Eberspaecher, H. I., 305 Evang, A., 368 Ebisuzaki, K., 260 Eylar, E. H., 317, 318(112) Eby, D., 29, 30(126) Edelhoch, H., 101, 189 Edelstein, S. J., 101 F Edmondson, D., 222 Faeder, E. J., 170, 287, 288, 290/415), 292 Edwards, J. O., 399 Edwards, J. T., 401 (415) Fahien, L. A., 26, 31(110) Egan, R. S., 377 Ehrenberg, A., 200, 235, 330, 331(174), Fairs, K., 302 346, 348(22), 351(22), 352(22), 353 Fajer, J., 356, 396, 397(173) Feeney, R. E., 2(22), 3, 26(22) (20,22), 356,387,388,400(143)

.

415

AUTHOR INDEX

Fehrniann, H., 106, 107(106), 112(106) Feigin, L. A,, 366 Feinberg, R. H.,262 Feinstein, R. X., 365 Feldberg, N. T.,249 Feldberg, R.,90,282 Felsenfeld, G., 330 Felton, R. H.,356, 396,397(173) Felton, S. P.,179, 188(37), 189, 195(63), 203(63) Fenselau, A.,23,42,43(202) Ferri, G., 22,44(76),101 Fife, T.H.,39,45(181) Filmer, D.,32,33(149) Fisher, H.F., 29 Fisher, R. J., 64, 65, 67, 68(61), 72(61), 79 Fisher, R. R., 68, 69(112), 71(109, 110, 111, 112), 76, 77(118), 78(109, 110, 111, 112), 207, 213 Fitzgerold, B., 48 Flashner, M.I. S., 101 Fleischer, S.,180,312,313 Flohe, L.,130 Fluharty, A. L.,105 Forster, T.,359 Fogo, J. K.,202 Fonzo, D., 84 Forchielli, E.,83 Forcina, B. G., 22,44(76) Ford, G. C.,9, 10(46), 11(46, 48), 24(47), 29(47), 39(47, 48), 44(46, 47) Forestier, J. P., 268,269(302) Forman, A,, 396,397(173) Forti, G., 100 Foster, R., 384 Foust, G. P.,90,94,98(50), 146, 147(284). 282 Fowler, L. R., 179, 258(33), 306(50), 307, 308(50) Fox, J. B., Jr.. 24 Frampton, V. L.,366,367 Francavilla, A,. 63,86(50), 87(54) Francis, S. H., 24,42, 44, 45(90), 48(200) Franklin, M.R.,168 Fraser, D.R.,83 Frech. M.E.,62,65(31) Fredericks, W. W.,54, 59(13). 66(13) Fricke, H.,302 Friedrirh, P.,23

Frisell, W. R., 259, 260(243), 262(243) Furhs, S.,132 Fuchsman, W. H., 304, 305(40), 314, 334 (941,338(40, 94) Fuhrhop, J. H., 396 Fujihara, Y.,320, 325(125), 326(125), 331 (125),332(125) Fukuyoski, Y.,108, 109, llO(132) Furfine, C.,3, 19,31(12), 34(137),40(60), 41(60), 42(60), 44(60), 48(60) Furuta, H., 366,367(50)

G Caber, B. P., 105 Gal, E. M.,106, 107(94) Galante, Y.,178, 214, 246, 247(191), 296, 297 Gallego, E., 31 Gallop, P.M., 379 Galston, A. W.,366 Gang, H., 402 Ganther, H.E.,132 Garhe, A.,24 Garland, P. B., 63, 86, 87(52), 88, 204, 217,219(131), 220(135) Garrett, R. H., 274,275(351),275(350) Garwood, D.C.,119 Garwood, D.S.,119 Gaylor, J. L.,151 Gehl, J. M.,54, 59(13), 66(13) George, P.,304, 346, 361% 9), 364, 369, 372, 375(99), 389(9), 392 Gerari, G., 333 Gerth, E., 48 Ghalambor, M. A., 222, 224(141), 225 (141), 230(141), 232(166), 236, 237 (1661, 240(166), 241(166), 242(166), 243(166), 248(166), 253( 166) Ghazarian, J. G., 83 Ghiretti-Magaldi, A.,271, 273 Ghisla, S.,97, 235 Gibson, F., 64, 68, 78(73), 79, 81(73) Gibson, Q . H., 92,94(24), 97(24),98(24), 107(24), 109, 111(24), 112(24), 113 (24, 1371, 115(24, 137), 116(24), 136, 167(245), 168(245, 386), 169(245), 170(245,3861, 172(245,398), 286, 287 (3941, 290(394), 291(394), 308, 322, 324, 327, 333, 335(153), 336(153)

416 Gierisch, W., 367 Gieseman, H., 383 Gilboa-Garber, N., 286, 287(391) Gillette, J. R.,153 Gilmour, M. V., 311, 312(78), 324, 335 Gioeli, R. P.,65 Giordano, M.,237, 247 Giovenco, M.A.,237,247 Giovenco, S.,247 Girotti, A. W.,372 Giuditta, A.,246,247(189) Givol, D.,37 Glatzle, D.,131 Gleason, F.K.,143 Glenn, J. L.,199,260 Goldman, D.S.,106, 107(111) Gonze, J., 63, 186, 203(48), 262 Goodall, D.,31 Gordon, M.S.,300 Gorjunov, A. I., 9 Goto, M.,150 Gottesman, D.P.,110 Goulding, E.,399 GouLian, M.,143 Grab, D.J., 365 Gralen, N.,366 Grandchamp, S.,217 Grande, H. J., 126 Grant, J. K.,83,84(192) Grataer, W.B.,32 Gray, H.B.,385,386(137) Gray, R. W.,83 Grebner, D.,310,319 Green, A. P.,65 Green, D.E.,2, 75,222,237,256,257(223) Greenbaum, A. L.,46,47(211), 81,82 Greene, J. C.,2(22), 3,26(22) Greenfield, R.E.,366 Greengard, P.,83 Greenstein, D.S.,388 Greenwood, C., 315, 316(99), 322, 323 (99), 324, 327, 335(153), 336(99, 153, 192), 389 Gregolin, C., 270, 271, 272(312) Greville, G. D.,87, 273 Griffith, J. S.,372,375(99) Griffiths, D. E.,64, 68(63), 69, 72(63), 73(63), 178, 179(27, 281, lSl(281, 183 (28), 184(28), 190(27, 281, 245(27),

AUTHOR INDEX

258(33), 306(45), 307, 308(45, 53), 327(53), 330(53) Grinius, L. L., 69,74(120), 75(120, 148) Groot, G. S.P., 73,312 Grossman, S., 219, 220, 221(136) Groudinsky, O.,264, 265, 267 Gudat, J. C.,275 Guerra, F.,84 Guerrero, M. G., 274, 276 (342, 345) Guest, J. R.,110 Guiard, B.,267, 296 Guillory, R. J., 68, 69(112), 71(109, 110, 111, 112, 113, 117), 78(109, 110, 111, 112), 213 Guindon, A. H., 106, 107(97) Gumaa, K.A.,46, 47(211), 81 Gunsalus, I. C.,91,92, 106, 107(110, 115), lOS(lS), 172 Gupta, R. K.,347, 348(38), 357(38) Gurd, F. R.N., 317, 318(112) Guseva, M.K.,2(24), 3,25(24), 30(24) Gustafsson, J., 83 Guthenberg, C.,133 Gutierrez, J., 133 Gutman, M.,69, 177, 186(23), 188(19, 23), 200(19), 201(19, 23), 203(19, 231, 204 (19,23), 205(19, 23,881,206(19), 214, 216(116), 223(23), 224(23), 235(23), 236(23), 238(23), 247(20, 231, 248(23, 24), 249(23, 197), 250(23, 195), 251 252 Gutnick, D. L., 66,79

H Haaker, H., 106, 107(114), 114(114) Haas, E.,167 Haavik, A. G., 178, 179(27, 28), 181(28), 183(28), 184(28), 190(27,28), 224(27), 245(27), 258(33) Haber, A., 398 Hachimori, A.,366, 367(50) Hackert, M.L.,24 Hageman, R. H., 274 Hager, L. P.,377, 378(118), 397(118), 401 (118) Hagihara, B.,323 Hagman, L. O.,348, 364 Haight, G. P.,Jr., 407 Hainfeld, J., 126

AUTHOR INDEX

Halkerston, I. D. K., 83 Hall, D.E.,93,143(46), 144(46) Hall, P.F.,83, 85(209) Halsey, Y.D.,130 Hamada, M.,108, 109, llO(132) Hamberg, M.,83 Hamilton, G. A., 396, 397(167) Hamilton, L.,108 Hammes, G. G.,126 Hanania, G.I. H., 372, 373(96) Haniu, M.,100 Hansen, R. E.,179, 180, 181(41), 184(34), 185(34, 461, 186(46), 187(34, 46), 193 (461, 215(46, 54), 221(46), 309, 316 (66),335(66), 336(66, 194) Hanstein, W. G., 78, 178, 179, 181(80), 191, 192, 193(32), 199(68), 200, 203 (68),206(42), 207(42), 208, 209, 210, 211(80), 212(80), 214(80), 222, 224 (141, 1421, 225(72, 141, 1421, 226 (321,230(141, 1421, 232(71), 236, 237 (1661,240(166), 241, 242,243, 248(70, 71, 721, 253(166) Harano, Y., 84 Harding, B. W.,65, 83(84), 84(84, 211) Hardman, K. D.,317, 318(112) Hargrave, K. R.,395 Harlow, D.R.,66 Harmsen, B. J. M., 32, 33(151) Harrer, C.J., 167 Harrigan, P. J., 21, 23, 35(69), 37(83), 39(69, 831, 40(83), 41(69, 1901, 43 (831, 44 Harrington, W. F, 5, 24 Harris, J. I., 2(18, 19, 211, 3, 4(18, 19), 5, 9(18,33), 14(35, 371, 18, 19, 20(19, 29, 33, 371, 21(29, 30, 34, 37), 22(35, 37), 23, 24(35, 85), 25, 26, 27(103), 34, 36(35), 39, 45(29) Harrison, P. M., 100, 103(68) Harte, E.M.,91, 143(6) Harting, J., 30,39,45(184) Hartree, E.F.,301, 346, 366,367(30), 382 (30),387(30), 388 Hartzell, C. R.,309, 316(66), 317, 320, 323, 325(107, 125), 326(107, 125), 327 107), 330(165), 331(125), 332(125, 126), 335(66, 107, 1261, 336(66, 194) Hasson, E. P.,66 Hatano, S., 274

417 Hatch, M. D., 143 Hatefi, Y., 78, 178(30), 179(27, 291, 180, 181(29), 183, 184(28, 291, 190(27, 28), 191, 192(71, 73), 193(69), 194(40, 671, 195, 196, 197, 198(40), 199(88), 200 (29,45,69),203,204, 205,206(40,42), 207(40, 421, 208, 209, 210, 211(80, 107), 212(80), 213, 214, 216(40, 108), 222, 224(141, 142, 1431, 225(72, 141, 142, 143), 226, 227, 228, 229, 230(73, 141, 142, 143), 231, 232(71, l66), 233, 234, 235(143), 236(156), 237(166), 239,240(166, 178),241(166), 242(166), 243(166), 244(143, 1561, 245(143), 246 (143, 156), 248(70, 71, 72, 1661, 253, 254, 255(220a), 256, 258(29, 331, 296, 297, 306(50), 307 308(50) Hattori, A.,274 Hauber, J., 237,240,247,255 Havsteen, B. H., 31 Hayaishi, O., 189 Hayakawa, T.,106, 107(96), 108, 109, 110 (132) Hayashi, A.,372, 373(92) Hayashi, H., 311 Hayaski, T., 108 Hayes, J. E.,30 Hechter, O.,83 Heidema, J., 149 Heim, W.G.,376 Heimberger, G.,387 Heineman, W.R.,325, 330 Helleman, P. W.,131 Hemmerich, P.,170, 235, 308 Hemmes, R. B., 54, 55, 56(16), 58(16), 59(16), 80 Hems, D. A., 46 Henderson, R.W.,325,326(162) Henley, K. S., 82 Henning, U.,106, 107(107), 108(107) Heppel, L.A.,388 Herbert, D.,366, 367(45, 46), 398, 403 Hershberg, R. L.,364, 374, 385(102) Heusler, K.,407 Hewitt, E.J., 274 Hildebrandt, A., 151,152(321) Hill, E.,10 Hill, F. L.,48 Hilvers, A. G.,33,39,41(159) Hinkle, P.C.,88

418

AUTHOR INDEX

Hiraga, M., 367, 368, 393(69), 394(69) Howard, R . L., 191 Hirayama, K.,165 Howell, L.G.,90,91,146, 147(284), 282 Hiromi, K.,268 Howland, J. L.,63 Hoagland, V. D., 25 Hou, C., 64, 68(62), 72(62), 79, 80 Hoard, J. L.,376 Hrycay, E.G., 150,152 Hoberman, H. D.,69 Huang, J. J., 172 Hochberg, R . B.,85 Huang, P. C.,189, 190(59), 379 Hocking, J. D.,2(19), 3, 4(19),5, 14(37), Hucho, F., 106, 107(92), 108(92) 20(19, 37), 21(37), 22(37) Hudson, B.,91, 133, 143(6) Hockstein, P., 168 Huennekens, F. M.,106, 107(112), 108 Hodgins, D.S., 143 (112), 112(112), 179, 188(37), 189, Hodson, R. C.,282, 283 195(63), 203(63), 216 Hojeberg, B., 53, 58(8), 61(8, 231, 69(8), Huheey, J. E., 334 Hultquist, D.E.,165 70(8), 71(8), 7603) Hoek, J. B., 53, 58(8), 61(8, 231, 68, Humphrey, G. F.,62, 65(32), 67, 69(32), 69(8), 70(8), 70(107), 71(107),76(8), 70(32), 76(32) 78(107), 82,86(191), 87(184), SS(130) Hundal, T.,68,70(107), 71(107), 78(107) Hoeksema, W. D., 286 Husain, M.,297 Hoffman, B. M,360 I Hofmann, T.,92, 100(32), 103(32, 68), 124(32) Ibers, J. A.,302 Hogeboom, G. H., 67 Ibne-Rasa, K.M.,399 Hogenkamp, H. P.C., 143 Icen, A.,93, 94(39), 100(39), 129(39), 132 Hogness, T.R., 167,345,346(1, 21, 347(1, (39), 138(39), 140(39), 141 2),348(1,2),353(1,2) Ichihara, K., 149 Hogue, P.,248 Ide, S.,106, 107(96) Hoguem, P.K.,109, llO(130) Ii, I, 129 Holleman, W.H., 25 Iizuka, T.,347,351, 356(55), 360,372,376, Hollocker, T. C.,249 382(107), 387(100) Holloway, M.R., 25,26; Imahori, K.,2(20), 3 Holloway, P.W., 151 Imai, K.,66, 72(89), 73(89), 76(89), 372, Hollunger, G.,207 373(92) Holmes, R. S.,365 Imai, Y., 151 Holmgren, A., 93, 119(45, 471, 143(46, Ingraham, L. L.,397 47), 144(46,47) Inomata, H., 165 Holtaman, J. L.,150 Irukulla, R., 365 Hommes, A.,70,77(126) Isaacs, G. H., 259 Hommes, F.A.,207 Isaacson, E. L.,150 Hommes, R. W., 63 Isherwood, F.A., 129, 130(191), 138(191) Hong, J., 243 Ishimoto, M.,286,295(395) Hood, W.,2(27), 3 Ishizawa, S.,165 Hooper, M.,65 Ito, A.,154 Hopkins, F.G.,245 Iwatsubo, M.,268, 269(299), 272, 273 Horecker, B. L.,166,167(375) (329) Horgan, D.J., 177, 205(22), 206(22) Iyanagi, T.,165, 166(373), 167(373), 170 Horie, S.,306(47), 307, 322 (373), 171(373), 172(373, 402) Horio, T.,323 J Horney, D.L.,108 Hoskins, D.D.,164 Jacobs, E. E., 306(48), 307,308(48), 313 Hosoya, T.,346 Jacobs, N.J., 260

AUTHOR INDEX

419

(390, 397), 289(390, 3971, 290(394, 413), 291(394), 292(390, 397, 4131, 293(390), 295(412) Kaneda, T., 108 Kanner, B. I., 79 Kanzaki, T., 108 Kaplan, N. O., 2(15), 3, 20, 28(15), 29, 30, 52, 53(1), 54(1), 56(1, 17), 57(6, 71, 58(1, 2, 3, 5, 7, 21, 221, 59(17), 60(7), 61, 62(21), 65(30, 311, 67, 69 (30, 78, 96, 971, 70(30, 961, 72(78), 73(30), 76(30), 77(118), 78(97), 86 (75), 87(24), 110, 167, 168, 169(388), 207 Karpukhina, S. Ya., 366 Karr, G. M., 5, 24 Karuzina, I., 153 Karyakin, A. V, 153 Kaschnitz, R. M., 153, 206 Kaufman, B. R., 67, 69(96), 70(96), 110 Kaufmann, H., 367, 379(63), 380(63) Kawahara, Y., 106, 107(102, 104), 126 (104) Kawakita, M., 187 Kawasaki, T., 65, 69(78), 72(78) Kearney, E. B., 69, 176, 177, 188, 189, 190 (58, 83), 202, 203, 217, 222, 223(25, 140), 224(25), 234(25), 235(25), 236 (25), 237(25), 238(25), 245, 24700, 25), 248(24, 25, 261, 249(25, 26, 197, 199), 250(25, 195, 196, 1971, 251(197), 252(197), 253(184), 254(16, 251, 270 Keefer, R. M., 384 Keenan, T. W., 312, 313(84) Keilin, D., 222, 223, 240(147), 264, 300, 301, 315, 338(14), 346, 363(4), 364, 366, 367(30), 382(30), 387(30), 388, K 398 Keirns, J. J., 311, 312(78) Kadlubar, F, F., 153, 154 Keister, D. L., 54, 55, 56(11, 16), 58(16), Kadziauskas, Y.P., 69, 74(120), 75(120) 59(11, 16), 64, 66(11, 64, 65), 68(64, Kagawa, T., 263 65), 69(65), 72(64, 65), 73(65), 76 Kajiwara, S., 346 Kallai, 0. B., 371(85), 372 ( 6 9 , 80 Keleti, T., 24, 25, 40 Kalra, V. K., 65 Keller, R., 267 Kalse, J. F., 124, 125(172, 173). 238 Kelso, J . R., 300 Kamen, M. D., 371(84), 372 Kamin, H., 91, 136, 166(10), 167(10, 245), Kendrew, J. C., 349, 364, 372 168(10, 245, 384, 3861, 169(245), Kenney, W. C., 109, 110(130), 177, 222, 223(25), 224(25), 225, 226(158), 230 170(245, 3861, 172(245, 398), 176, 278 (158), 234(25, 158), 235(25), 236(25), (3), 286, 287(390, 394, 397, 412), 288

Jacobson, M., 149 Jacq, C., 264, 265, 266, 268(290, 291), 296 Jacquot-Armand, Y., 267 Jaenicke, R., 25, 32 Jajczay, F. L., 372, 373(90), 379(90), 380 (1251, 381(125), 382(90, 125), 383, 384, 385(125) Jalling, O., 261 James, B. R., 302 Jasaitis, A. A., 69, 74(120), 75(120, 148) Jecsai, G., 25 Jedeikin, L. A., 46, 49(214) Jefcoate, C. R., 83, 151 Ji, S., 75 Jick, H., 150 Jocelyn, P. C., 129 Jornvall, H., 5 Johansson, B. C., 254, 255(220), 256(220) Johnston, J. M., 151 Jollow, D., 153 Jones, E. T., 92, 94(35), 100, 103(35), 104 (35), 119(35), 120(61), 123(61), 141 (35) Jones, G. L., 365 Jones, G. M. T., 5, 14(35), 22(35), 24(35), 36(35) Jones, 0. T. G., 274, 275(346) Jones, P., 151, 368, 369, 388, 389, 393, 394 (1621, 395(75), 397(75) Jose, J., 2(16), 3, 4(16) Joy, K. W., 274 Joyner, T. B, 301 Junge, J. M., 2(10), 3 Junk, K. W., 149, 165(286) Juntti, K., 65, 72, 212

420 237(25), 238(25), 247(25), 24805, 158), 249(25), 250(25), 254(25) Kensler, C. J., 260 Kepler, C., 106, 107(97) Kessler, E., 274 Kielly, W. W., 62, g5(34), 67(34) Kierkegaard, P., 348,364 Kim, I. C., 217 Kim, K. H., 130 Kimura, T., 172, 240, 247, 260, 261(256), 262(262), 263(266) King, T. E., 78, 176, 182, 187, 188, 189, 190(43, 43a), 191, 222, 223, 226(151), 235, 236, 237, 240(147, 148, 1671, 242, 244(164), 245(167, 1731, 246, 248, 253 (164), 296, 306(51), 307, 308(51), 310 (51), 312, 319, 320, 322(124), 323 (124), 324, 325(124), 326(124), 331 (124), 332(124), 333(131), 334 Kinon, B. J., 131 Kirkman, S. R., 86 Kirkpatrick, F. H., Jr., 313 Kirsohner, K., 25, 26, 31(101), 32, 33 (101), 41(146) Kirtley, M. E., 29, 30(126) Kita, M., 375 Kitamura, T., 108 Klaaae, A. D. M., 237, 238(172)

AUTHOR INDEX

Konings, A. W. T., 68, 71(113, 117) Kopko, F., 129 Korgenovsky, M., 260 Koritz, S. B., 83 Koshland, D. E., Jr., 26, 31, 32, 33(149),

34(143), 35(164), 36(113), 37(113, 172), 42(143) Kosow, D. P., 47 Kosower, E. M., 28, 97, 131, 379 Kosower, N. S., 131 Koster, J. F., 124, 125(172), 126(180), 238 Kotani, M., 347, 351, 356(55), 366 Kowal, J., 84 Koyama, J., 286 Kramer, R., 67, 69(100), 70(98), 78(100) Kratky, O., 32 Krebs, E. G., 2(10), 3 Krebs, H. A., 46, 47, 49, 63, 79, 81(175), 82(175), 86 Kremer, M. L., 305,366,385 Krimsky, I., 13,28,39(54), 40,41, 42(196) Kronman, M. J., 25 Krul, J., 106, 107(114), 114(114) Krupas, M., 269 Kubose, A., 106, 107(94) Kuboyama, M., 306(51), 307, 308(51), 310, 334 Kuma, F., 165 Kumar, S. A., 189, 195(63), 203 Klein, K. O., 84 Kupriyanov, V. V., 70 Klein, S. M., 106, 107(113), 108(113) Kustin, K., 407 Kleineke, J., 82 Kusunose, E., 149 Kleinsek, D. A., 88 Kusunose, M., 149 Kleppe, K., 101 Kuwana, T., 317, 320, 325(107, 125), 326 Klima, J., 167 (107, 125), 327(107), 330(165), 331 Klingenberg, M., 56, 57(20), 63, 66(20), (125), 332(125), 335(107) 81(20, 38), 86(38, 45, 461, 258, 259 Kuenetsova, G. P., 153 (228) Klouwen, H., 180, 312 1 Klybas, V., 44 Knof, S., 25 Labeyrie, F., 265, 267, 268,269, 272(306) Knutson, J. C., 83 Ladany, S., 85 Kobayashi, K., 286, .295(395) Lafferty, M. A., 274, 275(351) Kodicek, E., 83 Lakatos, S., 26 Koeppe, 0. J., 31 Lakshmanan, M. R., 88 Koike, K., 108 Koike, M., 93, 106, 107(96, 109), 108, 109, Lam, K. W., 79 Lambert, J. M., 45 110(132), 114(109) Lambeth, D. O., 375 Kolb, E., 2(21), 3,5 Lambowitz, A. M., 215 Kolvoda, J., 407 Lamprecht, W., 24 Komai, H., 311, 312(75)

AUTHOR INDEX

Land, G., 143 Landau, B. R.,402 Landriscina, C.,88 Lands, W.,168 Lang, G.,331,356 Langdon, R. G., 91,129,130, 142, 166(11), 167(11, 380), 168(11) Lange, L. G., 111,213 Lange, R.,20, 368 Langemann, H.,260 Langford, C. H., 385,386(137) Lardy, H.A., 110, 256, 259, 263 Lardy, M. A., 62, 65(35), 67(35) Larsson, A., 143, 144 Larsson, L. L., 364 Larsson, L. O., 348 Laskawska-Klita, T.,310 Laughrey, E.G.,82 Launay, A. N.,84 Laurent, T. C., 92, 142(23), 143(23) Lazdunski, M.,39 Lazzarini, R.A,, 274,277(339), 286(339) Leadbetter, E.,274, 276(342) Lean, J. D., 57,58(21),62(21) Lebeault, J. M., 149,150(295), 151(295) Lebherz, H.G., 9,26 Lederer, F., 264, 265, 266, 267, 268(290, 2911, 296 Lee, C.P., 64,67,68(66), 69(106), 70(59), 72(59, 102, 103, lW),73(106, 135, 136, 1371, 74(106, 136, 137, 166), 76(59), 77(59, 106, 135, 136, 1371, 78, 87(74), 204,207,214,246 Lee, C.Y., 29 Lee, J. P.,286,288,295(396) Lee, K.L., 259 Lee, R., 168 Lee, Y.P.,259 Lees, B.D,329 Le Gall, J., 281, 286, 288, 295(373a, 389, 396) Lehninger, A. L., 65, 92 Leigh, J. S., 187, 296, 320, 322(124), 323 (124), 324, 325(124), 326(124), 327, 331, 332(124, 168), 333(168), 336 (155),350,360,365, 372,376(88), 389, 390(150), 391(150) Leinweber, F. J., 286,287, 288(404) Lemberg, R.,301, 315, 316(18, 1001, 320 (18), 322, 323(100), 324, 335(18)

421 Lenaz, G., 70 Lenhoff, H.M.,168,169(388) Lents, P.J., 24 Leonard, J., 347, 348(39), 349(39), 359 (39) Lerner, D. A.,126 Leterrier, A. F.,317,318(109) Levenberg, M.I., 377 Levin, W.,149, 152 Levitzki, A.,34,35,36 Levy, H.R.,39 Lewis, J. A.,396 Lewis, S. N.,399 Liberman, E. A., 69, 74(120), 75(120) Libor, S., 23, 25 Lichtenberger, F.,153 Lieber, C. S.,81(176) Lieberman, S.,83,85 Light, P. A., 204, 217, 219 Lilienthal, H.R.,314,334 Liljas, A., 10,11(51), 12(51) Lim, J., 235, 244(164), 253(164), 296 Lindberg, O.,261 Lindsay, J. G.,302, 321, 322(138), 323, 326(138), 327(128), 331(138), 333 ( 1381, 335(138) Linn, T. C., 106, 107(92), 108(92) Linnane, A. W.,216 Lipmann, F.,38,74, 279 Lippard, S. L., 331 Lipscomb, J. D.,172 Lipscomb, M.D., 83, 84(210) Listowsky, I., 31, 34037) Littler, J. S.,407 Lo, S., 361 Low, H.,261 Loewus, F. A., 39 Lombardino, J. G.,379 Long, R.W.,88 Lopez-Colome, M., 133 Losada, M.,274,276(342, 345) LoSpalluto, J., 150 Louie, D.D.,53,57(7), 58(21, 22), 60(7), 62(21) Love, B., 310, 311(69) Lovenberg, W.,176,243 Loverde, A., 156,164(349) Lovrien, R.,367 Lowe, G., 43 Lowry, C., 24

AUTHOR INDEX

Lowry, 0. H., 47 Lu, A. Y.,149,152,165(286) Lund, P.,46,82,86(185) Lundsgaard, E.,2 Lusty, C. J., 106, 107(91, 93), 109(91), 202, 262, 263(266) Luthy, J., 69, 203 Lutwak-Mann, C.,245 Luzikov, V. N.,70 Luzzati, M., 264 Lyanagi, T., 169 Lyric, R. M., 281, 282(375), 284 Lyster, R.J. L., 369

M McAllister, J. K., 92, 94(36), 100(36), 103(36), l06(36), 107(36), 143(36), 144(36) McCann, L. M., 68,79 McCarthy, J. L., 84 McCarthy, K., 243 McCay, P. B.,168 McCleverty, J. A., 396 McCormick, D. B.,101, 125 McCoy, S.,304, 305(40), 317, 321, 322 (114),323(114), 337(115), 338(40) McDaniel, M. C.,334 McDonald-Gibson, R. G.,247 McGraw, J. C., 164 Machinist, J. M., 78, 190(84), 202 Machleidt, W.,311 McIntosh, E.N.,65 McKee, E. M., 154 Mackerer, C. R.,87 Mackey, L. N.,317, 325(107), 326(107), 327(107), 335(107) Mackler, B., 179,188(37),189,195(63), 203 (63),216 McLean, P.,46, 47(211), 81 McManus, I. R.,100, 103(65), 106, 107 (1051,1O9(65) MacMunn, C. A.,300, 315 McMurray, C. H.,41 McMurray, W. C., 62, 65(35), 67(35) McPherson, A., 10,24 MacQuarrie, R. A., 34, 36, 37(175), 42 (165),48(165), 49(165) Magar, M. E.,14,25(57) Mager, J., 286,287(386, 391)

Magni, G., 247 Mahler, H. R., 188, 189, 199, 217, 307, 308(59) Mahoney, A. J., Jr., 261 Major, J. P.,21 Makhlis, T.A., 70 Makino, N.,171, 172(402) Maley, G. F.,62,65(35),67(35) Malhotra, 0. P.,33, 35(160), 36(160), 37(174), 41(160) Mallet, A. I., 407 Malmstrom, B. G., 300, 301, 316(11), 317 (ll), 326, 328, 330,334(11), 335(108), 336(108, 171) Maltempo, M., 365 Malviya, A. N.,335 Mandula, B., 131 Mann, P.J. G., 260,261(248) Mann, T., 346 Mannervik, B., 132, 133, 139, 140, 141 (253, 254) Manocha, S.,300 Mansley, G. E.,322 Manzocchi, A., 248 Mapson, L. W.,129, 130(191), 138(191) Margoliash, E.,368,371(85), 372,376,377 (lll), 378(115), 380(111), 383(116) Margolis, J., 402 Margolis, S. A,, 70 Marklund, S.,393, 394(164) Markovich, D.S.,25,31(105) Martell, A. E.,407 Martin, R. B.,304 Martinez, J., 132 Marver, H.S., 150 Maskasky, J., 307, 314(60), 334(60), 338 (60), 343(60) Mason, H. S., 165, 166(373), 167(373), 170 (373), 171(373), 172(373, 4021, 176 Mason, T.L.,307, 308(58), 311, 312 Massey, V., 90, 91, 92, 93, 94(15, 24, 27, 29),95(27), 96(27), 97(1, 24, 29, 55), 98(1, 24,50, 55), 100(29, 32), 101, 103 (29,32),106(4,27),107(4,24,97,101), io8(4i), 109,111, ii2(24,55,ioi, 118, 150), 113(4, 24, 27, 29, 59, 137), 114 (27, 55, 150), 115(24, 137), 116(4, 24 27), 117(27, 1171, 120(83), 122(150, 154, 155), 123(154, 1551, 124(32), 125, 126, 133, 134(27, 1181, 135(29), 136

423

AUTHOR INDEX

(118), 137(1, 29, 59, 244), 138(29), 139(244), 140(244), 141(29), 146, 147 (284), 148(58), 170(1), 177, 153(23), 188(23), 200, 201(23), 203(23), 204 (23), 205(23), 223(23), 224(23), 235 (23), 236(23), 238(23), 246, 247(23), 248(23), 249(23), 250(23), 254, 282 Masters, B. S. S., 136, 150, 151, 154, 165 (337), 166, 167(245, 3371, 168(245, 386), 169(245), 170(245, 386), 172 (245,337,398) Masters, C. J., 365 Mathew, E.,11, 21, 22(52, 701, 42(70), 45(70) Mathews, C.T., 302,303(25) Matsubara, H.,310 Matsubara, S., 365 Matsurnura, Y.,311 Matthews, J., 106, 107(99), 108, 112(99), 114(99) Matthews, R. G., 90,91, 100, 103, 104(63), 114, 118(63, 156), 119(63, 156), 120 (63),121(63), 122(156), 146, 147(284), 282 Mauk, M. R., 372 Mauleon, P.,300 Mauzerall, D.,396 Mavis, R.D., 92, lOO(34) Maxwell, J. C.,319, 321, 322(142), 323 140), 337(117, 118), 339(117, 118), 340, 389 May, H.E.,168 Mayhew, S. G.,90, 91, 100, 123, 282 Mayr, M, 247. 249(199) Medina, A.,274,275(346),276(367) Mehler, A. H.,9 Meighen, E. A., 25, 26(99) Meigs, R. A,, 83 Meijer, A. J., 80,88 Melandri. B. A..2(28), 3 Meldrum, N. U.,92 Menon. K.M. J., 83 Mercer, W. D., 9, lO(45) Meriaether. B. P.,5. 11. 20(29), 21(29), 22(52, 70), 24, 39(29), 42(70), 44(90), 45(29, 70, 90, 185), 48(200) Merola, A. J., 180, 181(41), 312, 313(85) Mersmann, H., 69, 203 Mevel-Ninio, M.,269 Meyer, A.J., 88

Meyerhof, O., 2, 28(la) Michaels, G,B.,282 Michejda, J. M.,84 Middleditch, L. E.,55, 56(18), 57(18), 58(18) Middlemiss, D. N., 393,394(162) Midelfort, C.F.,9 Mihara, K., 151 Mildvan, A. S.,374, 385(100a) Millard, S. A., 106, 107(94) Miller, Z. B.,261 Mills, G. C.,48 Minakami, S.,176, 187, 188(56), 189(56), 190(56) Minchiotti, L.,100, 103(66), 143(66) Minnaert, K.,307, 325 Misaka, E.,106, 107(102, 1041, 122 Mitchell, C. H.,153 Mitchell, J. R.,153 Mitchell, P.,74, SO(155, 157), 81(157), 87 Mize, C.E.,129, 142 Mochan, E.,334,357 Moe, 0.A,, Jr., 126 Moir, N. J., 151 Molbert, E.,366, 367(48) Moleski, C.,334 Monod, J., 31 Monroy, G.L.,72 Montal, M., 72 Monteilhet, C., 264 Monty, K.J., 286,287(387), 288(404) Moore, E. C.,91, 92(8), 93(8), 99(8), 142 (23), 143(8, 231, 144(8), 145(8). 156 (9)

Moore, J., Jr., 23 Moran, T.,312 Moras, D.,9, 10(46), ll(46, 481, 18, 24 (47),29(47), 39(47, 48), 44(46, 47), 129, 141(188) Moreno, C. G., 274 Morgan. E.,245 Mori, M., 304 Morikofer-Zwex, S.,367, 379(63), 380 Morimoto, H., 372,373(92) Moroff, G.,141 Morrison, G.,83 Morrison, M., 176, 306(47), 307, 322 Morton, R.K.,264,269(300) Moss, T.H., 314, 334(94), 338(94), 356 Moyle, J.,74, 75(157), 81(157), 87

424

AUTHOR INDEX

Mozolovsky, A., 25 Muller, F., 282 Muller, M., 67, 69(100), 78(100) Muesing, R. A., 88 Muijsers, A. O., 308, 315, 316(101, 1021, 317, 318(111), 320(101, 102), 321(102, 132), 324,325,326(162), 335(157) Muiswinkel-Voetberg, H., 117, 118(160), 119(160), 124, 125, 128(181, 182) Mukherjee, B. B., 108 Muller, F., 90, 123, 126 Mulrow, P. J., 84 Munk, P., 108 Muraoka, S., 390 Murdock, A. L., 31 Murphy, M. J., 286, 287(397), 288(397), 289(397), 292(397), 295(410, 412) Murphy, T. A., 259 Murthy, P. S., 64, 65, 66(60), 68(60), 72

(60) Myer, Y. P., 319

N Nagahisa, M., 367 Nagai, Y., 286 Nagradova, N. K., 2(24), 3, 25(24), 30 (241, 48

Naiki, N., 286, 287(399), 288,292(399,424) Najjar, V. A., 274 Nakajima, H., 165 Nakamura, H., 217 Nakamura, M., 122 Nakamura, T., 286,295(402, 403) Nakanishi, K., 106, 107(102, 104), 122, 126 (104), 128(104)

Nakatani, M., 367 Namba, Y., 108 Narashimhulu, S., 166 Naslin, L., 267, 272 Nason, A., 274, 275(344), 277(337), 278 (356)

Navazio, F., 86 Nawa, H., 93 Needham, D. M., 2 Negelein, E., 301 Nelson, E. B., 150 Nelson, D. H., 65, 83, 84(211) NkmBthy, G., 32, 33(149) Nepokroeff, C. M., 88 Nesbakken, R., 130

Neas, G. C., 88 Neufeld, E. F., 52, 53(1), 54(1), 56(1), 58(1, 2, 3), 62(1), 65(30), 67(30), 69 (30), 70(30), 73(30), 76(30), 167

Newsholme, E. A., 47 Nicholas, D. J. D., 274, 275(346), 276 (367), 286, 287(401), 288, 292(401, 4221, 293(401)

Nicholls, D. G., 63,86,87(52) Nicholls, P., 237, 301, 320, 321(133), 334, 335(21), 346, 357, 386, 369(17), 370 (17, 721, 374(17, 721,377, 384(17), 385 (171, 389(17), 391(17), 395(17), 397 (17), 398, 400(175) Nieuwenhuis, F. J. R. M., 69, 74(119), 80 (1191, 214 Niluson, S., 384 Nise, G., 132 Nisley, S. P., 63, 66(37), 81(37), 86(37) Noeler, H. F., 5, 9(33), 20(33) Norling, B., 246, 249(188) Northrop, D. B., 141 Notani, G. W., 106, 107(110) Novogrodsky, A., 376, 377(111), 378(115), 380(111) Nurnberger, H., 368 Nygaard, A. P., 38, 44(179), 162, 264, 267 (280), 270, 271

0 O’Brien, P. J., 150, 152 Oda, T., 237 Oesper, P., 45 Oestereicher, G., 248 Oguchi, M., 48 Ogura, Y., 187, 372, 373(89), 376(89), 382 (89, 1071, 387(100), 388, 390(144)

Ohlsson, P. I., 365 Ohnishi, T., 187, 204, 215, 219, 235, 244, 253, 296, 346, 347(19), 353(19), 361 (19)

Ohno, H., 205 Ohta, Y., 366, 367(50) Okabe, K., 106, 107(96) O’Keeffe, D. H., 307,314(60), 315(93), 329 (931, 334(60, 94), 338(60, 941, 343(60)

Okunuki, K., 306(46), 307, 308(46), 274, 275(364, 365), 276, 310, 311, 315, 316 (1031, 323, 334

425

AUTHOR INDEX

Olah, G., 384 Olcott, R.J., 400 Oldham, S. B.,65, 83(84), 84(84) Olsen, K.W.,9, 10(46), ll(46, 481, 18, 24 (47), 29(47), 39(47, 481, 44(46, 471, 129, 141(188) Olson, E.J., 23, 45(81) Olson, J. A.,86 Olson, M. S., 110 Omdahl, J. L.,83 Omura, H., 274 Omura, T.,91, 150, 154, 165, 166(372), 167(372) Ondarza, R. N.,132, 133 Oosthuizen, C.,33 Oppenheimer, C.,363 Oppenheimer, N.J., 29 Orii, Y., 310, 311, 315,316(103), 320(103), 321(128), 324, 334, 336(134) Orlando, J. A., 66, 68, 71(114, 115), 72 (871, 79(115) Orme-Johnson, N. R., 179, 184(34), 185 (34), 186, 187(34, 461, 193(46), 215, 221 (46) OrmeJohnson, W. H.,83, 179, 184(34), 185(34), 187(34), 226(218), 253, 323 Orr, M.D.,143 Orrenius, S.,150, 152, 166 Orsi, B.A.,40 Osajima, Y.,274 Osborn, M.,366 Osenga, G., 222, 225(150) Oshino, N.,151, 365 Ostroumov, S. A.,69,74(121) Ota, A.,274,275(364) O’Toole, M.C.,308,314,315(93), 317(65), 318(65), 322(65), 323(65), 325(65), 328(65), 329(65, 93), 337(65), 338(65) Otsuka, J.,366 Otsuka, K.,108 Otto, J., 311 Ovadi, J.,24,25 Overberger, C.G.,379 Owen, C. S.,320, 322(124), 323(124), 325 (124),326(124), 331(124), 332(124) Ozawa, T.,234, 335

P Packer, L., 259,261, 262(262) Pain, R.H., 368

Pajot, P., 264,265,269 Palmer, G., 90,92,95,97(l,55), 98(1, 55), 100(32), 101, 103(32), 112(55), 113 (591, 114(55), 120(83), 124(32), 137 (1, 591, 170(1), 205, 322,324, 330, 335 (1531,336(153), 375 Paltauf, F., 151 Panebianco, P., 219 Paneque, A.,274,276(345) Paniker, N.V.,131 Panov, A. V., 70, 88(129), 212 Papa, S.,63, 73, 86(50), 87(53, 54, 55) Paradies, G.,70,88(129), 212 Park, J. H.,3, 5, 11, &(29), 21(29), 22 (52, 701, 23, 24, 28(13), 29(13), 31, 38(13), 39(13, 291, 40(13), 42(70), 44 (13, QO), 45(29, 70, 81, 90, 1851, 48 (200) Park, R., 84 Parker, D.J., 20, 21(66), 30 Parkhouse, R. M. E., 132 Parkhurst, L. F.,333 Passon, P. G.,165 Passoneau, J. V.,47 Patchornik, A., 379 Paul, K.G., 365 Pauling, L.,364 Payne, M.,33, 35(160), 36(160), 41(160) Payne, W.J., 274 Pazur, J. H., 101 Pearson, R.G.,375 Peck, H.D.,Jr., 279, 281, 282, 284,285, 286(372), 288,295(396), 297 Peczon, B. D., 33, 41(161) Pederson, T. C., 165, 166(369, 374), 167 (374), 168(374), 169(374) Peisach, J., 368, 369, 370(77, 781, 376(77), 380, 381(126b), 390(126b) Pelley, J. W.,106, 107(92), 108(92) Perham, R. N.,5, 24, 21(30, 34), 45, 104, 118(86), 119(86),120, 121(86) Peron, F. G., 84,88(225) Persson, B.,246,249(188) Perutz, M.F.,349,372,373(91) Pesch, L. A.,70 Petering, D.H.,360 Peters, J. M., 92,97(28),104(28), 114(28), 127(28) Peterson, J., 70

426 Pette, D., 48 Pettit, F. H.,106, 107(92), 108(92), 110 Pfennhger, O., 33, 35(160), 36(160), 41 ( 160) Pfleiderer, G., 24 Pharo, R. L., 70, 76(127), 77(127), 189, 190(59), 195(64), 198(64), 205 Phillips, A. H.,91, 166(11), 167(11), 168 (11) Pihl, A,, 20, 368 Pillai, P., 2 Pincus, G., 83 Pinsent, J., 366, 367(45, 46) Plaut, G.W.E., 86,87 Plumley, H.,130 Pnafili, E.S.,155 Pocker, Y.,407 Podack, E.R., 88 Poff, K.L.,239,240(178) Pogson, C.I., 41 PolgBr, L., 21, 22(73, 741, 23, 43, 45(73, 74) Poole, B., 365 Popowsky, M.,202 Porque, P. G.,143,144(275) Porter, J. W.,88 Porterfield, U.T., 388 Postma, P. W.,79 Poulsen, L. L., 106, 107(100), 153 Poyton, R. O.,307,308(58), 311 Prabhakararao, K., 286, 287(401), 288, 292(401, 422), 293(401) Prados, R., 304 Prager, G., 255 Prakash, O.,274,277,278,297(341) Presswood, R. P., 170, 286, 287(394), 290 (3941,291(394) Preabindowski, K.S.,313 Price, N.C.,33 Price, V. E., 366 Pronk, J., 120, 148(163) Prough, R. A., 151,166,169,172 Psychoyos, S.,83 Puchwein, G.,32 Pullman, M.E.,72, 88 Pulsinelli, P. D.,372,373(91) Pupillo, P., 2(28), 3 Purvis, J. L.,84 Pyfram, H.T., 376

AUTHOR INDEX

0 Quagliaridllo, E., 63, 73, 87(53, 54), 88 Quastel, J. H.,260, 261(248)

Rabinowitz, J. C., 243 Racker, E., 13, 28, 38, 39(54), 40, 41, 42 (196), 44, 66, 179, 180(36), 181(36), 183, 184(36), 186, 187, 201(35), 214, 239,244,246,302 Radcliffe, B. C.,274 Radda, G.K.,33 Rafter, G. W.,2(10), 3 Ragan, C. I., 78, 179, 180(36), 181(36), 182, 183, 184(36), 188, 187, 190(43a, 133), 201(35), 204, 214, 217, 218(133), 219( 1311, Raijman, L., 46 Rall, T. W., 92 Ramasarma, T., 247 Randall, D.D.,106, 107(92), 108(92) Ranney, H.M.,372,373(91) Rao, N. A,, 179, 188, 189, 195(63), 203 (631,216 Rapkine, L., 2,20(5) Rasmussen, 0.L.,302 Rawitch, A. B.,101 Ray, D.K.,317, 318(112) Ray, G. S.,334, 335(185), 336(185), 346, 348(14), 352(17), 353(14) Recheigl, M., 365 Redfield, A. G.,357 Redline, R., 148, 151 (284a) Redman, C.M., 365 Reed, D.W.,165 Reed, G.,350, 360 Reed, G.H., 372, 376(88) Reed, J. K.,106, 107(95), 110, 116(95), 117(95), 118(95), 119(95), 139(95) Reed, L. J., 92,93, 106, 107(92, 99, 1091, 108(19, 43, 92),109(03), 110,112(99), 114(99,log), 126(126) Reichard, P., 91,92(8), 93031, 99(81, 142 (23),143(8,23,261,2621, 144(8, 275), 145(8), 156(9) Reiske, J S., 310 Rendina, G.,260,261(254, 255) RBtey, J.,252

427

AUTHOR INDEX

Richardson, S. W.,306(50), 307, 308(50) Righetti, P.,225, 230(157), 231,234 Rikihisa, T.,39,45(181) Ringler, R. L.,176, 187, 188(56), 189, 190 (56),238, 257, 258(226) Riordan, J. F., 213 Rippa, M.,270 Rider, J. L.,264 Rivas, J., 274, 276(345) Robberson, B., 366, 367(41), 370(41), 371 (41) Robbins, P. W.,279 Roberts, K.D.,83 Robertson, A. M., 69 Robins, R. G.,302,303(25) Robinson, J., 65, 85(86) Robinson, J. R., 108 Rocca, E., 177, 217,254(16) Rodkey, F. L.,113 Rodman, H.M.,402 Rogers, M. J., 148, 151(284a), 154, 156 (341, 342), 161(341, 342, 344), 161 (341,,342) Rogers, L.A., 285 Rogers, W.I., 261 Rolleston, F. S.,47 Ronchi, S.,2!2, 44(76), 100, 101, 103(62, 661, 104(61,62), 105(62), 120(61), 123 (611, 143(66), 144(62), 145, 146, 147 (284) Roos, D., 73 Roper, M.,150 Rose, I. A., 47 Rosemeyer, M.A,, 131 Roskn, S.,331 Rosenthal, O.,83, 91 Rosenthal, S.,152,166 Rosing, J., 74 Rosman, J., 310 Ross, E.,66 Rossi, C., 78, 190(84), 202, 222, 225(150) Rossi, E.,246, 249 Rossi, F., 222, 225(150) Rossi-Fanelli, A.,349 Rossmann, M. G.,9, 10(46), ll(46, 48, 51), 12(51), 18,24(47), 29(47), 39(47, 48),44(46,471,129, 141(188) Rothschild, H. A.,261 Rotilio, G.,304 Roughton, F.J. W., 388

Rouslin, W.,312 Roussos, G.G.,274,278(356) Rubin, E.,81(176), 402 Rubin, M.S.,307,308(57),311(57) Rueger, D. C., 170, 286, 287(394), 290 (3941,291(394) Rumack, B.H.,150 Rumen, N. M., 374 Rutter, W.J., 9 Ruzicka, F.J., 245,297 Ryan, D.,149, 152 Ryan, K.J., 83 Ryback, G.,252 Rydstrom, J., 53, 58(8), 61(8), 64, 66, 68, 69(8, 68, 69, 70, 71, 721, 70(8, 67, 70, 72, 107), 71(8), 72(67), 75(67, 68, 69, 70,711,76(8, 67,68,70,71,721, 77(67, 68, 69, 70, 71, 72), 78(70, 1071, 87, 88 (70,71,129, 1301,207,212 Rytka, J., 272

S Sabeson, M. N., 11 Sabo, D.,66, 72(87) Sacktor, B.,258, 259(229, 233, 234) Sadana, J. C.,274,277,278, !297(341) Sadasivan, N.,305 Sagers, R. D.,106,107(113), 1W113) Saggerson, E. D.,47 Sajg6, M.,5,9(33),20(33) Sakai, H., 129 Sakamoto, Y.,150 Sakurai, Y.,108,109,llO(132) Salach, J. I., 222, 235, 247, 249(200), 258 Salhanick, H.A.,65 Salmon, D.M.W., 46 Saltzgaber, J., 312 Salvenmoser, F., 67, 69(100), 70(98), 78 (100) Samejima, T., 366,367(50),375 Samson, L.,371 (85),372 Samuelsson, B.,83, 168 Samuilov, V. D.,69, 74(121) Sanadi, D. R., 64, 65, 67, 68(31), 70, 72 (61), 76(127), 77(127), 79, 91, 92, 97 (26, 281, 104(28), 106, 107(25), 108, 113, 114(26, 28), 116(116), 117(151), 127(28), 189, 195(64), 198(64), 205 (64)

428 Sandberg, H. E., 369, 370(74, 791, 372(74, 791, 376(79) Sandborn, B. M.,249 Sanders, E., 91 Sands, R. H., 98, 113(59), 137(59), 235, 244, 330 Sani, B. P., 67 San Pietro, A., 53, 54, 55(11), 56(11), 58 (5), 59(11), W11) Santema, J. S., 55, 56(19), 57(19), 58(19), 59(19), 62, 80(19), 106, 107(114), 114 (114) Sarkar, N. K., 188, 189(57), 199(57) Saronio, C., 324, 336(155), 389, 390(150), 391(150) Sasame, H. A., 153 Ssto, R., 66, 72(89), 730391, 76(89), 151, 154, 288, 287(400), 288, 292(400, 421, 423, 424), 293, 294, 295(402, 403) Satoh, K., 65,69(78), 72(78) Sauer, L. A., 84 Savage, B., 26 Savage, N., 94, 98(54) Schachman, H. K., 25,26(99), 34(112) Schacter, B. A., 150 Schatz, G., 66, 167, 183, 214, 307, 308(58), 311, 312 Schejter, A., 377, 378(115) Scherz, B., 367 Schevitz, R. W., 10, 24 Schiff, J. A., 279, 280, 282, 283 Schindler, F. A., 390 Schlessinger, J., 34 346s 348(ZZ)3 351(n)3 Schleyer, (n), 353(20, '22) Schmid, D., 25 Schneider, W. C., 67 Schoenhard, D. E., 286 Schollmeyer, P., 63,81(38), 86(38) Scholz, R., 88 Schonbaum, G. R., 361, 365, 369(17), 370 (17), 372, 373(98), 374(17), 377, 378 (117), 379(118b), 2"25), 381(125, 126b), 382(125), 384(17)9 385(17, 100at llsb, 125), 387(118b), 389(17), 390 (lola), 391(17, lola), 392, 393(101a, 159), 394(101a), 395(17, 159, 159a), 397(17, lola), 398(101a), 399(101a, 118b), 400, 401, 402(118b, 159), 403 (118b), 404, 405

AUTHOR INDEX

Schramm, M.,44 Schrauzer, G. N., 396 Schroeder, W. A., 366, 367(41), 370(41, 63a), 371(41, 63a), 372(63a), 377, 378 Schultz, J., 378 Schuman, M.,90,91, 101,282 Schuster, I., 25,26,31(101), 32,33(101) Schutte, H. R., 368 Scott, E. M.,93, 94(40), 138(40), 139(40), 140(40), 164 Scouten, W. H., 106, 107(105), 109 Seamonds, B., 346, 351(21) Searls, R. L., 92, 97(26, 281, 104(28), 106, 107(25), 108, 114(26, 281, 116(116), 127(28) Sebald, W., 311 Segal, H. L., 39, 40 Segel, I. H., 129,132(193), 138(193) Sehested, K., 302 Seibl, J., 252 Seifried, H. E., 151 Sekuzu, I., 306(46), 307, 308(46), 323, 334 Sellinger, 0. Z., 259 Sels, A. A., 347 Seng, R., 222 Setlow, B., 148, 151(284a) Seubert, W., 88 Severina, I. I., 74, 75(148) Seydoux, F., 33, 35(160), 36(160), 41(160) Shah, P. C., 106, 107(109), 114(109) Shakespeare, P. G., 307, 308(59) ShaltiB1, S., 20, 22 Shaw, D. C., 11, ZZ(52) Shelton, J. B., 366, 367(41), 370(41), 371 (41) Shelton, J. R., 366, 367(41), 370(41), 371 (41) Shepley, K., 269 Shibata, H., 150 Shibata, Y., 25 Shifrin, s.,28 Shimakata, T.,151 Shin, B. c.,48 Shin, M., 54, 55, 58(12), 66(12) c. E**47 Shusterf L.*59f 150 Siege19 L. M.9 170, 286, %7(390, 394, 3979 412), 288(390, 397, 404), 289(390), 290 (394,413,414,415), 291(414), 292(390, Shonkj

AUTHOR INDEX

397, 415), 293(390), 294(414), 295(410, 412) Sies, H., 130, 365 Sih, C. J., 83 Simard-Duquesne, N., 69 Simon, A. M., 265 Simon, I., 34 Simplicio, J., 303 Simpson, E. R., 84 Simpson, R. J., 2(25), 3 Singer, T. P., 69, 78, 106, 107(91), 109(91), 110(130), 176, 177, 184, 186(23), 187, 188(19, 23, 561, 189(56), 190(56, 83, 841, 200(15, 191, 201(14, 15, 19, 231, 202, 203(19, 2 3 , 204(19,23), 205(19, 23), 205(19, 22, 23, 88), 206(19, 221, 214, 216(116), 217, 219, 220(136), 221 (1%) 222, 22303, 25, 140, 144), 225 (23, 25, 1441, 226(158), 230(158), 234 (25, 158), 235(23), 236, 237(15), 238 ( 2 3 , 240(15), 245, 246, 247(15, 20, 23, 25, 189), 248(23, 24, 25, 26, 249(23, 25, 26, 197, 199, 200), 250(23, 25, 195, 196, 1971, 251(197), 252(197), 253(1&1), 254(15, 16, 251, 255, 257, 258(=), 260, 261(%7, 254, 2557 256)* 262(227, 2621, 263(266), 284, 2709 271, 272(312), 273 Singh, A. P., 79 Singh, J., 275 Sivak, A., 261 Sjoberg, B. M., 93, 119(47), 143(47), 144 (47) Skulachev, P., 214 Skulachev, v. P., 69, 74(120, 121), 75(120, 146, 147, 1481, SO(146, 147) Skvaril, F., 367 ‘later’ E’ “’ 28’ 31’ 33(144)’ 34(144)9 41 (159), 63, 74,86(39, 49), 176, 187, 237, 238(172), 251, 307, 390 Slein, M. W., 2, 3(8), 48 Slencaka, W., 56, 57(20), 63, 66(20), 81 (20), 86(20) Sloan, D. L., 32, 33(155) Slonimski, P. P., 217, 272 Sluse, P. E., 88 Smiley, I. E., 24 Smillie, L. B., 371(861, 372 Smith, C. M., 40, 41, 42, 45(194), 49(194), 110

429 Smith, D. W., 369, 370(76), 372(76), 374 (76) Smith, G. D., 26,34(112) Smith, J. E., 132, 142 Smith, M. H., 300 Smith, M. L., 307, 314(60), 334(60), 338 (60), 343(60) Smith, T. E., 40 Smith, W., 168 Smythe, G. A., 307, 314(60), 315, 334(60, 94),338(60,94), 343(60) Snyder, H., 378 Soling, H. D.,82 Somlo, M., 269,272(306) Sone, N.,260 Sordahl, L. A., 189, 195(64), 198(64), 205 (64) Sorger, G. J., 274 So&, S., 20 Sosfenov, N. I., 366 Spallhols, J. E., 321, 322(142) Spats, L., 100, 148, 151(284a), 154(74), 155, 156(74), 161(74), 166(74), 167(74) Spencer, D., 274,277(337) Spencer, R. L., 100, 103(67) Speranaa, M. L., 100, 103,143(66) 41(161) Spivey, H. O., 3, Sportorno, G. M., 25,26 Spyridakis, A., 265,268(289) Sreenivasan, A., 260 Srere, P. A., 88 Srivastava, s. K., 131, 132 Staal, G. E. J., 94, 125, 131, 137(51), 139 (51), 140(51) Stachiewicz, E., 272 Stadtherr, L. G., 304 Stadtman, T. C., 143 Stallcup, W. B., 26, 35, 36(113), 37(113, 172) Stanbrough, E. C., 143 Stanbury, J’, 315, 316(100), 322, 323(100) Stansell, M. J., 366,382(39) Stark, G. R., 383 Staudt, H., 153 Steele, W., 244 Stein, A. M., 65, 86(75), 109, 110, 114, 117, 118(153) Stein, J-H., 86,109, 1 1 4 3 118(153) Stellwagen, E., 92, 100(34)

430 Stempel, K. E.,180, 181(40), 191, 194(40, 67), 195, 196, 197, 198(40), 199(68), 203(68), 206(40, 42), 207(40, 42), 216 (40) Stern, K. G., 363,366,391,397(160) Stevenson, P. M.,65, 85(86) Stiggall, D.L.,178,297 Stockell, A.,31 Stolzenbach, F. E.,30, 54, 55(11), 56(11), 59(11), 66(11) Stotz, E.,310,311(69) Straub, F.B.,106, 107(90), 189 Strength, D.R.,261 Strickland, S., 91 Strittmatter, P.,91, 100, 124, 148, 151(9, 17, 284a), 15407, 74, 307, 308), 155 (347), 156(74, 307, 341, 3421, 157 (3501, 158(354), 159(308, 351), 160 (308,354, 355), 161(74, 341, 342, 343, 344, 3501, 162(171, 341, 342), 163 (171, 352), 164(347, 349, 352, 359, 360), 165(347), 166(74), 167(74), 168 (360) Strobel, H. W.,149, 153, 169 Stromberg, C., 317,335(108), 336(108) Strom, R.,237,247 Strother, G.K.,395 Stryer, L.,93,349,364 Studier, M.H., 377 Sturani, E.,100 Sturtevant, J. M., 33, 34(156), 268, 269 (300) Stynes, D.V.,302 Stynes, H.C., 302 Su, G., 117, llS(l61) Suematsu, T., 108 Suggett, A., 368, 369, 388, 389, 395(75), 397(75) Sugita, Y.,317,318(110), 349 Sullivan, P.A.,90,91,282 Sulmovici, S.,83 Sumner, J. B.,38,440791,366 Sun, F.F.,306(48), 307, 308(48), 313 Sund, H.,366, 367(48) Susheela, L.,247 Suter, H., 365 Sutin, N.,375 Suzuki, I.,281, 282(375), 284 Suzuki, K.,2(18, 20), 3, 4(18), 9(18), 19, 25, 26, 27(103)

AUTHOR INDEX

Suzuki, T., 372, 373(92) Swartz, M.N.,62,65(31) Sweat, M.L.,83, 84(210) Sweetman, A. J., 64, 65, 68(63), 72(63), 73(63) Swoboda, B. E. P., 98, 113(59), 137(59) Symons, R.H., 267 Szabolcsi, G.,23, 31 Szarkowska, L.,258 Szorhyi, E.,2(14), 3 Szumilo, T., 273 Szymona, M., 273

T Tagawa, K., 54, 55(12), 56(12), 66(12) Tager, J. M.,63, 73, 81(40), 82, 86(39, 40,49,191),87(53,54),88 Takahashi, E.,286, 295(395) Takahashi, H.,274, 277(337) Takahashi, Y.,335 Takano, T., 371 (85),372 Takemori, A.,259 Takemori, S.,237, 245(173), 306(46), 307, 308(46),334 Takesue, S., 154, 165, 166(372), 167(372) Tallan, N.H.,83 Tamaoki, B.,83 Tamura, G., 274, 286(359), 295(359, 405) Tanaka, M.,100 Tanaka, N.,108 Tanford, C., 367 Taqui Khan, M.M., 407 Tarr, H.L.A., 92 Tasaki, A., 366 Taube, H., 303 Tauber-Finkelstein, M.,22 Taylor, E.L.,39 Taylor, J. F.,24 Taylor, P. L.,85 Taylor, W.E.,150 Teale, F.W.J., 348, 380 Tedeschi, P.,28 Teeter, M.E.,205 Teipel, J., 34, 35(164) Teixeira da Cruz, A., 64, 69(68, 691, 70 (67), 72(67), 75(67, 69), 76167, 68, 691,77(67,68,69),207 Telegdi, M., 25 Teller, D.C.,25

431

AUTHOR INDEX

ter Welle, M. F., 63, 72(47), 73(47), 81 (47) Tepley, L. J., 189 Testolin, G., 222, 225(150) Thelander, L., 91, 92(8), 93(8), 98(31), 99(8, 311, 100(31), 101(31), 103(31), 104, 142, 143(8, 262, 2631, 144(8, 38), 145(8, 31), 156(9) Theorell, H., 162, 200, 356, 364, 366, 369, 370(6), 385, 388, 400(143) Thomas, J. O., 23 Thompson, T. E., 142 Thor, H., 150 Thorgeirsson, S. S., 153 Thorn, M. B., 247 Thornber, J. M., 109 Thorpe, C., 123 Thurman, R. G., 88 Tiesjema, R. H., 317, 318(111), 319, 324, 325, 326(162), 335(157) Tietre, F., 130, 143(202) Tisdale, H., 205, 222, 225, 226(158), 230 (158), 234(158), 248(158), 255 Tolbert, N. E., 274 Tomizawa, H. H., 130 Topali, V. P., 69,74(120), 75(120) Topper, D. L., 407 Toren, D., 83 Torii, K., 279, 372, 373(89), 376(89), 382 (89, 107) Torndal, U. B., 65, 73(166), 78, 207, 212 Tosi, L., 264 Tottmar, S. 0. C., 190(133), 217, 218 (133) Tove, S., 287(412), 288,295(412) Trentham, D. R., 21, 23, 28, 35(69), 37 (83, 1171, 39, 40(83, 117), 41(69, 189, 190), 42(116, 117), 43(83, 1171, 44, 45 Trudgil, P W., 91 Trudinger, P. A., 286, 295(392) Triiper, H. G., 285 Tsai, C. S., 117 Tsai, P., 150 Tsao, M. S., 302 Tsernoglou, D., 10 Tsofina, L. M., 69,74(120), 75(120) Tsou, C. L., 222, 223(146), 226(146), 246 (146) Tsuchihashi, M., 367 Tsudzuki, T., 325, 326(163), 327

Tu, S. C., 101 Tubbs, P. K., 88, 273 Tucker, A., 300 Tung, T., 256 'Turini, P., 255 Turkki, P. R., 262 Turner, J. F., 143 Tyler, D. D., 186,203, 262 Tysarowski, W., 272 Tyson, C. A., 172 Tzagoloff, A., 307, 308(57), 310, 311(57), 317

U Ueda, T., 91 Uesugi, I., 274 Ullrich, V., 152, 153 Urnes, P., 372, 373(95) Urry, D. W., 319 Uigiris, V. I., 65

v Vanngard, T., 317, 330, 335(108), 336(108, 171) Vainshtain, B. K., 366 Vallee, B. L., 213 Van Ark, G., 332 Van Buuren, K. J. H., 308, 315, 316(101, 102), 320(101, 102), 321(102, 132,133), 335 van Dam, K., 63, 69, 72(47), 73(47, 481, 74(119), 80(119), 81(47), 86(49), 214 Van Demark, P. J., 260 van den Brock, H. W. J., 53, 55, 56(19), 57(9), 58(9), 59(9, 19), 60(9), 62, 80, 143 Van der Hoeven, T. A., 153, 165, 166 (3701, 170(370) van de Stadt, R. J., 69,74, 80(119), 214 Van Drooge, J. H., 332 Van Eys, J., 3, 28(13), 29(13),.38(13), 39 (13), 40(13), 44(13) Van Gelder, B. F., 307, 308, 315, 316(101, 1021, 317, 318(1111, 319,320(101, 102), 321(102, 132, 133), 324, 325, 326(162), 331(139), 332(139), 333(139), 335 (157) van Haefen, H., 63, 86(45)

432 Van Heerikhuizen, H., 187 Vanko, M., 260 Van Lis, M. J., 33,41(159) Vanneste, W. H., 315, 316(98), 317(98), 320(98), 322(98) Varandani, P. T., 130 Varshavsky, Y. M., 14,3166) Veech, R. L., 46, 79, 81(175), 82(175), 86(175) Veeger, C., 63, 56, 58(19), 57(9, lg), 58(9, 191, 59(9, 191, 60(9), 62, 80(19), 92, 94(24, 27), 95(27), 96(27), 97(24), 98(24), 106(27), 107(24, 97, 106, 114), 111(24), 112(24, 108, 1181, 113(24, 27), 114(27, 114), 115(24), 116(24, 27), 117(27, 157), 118(157, 160), 119 (1601, 120, 122(154), 123(154), 124, m ( i 7 2 , 173, 174, 1751, i26(174, i79, 180, 181, 1821, 131, 133, 134(27, 1181, 136(118), 137(51), 139(51, 157), 140 (51), 143, 148(163), 222, 224, 226 (218), 236(149), 237, 238(149), 251, 252, 253 Vega, J. M., 274, 276(342) Velick, S. F., 3, 9, 19, 29, 30(125), 31 (12), 32(127), 33(155), 34(156), 39, 40(60), 41(60), 42(60), 44(60), 45 184, 194), 48(60), 49(194), 91, 92, 151(9) Vennesland, B., 39 Vennesland, J. W., 92 Vermilion, J. L., 165, 166(371), 167(371) Vernon, L. P., 188, 189(57), 199(57) Vignais, P. V., 84 Vinogradov, A., 248 Visser, J., 116, 117(157), 118(157), 120, 124, 125,(175), 126(179, 180, 1811, 139 (1571, 148(163) Vitols, E., 143, 216 Vladimirova, M. A., 69, 74(120), 75(120) Voetberg, H., 116, 117(157), 118(157), 120, 125, 126(180), 139(157), 148(163) Vogel, O., 106, 107(107), lOS(l0t) Voigt, B., 31 , Volkstein, M. V., 25,31(105) Volpe, J. A., 306(52), 307, 308(52), 313 (52), 315(52), 316, 317(52, 651, 318 (52, 65), 319, 321, 322(65), 323(65, 1401, 325(65), 328(65), 329(65), 333, 337(65, 118), 338(65), 339(118), 389

AUTHOR INDEX

von Ellenrieder, G., 32 von Wartburg, J. P., 367,379(63), 380(63) Vore, M., 152 Vorona, M. K., 48 Vyas, S. R., 189, 195(64), 198(64), 205 (64)

W W d a m , F., 150 Waentig, P., 367 Wainio, W. W., 306(49), 307, 308(49), 310, 319, 322(49), 324 Wainwright, T., 286, 287(398), 288, 292 (398, 420) Wakil, S. J., 88, 151 Walajtys, E. I., 110 Walker, G. C.,274, 275 Walker, W. E., 222, 235 Wallace, W. J., 319, 321, 323(140), 337 (1171, 339(117), 340, 389 Wallenfels, K., 28, 33 Waller, H. D., 130, 131 Wang, L., 109 Wang, T. Y., 222, 223, 226, 246(146) Wang, Y. L., 222, 223(146), 226(146), 246(146) Warburg, O., 2, 301, 315, 363, 364(3) Wartofsky, L., 91, 14303 Warnarman, P. M., 19,21, 28(61) Wassink, J. H., 55, 56(19), 57(19), 58(19), 59(19), 80(19), 108, 107(114), 114 (114) Watari, H., 190(83), 202,267, 372, 373(922) Waterman, M. R., 360 Waters, W. W., 407 Watson, H. C., 9, 10(45), 19, 21, 28(61), 35, 364, 372 Webb, L., 10 Weber, F., 131 Weber, K., 366, 367(48) Weber, M. M., 110, 168, 169(388) Wedding, R. T., 106, 107(100) Weenen, J. H. M., 39 Weil, J. A., 304 Weinbach, E. C., 66 Weinhouse, S., 46, 49(214) Weiss, L., 88 Welch, M., 85 Welinder, K. G., 371(86, 871, 3?2

AUTHOR INDEX

Wells, I. C., 261 Welton, A. F.,165, lsS(369) Wendel, A., 130 Wendell, P. L.,133 Wenske, G.,63, 86(45) Werner, A,, 303 West, C.A.,66 West, S., 149, 152 Wever, R.,315, 316(102), 320(102), 321 (1021, 332 Wharton, D. C., 275, 301, 306(45), 307, 308(45), 310, 312, 322, 324, 325, 327 (53), 330(53), 335(153), 336(153) White, H.B., 111, 39 White, R. P., 378, 379(118b), 385(118b), 387(118b), 399(118b), 401, 402(118b), 403(118b), 404, 405 Wiberg, K. B., 407 Widger, W. R.,78, 182, 190(43a) Wieland, O.,88 Wikstrom, M. K. F., 215 Wilken, D.R., 133, 263 Wilkins, R. G.,303 Williams, C. H, Jr., 91,92,93, 94(29, 35, 36), 97(29), 98(30, 501, 99(30), 100 (29, 33, 36, 37), 101, 103(29, 631, 104(33, 35, 61, 62, 63), 105(33, 62, 85), 106(36), 107(36, 103, 108), 113 (29, 59), 114(108), 118(63, 156), 119 (33,35, 63,85, 1561, 120(61, 631,121 (631, 122(156), 123(61), 129, 135(29, 601, 136(60), 137(29, 59, 60, 244), 138 (291, 139(244), 140(244), 141(29, 351, 143(36), 144(36,62), 145(30), 146(30), 147(284), 148(58), 166(10), 167(10, 245), leS(l0, 245, 3861, 169(245), 170 (245), 172(245) Williams, G.R., 263 Williams, J. N.,Jr., 260, 334 Williams, J. R.,81 Williams, R.J. P., 317,318(109), 351,369, 370(76), 372(76, 811,374(76, 811, 390 Williamson, D. H.,46, 82, 86(185, 186) Williamson, J. R., 46, 47(215), 48(219), 110 Willms, B., 82 Willms, C. R., 108 Wilmarth, W. K.,302, 304 Wilson, D.F.,215,302, 320,321, 322024, 138), 323(124, 137), 325(124), 326(124,

137, 138, 1631, 327, 328(1379, 331(124, 138), 332(124, 168),333(137,138,168), 335(138), 346, 351(21) Wilson, J. E., 100, 101(64), 103(64), 109 (64), 117, 118(161) Wilson, L. D.,83, 84(211) Wilson, L.G.,91,133(7), 143(7), 279 Wilson, M.T.,315, 316(99), 323(99), 324 (99),335,336(99,192),389 Wilting, J., 335 Winell, M.,133 Winter, D. B., 235, 244(164), 248, 253 (164), 296 Wiss, O., 131 Witkowski, P.E.,262 Wit-Peeters, E. M.,88 Woenckhaus, C., 24 Wold, F.,100,103(67) Wolf, B., 109 Wolman, Y., 379 Wolny, M., 9 Wonacott, A. J., 10, 19, 20 Wong, D.,204 Wong, S.H., 83,84(211) Wood, P. M.,303 Woodin, T. S., 129, 132(193), 138(193) Woodrow, G.V.,111,360 Woodward, H.E.,260,261(248) Woolf, A. A.,384 Worthington, D. J., 131 Wren, A., 106, 107(101), 112(101), 117 ( 117)

Wiithrich, K.,267, 357 Wyman, J., 31 Wyss, S.R.,367

Y Yagi, K.,176,234,335 Yamafuji, K.,274 Yamamoto, H.,350, 360, 369, 370(80), 372(80), 376(88) Yamamoto, T., 311 Yamanaka, T., 274,275,276 Yamasaki, I., 122, 169 Yang, C.S.,198,311,312(78) Yang, J., 366,367 Yang, P. C.,310 Yang, S. T.,25, 30(108), 48(108) Yasunobu, K.T.,100

AUTHOR INDEX

Yeghiayan, A,,372,373(96) Yike, N. J., 64, 66(64, 651, 68(64, 65), 69(65), 72(64, 65), 73(65), 76(65) Yonetani, T.,306(44,46), 307,308(44,46), 320, 330, 331(174), 334, 335(185), 336 (1851, 346, 347(19), 348(14, 26, 27, 28, 29, 30, 31, 32, 33, 37, 38, 39), 349 (26, 28, 30, 32, 39, 461, 350, 351(21, 22), 352(17, 22), 353(14, 16, 18, 19, 20, 22, 34), 355(34, 361, 356(55), 357 (17,38), 359(39), 360(27, 31), 361(17, 19), 364, 369, 370(80), 372(80), 376 Yoneyama, Y., 317, 318(110), 349 Yong, F. C.,306(51), 307, 308(51), 310 (511, 319 Yoshida, K., 372,387 Yoshikawa, S.,320, 321(128), 336(134) Yoshimoto, A., 286, 287(400), 288, 292 (400, 421, 423, 424), 293, 294, 295 (402, 403) Yoshiaawa, K.,108 You, K. S.,179, 193(32), 214, 226(32) Yu, C.,312 Yu, L.,312

Z Zahler, W. L., 313 Zakim, D., 109, llO(130) Zanetti, G.,92, 94(36), 97, 98(30, 501, 99 (30), 100(36), 103(36), 106(36), 107 (36), 143(36), 144(36), 145(30), 146 (30), 147(284), 148(58), 222,225(150), 216, 247(191) Zapponi, M. C., 22,44(76) Zatman, L.J., 53 Zawodsky, P.,14, 25, 26, 31(56, 105) Zerfas, L. G.,263 Zeszotek, E.,222 Zeylemaker, W. P., 222, 224(149), 236 (149), 237(149), 238(149, 1721, 251, 252, 253(149) Zherebkova, N. S., 153 Zidoni, E.,305 Ziegler, D. M.,153, 154, 165(337), 167 (3371, 172(337), 224, 236(153, 1541, 239 Zumft, W. G., 274 Zuurendonk, P. F.,308, 320, 321(132)

Subject Index A

glyceraldehyde-3-phosphate dehydrogenase and, 30 Absorption spectra small NADH dehydrogenases and, 196 adenylyl sulfate reductase, 282, 284, 285 transhydrogenases and, 59,69 catalase, 372-374, 381, 382, 397 ubiquinone reductase and, 181, 184, 186, cytochrome b?, 265, 267 215-216 cytochrome b, reductase, 155-156, 157Acetylpyridine adenine dinucleotide 158 phosphate, sulfite reductase and, cytochrome c oxidase, 315-319, 322,323, 288, 290, 292 327 Achromobacter fischeri, nitrite reductase, cytochrome c peroxidase, 351,353,354 physical properties, 277-279 glutathione reductase, 95, 135-136, 137Active site, lipoamide dehydrogenase, 105 138 Acyl hydrazides, catalase and, 379 a-glycerophosphate dehydrogenase, 257 Acyltransferase activity, glyceraldehydelipoamide dehydrogenase, 118, 122, 123, 3-phosphate dehydrogenase, 44-45 126 Adenine nucleotides nitrite reductase, 278 glyceraldehyde-3-phosphate dehydrosmall NADH dehydrogenase, 193, 194 genase and, 2, 45, 46, 48 succinate dehydrogenase, 232, 233 transhydrogenase and, 70 sulfite reductase, 288, 289,291, 293 Adenosine, transhydrogenase and, 71 thioredoxin reductase, 98 Adenosine diphosphate ubiquinone reductase, 183-184 choline dehydrogenase and, 262,263 Acatalasemia, form of, 367 lipoamide dehydrogenase and, 125 Acetaldehyde, catalase and, 391-392, 406 NADH dehydrogenase and, 207 Acetate succinate dehydrogenase and, 250 catalase and, 383, 385 transhydrogenase and, 71 small NADH dehydrogenases and, 192 Adenosine diphosphate ribose, cytoAcetoacetate, succinate dehydrogenase chrome b, reductase and, 163 and, 238 Adenosine diphosphate sulfurylase, Acetyl coenzyme A, transhydrogenase reaction catalyzed, 282 and, 71 Adenosine monophosphate Acetyl dephospho coenzyme A, transadenylyl sulfate reductase and, 282, hydrogenase and, 71 283, 284 N-Acetylimidazole, catalase and, 367 cytochrome P-450 reductase and, 167 Acetyl phosphate, glyceraldehyde-3-phosNADH dehydrogenase and, 188, 207 phate dehydrogenase and, 21, 28, 38transhydrogenase and, 71 39,43, 44-45 Adenosine 2’-monophosphate Acetylpyridine adenine dinucleotide sulfite reductase and, 293 cytochrome bs reductase and, 156, 157, transhydrogenase and, 57,58,59,60, 159, 160, 163 61, 71 435

436 Adenosine 3'-monophosphate, transhydrogenase and, 71 Adenosine 3',5'-monophosphate, transhydrogenase and, 71 Adenosine 5'-phosphosulfate, formation of, 279 Adenosine triphosphatase cytochrome c oxidase and, 321 transhydrogenase and, 79 Adenosine triphosphate choline dehydrogenase and, 263 cytocrome c oxidase and, 300, 302, 343 glyceraldehyde-3-phosphate dehydrogenase and, 25,26, 48,49 lipoamide dehydrogenase and, 125 NADH dehydrogenase and, 207 ,redox potentials and, 215, 216 succinate dehydrogenase and, 247, 248, 249 sulfate reduction and, 279 transhydrogenation and, 63-64, 65, 6768, 72, 73-74, 77, 80, 81, 207, 214 Adenylyl sulfate reductase(s) occurrence of, 282 properties, 279-286 Adipose tissue glyceraldehyde-3-phosphate dehydrogenase in, 47, 48 glycolytic enzymes, diabetes and, 47 Adrenal cortex transhydrogenase of, 65 function, 83-85 Aerobacter aerogenes, sulfate reduction by, 281 Aeromonas punctata, sulfate reduction by, 281 Alanine residues, glyceraldehyde-3-phosphate dehydrogenase, 11, 12 Alcohols, catalase and, 388, 398, 401 Aldehydes, glyceraldehyde-3-phosphate dehydrogenase and, 39 Algae, sulfate assimilation by, 279, 280 Alkali cytochrome c oxidase and, 311 respiratory particles and, 240-241, 248 Alkyl bromides, succinate dehydrogenase and, 246 Alloxan diabetes, cytosolic redox state and, 46-47

SUBJECT INDEX

Ally1 alcohol, catalase and, 401-402, 403, 404 Amine oxidase, mixed function, 153-154 Amino acid (s) conservation, glyceraldehyde-3-phosphate dehydrogenase, 14-19 cytochrome bs reductase composition, 155 cytochrome c oxidase composition, 310 cytochrome c peroxidase composition, 348 lactate dehydrogenase, 266 pyridine nucleotide-disulfide oxidoreductases, composition, 100, 102 sequences glyceraldehyde-%phosphate dehydrogenases, 6-8 hemoproteins, 371 thioredoxins, 144 small NADH dehydrogenase composition, 195 synthesis, transhydrogenase and, 80-81 3-Arnino-1H-1,2,4-triazole, catalase and, 376-378 Ammonia, catalase and, 387 Ammonium sulfate glyceraldehyde-3-phosphate dehydrogenase and, 4 lipoamide dehydrogenase and, 124,125, 126 succinate dehydrogenase and, 229-231 Amytal choline dehydrogenase and, 261-262, 263 respiratory particles and mitochondrial, 199 yeast, 217 ubiquinone reductase and, 181,182, 204, 205 Anaerobes, succinate dehydrogenase of, 254 Aniline, hydroxylation of, 150 Anilino-naphthalene sulfonate cytochrome c peroxidase and, 349,359 transhydrogenase and, 69 Antimycin A cholesterol side chain cleavage and, 85 choline dehydrogenase and, 262 a-glycerophosphate dehydrogenase and, 268

437

SUBJECT INDEX

L-lactate dehydrogenase and, 269 small NADH dehydrogenase and, 199 succinate dehydrogenase and, 239, 250 ubiquinone reductase and, 182, 197 yeast NADH dehydrogenase and, 219 Antiparallel sheet, glyceraldehyde-3phosphate dehydrogenase, 13, 14 Arginine residues catalase, 395 glyceraldehyde-3-phosphate dehydrogenase, 12, 22, 24 transhydrogenases and, 213-214, 296 Arsenate, glyceraldehyde-3-phosphate dehydrogenase and, 38, 44-45 Arsenite lipoamide dehydrogenase and, 95-97, 110, 111, 113-114 sulfite reductase and, 289 thioredoxin reductase and, 99 Arsenocholine, choline dehydrogenase and, 261 Arterial tissue, transhydrogenase in, 65 Ascites cells, glyceraldehyde-3-phosphate dehydrogenase in, 47 Ascorbate, cytochrome c peroxidase and, 353 Asparagine residues glyceraldehyde-%phosphate dehydrogenase, 11, 12, 30 lipoamide dehydrogenase, 101 Aspartate residues, glyceraldehyde-3phosphate dehydrogenase, 11, 12, 30 Atebrin, L-lactate dehydrogenase and, 264 Azide catalase and, 376,385,400 cytochrome c oxidase and, 320-321,328, 333 cytochrome c peroxidase and, 350,353 Azotobacter lipoamide dehydrogenase of, 114 transhydrogenase molecular properties, 57-59 reaction mechanism and regulation, 59-62 Azotobacter anile, transhydrogenase of, 54 Azotobacter chroococcum nitrite reductase of, 275,276-277 transhydrogenase of, 54 Azotobacter vinelandii NADH dehydrogenase of, 221

nitrite reductase of, 275, 277 transhydrogenase of, 53,54 function, 80 purification, 55, 56

B Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase of,2, 4, 9, 23 apoenzyme, 19 pyridine nucleotide binding, 34 Bacteriophage T4, thioredoxin and, 144 Barbiturates, NADH dehydrogenases and, 203, 204, 206 Bathocuproin sulfonate, cytochrome c oxidase and, 308 Bathophenanthroline, succinate dehydrogenase and, 246 Bathophenanthroline sulfonate, NADH dehydrogenase and, 206 3,4-Benzpyrene hydroxylation, pyridine nucleotides and, 152 Benzyl viologen, nitrite reductase and, 275, 276 Betaine, formation of, 260 Betaine aldehyde, choline dehydrogenase and, 261, 262 Betaine aldehyde dehydrogenase, occurrence of, 260 Bicarbonate, succinate dehydrogenase and, 238 Borohydride, glutathione reductase and, 136, 140 Brain glyceraldehyde-3-phosphate dehydrogenase in, 47, 48 a-glycerophosphate dehydrogenase of, 256-258 Bromelain, cytochrome P-450 reductase and, 166, 167 Bromide small NADH dehydrogenases and, 192 succinate dehydrogenase and, 247, 248 3-Bromoacetylpyridine, glyceraldehyde-3phosphate dehydrogenase and, 24 Bromopyruvate glyceraldehyde-3-phosphatedehydrogenase and, 24 succinate dehydrogenase and, 249

SUBJECT INDEX

Bumilleriopsis filiformis, transhydrogenase of, 54 2,3-Butanedione, transhydrogenation and, 296 n-Butanol, catalase and, 401402 Butylhydroperoxide, catalase and, 392 Butyraldehyde, catalase and, 406

C Cadmium, lipoamide dehydrogenase and, 114 Calcium choline dehydrogenase and, 263 transhydrogenases and, 58, 61, 70 Candida utilis, NADH dehydrogenase of, 216-218, 219, 221 Carbohydrate degradation, a-glycerophosphate dehydrogenase and, 259 synthesis, transhydrogenase and, 80 Carbon monoxide catalase and, 401 cytochrome c oxidase and, 301, 317418, 322, 327-328,329, 333, 336-337, 338 cytochrome c peroxidase and, 353 heme iron and, 321-323 nitrite reductase and, 275, 278-279 sulfite reductase and, 289, 293 Carboxylate groups, catalase, 395 Cardiolipin cytochrome c oxidase and, 313 ubiquinone reductase and, 183 Carotenoids, transhydrogenase and, 69 Catalase active site distal ligand identity, 376-385 ligand exchange reactions, 385-388 ligand identity at fifth and sixth coordination positions, 369-376 apoprotein, selective modifications, 376-385 general properties, 366-369 historical background, 363-366 hydroperoxide and, 356 redox reactions, 388-389 nature of Compound I, 389-390 reaction mechanism, 390-408 Catalytic domain, glyceraldehyde-3phosphate dehydrogenase, 16

Cetyldimethylethylammonium bromide, succinate dehydrogenase and, 234 Chemiosmotic hypothesis, transhydrogenase and, 74-75 Chloramphenicol, yeast NADH dehydrogenase and, 217 Chlorella pyrenoidosa, adenylyl sulfate reductase in, 282, 283 Chloride, succinate dehydrogenase and, 247, 248 Chlorobium limicola, adenylyl sulfate reductase of, 286 5-Chloro-3-t-butyl-2-~11loro-4’-nitrosalicylamide, cytochrome c oxidase and, 321 2-Chloroethanol, catalase and, 401 p-Chloromercuriphenyl sulfonate glutathione reductase and, 141 small NADH dehydrogenase and, 203 thioredoxin reductase and, 146 ubiquinone reductase and, 197 Chloroplasts, transhydrogenase of, 66 Chlorosuccinate, succinate dehydrogenase and, 237-238 Cholate NADH dehydrogenase and, 189 ubiquinone reductase and, 178, 182, 183 Cholesterol, side chain cleavage, 83, 84-85 Choline, oxidation to betaine, 260 Choline dehydrogenase electron transport system and, 261-263 properties, 260-261 Chromatium sulfate reduction by, 281 transhydrogenase of, 54 function of, 80 molecular properties, 58, 59 purification, 55, 56 Chromium complexes, oxygen and, 304 Chymotrypsin, cytochrome b5 reductase and, 155 Circular dichroism catalase, 382 cytochrome c oxidase, 319 transhydrogenase and, 62 Clostridium kluyveri, sulfate reduction by, 281 Clostridium nigrijicans, sulfate reduction by, 281

SUBJECT INDEX

Clostridium pasteurianum, sulfate reduction by, 281 Cobalt ammine complexes, oxygen and, 303304 Coenzyme A, transhydrogenase and, 70, 71 Complex I, see under Nicotinamide adenine dinucleotide Complex 1-111 properties of, 197 Complex 11, see also Succinate dehydrogenase properties of, 239 Compound I, nature of, 389-390 Configuration, pyridine nucleotide binding and, 31 Conformation hypothesis, transhydrogenase and, 75 Copper complexes, oxygen and, 304 cytochrome c oxidase and, 302,307309, 314, 315, 317,319, 322, 329-330, 338 lipoamide dehydrogenase and, 114, 122-123 nitrite reductase and, 275 Corpus luteum, transhydrogenase of, 65, 85 Cyanide catalase and, 376, 377, 382,385, 387, 399,401 cholesterol side chain cleavage and, 85 cytochrome c oxidase and, 301,308, 320-321, 335, 336 cytochrome c peroxidase and, 350,353 2-hydroxyacid dehydrogenase and, 272 L-lactate dehydrogenase and, 269 microsomal electron transport and, 148, 151 nitrite reductase and, 275, 276, 277,278 succinate dehydrogenase and, 223,246247, 250 sulfite reductase and, 289, 293 Cyanogen bromide, catalase and, 379-385 Cycloheximide, yeast NADH dehydrogenase and, 217,220 Cystamine, glutathione reductase and, 132 Cysteine, lipoamide dehydrogenase and, 122

439 Cysteine residues glyceraldehyde-3-phosphate dehydrogenase, 5, 12-13, 14, 22, 28, 29, 34, 38, 39, 44, 45, modification of, 20-21 pK. of, 43 Cystine, glutathione reductase and, 132 Cystine residues lipoamide dehydrogenase, 120-122 pyridine nucleotide-disulfide oxidoreductases and, 95, 101, 104 thioredoxin, 92 Cytochrome(s) ubiquinone reductase and, 179, 180 Cytochrome a, see Cytochrome c oxidase Cytochrome b choline dehydrogenase and, 261, 262 succinate dehydrogenase and, 224, 239, 244-245 Cytochrome b2, L-lactate dehydrogenase and, 263-264,266267,296 Cytochrome bs, cytochrome b, and, 267, 296 Cytochrome b5 reductase cytochrome P-450 reductase and, 151153 function of, 150-151 mechanism, microsome bound, 161-162 mechanism of Strittmatter, review, 156-161 methemoglobin reductase and, 164-165 molecular properties, amphipathic and soluble forms, 154-156 structural studies, 162-164 Cytochrome c adenylyl sulfate reductase and, 281, 285-286 choline dehydrogenase and, 261 cytochrome b2 and, 267, 268-269 cytochrome b5 reductase and, 156 cytochrome c oxidase interaction, 334335 356-360 cytochrome c peroxidase an!, cytochrome P-450 reductase and, 167, 168, 169, 170 dehydrogenases and, 90 D-lactate dehydrogenase and, 270 nitrite reductase and, 275 small NADH dehydrogenases and, 194, 195, 196, 198, 199-200, 203,206

440 succinate dehydrogenase and, 250 sulfite reductase and, 288, 290 transhydrogenase and, 68 ubiquinone reductase and, 181, 182, 197 yeast NADH dehydrogenase and, 217 Cytochrome c oxidase biological role, 299-300 chemical and physical properties, 301302, 313-314 electronic spectroscopy, 315-319 electron paramagnetic resonance, 329-334 interaction with cytochrome c, 334335 kinetic studies, 335-337 ligand binding studies, 319-325 models, 314-315 potentiometry, 325-329 historical background, 300-301 lipids of, 312313 mechanisms, 337-344 metal components, 307-309 occurrence of, 300 preparation, 305-307 protein of, 309-312 Cytochrome c peroxidase cytochrome c interaction, 356-360 enzymatic activity, 352353 general comments, 380361 historical background, 345-347 preparation and molecular properties, 347-348 reaction mechanism, 353-356 structural aspects, 348-351 Cytochrome c reductase succinate dehydrogenase and, 245 transhydrogenase and, 55 Cytochrome cI choline dehydrogenase and, 262 ubiquinone reductase and, 205 Cytochrome cs, adenylyl sulfate reductase and, 281 Cytochrome P-450, monooxygenases and, a3 Cytochrome P-450 reductase, 165-166 catalytic activities, 167-169 cytochrome bs reductase and, 151-153 general properties, 166-167 mechanism, 169-173 substrates and components, 149-150

SUBJECT INDEX

Cytoplasm, cytochrome c oxidase polypeptides and, 311

D Deaminonicotinamide adenine dinucleotide glyceraldehyde3phosphate dehydrogenase and, 30 transhydrogenase and, 59 Dehydrogenase(s), characteristics of, 90-91 Demerol, ubiquinone reductase and, 204 Deoxycholate ubiquinone reductase and, 178 yeast NADH dehydrogenase and, 218 Deoxyribonucleic acid, blactate dehydrogenase and, 264 Dephospho coenzyme A, transhydrogenase and, 70, 71 Desulfotomaculum, adenylyl sulfate reductase in, 282 Desuljovibrio, adenylyl sulfate reductase in, 282 Desuljovibrio desuljuricans, sulfate reduction by, 281 Desuljovibrio vulgaris, adenylyl sulfate reductase of, 281, 282484,285 Detergent, see also specific compounds cytochrome bs reductase and, 154-156, 161, 163 cytochrome P-450 reductase and, 166 glyceraldehyde-3-phosphate dehydrogenase and, 26 transhydrogenaae and, 70 Deuterium, cytochrome b6 reductase and, 159 Deuterium oxide small NADH dehydrogenases and, 192-193 succinate dehydrogenase and, 229-230 transhydrogenase and, 70 Deuteroethanol, catalase and, 405 Deuteroformate, catalase and, 404 Diamide, glutathione and, 131 Diaphorase, transhydrogenase and, 59 Dibromoacetone, glyceraldehyde-3-phosphate dehydrogenase and, 23 Dichloroacetate, small NADH dehydrogenases and, 192

441

SUBJECT INDEX

Dichlorohydroquinone, cytochrome c oxidase and, 325 2,6-Dichlorophenolindophenol choline dehydrogenase and, 261 cytochrome b, and, 267 cytochrome P-450 reductase and, 167 a-glycerophosphate dehydrogenase and, 257 2-hydroxyacid dehydrogenase and, 272 small NADH dehydrogenases and, 196, 198, 199, 203, 206 succinate dehydrogenase and, 250 sulfite reductase and, 288 transhydrogenases and, 59 ubiquinone reductase and, 181, 182, 197 yeast NADH dehydrogenase and, 217 2,3-Dicyano-5,6-dichoro-l,4-benzoquinone, ubiquinone reductase and, 182 N,N'-Dicyclohexylcarbodiimide, transhydrogenase and, 72 1,l-Dideuteroethanol, catalase and, 404, 405 1,5-Difluoro-2,4-dinitrobenzene,glyceraldehyde-3-phosphate dehydrogenase and, 22 2,2-Difluorosuccinate, succinate dehydrogenase and, 237 Digitonin, transhydrogenase preparation and, 67 Dihydrolipoamide, physiological form, 93 Dihydroxyacetone phosphate, a-glycerophosphate dehydrogenase and, 256257 1,2-Dihydroxybenzene 3,5-disulfonate, see Tiron 2,3-Dime thoxy-5,6dimethylbenzoquinone, ubiquinone reductase and, 181 2,3-Dimethoxy-5-methylbenzoquinone, ubiquinone reductase and, 181 2,4-Dinitrophenol dehydrogenases and, 63 succinate dehydrogenase and, 247 Dioxygen, see Oxygen Diphosphatidylglycerol, cytochrome c oxidase and, 312 Diphosphoglycerate, glyceraldehyde-3phosphate dehydrogenase and, 35,37 38, 41, 42, 44, 47, 48, 49

Disulfide (s) lipoamide dehydrogenase and, 122 pyridine nucleotidedisulfide oxidoreductases and, 92, 98, 100, 105 Disulfide groups, thioredoxin reductase, 145-146

5,5'-Dithiobis(nitrobenzoate) glutathione reductase and, 132 glyceraldehyde-3-phosphate dehydrogenase and, 21, 36 lipoamide dehydrogenase and, 120,123 Dithionite adenylyl sulfate reductase and, 284 catalase and, 367,375, 382 cytochrome ba,., and, 239, 240 cytochrome c oxidase and, 335, 338 cytochrome P-450 reductase and, 171172 iron-sulfur centers and, 216 lipoamide dehydrogenase and, 113 small NADH dehydrogenase and, 193, 194 semiquinone formation and, 90 sulfite reductase and, 288 thioredoxin reductase and, 145 yeast NADH dehydrogenase and, 219 Dithiothreitol, succinate dehydrogenase and, 227,240, 242,248 Dodecyl sulfate cytochrome c oxidase and, 311 lipoamide dehydrogenase and, 123-124 succinate dehydrogenase and, 227-228, 230-231,234,244,245 ubiquinone reductase and, 180 DT-diaphorase, transhydrogenation and, 52 Duroquinone, ubiquinone reductase and, 182

E Echinocystis macrocarpa seeds, transhydrogenase of, 66 Electromechanochemical model, branshydrogenase, 75 Electron economy, cytochrome c oxidase, 325-326 Electron paramagnetic resonance adenylyl sulfate reductase and, 284

442 catalase, 370, 372, 381, 396 cytochrome c oxidase and, 308309, 314, 316, 317, 321, 322, 327, 335-336 copper and, 329-330 iron and, 331 ligand binding effects, 332-333 p-oxobishemin and, 333-334 valence state changes and, 331332 cytochrome c peroxidase, 350,351,353, 355 fumarate reductase, 256 succinate dehydrogenase, 235, 214, 253, 254 sulfite reductase, 288 ubiquinone reductase, 184-187 yeast NADH dehydrogenase, 219, 220 catalase and, 389,396, 397 sulfite reductase and, 292, 294-295 Electron transport choline dehydrogenase and, 261-263 cytochrome c oxidase and, 301-302 a-glycerophosphate dehydrogenase and, 258 microsomal, 148-149 cytochrome b, reductase system, 150151 cytochrome P-450 reductase system, 149-150 mixed function amine oxidase, 153154 synergism between systems, 151-153 mitochondrial, components of, 178-179 succinate dehydrogenase and, 223-224, 245, 250 transhydrogenase and, 72-74 Emasol, cytochrome c oxidase and, 313 Energy, conservation and coupling by complex I, 214-216, 296 Energy-coupling, transhydrogenase and, 71-75 Enoyl coenzyme A reductase, transhydrogenase and, 88 Entnmoeba histolytica, transhydrogenase of, 66 Erythrocytes glutathione reductase of, 93,94, 138 kinetics, 139-140 substrates, 132

SUBJECT INDEX

glyceraldehyde-3-phosphate dehydrogenase in, 48 methemoglobin reductase of, 164-165 Escherichia coli glutathione reductase of, 102 dissociation constants, 135 glyceraldehyde-3-phosphate dehydrogenase, hybrids of, 26 lipoamide dehydrogenase of, 102, 104, 105, 110, 114, 115, 126 nitrite reductase of, 276, 277 ribonucleotide reductase of, 142-143 sulfate reduction in, 281 sulfite reductase of, 287-290 thioredoxin amino acid composition, 102 partial sequence, 93 thioredoxin reductase general properties, 144-145 specificity, 144 transhydrogenase of, 65, 66 energy and, 73 function, 80-81 molecular properties, 69 reconstitution, 79 Esterase activity, glyceraldehyde-3-phosphate dehydrogenase, 45 EthanoI, catalase and, 392,399,401402, 403, 404, 405 Ethylenediaminetetraacetate cytochrome c oxidase and, 308 a-glycerophosphate dehydrogenase and, 258 Zhydroxyacid dehydrogenase and, 272273 n-lactate dehydrogenase and, 271 lipoamide dehydrogenase and, 126 NADH dehydrogenase and, 206 succinate dehydrogenase and, 247 thioredoxin reductase and, 147, 148 Ethyl hydrogen peroxide, catalase and, 391-392, 395 N-Ethylmaleimide cytochrome b, reductase and, 163, 164 glutathione reductase and, 141 small NADH dehydrogenase and, 203 succinate dehydrogenase and, 246, 249 Ethylmorphine, demethylation of, 150

443

SUBJECT INDEX

Ethylpyridylketone, transhydrogenase and, 59 Euglena gracilis, transhydrogenase of, 54

F Fatty acid(s) cholesterol side chain cleavage and, 85 desaturation of, 151 w-hydroxylation of, 150 synthesis, transhydrogenase and, 88 Ferredoxin sulfite reductases and, 295 transhydrogenase and, 54, 59 Ferric compounds, sulfite reductase and, 293 Ferric ion, cytochrome P-450 reductase and, 168-169 Ferricyanide adenylyl sulfate reductase and, 281,282, 283, 285 bacterial NADH dehydrogenase and, 221 choline dehydrogenase and, 261 cytochrome bl and, 267, 268 cytochrome ba reductase and, 156, 159, 160, 161, 162 cytochrome c oxidase and, 321, 327,337 cytochrome P-450 reductase and, 168, 171, 172 dehydrogenases and, 90 u-glycerophosphate dehydrogenase and, 257 high molecular weight NADH dehydrogenase and, 188, 201,203-204 2-hydroxyacid dehydrogenase and, 272 methemoglobin reductase and, 165 small NADH dehydrogenase and, 194195, 196, 198, 199, 203, 206 succinate dehydrogenase and, 223, 236, 237, 254 sulfite reductase and, 288, 290 thioredoxin reductase and, 148 transhydrogenase and, 59 ubiquinone reductase and, 181, 182, 183, 186, 197, 199,203,207 yeast NADH dehydrogenase and, 217, 218,219, 220, 221 Ferrocyanide, cytochrome c peroxidase and, 353, 355

Ferrocytochrome c, cytochrome c peroxidaae and, 345, 352, 353,354-355, 357, 361 Ferrous ions, succinate dehydrogenase and, 243 Ferrous iron, oxygen reduction and, 304305 Flavin adenylyl sulfate reductase and, 279, 285 choline dehydrogenase and, 260 cytochrome bz and, 268-269 a-glycerophosphate dehydrogenase and, 256, 257, 260 high molecular weight NADH dehydrogenase and, 188,190 Zhydroxyacid dehydrogenase and, 272, 273 succinate dehydrogenase and, 223-225, 226,232234,237,241,243,244,253, 254 sulfite reductases and, 287 Flavin adenine dinucleotide adenylyl sulfate reductase and, 282 283, 284, 297 choline dehydrogenase and, 261 fumarate reductase and, 256 a-glycerophosphate dehydrogenase and, 257, 260 2-hydroxyacid dehydrogenase and, 273 n-lactate dehydrogenase and, 270,272 lipoamide dehydrogenase and, 123,124, 125, 127 mitochondria1 protein and, 297 NADH dehydrogenase and, 217 nitrite reductase and, 275,276, 277,278 pyridine nucleotide-disulfide oxidoreductases and, 92, 94, 95, 97,99, 100, 101, 105 succinate dehydrogenase and, 234-235 sulfite reductase and, 288, 290,292,294 transhydrogenases and, 57-58 Flavin rnononucleotide bacterial NADH dehydrogenase and, 221 cytochrome bs reductase and, 162 2-hydroxyacid dehydrogenase and, 272 L-lactate dehydrogenase and, 264, 265 lipoamide dehydrogenase and, 124-125 low molecular weight NADH dehydrogenases and, 191, 193-194, 198

444 nitrite reductase and, 275, 277, 278 succinate dehydrogenase and, 238, 247, 249 sulfite reductase and, 288, 289, 292, 293-294 ubiquinone reductase, 179-180, 182, 186 yeast NADH dehydrogenase and, 218 Flavoprotein ( s ) functions of, 175-177 mitochondrial, 297 sulfite and, 282 Fluorescence cytochrome bs reductase and, 162, 163 glyceraldehyde-3-phosphate dehydrogenase, 32 lipoamide dehydrogenase, 117-118, 123, 124, 126 thioredoxin, 93 Fluoride catalase and, 377, 383,385 cytochrome c oxidase and, 320421,333 cytochrome c peroxidase and, 350, 353 p-Fluorodinitrobenzene, glyceraldehyde3-phosphate dehydrogenase and, 20 p-Fluoro-m,m'-dinitrophenylsulfone, glyceraldehyde-3-phosphate dehydrogenase and, 37 2-Fluoroethanol, catalase and, 401, 403 Formaldehyde, catalase and, 406 Formamide, catalase and, 386, 399 Formate catalase and, 374-375,383, 385, 386,404 succinate dehydrogenase and, 238, 247, 248 Formic acid, catalase and, 388,395,398, 399, 403, 405-406 Freezing, lipoamide dehydrogenase, 125126 Fructose-l,&diphosphatase, glutathione reductase and, 130 Fumarate choline dehydrogenase and, 262 a-glycerophosphate dehydrogenase and, 258 succinate dehydrogenase and, 238-239, 247, 253 Fumarate reductase anaerobes and, 254 yeast, 255-256

SUBJECT INDEX

p- (2-Furylacrylolyl phosphath, glyceraldehyde3phosphate dehydrogenase and, 35, 36

G Glucokinase, activity, liver and, 47 Gluconeogenesis, glyceraldehyde-3-phosphate dehydrogenase and, 45-49 Glucose-6-phosphate dehydrogenase, glutathione reductase and, 131 Glutamate, redox couple, 82 Glutamate dehydrogenase, transhydrogenases and, 52, 82, 85-86 Glutamate residues, glyceraldehyde-3phosphate dehydrogenase, 30 Glutamine residues, lipoamide dehydrogenase, 101 bis-N,N(y-Glutamyl)cystine,glutathione reductase and, 132 Glutathione nitrite reductase and, 275 reduced : oxidized ratios, 129-130 reduction of, 87 reoxidation of, 93 Glutathione-cystine oxidoreductase, 130 Glutathione-homocystine oxidoreductase, 130 Glutathione-insulin transhydrogenase, 130 Glutathione peroxidase, 130 Glutathione protein disulfide oxidoreductase, 130 Glutathione reductase amino acid composition, 102, 104, 105 cystine residues, 104 kinetic studies, 138-141 mechanism, 94,97-98,134 metabolic functions, 129-133 reaction catalyzed, 92 reduction of, 112, 113 specificity of, 92-93 coenzymes and, 91 thiol groups, 141-142 two-electron-reduced enzyme, properties, 133-138 Glyceraldehyde, glyceraldehyde-3-phosphate dehydrogenase and, 44 Glyceraldehyde-3-phosphatedehydrogenase catalytic properties,

SUBJECT INDEX

mechanism of action, 38-46 metabolic role, 45-49 pyridine nucleotide binding, 28-38 chemical modifications, 20-24 dissociation and hybridization, 24-27 distribution of, 3 historical background, 2-3 maximal activities in rat and human tissues, 47-48 metabolic role, 4549 molecular properties, isolation, 3-4 structure, 5-27 other activities of, 44-45 physiological activity, 38-44 preexisting asymmetry model, 35-38 pure, sources of, 2 reaction catalyzed, 1 ~-Glycerol-3-phosphatedehydrogenase, properties, 256-260 a-Glycerophosphate, succinate dehydrogenase and, 239, 250 a-Glycerophosphate cycle, function of, 259 Glycine catalase and, 387-388 oxidative decarboxylation of, 108 Glycine residues, glyceraldehyde-3-phosphate dehydrogenase, 12 Glycogen synthetase, glutathione reductase and, 130 Glycolate, succinate dehydrogenase and, 238 Glycolysis, glyceraldehyde-$phosphate dehydrogenase and, 4549 Glyoxylate, succinate dehydrogenase and, 238 Glyoxylate cycle, transhydrogenase and, 80 Guanidine, glyceraldehyde-3-phosphate dehydrogenase and, 24 Guanidinium chloride cytochrome bz and, 268 lipoamide dehydrogenase and, 123, 124 NADH dehydrogenases and, 199,207 succinate dehydrogenase and, 232 Guanosine triphosphate, oxidation of NAD+-linked substrates and, 110 Guiacol, cytochrome c peroxidase and, 353

H Halogenacetic acids, glyceraldehyde-3phosphate dehydrogenase and, 2 Hansenula anomala, L-lactate dehydrogenase of, 265, 268, 269 Heart cytochrome c oxidase of, 311 glyceraldehyde-3-phosphatedehydrogenase in, 47, 48 lipoamide dehydrogenase of, 102, 104 111-114, 116 transhydrogenase of, 62-63, 64, 65 kinetic constants, 76-77 molecular properties, 70-71 preparation, 67-68 Helix, glyceraldehyde-3-phosphate dehydrogenase, 13-14 Heme cytochrome bf and, 268-269 sulfite reductases and, 287, 288,289, 290, 292,293,294,295 Heme az, nitrite reductase and, 275,297 Heme A, cytochrome c oxidase and, 307, 309, 314-315 Heme G adenylyl sulfate reductase and, 285, 286 nitrite reductase and, 278 Heme oxygenase, cytochrome P-450 reductase and, 150 Hemoglobin, absorption spectra, 317-319 Hemoprotein(s), amino acid sequences, 371 Hepatoma, transhydrogenase in, 65 Hexokinase, activity in muscle, 47 Histidine residues catalase, 370-372,375,377-378,381,383, 395 cytochrome c peroxidase, 350 glyceraldehyde-3-phosphate dehydrogenase, 14, 44 modification of, 23-24 lipoamide dehydrogenase, 101 succinate dehydrogenase, 235 Homocystine, glutathione redubtasq and, 132 Horseradish peroxidase, reaction cycle, 356 Human, glyceraldehyde-3-phosphate dehydrogenase of, 9

446

SUBJECT INDEX

Hydrazoic acid, catalase and, 388 Hydrogen bonds, glyceraldehyde-3-phosphate dehydrogenase, 11, 14, 15,25, 43 Hydrogen peroxide catalase and, 388,395,398,403 cytochrome c peroxidase and, 345-346 Hydrogen sulfide, formation of, 279 Hydroperoxides catalase and, 398, 406 cytochrome c peroxidase and, 352,353, 354 Hydrophobic bonds, catalase and, 367 Hydroquinone, cytochrome c peroxidase and, 353 D-2-Hydroxyacid dehydrogenase, properties, 272-273 p-Hydroxybutyrate, redox couple, 82 ~-2-Hydroxybutyrate,D-laCtate dehydrogenase and, 270 25-Hydroxycholecalciferol,hydroxylation of, 83 Hydroxylamine catalase and, 388, 398 formation of, 153 nitrite reductase and, 275,276, 277,278, 279 sulfite reductase and, 288-289,290, 292, 293, 294, 295 p-Hydroxymercuribenzoate, transhydrogenase and, 58 cu-Hydroxymonocarboxylic acids, L-lactate dehydrogenase and, 267 5-Hydroxy-1,4-naphthoquinone,see Juglone 17-Hydroxyprogesterone, hydroxylation of, 150 Hyponitrite, nitrite reductase and, 275

I Imidazole, catalase and, 370 Infrared spectroscopy, cytochrome c oxidase and, 321,322, 323 Inosine diphosphate, succinate dehydrogenase and, 247, 248 Inosine triphosphate, succinate dehydrogenase and, 247, 248 Insulin glycolytic enzymes and, 47

phenylalanyl chain, glutathione reductase and, 130 Iodide, succinate dehydrogenase and, 247, 248 Iodine cytochrome ba reductase and, 163 glyceraldehyde-3-phosphate dehydrogenase and, 22-23, 24 Iodoacetamide glyceraldehyde-3-phosphate dehydrogenase and, 35, 36-37,38,42,43 lipoamide dehydrogenase and, 118-119, 122 Iodoacetate glyceraldehyde-3-phosphate dehydrogenase and, 20,28,35, 36-37,38, 39, 42-43, 45 lipoamide dehydrogenase and, 120, 123 a-Iodopropionamide, glyceraldehyde-3phosphate dehydrogenase and, 33 a-Iodopropionic acid, glyceraldehyde-3phosphate dehydrogenase and, 33 Iodosobenzoate, glyceraldehyde-3-phosphate dehydrogenase and, 20, 21 Ionic strength cytochrome P-450 reductase and, 167 glutathione reductase and, 139-140 glyceraldehyde-3-phosphate dehydrogenase and, 25, 26,43 Iron, see also Nonheme iron adenylyl sulfate reductase and, 279, 297 carbon monoxide and, 321-323 catalysis and, 363-364 cytochrome c oxidase and, 302,307309, 321 electron paramagnetic resonance, 331 deficiency, NADH dehydrogenases and, 219, 221 a-glycerophosphate dehydrogenase and, 256, 257, 260 high molecular weight NADH dehydrogenases and, 188, 190,201-202 low molecular weight NADH dehydrogenases and, 191, 193-194,198 mitochondria1 flavoprotein and, 297 nitrite reductase and, 275 sulfite reductase and, 288, 290, 292,293, 295 ubiquinone reductase and, 179, 180, 182, 183-184, 186-187, 204, 209-210

447

SUBJECT INDEX

Iron-copper coupling, cytochrome c oxidase, 326-327, 338-343 Iron sulfide, succinate dehydrogenase, 230-232,235,243,244, 245,246,253, 254, 296 Iron-sulfur centers bacterial NADH dehydrogenase and, 221 energy conservation and, 214-216, 296 yeast NADH dehydrogenase and, 217, 218, 219 Isocitrate dehydrogenase, transhydrogenase and, 52-53,81,84,86-88 Isoleucine residues, disulfide oxidoreductases, 104 Isonitriles, catalase and, 401 Isozymes, lipoamide dehydrogenase,. 109

J Juglone ubiquinone reductase and, 182 yeast NADH dehydrogenase and, 219, 220

K a-Keto acids decarboxylation of, 108 2-hydroxyacid dehydrogenase and, 272 a-Ketoglutarate, reductive carboxylation of, 87 Kidney transhydrogenase in, 64 function, 83 Kinetic studies, cytochrome c oxidase, 335-337

1 Lactate dehydrogenase(s1, types of, 263 n ( -)-Lactate dehydrogenase, 269-270 enzymic properties, 270-272 physical properties, 270 L(+)-Lactate dehydrogenase cytochrome b, core, 266-267 enzymic properties, 267-269 historical background, 263-264 physical properties, 264-266

Lactobacillus leichmannii, ribonucleotide reductase of, 142 Lecithin, transhydrogenase and, 70-71 Leucine residues disulfide oxidoreductases, 104 glyceraldehyde-%phosphate dehydrogenase, 11, 12 Leucomethylene blue, nitrite reductase and, 275 Leucomethyl viologen, 2-hydroxyacid dehydrogenase and, 272 Lipase, cytochrome P-450 reductase and, 167, 168,169,170-171, 172 Lipid cytochrome bs reductase and, 151, 158 cytochrome c oxidase and, 309, 311, 312-313 high molecular weight NADH dehydrogenase and, 188 peroxidation, cytochrome P-450 reductase and, 168,169 transhydrogenase and, 70 Lipoamide dehydrogenase(s1 amino acid composition, 102, 104,105 apoenzymes, 124 coenzyme specificity, 94 cystine residues, 104, 120-122 distribution, 106107 kinetic studies, 115-117 mechanism, 94-97, 126-129 mechanism of Massey and Veeger, review of, 111 metabolic functions, 107-1 10 reaction catalyzed, 92 role of NAD' as modifier, 117-120 structural studies, 120-126 two-electron-reduced enzyme, properties, 111-115 Lipoate glutathione reductase and, 93, 132, 140 ubiquinone reductas? and, 181 Lipoprotein, cytochrome c oxidase and, 302 Liver glyceraldehyde-3-phosphate dehydrogenase in, 47, 48 glycolysis, cytosolic redox state and, 46 lipoamide dehydrogenase of, 116-117 transhydrogenase in, 64

SUBJECT INDEX

Lobster muscle glyceraldehyde-3-phosphate dehydrogenase cysteine residues, 20,28 hybrids of, 26-27 lysine residues, 22 primary structure, 5, 6-8, 9, 18 pyridine nucleotide binding, 33 tyrosine residues, 22-23 Lubrol, NADH dehydrogenase and, 189 Lysine residues cytochrome bs reductase, 164 dihydrolipoate and, 108 glutathione reductase, 105, 119 glyceraldehyde-3-phosphatedehydrogenase, 11, 14, 44, 45 modification of, 21-22,3748 lipoamide dehydrogenase, 119, 126 thioredoxin, 119 Lysolecithin, transhydrogenase and, 68,71 Lysophosphatidylethaolamine, cytochrome P-450 reductase and, 150 Lysosomes, cytochrome bs reductase and, 154-156

M Magnesium choline dehydrogenase and, 263 transhydrogenase and, 70,77 Magnetic susceptibility, catalase, 382,396 Malate choline dehydrogenase and, 262 succinate dehydrogenase and, 237 Maleate, succinate dehydrogenase and, 238 “Malic” enzyme, steroid hydroxylations and, 83-84 Malonate choline dehydrogenase and, 263 succinate dehydrogenase and, 238, 247, 248 Manganese nitrite reductase and, 277 transhydrogenase and, 70 Membrane(s) integrity, glutathione and, 131 Menadione bacterial NADH dehydrogenase and, 221

cytochrome P-450 reductase and, 168, 169 2-hydroxyacid dehydrogenase and, 272 small NADH dehydrogenases and, 194195, 196, 206 sulfite reductase and, 288, 290 ubiquinone reductaae and, 181,182, 197, 207 yeast NADH dehydrogenase and, 218, 220 Menadione reductase, transhydrogenase and, 55 Mercaptoethanol cytochrome c oxidase and, 311 small NADH dehydrogenase and, 198 succinate dehydrogenase and, 243 Mercurials glyceraldehyde-3-phosphate dehydrogenase and, 21 large NADH dehydrogenase and, 203204 lipoamide dehydrogenase and, 114, 122 low molecular weight NADH dehydrogenases and, 191, 199, 203 nitrite reductase and, 276 succinate dehydrogenase and, 246 sulfite reductase and, 292 ubiquinone reductase and, 181, 186, 199, 203, 204 p-Mercuribenzoate choline dehydrogenase and, 261 cytochrome ba reductase and, 163 cytochrome c peroxidase and, 348 cytochrome P-450 reductase and, 168 L-lactate dehydrogenase and, 264 nitrite reductase and, 275, 276, 278 sulfite reductase and, 293 p-Mercuriphenyl sulfonate n-lactate dehydrogenase and, 27l’ sulfite reductase and, 288, 289 yeast NADH dehydrogenase and, 217 Mersalyl cytochrome bs reductase and, 163, 164 cytochrome P-450 reductase and, 168 small NADH dehydrogenase and, 193 succinate dehydrogenase and, 246 Metal(s) transition, oxygen reduction and, 303 Methanol, catalase and, 391, 392, 401, 403,404,405,406

SUBJECT INDEX

Methemoglobin reductase, cytochrome ba reductase and, 164-165 Methionine residues glyceraldehyde-3-phosphate dehydrogenase, 11 lipoamide dehydrogenase, 118 5-Methoxyindole-2-carboxylate, lipoamide dehydrogenase and, 110 Methylene blue choline dehydrogenase and, 261 cytochrome b, and, 267 a-glycerophosphate dehydrogenase and, 257 2-hydroxyacid dehydrogenase and, 272 nitrite reductase and, 275 succinate dehydrogenase and, 246 ubiquinone reductase and, 182 Methylene succinate, succinate dehydrogenase and, 238 3-Me thylflavin adenine dinucleotide, lipoamide dehydrogenase and, 125 Methyl hydrogen peroxide, catalase and, 390-391, 398, 405 Methyl hydroperoxide, catalase and, 392 N-Methyl hydroxylamine, catalase and, 399 o-Me thy1 hydroxylamine, catalase and, 399 2-Methylnaphthoquinone, see Menadione Methyl succinate, succinate dehydrogenase and, 237-238 Methyl viologen adenylyl sulfate reductases and, 281 nitrite reductase and, 276 sulfite reductases and, 287, 288-289, 290, 292, 293 Micrococcus denitrifians, transhydrogenase of, 73 Microorganism(s) , succinate dehydrogenase of, 254-256 Microsomes electron transport, 148-149 cytochrome b, reductase system, 150151 cytochrome P-450 reductase system, 149-150 mixed function amine oxidase, 153154 synergism between systems, 151-153 hydroxylation reactions in, 87

Mitochondria choline dehydrogenase and, 260-263 fatty acid synthesis in, 88 glutamate and isocitrate metabolism, 85-88 a-glycerophosphate dehydrogenase of, 256-259 membranes, cytochrome c oxidase and, 302 monooxygenase reactions, 83-85 nicotinamide adenine dinucleotide dehydrogenases, 177-178 energy conservation and, 214-216 high molecular weight, 187-189 inhibitors of, 203-207 low molecular weight, 189-198 relevance of low and high molecular weight dehydrogenases, 198-203 transhydrogenation and, 207-214 ubiquinone reductase (Complex I), 178-187 nicotinamide nucleotides, redox state, 81-82 transhydrogenase of, 62, 65 assay, 66-67 preparation, 67-68 Models, cytochrome c oxidase, 314-315 Molecular weight, succinate dehydrogenase, 232-234 Molybdenum bacterial NADH dehydrogenase and, 221 ubiquinone reductase and, 187 Monochloroacetate, small NADH dehydrogenases and, 192 Monofluorosuccinate, succinate dehydrogenase and, 237 Monooxygenase ( s ), transhydrogenases and, 83-85 Musca domestica, a-glycerophosphate dehydrogenases of, 258-259 Muscle glyceraldehyde-3-phosphate .dehydrogenase in, 47,48 transhydrogenase in, 64 Mycobacterium, 2-hydroxyacid dehydrogenase of, 273 Mycobacterium phlei, transhydrogenase of, 65-66

450

SUBJECT INDEX

Myoglobin, absorption spectra, 317-319 Myohematin, 300

N l,2-Naphthoquinone 4-sulfonate1 cytochrome b, and, 267 Neotetrazolium, cytochrome P-450 reductase and, 168 Nerve transmission, glutathione and, 131 Neurospora crassa cytochrome c oxidase of, 311 nitrite reductase of, 275-276 Nicotinamide adenine deoxydinucleotide, glyceraldehyde-3-phosphate dehydrogenase and, 30 Nicotinamide adenine dinucleotide choline dehydrogenase and, 261,262 cooperativity of binding, 30-35 cytochrome c oxidase and, 335 glyceraldehyde-3-phosphate dehydrogenase and, 3, 4, 26,42,45, 48 amino acid modification and, 21, 22 binding, 10-16, 28-38, 49 pK. and, 43 inhibition by, 206-207 lipoamide dehydrogenase and, 117-120, 125, 126, 128 nitrite reductase and, 275, 277, 278 oxidized : reduced ratio, glycolysis and, 46, 49 succinate dehydrogenase and, 252 Nicotinamide adenine dinucleotide dehydrogenase Azotobacter vinelandii, 221 high molecular weight, 187-189, 190 relevance to mitochondrial enzyme, 198-203 inhibitors of, 203-207 low molecular weight, 189-198, 295 relevance to mitochondrial enzyme, 198-203 transhydrogenase and, 78 yeast, 216-221 Nicotinamide adenine dinucleotide 3'phosphate, transhydrogenase and, 59, 61, 69 Nicotinamide adenine dinucleotide phosphate cytochrome bs reductase and, 156, 157, 160

glutathione reductase and, 134-137 large NADH dehydrogenase and, 202 nitrite reductase and, 275,276, 278 respiratory particles and, 200 sulfate reduction and, 281,283 sulfite reductases and, 287-295 transhydrogenation, complex I and, 207-214,296 ubiquinone reductase and, 181 Nicotinamide adenine dinucleotide-ubiquinone reductase, 178-179 activities, 180-183 composition, 179-180 spectral properties, 183-187 Nicotinamide bdeaminoadenine dinucleotide, glyceraldehyde-3-phosphate dehydrogenase and, 30 Nicotinic acid hydrazide adenine dinucleotide, glyceraldehyde-3-phosphate dehydrogenase and, 30 Nicotinamide mononucleotide, transhydrogenase and, 59 Nicotinamide nucleotide transhydrogenase AB-specific historical, 62-64 kinetics and reaction mechanism, 7578 molecular properties, 69-71 occurrence, 64-66 preparation and assay, 66-69 reconstitution, 78-79 relationship to energy-coupling system, 71-75 BB-specific historical, 52-53 molecular properties, 57-59 occurrence, 63-54 purification and away, 54-57 reaction mechanism and regulation, 59-62 definition, 5162 physiological roles, 79-81 fatty acid synthesis, 88 mitochondrial glutamate and isocitrate metabolism, 85-88 mitochondrial monooxygenase reactions, 83-85 redox state of mitochondrial nicotinamide nucleotides, 81-82

45 1

SUBJECT INDEX

Nicotinylhydroxamic acid adenine dinucleotide, glyceraldehyde-3-phosphate dehydrogenase and, 30 Nigericin, transhydrogenase and, 72 Nitrate reduction of, 273 succinate dehydrogenase and, 227, 247, 248 Nitric acid, ferrocytochrome c peroxidase complex, 350 Nitric oxide catalase and, 372,375,376 nitrite reductases and, 274,275 Nitrite catalase and, 391, 392,400 sulfite reductase and, 288-289,290,292, 293, 294, 297 Nitrite reductase (s) occurrence of, 274 properties of, 275-279, 297 Nitrogen, nitrite reductases and, 274 Nitrogen mustard, choline dehydrogenase and, 261 Nitrones, formation of, 153 p-Nitrophenylacetate, glyceraldehyde-3phosphate dehydrogenase and, 21,45 Nitrous acid, catalase and, 388,398 Nitrous oxide, catalase and, 400 Nonheme iron, see also Iron adenylyl sulfate reductase and, 282, 284, 285 choline dehydrogenase and, 260 succinate dehydrogenase and, 223-225, 226 sulfite reductases and, 287 Nuclear magnetic resonance, ferrocytochrome c, 357-359

0 Oleate, gluconeogenesis and, 46, 47 Oligomycin succinate dehydrogenase and, 249 transhydrogenase and, 67-68, 72 Optical rotatory dispersion glyceraldehyde-3-phosphate dehydrogenase, 14 transhydrogenase, 62 Ovary transhydrogenase, function, 83,85

Oxalate 2-hydroxyacid dehydrogenase and, 272 n-lactate dehydrogenase and, 271 Oxaloacetate choline dehydrogenase and, 262 D-lactate dehydrogenase and, 271 succinate dehydrogenase and, 237,238, 248-249 Oxidase(s), characteristics of, 90-91 Oxidative phosphorylation, transhydrogenase and, 73 Oximes, catalase and, 398 p-Oxobishemin A, cytochrome c oxidase and, 333-334 Oxygen adenylyl sulfate reductase and, 282,284 choline dehydrogenase and, 261, 262 cytochrome b, reductase and, 156 cytochrome c oxidase and, 323-325, 336-337, 338-343 cytochrome P-450 reductase and, 170 electronic structure, 303 a-glycerophosphate dehydrogenase and, 260 heme absorption spectra and, 317419 lactate dehydrogenases and, 269,270 microsomal mixed function oxidations and, 149, 152 reduction, chemistry of, 302-305 succinate dehydrogenase and, 236 sulfite reductase and, 288

P Palmitylcoenzyme A, transhydrogenase and, 70,78, 88, 212 Palmityldephosphocoenzyme A, transhydrogenase and, 70, 71 Pantothine, glutathione reductase and, 132 Peas, glutathione reductase of, 138 Pea seed, glyceraldehyde-3-phosphate dehydrogenase of, 40 Penicillium chrysogenum, glutathione reductase of, 138 Peptococcus glycinophilus glycine decarboxylation by, 108 lipoamide dehydrogenase of, 112 Peracetic acid, catalase and, 392-393, 395, 397

452 Perchlorate small NADH dehydrogenases and, 191, 192, 203 succinate dehydrogenase and, 227-228, 229,231,232, 247,248 Perfluoro-n-hexane, oxygen consumption and, 152-153 Peroxide, see also Hydrogen peroxide oxygen reduction and, 303,305 PH cytochrome bs reductase and, 163,164 cytochrome c peroxidase and, 350-351 cytochrome P-450 reductase and, 167, 168 glutathione reductase and, 134, 140, 141 glyceraldehyde-3-phosphate dehydrogenase and, 41,48,49 lipoamide dehydrogenase and, 116, 119, 125, 128 small NADH dehydrogenases and, 194195, 203 succinate dehydrogenase and, 247, 248 thioredoxin reductase and, 144 transhydrogenases and, 58, 76,208,210211, 213 o-Phenanthroline 2-hydroxyacid dehydrogenase and, 272 n-lactate dehydrogenase and, 271-272, 273 NADH dehydrogenases and, 206 succinate dehydrogenase and, 246 Phenazine methosulfate choline dehydrogenase and, 261, 262, 263 a-glycerophosphate dehydrogenase and, 257, 258 n-lactate dehydrogenase and, 270 succinate dehydrogenase and, 223,225, 227, 232,236,237, 238, 239,242, 243, 246, 249-250, 253, 254 Phenobarbital, cytochrome P-450 reductase and, 150 Phenols, catalase and, 398 Phenylalanine residues, glyceraldehyde-3phosphate dehydrogenase, 11, 33 Phenyl mercuric acetate, glutathione reductase and, 141 Phenylmethylsulfonyl fluoride, L-lactate dehydrogenase and, 265,266

SUBJECT INDEX

Phosphate choline dehydrogenase and, 263 glyceraldehyde-3-phosphate dehydrogenase and, 44,48 nitrite reductase and, 275 succinate dehydrogenase and, 247 Phosphatidylcholine cytochrome c oxidase and, 312, 313 cytochrome P-450 reductase and, 149 Phosphatidylethanolamine, cytochrome c oxidase and, 312, 313 Phosphatidylinositol, cytochrome c oxidase and, 312, 313 3'-Phosphoadenosine 5'-phosphosulfate, formation of, 279 Phosphocreatine, glyceraldehyde-3-phosphate dehydrogenase and, 48 Phosphofructokinase activity in adipose tissue, 47 glycolysis and, 49 3-Phosphoglycerate, a-glycerophosphate dehydrogenase and, 258 Phosphoglycerate kinase glycolysis rate and, 46,49 pyruvate kinase and, 47 Phospholipase (9) choline dehydrogenase and, 260 transhydrogenase and, 70 Phospholipase A cytochrome c oxidase and, 313 a-glycerophosphate dehydrogenase and, 257 n-lactate dehydrogenase and, 270,271 NADH dehydrogenase and, 187 Phospholipase C low molecular weight NADH dehydrogenase and, 191 yeast NADH dehydrogenase and, 218 Phospholipid amine oxidase and, 153 cytochrome bs reductase and, 161 succinate dehydrogenase and, 244,245, 247 synthesis, a-glycerophosphate dehydrogenase and, 259 ubiquinone reductase and, 179, 180, 182-183, 201 Photoirradiation, semiquinones, 90

453

SUBJECT INDEX

Photooxidation, glyceraldehyde-3-phosphate dehydrogenase and, 24 Photosynthesis, transhydrogenation and, 64, 66

Piericidin A respiratory particles and, 199 small NADH dehydrogenase and, 199 transhydrogenation and, 211-212 ubiquinone reductase and, 181,203, 204-206, 214

yeast NADH dehydrogenase and, 219, 220

Pig muscle, glyceraldehyde-3-phosphate dehydrogenase of, 6-8, 18, 24 hybrids of, 26-27 tyrosine residues, 23 Plants, transhydrogenases of, 54 Plasmalogen, biosynthesis, 151 Polyacrylamide gel electrophoresis, succinate dehydrogenase, 227-228,230, 245

Potassium bromide, cytochrome bs reductase and, 162 Potassium ions, transhydrogenase and, 72

Potentiometry cytochrome c oxidase and, 322 electron economy, 325-326 interpretation and summary, 328-329 iron-copper coupling, 326-327 ligand binding, 327-328 Progesterone, hydrdxylation of, 150 Proline residues, glyceraldehyde-3-phosphate dehydrogenase, 11 n-Propanol, catalase and, 401-402 Propargyl alcohol, catalase and, 401-402, 403

Propionibacterium arabinosum, a-glycerophosphate dehydrogenase of, 260 2-Propyn-1-01, catalase and, 404 Prostaglandin, biosynthesis, 168 Prosthetic groups, succinate dehydrogenase, 234-235 Protease, D-lactate dehydrogenase and, 270

Protein(s) catalase function and, 369-370 cytochrome c oxidase and, 309-312 synthesis, glutathione and, 131

Protein disulfide isomerase, glutathione and, 132 Proteus mirabilis, sulfate reduction by, 281

Proteus vulgaris, sulfate reduction by, 281 Protoheme, cytochrome c peroxidase and, 345,346, 348,349

Protons, transhydrogenase and, 77-78 Protoporphyrin IX, catalase and, 366 Pseudomonas transhydrogenase function, 80 molecular properties, 58-59 reaction mechanism and regulation, 59-61

Pseudomonas aeruginosa nitrite reductase of, 274, 275 transhydrogenase of, 53 molecular properties, 57 purification, 54, 56 Pseudomonas denitrificans, nitrite reductase of, 274275 Pseudomonas fluorescena transhydrogenase of, 52-53 purification, 54, 56 Pseudomonas hydrophila, sulfate reduction by, 281 Pseudomonas stutzeri, nitrite reductase of, 274 Pyocyanine, nitrite reductase and, 275 Pyridine adenine dinucleotide, transhydrogenase and, 59 Pyridine aldehyde adenine dinucleotide cytochrome bs reductase and, 156,158159

glyceraldehyde-3-phosphate dehydrogenase and, 30 transhydrogenase and, 59 Pyridine nucleotidedisulfide oxidoreductases mechanism, similarities and contrasts, 94-99

reaction catalyzed-chemical similarities and cross-reactivity, 92-94 structure, similarities and contrasts, 99-105

Pyridoxal phosphate, glyceraldehyde-3phosphate dehydrogenase and, 22 Pyrogallol, cytochrome c peroxidase and, 353

SUBJECT INDEX

Pyrophosphate lipoamide dehydrogenase and, 125 transhydrogenase and, 72 Pyruvate gluconeogenesis and, 47,48 oxidation, transhydrogenase and, 80 Pyruvate dehydrogenase lipoamide dehydrogenase and, 126 transhydrogenase and, 55 Pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and, 47 Pythium ultimum, lipoamide dehydrogenase of, 112

0 Quinoline oxide, choline dehydrogenase and, 262 Quinones, sulfite reductase and, 293

R Rabbit muscle glyceraldehydea-phosphate dehydrogenase, 25 amino acid modification, 22,24 hybrids of, 26-27 pyridine nucleotide binding, 33-35 Rat muscle, glyceraldehyde-3-phosphate dehydrogenase of, 25 Redox potentials cytochrome c oxidase, ligand binding and, 327-328 cytochrome P-450 reductase and, 172 Reductase(s), characteristics of, 90-91 Respiration, transhydrogenation and, 64 65, 72 R hodopseudomonas palustris sulfate reduction by, 281 transhydrogenase of, 66 R hodopseudomonas spheroides sulfate reduction by, 281 transhydrogenase of, 66 molecular properties, 71 preparation, 68 reconstitution, 79 Rhodopseudomonas viridis, transhydrogenase of, 66 Rhodospirillum moliachianum, transhydrogenase of, 66

Rhodospiiillum rubrum succinate dehydrogenase of, 254-255 transhydrogenase of, 66,69, 213 energy and, 73,74 molecular properties, 69,71 preparation, 68 reconstitution and, 78 Riboflavin cytochrome ba reductase and, 162 deficiency, glutathione reductase and, 131 nitrite reductase and, 275 Ribonucleotide reductase, thioredoxin and, 142-143 Rose Bengal, cytochrome c peroxidase and, 350 Rotenone cholesterol side cleavage and, 85 choline dehydrogenase and, 262-263 small NADH dehydrogenases and, 196, 199 transhydrogenation and, 211 ubiquinone reductase and mitochondrial, 181,182,183,197,204206,214-215 yeast, 217,219

S Saccharomyces carlsbergensis, NADH dehydrogenase of, 216, 219-221 Saccharomyces cerevisiae, see also Yeast adenylyl sulfate reductase of, 283 lactate dehydrogenase of, 269-270 L-lactate dehydrogenase of, 264-266, 268, 269 NADH dehydrogenase of, 216,217, 219-221 succinate dehydrogenase of, 254 sulfite reductase of, 292-295 transhydrogenase of, 66 Salmonella typhimurium, sulfite reductase of, 290-291 Seconal ubiquinone reductase and mitochondrial, 204 yeast, 217 Semicarbazide catalase and, 379 choline dehydrogenase and, 262

SUBJECT INDEX

Semiquinone cytochrome P-450 reductase, 170, 172 dehydrogenases and, 90, 97-98 glutathione reductase and, 137-138 oxidases and, 90 thioredoxin reductase, 147-148 Serine residues, pyridine nucleotide-disulfide oxidoreductases, 101 Serum albumin, lipoamide dehydrogenase and, 126 j3-Sheet, glyceraldehyde-3-phosphate dehydrogenase, ll, 14 Soybean leaves, nitrite reductase of, 277-278 Spinach lipoamide dehydrogenase of, 112 transhydrogenase molecular properties, 59 purification, 54-55, 56 Spleen, glyceraldehyde-3-phosphate dehydrogenase in, 48 Starvation, cytosolic redox state and, 46 Steroid(s) metabolism, transhydrogenase ahd, 65, 83-84 Steroid 17,20-lyase, cytochrome P450 reductase and, 150 Streptococcus jnecalis, a-glycerophosphate dehydrogenase of, 260 Sturgeon muscle, glyceraldehyde-3-phosphate dehydrogenase of, 28, 33, 42 Subunits catalase, 366 cytochrome b?, 264-266 cytochrome c oxidase and, 311 glyceraldehyde-3-phosphate dehydrogenase, primary structure, 5 succinate dehydrogenase, 230-232 Succinate cholesterol side chain cleavage and, 85 succinate dehydrogenase preparation and, 223-224, 227,242-243, 248 Succinate dehydrogenase a-glycerophosphate dehydrogenase and, 258 mammalian, 222-223 enzymic properties, 236-245 inhibitors and modifiers, 245-247 mechanism, 251-254

455 molecular properties, 223-226, 296 regulatory properties, 247-251 microorganisms and, 254-256 Succinyl coenzyme A, succinate dehydrogenase and, 247 Sulfate nitrite reductase and, 275 reduction, pathways, 279,281 succinate dehydrogenase and, 247 Sulfhydryl groups, see also Thiol groups catalase, 368 glyceraldehyde-3-phosphate dehydrogenase and, 2 Sulfhydryl reagents, transhydrogenase and, 69-70 Sulfide acid-labile, ubiquinone reductase and, 179, 180, 182, 184, 186-187, 204, 209-210 adenylyl sulfate reductase and, 284285, 297 high molecular weight NADH dehydrogenases and, 90,201-202 low molecular weight NADH dehydrogenases and, 191, 193-194, 195,198, 206 mitochondria1 flavoprotein and, 297 succinate dehydrogenase and, 224, 226, 243 sulfite reductase and, 287, 288, 289, 290, 292, 293, 295 Sulfite adenylyl sulfate reductase and, 282, 284 lipoamide dehydrogenase and, 122 oxidases and, 90 Sulfite reductaseb) NADPH-dependent occurrence, 286,287-288 properties, 288-295 reduced methyl viologen-dependent, 295 types of, 286-287 Superoxide catalase and, 398 cytochrome P450 reductase and, 169 dehydrogenases and, 90 mixed function oxidations and, 153 oxygen reduction and, 303

456

SUBJECT INDEX

T Temperature glyceraldehyde-3-phosphate dehydrogenase and, 25 lipoamide dehydrogenase and, 116, 118, 123, 125 NADH dehydrogenases and, 203 succinate dehydrogenase and, 228-229, 231-232 Tetrathionate, glyceraldehyde-3-phosphate dehydrogenase and, 20, 21 2-Thenoyltrifluoroacetone, succinate dehydrogenase and, 246, 250, 255 Thermua aquaticus, glyceraldehyde-3phosphate dehydrogenase of, 2, 4,6, 20, 21, 22 Thiamine pyrophosphate, lipoamide dehydrogenase and, 126 Thiobacilli, adenylyl sulfate reductases in, 282 Thiobacillus denitrificans adenylyl sulfate reductase of, 283 sulfate reduction by, 281 Thiobacillus thiooxidans, sulfate reduction by, 281 Thiobacillus thioparus adenylyl sulfate reductase of, 283, 284 sulfate reduction by, 281 Thiocapsa roseopersicina, adenylyl sulfate reductase of, 282,283, 285-286 Thiocyanate catalase and, 387 small NADH dehydrogenases and, 192 succinate dehydrogenase and, 232 Thiol(s) adenylyl sulfate reductase and, 283 catalase and, 399-400, 401 cytochrome bs reductase and, 160-161, 162-164 lipoamide dehydrogenase, 118, 119, 120, 123 nitrite reductase and, 275, 277 Thiol groups, see also Sulfhydryl groups cytochrome c peroxidase and, 348 glutathione reductase, 141-142 NADH dehydrogenases, 203-204 succinate dehydrogenase, 245-246 ubiquinone reductase, 188, 203

Thionicotinamide adenine dmucleotide glyceraldehyde-3-phosphate dehydrogenase and, 30 transhydrogenase and, 57,59,60,6142, 69 Thioredoxin Escherichia coli, general properties, 144-145 nature of, 92, 93 Thioredoxin reductase amino acid composition, 102 cystine residues, 104 light-activated reduction-neutral semiquinone, 147-148 mechanism, 94, 98-99 metabolic functions, 142-144 reaction catalyzed, 92 reduced states, mechanism, 145-147 specificity of, 92-93 coenzymes and, 94 specificity of, 144 Threonine residues, lipoamide dehydrogenase, 105 Threose 2,4-diphosphate, glyceraldehyde3-phosphate dehydrogenase and, 44 Thyroid gland, a-glycerophosphate dehydrogenase and, 259 Tiron NADH dehydrogenase and, 206 succinate dehydrogenase and, 246 a-Tocopherylquinone, ubiquinone reductase and, 181-182 Torulopsis nitratophila, nitrite reductase of, 275, 276 Transacetylase dihydrolipoate and, 108 transhydrogenase and, 55 Transsuccinylase, dihydrolipoate and, 108 Tribromoacetate, small NADH dehydrogenases and, 192 Trichloroacetate small NADH dehydrogenases and, 192 succinate dehydrogenase and, 227,232, 234 Trideuteromethanol, catalase and, 404 Trifluoroacetate, small NADH dehydrogenases and, 192 2,2,2-Trifluoromethyl ethanol, catalase and, 401

457

SUBJECT INDEX

Triiodothyronine a-glycerophosphate dehydrogenase and, 259 transhydrogenase and, 70 Triton X-100 cytochrome c oxidase and, 313 o-lactate dehydrogenase and, 270-271 NADH dehydrogenase and, 187, 188189 succinate dehydrogenase and, 234 ubiquinone reductase and, 182-183 yeast NADH dehydrogenase and, 218 Trout, glyceraldehyde-3-phosphate dehydrogenase of, 26 Trypsin cytochrome b2 and, 266-267 cytochrome b5 reductase and, 164 cytochrome P-450 reductase and, 166, 167,169, 170-171, 172 transhydrogenases and, 58,65,71,212, 213, 296 yeast NADH dehydrogenase and, 217 Tryptophan residues cytochrome b, reductase, 164 cytochrome c peroxidase, 350,355 glyceraldehyde-3-phosphate dehydrogenase, 29 lipoamide dehydrogenase, 126 reductases and oxidases, 101 thioredoxin, 93 Turnover number, succinate dehydrogenase, 236-237 Tyrosine residues cytochrome b5 reductase, 163 cytochrome c peroxidase, 355 glyceraldehyde-3-phosphate dehydrogenase, 21 modification of, 22-23 reductases, 101

U Ubiquinone(s1 choline dehydrogenase and, 262 energy conservation and, 214 a-glycerophosphate dehydrogenase and, 258 large NADH dehydrogenase and, 201 small NADH dehydrogenases and, 194, 196, 206

solubility, ubiquinone reductase and, 180-181, 182 succinate dehydrogenase and, 224,225, 236,239,243,245,246,247,244-251 yeast NADH dehydrogenase and, 218 Ubiquinone reductase, see under Nicotinamide adenine dinucleotide Urea glyceraldehyde-3-phosphate dehydrogenase and, 21,24 L-lactate dehydrogenase and, 269 lipoamide dehydrogenase and, 122,124 nitrite reductase and, 279 small NADH dehydrogenase and, 203 succinate dehydrogenase and, 227 sulfite reductase and, 290,291 transhydrogenase and, 57

v Valence changes, cytochrome c oxidase, 331-332 Valine residues, disulfide oxidoreductases and, 104,105 Valinomycin, transhydrogenase and, 72 Venom, cytochrome b5 reductase and, 154 Vibro cholinicus, sulfate reduction by, 281 Vitamin K,, ubiquinone reductase and, 181, 182

W Water, catalase and, 374,385, 387

X Xanthine oxidase, cytochrome P-450 reductase and, 169 X-ray(s) glyceraldehyde-3-phosphate dehydrogenase apoenzyme structure, 19-20 holoenzyme structure, 9-19 Y

Yeast, see also Saccharomyces cytochrome c oxidase of, 311

458 cytochrome c peroxidase in, 347 glutathione reductase of, 94, 102, 138 diasociation constants, 135 kinetics, 140-141 substrates, 132 glyceraldehyde-3-phosphate dehydrogenase of, 6-8,18,26 amino acid modification, 22,23 half-site reactivity, 37 hybrids of, 26-27 pyridine nucleotide and, 28,31-33

SUBJECT INDEX

lipoamide dehydrogenase of, 112,117, 126 sulfate reduction in, 281 thioredoxin reductase of, 102

Z Zinc 2-hydroxyacid dehydrogenase and, 272-273 n-lactate dehydrogenase and, 270, 271-272

Topical Subject Index VOLUMES I-XI11 A

Acetamidyllysine residues, proteinase inhibitors, 111, 451452 Acetate: coenzyme A lipase catalytic properties cation requirements, X, 479-480 estimates of substrate affinity, X, 481 formation of enzyme-bound acetyl adenyla te, X, 478 selective modification of amino acid residues, X, 480-481 steady state kinetics and reation mechanism, X, 481-483 substrates and inhibitors, X, 477-478 molecular properties, X, 474-475 Acetoacetate decarboxylase historical background, VI, 255-256 inhibition studies borohydride, VI, 267-269 p-chloromercuriphenyl sulfonate, VI, 269-270

8-diketones, VI, 265-266 hydrogen cyanide, VI, 267 monovalent anions, VI, 266-267 2-oxopropane sulfonate, VI, 264 kinetic properties, VI, 263-264 mechanism, VI, 261-263 properties assay, VI, 256-257 latency, VI, 258-259 molecular weight, subunits and amino acid composition, VI, 260261

purification, VI, 257-258 stability, VI, 261

Acetylcholinesterase acceleration, V, 111-114 esteratic site, V, 95-97 historical background, V, 87-88 inhibitors anionic site, V, 98-100 esteratic site, V, 100-110 fluoride, V, 110-111 physical properties, V, 90-93 purification, V, 89 substrate binding, anionic site, V, 93-95 Acetyl coenzyme A-acyl carrier protein transacylase catalytic properties assays, VIII, 187 mechanism, VIII, 187-188 p H optimum and substrate specificity, VIII, 187 historical background, distribution and metabolic significance, VIII, 185186

molecular properties, VIII, 186-187 Acetyl coenzyme A carboxylase distribution, VI, 54-56 historical background and metabolic significance, VI, 53-54 molecular characteristics, VI, 58-59 reaction catalyzed, VI, 53 regulation of, VI, 79-82 substrate specificity, VI, 56-58 subunit structure and function active subunits in Escherichia coli, VI, 60-64 biotin carboxylase, VI, 70-71 biotin carboxyl carrier protein, VI, 64-70 459

460

TOPICAL SUBJECT INDEX

reconstitution and, VI, 72-78 structure of liver and wheat enzymes, VI, 78-79 transcarboxylase, VI, 71-72 N-Acetyl-n-glucosamine repimerase, properties, VI, 377-378 N-Acetyl-n-glucosamine 6-phosphate 2'epimerase, properties, VI, 377-378 N-Acetylglutamate-5-phosphotransferase allosteric inhibition kinetics, IX, 516-518 temperature effect, IX, 519-520 catalytic reaction assays, IX, 514-515 kinetics, IX, 515-516 pH optima and activating ions, IX, 514

stoichiometry, IX, 513-514 substrate specificity, IX, 514-515 historical background, IX, 511412 purification allosteric enzyme of Chlamydomonas, IX, 513 nonallosteric enzyme of Escherichia, I X , 513 N-Acetylneuraminate aldolase, properties, VII, 298-299 0-Acetylserine sulfhydrase, properties, VII, 54 Acidic nuclear protein kinases, properties, VIII, 580 Acid phosphatase(s) amebic, IV, 498 assay, IV, 457 problems, IV, 454 bone, IV, 496-497 distribution, IV, 450 Drosophila melanogaster, IV, 498 electrophoretic behavior, IV, 454455, 468-469

Escherichia coli, IV, 498 functional groups and group reagents iodination, IV, 469-471 sulfhydryl groups, IV, 469 tyrosine and tryptophan, IV, 471-472 Gaucher, IV, 496 general, IV, 455457 historical, IV, 450 kine tics fluoride inhibition, IV, 459-462

a-hydroxycarboxylic acid inhibition, IV, 462465 ion effects, IV, 466 pH and substrate effects, IV, 457-458 surface inactivation, IV, 459 liver bovine, IV, 491493 mouse, IV, 489-491 rat, IV, 484-489 Neurospora crassa, IV, 497 physical properties, IV, 476 plant, IV, 497 preparation, IV, 466-468 red cell general properties, IV, 477 purification and separation of genetic types, IV, 477-484 Saccharomyces, IV, 497 serum, IV, 495-496 specificity, alkaline phosphatase and, IV, 450-454 spleen, IV, 493495 staphylococcal, IV, 498 transphosphorylation, IV, 472473 use as reagent, IV, 473-476 Acid proteinase(s) pepsinlike chemical properties, 111, 728-730 distribution and isolation, 111, 724728

enzymic properties, 111, 734-740 physical properties, 111, 731-733 renninlike, 111, 740-741 distribution and isolation, 111, 741742

enzymic properties, 111, 743-744 physical and chemical properties, 111, 742-743 Aconitase catalytic properties assay, V, 423 equilibrium concentrations, V, 424 mechanism of action, V, 433-439 pH optima, V, 423-424 single vs. dual catalytic site, V, 432-433

specificity, V, 4-22 cofactors ferrous iron requirement, V, 422 role of reducing agent, V, 423


function, V, 413414 historical background, V, 414-415 inhibitors fluorocitrate, V, 4 W 3 0 iron-binding agents, V, 428 other carboxylic acids, V, 430-431 other inhibitors, V, 431432 intracellular distribution, V, 416-417 kinetics, latent period, V, 424425 Michaelis constants, V, 425426 relative reaction rates, V, 425 scheme, V, 426-428 mechanism of, 11, 302-304 molecular properties factors affecting stability, V, 419420 physiochemical properties, V, 418-419 purification, V, 417418 occurrence, V, 415416 reactions catalyzed, stereospecificity, 11, 164-168 role of metals, 11, 516-518 Aconitate, cis-trans isomerization, VI, 394-395 Active site(s) alkaline phosphatase, IV, 404-406 amino acid decarboxylases, VI, 245-248 8-aminolevulinate dehydratase, VII, 331433 aspartate transcarbamylase, IX, 262268 carbonic anhydrase, V, 617422, 643646 carboxylesterases, V, 61434 catalase, XIII, 3694388 chemical modification, I, 194-196 a-chymotrypsin, 111, 1!%-202 y-chymotrypsin, 111, 202-204 chymotrypsinogen, 111, 179-182 creatine kinase, VIII, 439442 deoxyribonuclease I, IV, 297-299 enolase components, V, 532-534 mapping with substrate analogs, V, 526-529 number, V, 530-532 fumarase, affinity labeling, V, 563-564 p-galactosidase, VII, 6574358 glucose-6-phosphate isomerase, VI, 285-287

461 glutamine synthetase, X, 720-733 j3-hydroxydecanoyl thioester dehydrase, V, 453455 invertase, V, 300301 lipase, VII, 593-595 papain, 111, 496499 activation, 111, 511414 chemical modification, 111, 51.5-516 half-cystine content, 111, 509-511 location of thiol group, 111, 514 staphylococcal nuclease, IV, 195-196 streptococcal proteinase, 111, 626-627 subtilisin, 111, 553-560, 575-584 thiolase, VII, 404-405 triosephosphate isomerase, VI, 330-333 trypsin, 111, 260-262 urease, IV, 20-21 Active-site-directed reagents as adjuncts to physical methods crystallography, I, 143 other spectroscopic methods, I, 145 spin labels, I, 143-145 characterization of functional groups and, I, 142 chymotrypsin and chloromethyl ketone from tosylphenylalanine, I, 94-96 other alkylating agents, I, 97-99 other neutral proteases, I, 102-103 other studies, I, 99-102 consecutive covalent modifications displacement, I, 115117 elimination, I, 115-116 intramolecular alkylation, I, 113-114 photolysis, I, 114-115 heme proteins and, I, 142 hydrolytic enzymes and, amidases, I, 130-131 deaminases, 1, 127-128 esterases, I, 124-127 glycosidases, I, 128-129 nucleases, I, 130 proteolytic enzymes, I, 118-124 lyases and carbonic anhydrase, I, 139-140 3-deoxy-o-arabino-heptulosonate-7phosphate synthase, I, 139 fumarase, I, 140-141 6-hydroxydecanoyl thioester dehydrase, I, 141


2-ke to-3-deoxy-6-phosphogluconic aldolase, I, 139 oxidoreductases and alcohol dehydrogenase, I, 136 glutamate dehydrogenase, I, 137 guanosine5’-phosphate reductase, I, 137 inosinic acid dehydrogenase, I, 137138 lactate dehydrogenase, I, 136 as selective enzyme inhibitors, I, 146 transferases and acyltransferases, I, 134-135 adenylyltransferase, I, 135 amidotransferase, I, 131-134 triosephosphate isomerase and, I, 138 trypsin and related enzymes, I, 103112 Acyl carrier protein distribution and intracellular Iocalization, VIII, 158-164 function in fatty acid biosynthesis, VIII, 164-165 molecular properties physical properties, VIII, 166 prosthetic group and primary sequence, VIII, 166-170 structure-activity relationships, VIII, 170-173 historical background, VIII, 155-158 prosthetic group synthesis and turnover, VIII, 173-176 Acyl coenzyme A ligases assays, VII, 411 general aspects of reaction, VII, 409410 reaction catalyzed, VII, 407-409 Acyl transfer carboxyl anions and, 11, 226-235 tertiary amino groups and, 11, 235-238 Acyltransferases, modification of, I, 134135 Adenine aminohydrolase, IV, 51-54 Adenine nucleotide aminohydrolase, IV, 75-76 Adenosine phosphodiester derivative biosynthesis adenyl cyclase, VIII, 26-27 adenylylation of glycoside antibiotics, VIII, 27-30

ribonucleic acid biosynthesis, VIII, 20-26 Adenosine aminohydrolase catalytic properties mechanism, IV, 5943 nature of active site, IV, 58-59 reaction parameters, IV, 5 6 5 7 molecular properties chemical and physical, IV, 55-56 purification, IV, 54-55 physiological function, IV, 63-64 Adenosine aminohydrolase (nonspecific), IV, 73-75 Adenosine diphosphate synthesis of derivatives adenine-myonic acid dinucleotide and adenylyl diphosphoglycerate, VIII, 33-35 adenosine diphosphate glucose synthesis, VIII, 3 2 3 3 general features, VIII, 3&32 Adenosine diphosphate sulfurylase, X, 663-665 Adenosine diphosphoryl glucose pyrophosphorylase activator, general effects, VIII, 77-78 Aeromonas formicam, VIII, 108-109 Chlorella pyrenoidosa, VIII, 90 classification, VIII, 75-77 enterobacteriaceae activator effects on kinetic parameters, VIII, 97-99 activator-inhibitor interaction, VIII, 99-100 energy charge and, VIII, 104-107 inhibitor effect on kinetic constants, VIII, 101-102 manganese effects, VIII, 102-104 Entner-Duodoroff pathway and, VIII, 78-81 Escherichia coli, VIII, 102-107, 109-110 mutantb) SG5 and CL1138, VIII, 110-114 mutantb) SG14, VIII, 115-117 general background, VIII, 73-75 nonchlorophyllous plant t i m e , VIII, 93-94 other leaves, VIII, 89-90

463


3-phosphoglycerate activation and phosphate inhibition of, VIII, 9192

physical properties, VIII, 117-119 R hodospirillum rubrum, VIII, 81-83 reaction mechanism, VIII, 86 temperature and, VIII, 83-86 Serratia marcescens, VIII, 107-108 spinach leaf, VIII, -9 Adenosine kinase assay, IX, 51-52 distribution and purification, IX, 51 kinetic and molecular properties, IX, 52-53

substrate specificity, IX, 53-54 Adenosine monophosphate, fructose-l,& diphosphatase and, IV, 618-620,627628, 636-637

Adenosine triphosphatase(s) Alcaligenes jaecalis membrane catalytic properties, X, 429 molecular properties, X, 428 solubilization and purification, X, 428

Bacillus megaterium membrane catalytic properties, X, 427-428 purification, X, 426 reassembly, X, 428 release from membranes, X, 426 size, amino acid composition and morphology, X, 427 subunits, X, 427 Bacillus stearothermophilus membrane properties, X, 425-426 solubilization and purification, X, 425

bacterial membrane, X, 396-400 other bacteria, X, 416 Streptococcus faecalis, X, 400, 416429

chloroplast Euglena gracilis, X, 394 spinach, X, 389-394 Escherichia coli membrane deficient mutants, X, 419421 molecular weight and catalytic properties, X, 418 release from membranes, X, 416-418 subunit composition, X, 418-419 function of, X, 375-377

Micrococcus lysodeikticua membrane electron microscopy, X, 422 FMC and, X, 424425 interaction with antibodies, X, 423 localization, X, 422423 release from, X, 421-422 size and catalytic properties, X, 422 subunits, X, 423-424 mitochondria1 assay, X, 377 beef heart, X, 377-386 rat liver, X, 387-389 yeast, X, 386-387 Rhodopseudoomonas spheroides membrane, X, 429 sarcoplasmic membrane calcium-dependent, X, 445-450 calcium-independent, X, 444-445 historical background, X, 432-434 Streptococcus jaecalis membrane active transport and, X, 414-416 amino acid composition, X, 405-406 carbodiimide-resistant mutants, X, 413-414

dicyclohexylcarbodiimide inhibition, X, 411-413 electron microscopy, X, 405 isolation of membranes, X, 400 kinetics, X, 408-409 molecular weight, X, 404-405 nectin and, X, 410411 purification, X, 402404 reassembly, X, 409-410 release from membranes, X, 400-402 subunits, X, 406408 Adenosine triphosphate calcium-efflux dependent net formation, X, 457458 phosphate exchange, X, 458-459 metal complexes, electronic structure, 11, 479-481 Adenosine triphosphate citrate lyase, VII, 368-369 assay and isolation, VII, 369-370 catalytic properties control, VII, 372 equilibrium and kinetics, VII, 371372

specificity, VII, 371 stereospecificity, VII, 372-373

464


molecular properties cofactors, VII, 370 inhibitors, VII, 370-371 molecular weight, VII, 370 stability, VII, 371 sulfhydryl groups, VII, 370 reaction mechanism adenosine diphosphate-adenosine triphosphate exchange, VII, 373 citryl coenzyme A aa intermediary, VII, 376 citryl-enzyme as intermediary, VII, 375-376

citryl phosphate as intermediary, VII, 374-375 oxygen transfer to orthophosphate, . VII, 373 phosphoryl-enzyme as intermediary, VII, 373-374 reaction scheme, VII, 377 Adenosine triphosphate sulfurylase general properties, X, 656-658 mechanism, X, 658-662 occurrence and purification, X, 652654

reactions and assay, X, 655-656 Adenosyltransferase(s) , general background, VIII, 121-123, 152-154 Adenylate energy charge, metabolic regulation and, I, 470-476 Adenylate kinase biological aspects distribution, VIII, 279-282 function, VIII, 285-288 genetics and disease, VIII, 282-284 catalytic properties assay, VIII, 300-301 equilibrium constants, VIII, 302 mechanism, VIII, 302-305 metal requirement, VIII, 297-298 nucleotide specificity, VIII, 298-300 molecular properties composition, VIII, 291-293 physical properties, VIII, 295-297 preparation and purity, VIII, 288291

reactive groups, VIII, 293-296 role of metals in mechanism, 11,502 Adenyl cyclase, properties, VIII, 26-27

5’-Adenylic acid aminohydrolase catalytic properties kinetics, IV, 66-70 mechanism, IV, 70-71 physiological function, IV, 71-73 specificity, IV, 66 molecular characteristics chemical and physical properties, IV, 65-66

purification and homogeneity, IV, 6465

Adenylosuccinase 5-amino-4-imidazole-N-succinocarboxamide ribonucleotide and, VII, 182-183

mechanism of action, VII, 196-197 Neurospora, VII, 191-192 assay procedures, VII, 192 catalytic properties, VII, 192 purification, VII, 192 subunit structure, VII, 192-193 sterospecificity of additional or elimiination, VII, 195 yeast, VII, 185-186 assay procedure, VII, 187 function and distribution, VII, 186187

pH optima and equilibrium, VII, 191 purification, VII, 187 substrate affinity and product inhibition, VII, 189-190 substrate specificity, VII, 187-188 sulfhydryl reagents and, VII, 188189

Adenylylsulfate kinase, X, 662-663 Adenylyl sulfate reductase(s), properties, XIII, 279-286 Adenylyltransferase, modification of, I, 135

Adenylyl transfer reactions, general background, VIII, 1-6 Adipose tissue, hormone sensitive lipase, VII, 609-610 Adrenal gland, glycogen synthetase of, IX, 353 Aerobacter aerogenes, pullulanase of, V, 195-201

Aerobacter cloacae, phage polysaccharide depolymerase, V, 398

465


Aeromonas formicans, adenosine diphosphoryl glucose pyrophosphorylase of, VIII, 108-109 Affinity labeling, chemical modification in general and, 91-94 Agaricaceae, y-glutamyltransferase of, IV, 95-96 Alanine dehydrogenase, regulation of, I, 443444 Alanine racemase, spore germination and, VI, 506 Alcaligenes fueculis, membrane adenosine triphosphatase of, X, 428-429 Alcohol dehydrogenase(s1 chemical modifications, XI, 141-145 arginine residues, XI, 179 cobalt enzyme, XI, 180 cysteine residues, XI, 176177 denaturation, XI, 180-181 histidine residues, XI, 177-179 manganese enzyme, XI, 180 properties, XI, 179-180 uncharacterized, XI, 179 coenzyme analogs and, XI, 150-152 comparisons evolutionary aspects, XI, 140-141 structural and functional aspects, XI, 136-140 denaturation studies, XI, 147-148 fluorescence and phosphorescence, XI, 148-150

functional aspects, activity in ethanol metabolism, XI, 106

physiological substrates, XI, 105-106 gene duplication and, I, 309-311 horse liver multiple molecular forms, XI, 107108

primary structure, XI, 113-116 human liver, multiple molecular forms, XI, 109110

primary structure, XI, 116 inhibitor binding, binary complexes, XI, 152-157 ternary complex, XI, 158-160

inhibitor studies coenzyme competitive inhibitors, XI, 181-182 others, XI, 182-183 kinetic aspects coenzyme binding, XI, 160-163, 183184

dismutase reaction, XI, 166 half-site reactivity, XI, 166-167 ordered mechanism, XI, 165-166 reaction mechanism, XI, 185-186 substrate binding, XI, 163-165 substrate specificity; XI, 184-185 liver, XI, 5 6 5 7 kinetic studies, XI, 20-22 mechanism for catalysis, XI, 168-171 metal content changes, XI, 145-147 modification of, I, 136 other sources, XI, 186-187 bacterial, XI, 187-188 insect, XI, 189-190 plant, XI, 188-189 rat liver, multiple molecular forms, XI, 111112

primary structure, XI, 114-117 tertiary structure crystallization and preliminary Xray studies, XI, 117-118 electron density maps, XI, 118-119 three-dimensional structure, XI, 1% 136

yeast chemical modifications, XI, 176181 inhibitor studies, XI, 181-183 kinetic aspects, XI, 22-23, 183-186 primary structure, XI, 173-176 purification and molecular properties, XI, 171-173 Aldehyde oxidase, metal complexes and, 11, 533-534 Aldolase(s) historical review, VII, 213-215 properties in bacteria and fungi, VII, 215-216

Schiff bases and, 11, 359-380 yeast, role of metals, 11, 515-516 Aldolase(s) (Metallo) distribution and general properties catalytic properties, VII, 253-254

466


multiple forms in microorganisms,

VII, 255-256 purification and metal content, VII, 254-255

reaction mechanism other functional groups, VII, 257258

possible molecular homology in yeast and muscle enzymes, VII, 258

role of metal ion, VII, 256-257 Aldolase(s) (Schiff base-forming) developmental aspects embryonic tissues and, VII, 249-251 modification of structure, VII, 252253

tumors and, VII, 251-252 mechanism of catalysis isotope exchange reactions, VII, 216-217

Shiff base formation, VII, 217-219 organ-specific molecular properties and subunit structure, VII, 221-223 nonidentical subunits and microheterogeneity, VII, 223-224 occurrence of isozymes in vertebrate tissues, VII, 220-221 specificity, VII, 219-220 phylogenetic studies comparison of active site peptides,

VII, 248-249 isolation from other species, VII, 24k248

rabbit liver and brain isolation and general properties, VII, 241-243

primary structure of active site, VII, 243-244

rabbit muscle active site sequence, VII, 236 amino acid composition, VII, 224 functional groups, VII, 224-232 isolation of crystalline enzyme, VII, 224

primary structure, VII, 236-239 reaction mechanism, VII, 232-236 X-ray crystallography and electron microscopy, VII, 239-241

Aldose-ketose isomerases general considerations, VI, 271-272 nonphosphorylated sugars and, VI, 340-354

Algae blue green, fructose-l,6diphosphatases of, IV, 640-642 Alkaline phosphatase active sites, number of, IV, 404-406 chelating agents and, IV,426-427 chemical modification, IV, 391-392 arginine, IV, 390 histidine, IV, 390 leucine, IV, 390 methionine and cystine, IV, 390391 phenylalanine, IV, 389-390 tryptophan, IV, 390 competitive inhibitors, IV, 394-396 composition, IV, 423-425 analysis, IV, 378-380 sequence work, IV, 380 crystal structure, IV, 389 distribution, IV, 374-376 histochemical and gel localization, IV, 433-434

historical background, IV, 373374 in vitro assay, IV, 432-433 isozymes, IV, 384387 kinetic studies, IV, 409-416 factors affecting, IV, 434-436 inhibition and, IV, 442443 Kim and 'Vim.,,IV, 436-439 metal ions and, IV, 440-441 phosphoryl enzyme formation and,

IV, 439 transferase activity, IV, 439-440 mammalian assay techniques, IV, 432-434 chemical modification, IV, 427-428 distribution, IV, 420-421 function, IV, 421-422 general survey, IV, 417-420 kinetic studies, IV, 434-443 mechanism, IV; 443-447 physical properties, IV, 423-427 purification procedures, IV, 422-423 reaction catalyzed, IV, 430-432 substrate specificity, IV, 428-430 mutations and, I, 251-254 phosphoryl enzyme, IV, 396-401


physical properties, IV, 387-388 purification, IV, 377-378 specificity, IV, 392-394 stability, IV, 425426 subunits, IV, 380-384 transphosphorylation and, IV, 406-409 zinc and, IV, 401404 Alkaline proteinase(s) diisopropylfluorophosphate-sensitive, 111, 744-745 chemical properties, 111, 749-754 distribution and isolation, 111, 745749 enzymic properties, 111, 758-763 keratinase, 111, 763-765 physical properties, 111, 754-758 8-Alkyl-L-cysteine sulfoxide lyase, properties, VII, 52 Alkylsulfatase(s), V, 15 Allylases, proton shifts and, 11, 299302 D-Altronate dehydrase, properties, V, 579 Ameba, acid phosphatase of, IV, 498 Amidases, modification of, I, 130-131 Amidinotransferase(s), reactions catalyzed, IX, 497-498 Amidotransferases, modification of, I, 131-134 Amine oxidase (s) , active site, substrate interaction, XII, 524-525 catalytic mechanism, XII, 525-526 definition and classification, XII, 511513 inhibitor reactions, XII, 523-524 metabolic function, XII, 513 microsomal, properties of, XII, 228. 230 other copper-containing, XII, 526-527 prosthetic groups, XII, 519-523 purification, molecular weight and substrate specificity, XII, 513-518 spectral properties, XII, 518-519 Aminoacetone, formation of, VII, 355 Amino acid(s) activation, VIII, 6-11 acyl carrier protein sequence, VIII, 166-170 amylase composition, microbial, V, 239-244 analogs, mutations and, I, 262265

aromatic aspartate transcarbamylase, IX, 267-268 biosynthesis, I, 228-237 asparaginase composition, IV, 111-113 aspartokinase(s) composition, VIII, 520, 542, 543 carboxylesterase composition, V, 52-53 creatine kinase composition, VIII, 390-392 degradation, VI, 504-506 elastase sequence, 111, 341-343 fumarase and, V, 544-545 a-glucan phosphorylase composition, VII, 446-447 glucose-6-phosphate isomerase composition, VI, 27S279 guanidino kinase composition, VIII, 469-470 function, VIII, 477-482 hexokinase composition, IX, 41 inorganic pyrophosphatase and, IV, 512514 microbial proteinases and, 111, 749751, 770-772 pancreatic ribonuclease sequence, IV, 653-654 papain composition and sequence, IV, 507-509 pepsin composition, 111, 128-130 sequence, 111, 1W133 phospholipase A, content, V, 80-81 sequence, V, 81-82 prothrombin composition, 111, 313 pyridoxal reactions with, 11,339445 sequences, carboxypeptidase B, 111, 64-66 streptococcal proteinase active site, 111, 626-627 composition, 111, 624-625 N- and C-terminal, 11, 625-626 thrombin composition, 111, 285-286 sequences, 111, 287-290 Amino acid decarboxylases active site absorption spectra of pyridoxal phosphatedependent, VI, 245-247

468


pyridoxal phosphate binding site, VI, 247-248

distribution and general properties, VI, 224-237

general considerations, VI, 217-219 mechanism of action glycine decarboxylase, VI, 240-241 pyridoxal phosphate a-decarboxylation, VI, 237-240 pyridoxal phosphate p-decarboxylation, VI, 241-243 pyruvate-containing, VI, 244 metabolic importance bacterial, VI, 219-221 mammalian and plant, VI, 221-224 subunit structure, VI, 248-253 n-Amino acid oxidase, molecular properties and kinetic mechanism, XII, 445-456

L-Amino acid oxidase, molecular properties and kinetic mechanism, XII, 456-461

Amino acid racemases assay methods coupling to L- or n-specific enzymes, VI, 489 polarimetric, VI, 489-490 cofactors flavin, VI, 496 metal ions, VI, 496-497 no pyridoxal phosphate, VI, 495 pyridoxal phosphate, VI, 494-495 status uncertain, VI, 495-496 equilibrium position, VI, 490-491 history and survey, VI, 481488 kinetic features, VI, 491494 mechanism of action aminoacyl complex and, VI, 501-502 nonpyridoxal enzymes, VI, 498-500 pyridoxal enzymes, VI, 497-498 physiological aspects n-amino acids in animal tissues, VI, 508-507

biosynthesis and degradation of free amino acids, VI, 504-506 cell wall biosynthesis, VI, 502-503 peptide antibiotic biosynthesis, VI, 503-504

spore germination, VI, 506 substrate specificity, VI, 490

Aminoacyl transfer ribonucleic acid, enzymic deacylation, X, 509-510 Aminoacyltransfer ribonucleic acid synthetases amino acid activation, X, 505-506 assay, X, 506 binding parameters, X , 507-508 reaction product, X, 506 substrate specificity, X, 506607 amino acid biosynthesis and, X, 536 chemical properties affinity labeling, X, 505 amino acid composition, X, 503 chemical modification, X, 505 proteolytic modification, X, 504 terminal amino acids and sequence analysis, X, 503-504 general considerations, X, 489492 genetics of, X, 529-534 mechanism of reaction general, X, 510-511, 517-515 isoleucyltransfer ribonucleic acid synthetase, X, 511-517 occurrence and distribution, X, 492-491 purification, X, 494-496 Crystallization, X, 496-497 regulation of biosynthesis, X, 534-535 size and subunit composition, X, 502503

multichain with dissimilar subunits, X, 502 multichain with similar subunits, X, 499-502

single chain, X, 497499 Amino group(s) aspartate transcarbamylase, IX, 265267

chemical modification, I, 175 elastase, 111, 386-373 ribonuclease, IV, 677-682 tertiary, acyltransfer to, 11, 235-238 trypsin, 111, 269-270 w-Amino group migrations, VI, 547-548 reaction mechanisms cobamide coenzyme as hydrogen carrier, VI, 560-561 general considerations, VI, 559-560 hydrogen transfer reaction, VI, 561 role in bacterial fermentations, VI, 562-563


Amino group transfer basic chemical features congruent nonenzymic models, IX, 387-391

general characteristics of intermediate steps, IX, 391-392 formally similar processes, IX, 384387

historical background, IX, 379-381 other substrates and, IX, 462463 w-amino and 0-0x0 acids, IX, 473474

glutamate-oxoglutarate or aspartateoxalacetate, IX, 463473 noncarboxylic acids, IX, 475-476 as side reactions, IX, 47-80 two a-amino-a-oxomonocarboxylic acids, IX, 473 recent developments, IX, 381-384 5-Amino-4-imidazole carboxamide ribonucleotide transformylase, properties, IX, 204-205 5-Amino-4-imidarole-N-succinocarboxamide ribonucleotide cleaving activity, VII, 183-184 assay, VII, 184-185 enzymic properties, VII, 185 function and distribution, VII, 184 purification, VII, 185 8-Aminolevulinate dehydratase active site, nature of, VII, 331-333 catalytic properties assay, VII, 330 kinetics, VII, 330-331 pH optima, VII, 331 cation requirements, VII, 328-330 mechanism of porphobilinogen synthesis, VII, 333-337 molecular weight, VII, 324326 aggregation and, VII, 326-327 reaction catalyzed, VII, 323-324 Schiff base and, 11, 361-362 subunits, quaternary structure and, VII, 327-328 6-Aminolevulinate synthetase analogous reactions, VII, 355356 cofactors, substrate specificity and kinetic constants, VII, 348-349 historical background, VII, 339-340

inhibitors amino acids and derivatives, VII, 349-350

carboxylic acids, VII, 351 sulfhydryl reagents, metals and porphyrins, VII, 350-351 mechanism, VII, 351-355 metabolic significance, VII, 343-344 molecular properties assay, VII, 347 high and low activity forms, VII, 344-315

isolation, VII, 346-347 stability, VII, 344 occurrence, VII, 341-342 Aminopeptidase A, 111, 111-112 Aminopeptidase B, 111, 112-113 Aminopeptidase M, 111, 102-105 Aminopeptidase P, 111, 115-116 Aminotripeptidase, 111, 117-118 Ammonia elimination enzymic general considerations, VII, 79-88 nomenclature, VII, 77-79 free energy, VII, 88-90 interpretation, VII, 92-94 ionic species, VII, 90-91 standard states, VII, 91-92 models for steric course, carbanion intermediate, VII, 114-116 concerted elimination, VII, 110-114 stereochernistry, VII, 94-95 configurations of amino acid chiral centers, VII, 95-98 configurations and conformations of olefinic products, VII, 109-110 specificity toward amino acid prochiral centers, VII, 99-109 Amphibolic pathways input signals and, I, 476-479 interaction with biosynthetic pathways, I, 481482 Amylase (8) action pattern effects of chain length, V, 173-182 hydrolysis and condensation specificity, V, 149-152 multiple attack, V, 165-173 single chain or multichain attack, V. 161-165

470


subsite model, V, 154-161 substrate structural requirements, V, 152-154

amino acid content, V, 127 assay methods, V, 117-120 biosynthesis, genetics and control, V, 182-189

chemical modification, V, 129-132 classification, V, 115-117 mechanism of action, 140-149 microbial amino acid composition, V, 239-243 assay, V, 265-266 calcium and, V, 247-250 carbohydrate components, V, 245-257 chemical modification, V, 261-263 denaturation and renaturation, V, 251-256

fragmentation, V, 257-258 mechanism, V, 268-271 mode of action, V, 266% molecular weights, V, 250-251 purification, V, 236-239 specificity, V, 263-265 sulfhydryl and disulfide groups, V, 244-245

terminal groups, V, 241-244 thermostable and acid stable, V, 258-261

origin, purification and molecular variants, V, 121-127 pH, temperature and salt effects, V, 132-140

size and shape, V, 128-129 subunits and multiple binding sites,

V, 127-128 Androstenolone sulfatase, V, 7-9 Anhydrochymotrypsin, elimination reactions and, I, 115-116 Animals, nuclear ribonucleic acid polymerase, X, 262-300 Anions, carbonic anhydrase and, V, 646-652, 658-660

Antibiotics amino glycoside, adenylylation, VIII, 27-30

Antithrombins, thrombin and, 111,305306

n-Arabinose isomerase, properties, VI, 346348

L-Arabinose isomerase, properties, VI, 348-349

Arabinose-bphosphate isomerase, properties, VI, 324-325 n-Arabonate dehydrases, properties, V, 582

L-Arabonate-n-fuconate dehydratases, properties, V, 581-582 Arene oxides, epoxidases and, VII, 211212

Arginine kinase, see also Guanidino kinases role of metals in mechanism, 11, 501502

Arginine monooxygenase, properties,

XII, 203-204 Arginine residues chemical modification, I, 174 chymotrypsinogen, 111, 176-179 ribonuclease, IV, 689490 Argininosuccinase (8) assay procedures, VII, 171 bovine kidney catalytic properties, VII, 179 purification, VII, 178-179 catalytic properties inhibitors, VII, 172 pH optimum and equilibrium, VII, 173

substrate specificity, VII, 172 function and distribution, VII, 170-171 historical background, VII, 169-170 mechanism of action, VII, 196 molecular structure subunit constitution, VII, 176-177 sulfhydryl groups and, VII, 177-178 Neurospora, catalytic and physical properties, VII, 180-181 number of binding sites, VII, 174-175 pea seeds, catalytic and physical properties, VII, 181-182 primary structure amino acid composition, VII, 179 antigenic properties, VII, 179 purification, VII, 171-172 regulation cooperative substrate effects, VII, 173-174

nucleotide stimulation, VII, 174

471


reversible cold inactivation and subunit dissociation, control of, VII, 175 kinetics, VII, 175-176 thermodynamic constants, VII, 176 stereospecificity of addition or elimination, VII, 194 Argininosuccinate, synthesis, VIII, 38-39 Arthropoda, glycogen synthetase of, IX, 358-359

Arylsulfatase(s), V, 23 type I, V, 3 4 , 23-26 type 11, V, 4, 26-39 Asparaginase (s) amylase content, V, 127 Escherichia coli isolation, IV, 107-108 properties, IV, 109-116 guinea pig serum isolation, IV, 105-106 properties, IV, 106-107 occurrence, IV, 102-105 other, IV, 116-117 physiological properties, IV, 117-121 properties amino acid composition, IV, 111-113 general, IV, 109-110 structure, IV, 113-116 substrate specificity and inhibitor effects, IV, 110-111 Asparagine synthetase, X, 578-580 bacterial sources, X, 568-572 glutamine-dependent, X, 572578 historical background, X, 561-568 Aspartate metabolism, regulation of, I, 457459 pyruvate carboxylase and, VI, 31-33 Aspartate ammonia-lyase catalytic process, VII, 135-137 distribution, purification and kinetic properties, VII, 116-118 size and constitution, VII, 119-121 Aspartate :oxoglutarate aminotransferase(s1 isoenzymes and multiple subforms, IX, 393-398 pig heart and animal tissues coenzyme analogs and, IX, 429435 dynamic spatial aspects, IX, 465-462 kinetics, IX, 424-429

optical properties, IX, 407416 physical parameters and macromolecular structure, IX, 398-406 primary structure and functionally important groups, IX, 416424 stereochemistry and active site topography, IX, 451455 substrates, quasi substrates and inhibitors, IX, 435-451 Aspartate residues, chymotrypsin, 111, 235-236, 243

Aspartate transcarbamylase active site functional groups, IX, 262-263

amino groups, IX, 265-267 aromatic amino acids, IX, 267-268 histidine residues, IX, 267 sulfhydryl groups, IX, 263-265 allosteric effectors bindine site, IX, 270-273 properties in presence of effectors, IX, 269-270 bacterial, IX, 297-302 biosynthesis and genetics control of, IX, 295-297 location of genes, IX, 292293 two chains-single operon?, IX, 293-294

catalytic subunit primary structure, IX, 232-234 size and substructure, IX, 231-232 comparison of native enzyme with subunits, IX, 277-278 cooperative properties of modified enzyme modification with partial specificity, IX, 279-280 other modified enzymes, IX, 284-285 specifically modified, IX, 280-284 urea and p H effects, IX, 278 cooperative substrate binding, IX, 268-269

detailed subunit structure, IX, 239-243 fungal, IX, 302-306 induced conformational changes allosteric effectors, IX, 276-277 substrates and substrate analogs, IX, 275-276 isolation and characterization away procedures, IX, 228-230


properties associated with regulation, IX, 227-228 purification, IX, 238 size and subunit composition, IX, 230-231

kinetics of ligand binding, I X , 285-287 mammalian, IX, 306-307 mechanism, IX, 243-282 mechanisms for cooperativity structural models, IX, 290-292 two-state major transition, IX, 287-290

plant, IX, 307-308 reconstitution importance of metals, IX, 237-238 metals other than zinc, IX, 238-239 methods, I X , 237 regulatory subunit metal binding site, IX, 236 primary structure, IX, 235 as regulatory protein, IX, 236-237 size and substructure, IX, 234-235 stoichiometry of ligand binding nucleotides, IX, 274-275 substrates and substrate analogs, IX, 273-274 Aspartokinase (s) assay of, VIII, 512-513 Escherichia coli, VIII, 513-544 historical background, VIII, 509-51 1 lysine-sensitive, adenylylation, VIII, 44-45

other coliform bacteria, VIII, 544 reaction catalyzed, VIII, 511-512 regulated by concerted feedback inhibition Bacillus polymyxa, VIII, 546-548 Bacillus stearothermophilus, VIII, 550

Bacillus subtilis, VIII, 548-550 other bacilli, YIII, 551 other genera, VIII, 552 other nonsulfur photosynthetic bacteria, VIII, 545446 pseudomonads, VIII, 551452 R hodopseudomonas capsulatus, VIII,

Aspartokinase I, VIII, 515-516 chemical properties, VIII, 520-522 conformational changes, VIII, 526-536 distribution of two activities on, VIII, 536-540

extinction coefficient, VIII, 517 kinetic parameters, VIII, 519-520 ligand binding, VIII, 523-525 molecular weight, VIII, 517-518 purification and criteria of homogeneity, VIII, 516 stability, VIII, 517 sulfhydryl titration effects, VIII, 525-526

tetrameric structure, VIII, 518-519 Aspartokinase I1 amino acid composition, VIII, 541-542 extinction coefficient, VIII, 541 kinetic parameters, VIII, 541 molecular weight, VIII, 541 purification and criteria of homogeneity, VIII, 540 stability, VIII, 540 subunit structure, VIII, 541 Aspartokinase I11 amino acid composition, VIII, $543 extinction coefficient, VIII, 542 inhibition, VIII, 544 kinetic parameters, VIII, 543 molecular weight, VIII, 543 purification and criteria of homogeneity, VIII, 542 Aspergillus, proteinases of, 111, 747, 769 Aspergillus nidulans molybdenum hydroxylases of, XII, 412414

Asymmetry molecular, notations of, 11, 129-134 Azotobacter phage polysaccharide depolymerase assay and purification, V, 397 properties, V, 397-398 Azotobacter agilis, glutaminase of, IV, 97-98

B

544-545

Rhodopseudomonas spheroides, VIII, 552-553

Saccharomyces cerevkiae, VIII, 553

Bacillus alkaline proteinases of, 111, 605-606 proteinases of, 111, 767-768


Bacillus cereus, phospholipase C of, V, 83-84 Bacillus megalerium membrane adenosine triphosphatase, X, 426-428 phage G-induced lytic enzyme bound, V, 409-410 soluble, V, 408-409 Bacillus polymyxa, aspartokinase of, VIII, 546-548 Bacillus stearothermophilus aspartokinasc of, VIII, 550 membrane adenosine triphosphatase of, X, 425-426 phage lytic enzyme, V, 410411 Bacillus subtilis aspartokinase of, VIII, 548-550 deoxyribonucleic acid polymerases, physiological role, X, 143-144 extracellular ribonuclease of, IV, 239-240 intracellular ribonuclease of, IV, 240 phage-induced exonuclease, IV, 258-259 Bacteria, see also specific organisms alcohol dehydrogenase of, XI, 187-188 aldolases of, VII, 215-216 amino acid decarboxylases of, VI, 219-221 amylases catalytic properties, V, 263-271 molecular properties, V, 236-263 aspartate transcarbamylases of, IX, 297-302 deoxyribonucleic acid methyltransferases, IX, 190-192 deoxyribonucleic acid polymerases of, X, 119-144 elongation factors, X, 55-67 endonuclease of, IV, 259-270 exonucleases of, IV, 252-259 ferredoxins of, XII, 37-46 fructose-1,6-diphosphatasesof, IV, 639-640 glutamate dehydrogenases bacilli, XI, 332 Escherichia coli, XI, 332-333 others, XI, 333-334 hyaluronidases of, V, 313-314 neuraminidases of, V, 324-325

5‘-nucleotidase of, IV, 338-340 photosynthesis in, XI, 509-516 pro teases acid, 111, 723-744 diisopropylfluorophosphate sensitive, 111, 744-765 metal-chelator sensitive, 111, 765-786 other, 111, 786-795 respiratory chains denitrifiers, XI, 521426 inorganic reductants, XI, 519-521 less-mitochondrion-like, XI, 517-519 mitochondrion-like,’XI, 516-517 sulfate respiration and, XI, 526-534 ribonucleic acid polymerases background, X, 333-335 catalytic properties, X, 344-374 molecular properties, X, 335-344 Bacterial cell wall lytic protease Myxobacter, 111, 786-788 other, 111, 789-790 Sorangium, 111, 788-789 Bacteriophage A endolysin catalytic properties, V, 391-392 chemical properties, V, 388-391 physiochemical properties, V, 387-388 purification, V, 385-386 induced exonuclease and, IV, 253-254 Bacteriophage F series polysaccharide depolymerase catalytic properties and biological significance, V, 393 enzyme assay, V, 392 partial purification, V, 392-393 stability, V, 393 Bacteriophage G lytic enzyme bound, V, 409-410 soluble, V, 408-409 Bacteriophage N20F’ lytic enzyme catalytic properties, V, 384-385 chemical properties, V, 383-384 physiochemical properties, V, 382-383 purification, V, 382 Bacteriophage P1,lytic enzyme, V, 399-400

474 Bacteriophage P14, lytic enzyme V, 399400 Bacteriophage PAL,lytic enzyme, V, 4W-401 Bacteriophage SP-3, induced exonuclease, IV, 258-259 Bacteriophage T2 induced exonuclease, IV, 255 lysozyme catalytic properties, V, 381382 chemical properties, V, 380-381 physicochemical properties, V, 380 purification, V, 379 Bacteriophage T 4 induced endonucleases I1 and IV, IV, 266-269 induced exonuclease, IX, 255 lysozyme catalytic properties, V, 369-374 chemical properties, V, 366-369 enzyme assays, V, 361-364 physicochemical properties, V, 366 purification, V, 364-366 role in life cycle, V, 376379 Bacteriophage T5, induced deoxyribonuclease, IV, 261 Bacteriophage T7, induced endonuclease, IV, 266-2436 Bacteriophage TP-I, lytic enzyme of, V, 410-411 Biosynthetic pathways input signals and, I, 474-481 interaction with amphibolic pathways, I, 481482 Biotin acetyl coenzyme A carboxylase and, VI, 64-71 activation of, VIII, 18 pyruvate carboxylase and, VI, 3-5 Blood coagulation, proteolysis and, I, 416-417 Blood cells, glycogen synthetase of, IX, 354-355 Blood platelets, thrombin and, 111, 300-301 Bone, acid phosphatase, IV, 4-97 Bovine pancreatic ribonuclease, see Ribonuclease Brain aldolase, VII, 241-244


creatine kinase of, VIII, 401-403 glycogen synthetase of, IX, 353-354 Bromelain, 111, 542545 y-Butyrobetaine hydroxylase catalytic properties, XII, 168-169 purification, XII, 167

C Calcium amylases and, V, 247-250 binding, staphylococcal nuclease and, IV, 163-171 hydrolases and, 11, 524-525 Calcium translocation and phosphoryl transfer backward reaction adenosine, phosphate and calcium binding, X, 462463 phosphoprotein formation, X, 463-465 forward reaction adenosine triphosphate-adenosine diphosphate exchange, X, 462 adenosine triphosphate and calcium binding, X, 459-460 phosphoprotein formation and, X , 460-462 Candida utilia fructose-1,6-diphosphatase inhibition by AMP, IV, 636-637 purification and properties, IV, 635-636 regulation, IV, 640 relation to SDPase, IV, 638 structure, IV, 637-638 Carbamate kinase assays, IX, 107-108 forward reaction, IX, 108-109 reverse reaction, IX, 109-110 distribution, IX, 101-102 function and metabolite control, IX, 115-119 historical background, IX, 97-100 molecular properties composition, size and subunit st’ructure, IX, 103-104 purification, IX, 102103 stability, IX, 104-105 sulfhydryl reagent effects, IX, 105


reaction catalyzed, IX, 105-107 specificity and cofactors, IX, 107 thermodynamics, kinetics and mechanism, IX, 110-115 Carbohydrate amylase composition, V, 245-247 prothrombin composition, 111, 313 thrombin composition, 111, 286-286 Carbon-hydrogen fission electron delocalization and, 11, 287-290 other catalyses, 11, 318-320 Carbonic anhydrase active-site-directed chemical modifications anionic reagents, V, 658-660 sulfonamides, V, 661 assay methods, V, 630-632 catalytic mechanism, V, 661-662 catalytic reaction, V, 662885 substrate binding, V, 662 conformation in solution hydrogen exchange, V, 628-829 spectroscopy of native enzymes, V, 622-625 stability and denaturation, V, 626-628 titration and chemical modification, V, 625-626 crystal structure investigations, V, 608-611 active site region, V,617-622 human enzyme structure, V, 611 secondary structures, V,611416 side chain locations, V, 616617 distribution and physiological function, v, 5 w 5 9 3 historical outline, V, 588490 inhibitors anions, V, 646-052 other, V, 658 sulfonamides, V, 652-658 kinetic properties hydration of aldehydes, V, 639-640 hydrolytic reactions, V, 636-639 interconversion of CO, and HCOo-, V, 632-636 metal ion cofactor, V, 640-641 cobalt as probe of active site, V, 643-646 specificity, V, 642-643

475 thermodynamics and kinetics of zinc binding, V, 641-642 modification of, I, 139440 molecular properties composition, V, 601-603 immunological properties, V, 598-599 methods of isolation, V,593-595 physical properties, V,599-601 polymorphism and nomenclature, V, 595-598 primary structure, V, 603-607 reactions catalyzed, V, 629-630 role of metals, 11, 522-524 structure fluorescence spectroscopy, 11, 425-426 physical probes, 11, 401406 Carbon-nitrogen cleavage, general features, VII, 168-169 Carboxyl anions, acyl transfer to, 11, 2’26-235 Carboxylesterase (s) active site, organophosphorus compounds and, V, 61-64 amino acid composition, V, 52-53 assay procedures, V, 64-85 equivalent weight, V, 50 inhibitors, V, 64 kinetics, V, 60-61 molecular weight, V, 48-50 multiple molecular forms, V, 4546 pH optimum, V, 59-60 physiological and pharmacological significance, V, 65-67 preparations and criteria of purity, V, 4748 substrate specificity acyl group transfer, V, 59 aromatic amides, V, 57-59 carboxyl esters, V, 53-57 thioesters, V,57 subunit structure, V, 51-52 Carboxyl group activation of, VIII, 6-20 chemical modification, I, 174 ribonuclease, IV, 675-677 Carboxypeptidase(s) bovine, homologies, 111, 66-67 fungal, 111, 790-791 gene duplication and, I, 308-309 role of metals, 11, 519-522

476


Carboxypeptidase A amino acid sequence, 111, 5 crystallography, 111, 17-18 description of structure, 111, 2146 determination of structure, 111, 18-21 esterase activity, 111, 7 kinetics, 111, 7-10 mechanism of action, 111, 14-17 ester cleavage, 111, 50-51 inhibition and activation, 111, 51-54 metal studies, 111, 54-56 peptide cleavage, 111, 46-50 metals and, 111, 11-12 side chain modification, 111, 1214 structure backbone conformation, 111, 29-31 correlation of sequence with, 111, 31-33

folding of chain, 111, 26-29 general features, 111, 21-23 helical segments, 111, 23-26 interpretation of substrate specificity, 111, 43-44 side chain conformation and interaction, 111, 33-36 pstructure, 111, 26 substrate binding changes, 111, 40-42 substrate and inhibitor complexes, 111, 3 7 4 0 success and failure of crystallography, 111, 44-46 substrate specificity, 111, 6-7 Carboxypeptidase B activation and inhibition, 111, 75-76 amino acid sequences and end groups, 111, 64-86

chemical composition, 111, 62-64 distribution, 111, 59-60 historical background, 111, 57-59 kinetics and competitive inhibition, 111, 71-75

mechanism, comment on, 111, 77 physical properties, 111, 61-62 purification and assay, 111,60431 specificity, 111, 69 esterase activity, 111, 71 peptidase activity, 111, 70-71 use in protein structural analysis and modification, 111, 77-79 Castor bean, lipase of, VII, 613-614

Catalase active site distal ligand identity, XIII, 376-385 ligand exchange reactions, XIII, 385-388

ligand identity at fifth and sixth coordination positions, XIII, 369376

apoprotein, selective modifications, XIII, 376-385 general properties, XIII, 366-369 historical background, XIII, 363-365 redox reactions, XIII, 388-389 nature of Compound I, XIII, 389-390 reaction mechanism, XIII, 390-408 Catalytic site, see Active site Cathepsin C, see Dipeptidyltransferase Catheptic endopeptidases cathepsin B, 111, 478-479 cathepsin D, 111, 476477 cathepsin E, 111, 477478 Catheptic exopeptidases cathepsin A, 111, 481482 cathepsin C, 111, 479-480 catheptic carboxypeptidases A and B, 111, 480 Cellobiose-2’-epimerase, properties, VI, 377

Cellulase (9) action on cellulose and related substrates, V, 287-289 applications, V, 289-290 assay and detection, V, 275-277 C1 factors and, V, 280-282 induction and repression, V, 277-278 physical and chemical properties, V, 282-285

production and isolation cultural conditions, V, 278 influence of cultural conditions on physical properties, V, 278-279 isolation methods, V, 279-280 significance and distribution, 8,274-275 substrate binding and catalytic properties, V, 285-287 Cellulose polysulfatase, V, 11-12 Cell wall structure and action of lytic enzymes chemical properties of cell walls, V, 353455

477


mode of action of enzymes, V, 355 morphogenesis of cell wall and membrane, V, 352-353 Cerebroside sulfatase, V, 13-14 Chain extension, protein structure and,

I, 293-300 Chain shortening, protein structure and, I, 292-293 Charge-transfer forces, propinquity effects and, 11, 254-264 Chemical modification alkaline phosphatase bacterial, IV, 389-392 mammalian, IV, 427-428 amylases, V, 129-130, 261-263 chymotrypsin, 111, 234, 238-239 control of selectivity, reaction conditions and, I, 168-170 reagent choice, I, 168 determination of degrees and sites of analytical methods, I, 170-171 instability of modified residues, I, 171-172

in elucidation of noncovalent structure intermolecular reactions, I, 191-194 intramolecular reactions, I, 183-191 functional group reactivity determinants field or electrostatic effects, I, 154 hydrogen bonding, I, 152-154 matrix effects, I, 155 microenvironment polarity, I, 151-152

miscellaneous effects, I, 155-156 steric effects, I, 154-155 general principles, I, 166167 immunochemistry and, I, 199-200 inorganic pyrophosphatase, IV, 514-518 insoluble enzymes and antigens, I, 200-201

lysozyme, I, 207-211 mechanism and reactivity kinetic considerations, I, 159-162 protein functional group nucleophilicities, I, 162-164 superreactivity, I, 164-166 papain, I, 205-207 active site, 111, 515-516 other reactions, 111, 516518 pepsin, 111, 133-137

as primary structure probe chemical cleavage and, I, 181-182 location of modified residues, I, 182 proteolysis and, I, 178-181 as probe of function, accessory sites, I, 196-197 active site, I, 194-196 multifunctional enzymes, I, 197-198 proteinase inhibitors, 111, 443447 reagent reactivity determinants, catalytic factors, I, 158 electrostatic interaction, I, 157 local environment polarity, I, 158-159 selective adsorption, I, 156 steric factors, I, 157-158 reversible, I, 172-173 amino groups, I, 175 arginine, I, 174 carboxyl groups, I, 174 histidine, I, 173 methionine, I, 173 serine and threonine, I, 173 sulfhydryl groups, I, 174-175 tryptophan, I, 173 tyrosine, I, 174 ribonuclease, IV, 675-697 subtilisin BPN’, I, 203-205 subtilisins, 111, 596-602 transient, I, 176 trypsin, 111, 269-273 unsuspected, I, 176-178 X-ray crystallography and comparisons of protein solutions and crystals, I, 202-203 isomorphous heavy atom replacements, I, 201-202 Chemical reaction rate equations, derivation, 11, 61-63 Chlamydomonas reinhardti, N-acetylglutamate-5-phosphotransferase of, IX, 513 Chlorella pyrenoidosa, adenosine diphosphoryl glucose pyrophosphorylase of, VIII, 90 Chloroplast ( s ) adenosine triphosphatase Euglena gracilis, X, 394 spinach, X, 389-394 ribonucleic acid polymerase of, X, 329-330

478


Choline dehydrogenase electron transport system and, XIII, 261-263

properties, XIII, 260-261 Choline sulfatase, V, 14 Chondroitin, hyaluronidase and, V, 310 Chondroitin sulfates, hyaluronidase and, V, 310 Chondrosulfatase, V, 12 Chymopapains, 111, 537-538 Chymotrypsin(s) active center amino acids, interaction, 111, 245-248 active-site-directed reagents and, I, 94103

individual amino acid function aspartic acid 102, 111, 235-236 aspartic acid 194, 111, 243 histidine 57, 111, 231-234 isoleucine 16, 111, 236-243 serine 195, 111, 217-231 ribonuclease and, IV, 674 structure physical probes, 11, 391-396 ultraviolet difference spectroscopy, 11, 414-417 fluorescence spectroscopy, 11, 421-424 substrate specificity acylamido interaction, 111, 208 amino acid side chain binding, 111, 205-208

locked substrates, 111, 209-212 stereo specificity, 111, 207-209 a-Chymotrypsin active center structure acyl enzyme, 111,20&202 enzyme-inhibitor complex, 111, 199200

enzyme-product complex, 111, 198199

native enzyme, 111, 190198 X-ray diffraction, 111, 190-195 7-Chymotrypsin, active center structure, 111, 202-204 8-Chymotrypsin, active center, 111,204 1.-Chymotrypsin, actiye center, 111, 204 Chymotrypsinogen activation, 111, 167-169 active site unblocking, 111, 244 X-ray crystallography, 111, 169-176 activation refolding, 111, 182-183

arginine 145, 111, 176-179 catalytic site, 111, 179-182 isoleucine 16, 111, 175-176 methionine 192, 111, 179 Chymotrypsinogen A, chemical structure, 111, 187-189 Chymotrypsinogens A and B, activation products, 111, 185-187 Circular dichroism protein structure and, 11, 381-382, 408 secondary, 11, 382386 tertiary, 11, 386-391 typical cases, 11, 381407 ribonuclease, IV, 719-723 Cis-trans isomerization about double bonds enzymic with bond migration, VI, 39@-395 without bond migration, VI, 381390 nonenzymic metals and metal ions, VI, 401406 nucleophilic catalysis, VI, 397-401 photoisomerization, VI, 395-397 reversible addition of radicals, VI, 406

Cistron, types of mutations and, I, 243245

Citramalate, cleavage and synthesis, VII, 431-432

Citrate lyase assay and isolation, VII, 378-379 catalytic properties control, VII, 387 equilibrium and kinetics, VII, 380381,386387

specificity, VII, 380, 385-386 stereospecificity, VII, 381, 387 molecular properties cofactors, VII, 379 inhibitors, VII, 380 molecular weight and subunits, VII, 379

sulfhydryl groups, VII, 379380 reaction mechanism, VII, 381382, 388389

Citrate synthase assay and isolation, VII, 358-360 catalytic properties control, VII, 362-383 equilibrium and kinetics, VII, 382


specificity, VII, 361-362 stereospecificity, VII, 363-364 molecular properties activators, VII, 361 cofactors, VII, 360 inhibitors, VII, 361 molecular weight and subunits, VII, 360 sulfhydryl groups, VII, 360-361 proton transfer and, 11, 316-317 reaction mechanism citric anhydride as intermediary, VII, 367-368 citryl coenzyme A as intermediary, VII, 367 enolization of coenzyme A, VII, 365366 inversion of configuration of acetyl coenzyme A, VII, 366-367 keto or enol .form of oxalacetate, VII, 365 Citric acid cycle reactions stereospecificity fumarase, 11, 171-176 isocitrate dehydrogenase, 11, 168-171 succinate dehydrogenase, 11, 176-179 synthesis of citrate, 11, 164-168 Clostridium histolyticum collagenases, 111, 662-663 catalytic properties, 111, 670-689 chemical and biosynthetic modification, 111, 669-670 composition, 111, M.Wf37 culture of organism and enzyme purification, 111, 663-665 molecular size, 111, 667-668 physical properties, 111, 669 possible subunits, 111, 668-669 Clostridium pasteurianum, phosphofructokinase of, VIII, 256 Clostridium perfringem phospholipase C of, V, 84-85 polynucleotide phosphorylase of, VII, 571-572 Clostripain as collagenase contaminant, 111, 715716 definition, 111, 699-700 -aeneral features

479 pH optimum and ion efficts, 111, 706-707 sulfhydryl requirement, 111, 707-708 historical perspective, 111, 700-703 inhibitors affinity labeling, 111, 714-715 competitive, 111, 712-714 origin of active site specificity, 111, 717-71 9 purification and assay, 111, 704 specificity proteins and polypeptides, 111, 710712 synthetic esters and amides, 111, 708710 structural properties amino acid composition, 111, 706 physical constants, 111, 704-705 use in sequence analysis, 111, 716-717 Cobalt carbonic anhydrase and, V, 643646 Coenzyme A transferase (s) catalytic properties assay, IX, 487 mechanism and kinetics, IX, 488496 specificity, IX, 486-487 thermodynamics, IX, 487488 properties, IX, 485436 reactions catalyzed, IX, 483485 Coenzyme B12-dependent reactions, stereochemistry, 11, 204-214 Collagenase(s) catalytic properties activation by transition metals, 111, 683 assay, 111, 684-685, 692 cofactors, 111, 677-678 evidence for intrinsic metal component, 111, 678-683 mechanism, 111,688-689 reaction catalyzed, 111, 670, 691 specificity, 111, 670-677, 691 substrate interaction, 111, 688 thermodynamics and kinetics, 111, 685-688 zinc containing peptides, 111, 683-684 Clostridium histolytlcum, 111, 662-689 clostripain in. 111. 715-716 definition, 111, 652-653

480


human synovial fluid, 111, 693-696 known enzymes and their functions, 111, 653-659 nature of substrate, 111,650-652 properties, generalizations, 111,659-660 R a m catesbiana, 111, 689-693 uses of, 111, 660-662 Compartmentalization, metabolic regulation and, I, 423426 Complementation, mutations and, I, 24925 1

Conformational states, frozen, I, 370-371 Control mechanisms compensatory antagonism of endproduct inhibition, I, 436437 metabolite activation, I, 434-436 precursor substrate activation, I, 438-439

Convergence, protein evolution and, I, 328-329

Cooperativity, molecular basis, I, 375379

Cooperativity index, enzyme regulation and, I, 358 Coordination schemes determination confirmatory techniques, 11, 468-470 provisional techniques, 11, 464-468 enzymes, metals and substrates, 11, 463464

experimentally determined, 11, 470476

techniques, 11, 464470 experimentally determined higher metal complexes, 11, 475476 higher substrate complexes, 11, 471475

ternary complexes, 11, 470-471 Copper-containing oxidase(s) blue biological distribution and function, XII, 558-559 history, XII, 557-558 oxidation-reduction properties, XII, 571-574

purification and molecular properties, XII, 560-563 catalytic properties reducing substrates, XII, 575-578

reduction of oxygen, XII, 578-579 specificity, inhibition and steadystate kinetics, XII, 574475 historical background, XII, 507-511 magnetic and spectroscopic properties definitions and distribution of copper forms, XII, 563-566 type 1 copper, XII, 566-567 type 2 copper, XII, 567-570 type 3 copper, XII, 570-571 reducing dioxygen to hydrogen peroxide amine oxidase, XII, 511-527 galactose oxidase, XII, 527-533 Cortisone sulfatase, V, 10 Creatine kinase active form of substrates guanidine, VIII, 414 nucleotide, VIII, 412413 adenosine polyphosphates as inhibitors, VIII, 422 anion effects, VIII, 423427 catalytic site, formation and topography, VIII, 439442 conformational changes, substrateinduced, VIII, 436-438 “essential” thiol group importance for catalytic activity, VIII, 443448 structural involvement, VIII, 44% 443

substrate effects, VIII, 448-451 equilibrium, VIII, 428-431 groups essential for activity cysteine residues, VIII, 431432 histidine, VIII, 434 lysine, VIII, 43S434 tyrosine, VIII, 434-436 historical background, VIII, 384485 hybrid, VIII, 403 mechanism, VIII, 451455 metal ions and, VIII, 409412 other brain-type, VIII, 402403 other muscle enzymes assay and specific activity, VIII, 4.p purification, VIII, 400 stability, VIII, 401 ox brain assay and specific activity, VIII, 401 purification, VIII, 401


stability, VIII, 401-402 rabbit muscle assay and specific activity, VIII, 395-398

purification, VIII, 395 stability, VIII, 398-399 role of metals in mechanism, 11, 499500

structure amino acid composition, VIII, 390392

isoenzymes, interspecific hybrids, conformers and genetic variants, VIII, 386-390 molecular weight, VIII, 395 primary, VIII, 392-393 secondary and tertiary, VIII, 393-394 subunit shape and organization, VIII, 394-395 substrate binding, VIII, 414-420 substrate specificity guanidines and organization of creatine binding site, VIII, 403-407 nucleotides and related inhibitors, VIII, 407-409 temperature and, VIII, 420-422 Crosslinks intramolecular, ribonuclease, IV, 696697

Crolalus adamanleus venom, phospholipase AI of, V, 75-76 Crotalus atroz venom, phospholipase A2 of, V, 7.7-78 Crotonase, properties, V, 568-571 Cyanate, inorganic pyrophosphatase and, IV, 516-517 Cyanogen bromide, inorganic pyrophosphatase and, IV, 514 Cyclic adenosine monophosphate protein kinase dependent mechanism, VIII, 568-572 nomenclature, VIII, 566 properties, VIII, 572-578 purification, VIII, 568 tissue and subcellular distribution, 567-568

Cyclodiene insecticides, epoxidases and, VII, 210-211 jj’-Cystathionase, properties, VII, 51-52 y-Cystathionase, properties, VII, 57159

Cystathionine p-synthetase, properties, VII, 54-56 Cystathionine y-synthetase, properties, VII, 60-61 Cysteamine oxygenase, properties, XII, 148-149

Cysteine lyase, properties, VII, 56-57 Cysteine oxygenase, properties, XII, 149150

Cysteine residues creatine kinase, VIII, 431432, 442-451 guanidino kinases, VIII, 477-480 Cysteine synthetase, properties, VII, 54 Cystine-disulfide groups, ribonuclease, IV, 690-696 Cystine peptides, microbial proteinases, 111, 752-754 Cytidine diphosphate-D-glucose oxidoreductase, properties, V, 478479 Cytidine kinas6 see Uridine kinase Cytidine monophosphate kinase, see Uridine monophosphs te kinase Cytidine triphosphate synthetase allosteric control, X, 546-547 cooperative effects, X, 547-548 6-diazo-5-oxonorleucine and half-thesites reactivity, X, 549-550 historical, X, 540-541 purification, X, 541-542 reaction catalyzed, X, 539-540 covalent chemistry, X, 543-546 related enzymes, X, 552-558 role of nucleoside triphosphates, X, 550-552

structure, X, 542-543 Cytochrome (s), absorption spectra, XI, 398-400 bacterial common methodology in research, XI, 506-508 evolution, XI, 540-547 patterns in electron transport pathways, XI, 508-509 sequence and structure, XI, 534-540 type b, XI, 591-593 Cytochrome b, mammalian, purification of, XI, 563564

occurrence and function, XI, 550-551 plant, XI, 587-591


reaction with substrates adenosine triphosphate-induced reduction, XI, 561 antimycin effect, XI, 562 kinetics, XI, 560-561 redox change, XI, 562-563 respiratory, XI, 551652 absorption spectra, XI, 554-558 different types, XI, 552-554 miscellaneous, XI, 563-564 oxidation-reduction potential, XI, 558-560 Cytochrome bl, occurrence of, XI, 579584 Cytochrome bl, preparation and properties, XI, 585-587 Cytochrome ba biological role, XI, 567 distribution, XI, 566 isolation, XI, 567-568 nomenclature, XI, 565-566 properties, chemical, XI, 668-569 physical, XI, 569 spectral, XI, 589-571 structure, XI, 571-572 amino acid sequence, XI, 572-573 ternary, XI, 573-576 Cytochrome bs reductase cytochrome P-450 reductase and, XIII, 151-153 mechanism, microsome bound, XIII, 161-162 mechanism of Strittmatter, review, XIII, 156-161 methemoglobin reductase and, XIII, 164-165 molecular properties, amphipathic and soluble forms, XIII, 154-156 structural studies, XIII, 162-164 Cytochrome b-662, distribution and preparation, XI, 584 properties and structure, XI, 584-585 Cytochrome(s) c, bacterial, XI, 497-506 evolution, XI, 64&547 function, XI, 506-509 photosynthesis, XI, 509316 respiratory chain, XI, 518634 structure, and sequence, XI, 534-540

eukaryotic, photosynthetic cytochromes f and CJSS,XI, 493497 respiratory cytochrome c, XI, 400489 respiratory cytochrome c,, XI, 489492 metal complexes and, 11, 534-538 principles of protein evolution and, I, 274-285 respiratory amino acid sequence, XI, 419429 evolution, XI, 429-450 molecular folding and structural integrity, XI, 450463 oxidation reduction mechanism, XI, 463489 structure, XI, 405-419 Cytochrome cl, respiratory, XI, 489-492 Cytochrome cm, photosynthetic, XI, 493497 Cytochrome c oxidase biological role, XIII, 299-300 chemical and physical properties, XIII, 301-302, 313-314 interaction with cytochrome c, XIII, 334-335 kinetic studies, XIII, 335-337 models, XIII, 314-315 chemistry of oxygen reduction, XIII, 302-307 electronic spectroscopy absorption spectra, XIII, 315-319 circular dichroic spectra, XIII, 319 electron paramagnetic resonance studies copper, XIII, 329-330 iron, XIII, 331 ligand binding effects, XIII, 332333 p-oxobishemin and, XIII, 333334 valence state changes and, XIII, 331-332 historical background, XIII, 300-301 ligand binding studies, XIII, 319-320 azide, fluoride and cyanide, XIII, 320-321 carbon monoxide, XIII, 321-323 dioxygen, XIII, 323-326 lipids of, XIII, 312-313 mechanisms, XIII, 337344


metal components, XIII, 307-309 potent iome try electron economy, XIII, 325-326 iron-copper coupling, XIII, 326-327 ligand binding effects, XIII, 327-328 summary, XIII, 328329 protein of, XIII, 309-312 Cytochrome c peroxidase cytochrome c interaction, XIII, 356360 enzymic activity, XIII, 352-353 general comments, 360-361 historical background, XIII, 345-347 preparation and molecular properties, XIII, 347-348 reaction mechanism, XIII, 353356 structural aspects, XIII, 348-351 Cytochrome f, photosynthetic, XI, 493497 Cytochrome 0,occurrence and properties, XI, 592-593 Cytochrome P-450 reductase, XIII, 165166 catalytic activities, XIII, 167-169 general properties, XIII, 166-167 mechanism, XIII, 169-173 Cytophaga, isoamylase of, V, 204-206

D Deaminases, modification of, I, 127-128 Debranching enzyme (s) characterization of, V, 223-226 classes of, V, 192-194 direct, V, 194-208 indirect, V, 208-210 assay of glucosidase-transferase activity, V, 211-212 effect on glycogen structure, V, 217219 glucosidase-transferase in glycogen storage disease, V, 221-222 other enzymes, V, 222-223 pH dependence, V, 215-217 purification and physical properties, v, 210-211 reversion reactions, V, 219-220 specificity, V, 213-215 in vivo roles, V, 226-228 structure determination and, 228-229

arrangement of unit chains, V, 230233 average chain length, V, 229-230 enzymic action pattern, V, 233-234 Debye forces, propinquity effects and, 11, 254-264 3-Decynoyl-N-acetylcysteamine,isomerization of, V, 459-461 Dehydratases, miscellaneous, VII, 53-54 Dehydration ( 8 ) metal ion-assisted L-arabonate-n-fuconate, v, 581-582 n-arabonate and p-xylonate, V, 582 galactonate, V, 578479 gluconate, V, 575-578 hexarate, V, 579-581 n-mannonate and n-altronate, V, 579 Schiff base-assisted glucosaminate dehydrase, V, 586 2-keto-3-deoxy-~-arabonatedehydratase, V, 583-585 5-keto-4deoxyglucarate dehydratase, v, 585-586 Dehydrogenases the bigger family general XI, 94 members, XI, 94-99 primordial mononucleotide binding proteins, XI, 101 structural relationships, XI, 99 time scale, XI, 99-101 characteristics of, XIII, 90-91 comparison of three-dimensional structure known structures, XI, 64-65 malate and lactate to alcohol and glyceraldehyde-3-phosphatedehydrogenase, XI, 65-69 mononucleotide binding unit, XI, 69-70 NAD binding structure, XI, 70-73 recognition of similar structural domains, XI, 73-74 dissociation constants of enzyme-coenzyme compounds, cooperative effects, XI, 46-47 initial measurements and binding studies, XI, 38-44 pH effects and role of histidine, XI, 44-46

484 domain and subunit assembly, conservation of contacts, XI, 91 gene fusion, XI, 89-90 quaternary structure, XI, 91-93 quaternary structure-evolution, XI, 93 equilibrium and kinetics of enzymecoenzyme reactions dissociation constants, XI, 34-42 kinetics, XI, 4247 flavodoxin and, XI, 94-96 functional aspects of dinucleotide binding domains, XI, 83-84 adenosine monophosphate, XI, 87-88 nicotinamide adenine dinucleotide, XI, 84-87 nicotinamide mononucleotide, XI, 88-89 some generalizations, XI, 89 inhibition and activation analogs, XI, 30-34 product, XI, 34-35 substrate, XI, 25-30 initial rate equations ordered mechanism with isomeric enzyme-coenzyme compounds : conformation change, XI, 10-11 ordered and random mechanisms for three-substrate reactions, XI, 13-15 preferred pathway mechanism, XI, 12-13 rapid equilibrium mechanism, XI, 11-12 simple ordered mechanism, XI, 7-10 kinases and, XI, 96-98 kinetics of enzyme-coenzyme reactions, conformational changes, XI, 50-52 velocity constants, XI, 47-50 kinetics of transient phase, XI, 47-48 integrated rate equations, XI, 48-50 lactate dehydrogenases, XI, 52-53 liver alcohol dehydrogenase, XI, 5052 other enzymes, XI, 53-55 kinetic studies with alternative substrates liver alcohol dehydrogenase, XI, 2022 other enzymes, XI, 23-24


yeast alcohol dehydrogenase, XI, 2223 nicotinamide adenine dinucleotidelinked, metal complexes and, 11, 525-528 preliminary generalizations, XI, 3-4 quaternary structure, XI, 91-92 alcohol dehydrogenase subunit association, XI, 93 evolution, XI, 93 P and R axis-cooperativity, XI, 9192 Q axis, XI, 91 rhodanese and, XI, 98 sequence comparisons based on structural alignments glutamate dehydrogenase comparisons, XI, 79 lactate, alcohol and glyceraldehyde3-phosphate dehydrogenases, XI, 77-79 statistics of comparisons, XI, 79-83 sequence comparisons in the absence of three-dimensional structural information significance, XI, 7677 suggested homologies or analogies, XI, 74-76 steady-state kinetics cooperative rate effects, XI, 31-34 inhibition and activation by substrates, substrate analogs and products, XI, 22-31 initial rate equations for ordered and random mechanisms, XI, 6-14 isotope exchange a t equilibrium, XI, 14-16 kinetic studies with alternate substrates, XI, 18-22 maximum rate and Haldane relations, XI, 16-18 phenomenological initial rate equations, XI, 4-6 subtilism and, XI, 98-99 Dehydroluciferin, adenylylation of, VIII, 19-20 Deletions, protein structure and, I, 293300 Deoxyadenosine kinase, properties, IX, 66-68

485


Deoxyadenosine monophosphate kinase, properties, IX, 86-87, 95-96 3-Deoxy-~-arabino-heptulosonate7-phosphate synthase, modification of, I ,

assay, X, 239-240 isolation of covalent intermediates deoxyribonucleic acid-adenylate,

139

ligase-adenylate, X, 245 mechanism of phosphodiester bond synthesis, X, 244-252 physical homogeneity, X, 241 physical properties, amino acid analysis, X, 242 molecular weight, X, 241 stoichiometry, X, 242-243 purification, X, 240-241 reversal of, 246-248 role in vivo bacteriophage-induced, X, 252-254 Escherichia coli, X, 254-259 steady state kinetics overall reaction, X, 248-249 partial reactions, X, 249-252 Deoxyribonucleic acid methyltransferases biological significance, IX, 194-195 occurrence, IX, 190 properties bacterial, IX, 190-192 eukaryotic, IX, 192-193 regulation, IX, 193-194 Deoxyribonucleic acid polymerase(s) catalytic reactions basic features, X, 123-124 fidelity of replication, X, 127-129 implications of mechanism, X, 134-

Deoxycytidine kinase, properties, IX, 62-66

Deoxycytidine monophosphate kinase, properties, IX, 88-89, 95-96 Deoxycytidylate hydroxymethyltransferase, properties, IX, 209-210 Deoxyguanosine kinase, properties, IX, 68-69

Deoxyguanosine monophosphate kinase, IX, 94-96, see Guanosine monophosphate kinase Deoxyribonuclease (s) adenosine triphosphate-dependent, IV, 259, 261-262

catalytic properties inhibitors, IV, 281-283 phosphodiesterase activity, IV, 283 substrate concentration, pH and ions, IV, 280-281 classification of, IV, 251-252 spleen components, IV, 275 dimeric structure, IV, 275-276 distribution, localization and role, IV, 285-287 features of degradation, IV, 276-278 general catalytic properties, IV, 280283

135

multiple sites in active center, X,

isolation, IV, 272-273 mechanism, IV, 278-280 methods of investigation, IV, 278 physical and chemical properties, IV,

124-125

polymerization step, X, 125-126 pyrophosphorolysis and pyrophosphate exchange, X, 126-127 ribonucleotides and, X, 133-134 specialized functions, X, 135-137 synthesis without template, X, 132-

273-275

specificity, IV, 283-285 Deoxyribonuclease I active center, IV, 297-299 chemical nature, IV, 292-297 historical background, IV, 289-291 inhibitor, IV, 299-302 ions and, IV, 302-303 kinetics, IV, 303-308 physiological role, I, 310 specificity, IV, 308-310 Deoxyribonucleic acid ligase(s) adenylyl transfer functions, VIII, 45-48

X,

245-246

133

synthetic product, X, 131-132 template-primer, X, 129131 classification native and denatured templates, X, 182-183

6

polyribonucleotide templates, X, 184 size, X, 183 comparison of properties, X, 144

486


definitions and measurements, X, 175176

exonucleases associated, IV, 255-258 fidelity of synthesis, X, 201-202 historical, X, 174-175 inhibitors and activators, X, 199-201 initiators, X, 203-204 intracellular distribution chloroplasts, X, 181 membranes and other structures, X, 181-182

mitochondria, X, 180-181 nuclei, X, 179-180 isolation and physicochemical properties, X, 120-123 kinetics extent of synthesis, X, 195-196 temperature and, X, 194-195 metal activators, X, 193-194 molecular properties antisera and, X, 192-193 homogeneity, X, 188-189 presence of nuclease, X, 189-190 sulfhydryl groups, X, 190-191 zinc, X, 191-192 occurrence, X, 176-179 pH and PI, X, 194 properties of purified viral enzyme, X, 218

size, X, 219-220 storage and stability, X, 219 proteolytic cleavage: two enzymes in one polypeptide, X, 138-139 purification chromatography, X, 185-186 extent, X, 186-188 stability, X, 185 subcellular fractionation, X, 184-185 substrates requirements, X, 198-199 specificity, X, 196-197 templates deoxyribonucleic acid, X, 204-205 ribonucleic acid, X, 206-207 variety of, X, 119-120 viral, purification, X, 216-218 solubilization, X, 215 virus purification and, X, 214-215

2-Deoxyribose-5-phosphate aldolase catalytic reaction activators and inhibitors, VII, 320 assay, VII, 319-320 equilibrium constant, VII, 320 pH optimum, VII, 320 Schiff base formation, VII, 321 substrate specificity, VII, 321 historical background, VII, 315 metabolic significance, VII, 316-317 molecular properties isolation, VII, 317-319 physical properties, VII, 319 occurrence, VII, 315-316 Deoxysugars synthesis, general considerations, V, 465467

Deoxy sugar aldolase (s) ,general, VII, 303-304

Deoxythymidine diphosphate-D-glucose oxido-reductase kinetic properties, V, 469-470 molecular properties, V, 467469 pyridine nucleotide and substrate release, V, 474476 subunit association, V, 476-478 reaction mechanism enzyme bound pyridine nucleotide,

V, 472-473 intramolecular hydrogen transfer, V, 470-472

isotope effects, V, 473-474 Deoxythymidine kinase distribution, purification and assay,

IX, 69-70 kinetic, molecular and allosteric properties, IX, 71-74 reaction mechanism; active site, IX, 74

substrate specificity, IX, 70 Deoxythymidine monophosphate kinase distribution and purification ; assay; stability, IX, 91-92 kinetic and molecular properties, IX, 93-96

substrate specificity, IX, 92-93 Deoxyuridylate hydroxymethyl transferase, properties, IX, 210 Dermatan sulfate, hyaluronidase and, V, 310-311

487


2,5-Diaminohexanoate, lysine mutase and, VI, 554 Diazonium-1H-tetrazole, inorganic pyrophosphatase and, IV, 517-518 3,&Dideoxyhexoses, synthesis of, V, 479480 7,&Dihydro-2-amino-4-hydroxy-6-hydroxymethylpteridine pyrophosphokinase, X, 627-628 Diisopropylfluorophosphate, papain and, 111, 516-517 Dioxygenase(s) biological function and general properties double bond cleavage, XII, 123-125 double hydroxylation, XII, 125 miscellaneous, XII, 125-127 sulfur-containing compounds, XII, 125 classification, XII, 121-123 heme-containing indoleamine 2,3-dioxygenase, XII, 130-132 tryptophan dioxygenase, XII, 127130 history and definition, XI, 120-121 a-ketoglutarate, XII, 151-152 y-butyrobetaine hydroxylase, XII, 167-169 p-hydroxyphenylpyruvate hydroxylase, XII, 179-183 lysyl hydroxylase, XII, 165-167 mechanism, XII, 183-189 prolyl hydroxylase, XII, 152-165 pyrimidines and nucleosides, XII, 169-179 nonheme iron-containing, XII, 132-133 cysteamine oxygenase, XII, 148-149 cysteine oxygenase, XII, 149-150 phenolic, XII, 133-148 phenolic extradiol, XII, 140-144 intradiol, XII, 133-140 others, XII, 144-148 Dipeptidases, 111, 116-117 Dipeptidyl aminopeptidase 1, see Dipeptidy 1-transferase Dipeptidyltransferase, 111, 105-111, see also Cathepsin C

Diphosphoglycerate mutase, properties, VI, 476477 Disaccharide phosphorylases general background, VII, 515-518 substrate specificity, VII, 526-528 Disulfide bridges elastase, 111, 339341, 348-349 p-hydroxydecanoyl thioester dehydrase, V, 452453 prothrombin, 111, 313 thrombin, 111, 290-291 trypsin, 111, 271 Disulfide groups, amylases, V, 244-245 Disulfide loop, proteinase inhibitor reactive site and, 111,420-422 Divergence protein evolution, I, 314-316 factors influencing rate, I, 317-321 speciation of homologous proteins: genetic drift, I, 321-328 Dopamine p-monooxygenase, properties of, XII, 294-295 Double bonds isolated, sterospecificity of reactions, 111, 179-186 Double reciprocal plots enzyme regulation and, I, 356-357 nonlinear, 11, 56-59 Drosophila melanogaster acid phosphatase of, IV, 498 molybdenum hydroxylase genetics of, XII, 406412 E

Ehrlich ascites cells, 5'-nucleotidase of, IV, 34-49 Elastase, see also Tosyl elastase activation, 111, 244-245 amino acid sequence determination complete sequence, 111,341-343 disulfide bridged peptides, 111,339341 assay methods elastin and, 111, 325-326 synthetic substrates and, 111, 326327 criteria of purity, 111, 32-29 crystals, activity of, 365-366 enzymic activity

488


inhibitors and activators, 111,337-338 irreversible inhibitors, 111, 338-339 proteins and, 111, 332-333 synthetic substrates and, 111, 333337

history and distribution, 111, 323-325 physicochemical properties, 111, 329331

proelastase and, 111, 331-332 purification methods, 111, 327-328 reactivity and pK. of amino groups,

111, 367472 competitive labeling technique, 111, 366-367

nitrous acid and, 111, 372 valine and aspartate residues, 111, 372-373

ribonuclease and, IV, 672-673 sequence homologies in serine proteinases activation peptides, 111, 343-344 B chains, 111, 344-348 disulfide bridges, 111, 348-349 hypothetical models, 111, 349-352 stability, 111, 332 X-ray crystallography, crystals and, 111, 353-354 Fourier synthesis, 111, 355-356 heavy atom derivatives, 111, 354-355 Electron delocaliration, enzymic-C-H fission and, 11, 287-290 Electron density maps, interpretation of,

I, 46-52 Electron microscopy, collagenase action and, 111, 693 Electron paramagnetic reaonance, ribonuclease, IV, 723-725 Electron-transferring flavoprotein catalytic properties, XII, 116-118 function, XII, 109-110 molecular properties oxidation-reduction, XII, 116 properties of chromophore, XII, 111-115

purification, molecular weight and amino acid composition, XII, 111 a, ,!) Elimination reactions aconitase, 11, 302-304 factors influencing, VII, 79-81 conjugation, VII, 81

delocaliration of ,!) charge, VII, 81-82

modification of X , VII, 82-84 solvent effects, VII, 85-86 stereoelectronic control, VII, 85 weak bases, VII, 86 fumarase, 11, 304-308 nonenrymic aspartate-fumarate interconversion, VII, 86-88 stereochemistry, 11, 309-312 ,!)-Elimination reactions mechanism, VII, 66-72 dehydratases, VII, 39-48 y-Elimination and replacement mechanism, VII, 72-73 pyridoxal phosphate and, VII, 59-62 y-Elimination reactions, pyridoxallinked, VII, 57-59 Elongation stringent response and, X, 78-79,82-83 effect of ppGpp, X, 81-82 synthesis of MSI and MSII, X, 79-81 Elongation factor(s) bacterial, X, 55-57 function of factor G, X, 64-67 function of factor Tu, X, 58-63 physical properties, X, 57-58 role of factor Ts, X, 63-64 Elongation factor G interaction with ribosomes, X, 66-67 protein synthesis and, X, 65-66 Elongation factor Tu complex interaction with ribosomes,

X, 62-63 guanine nucleotide binding, X, 58-59 ternqry complex, X, 59-62 Endolysin bacteriophage A catalytic properties, V, 391-392 chemical properties, V, 388391 physicochemical properties, V, 387388

purification, V, 385-386 Endonuclease (8) bacterial nonspecific, IV, 259-262 specific, IV, 262-270 Enolase active site components, V, 532-534


number, V, 530-532 chemical properties amino acid composition, V, 503 end groups and terminal sequences, V, 503-506 immunochemistry, V, 507 polypeptide chain identity, V, 506507 criteria of purity, V, 502-503 general conisderations, V,499-501 kinetic parameters, V,523-524 magnesium and, V,524-526 mechanism of dehydration reaction evidence for carbanion intermediate, v, 537 isotope effects, V, 535-536 molecular properties, summary, V, 518-519 monomer-dirner activity relationships, v, 537-538 physical properties, V,507-508 electrophoretic mobility, V, 508-510 subunit structure, V, 510-518 properties of reaction catalyzed assay, V, 519-523 equilibrium, V, 523 substrates, V, 519 rabbit muscle, glycidol phosphate and, v, 534 role of metals, 11, 508-509 substrate specificity, active site mapping with analogs, V, 526-529 yeast carboxymethylation, V, 533 photooxidation, V, 533-534 Enterochrome-566, properties, XI, 592 Enzyme (s) bridge complexes, 11, 477478 carbonyl containing other, 11, 356358 pyridoxal phosphate, 11, 346-356 covalent modification glycogen phosphorylase, I, 413-415 glutamine synthetase, I, 409413 development of novel properties, I, 334-335 intrachain repetitions, I, 335 multichain enzymes, I, 335-337 , inactive, proteinase inhibitors and, 111, 454-457

loss of function, survival value, I, 332-334 metal linkage coordination geometry, 11, 492494 nature of ligands, 11, 490-492 role of metals in mechanism, 11,498499 hydrolases, 11, 519-525 lyases, 11, 508-519 phosphoenolpyruvate carboxylation, 11, 507-508 phosphoryl and nucleotidyl transfer, 11, 499-507 specificity, historical background, 11, 119-129 structure in solution circular dichroism and optical rotatory dichroism, 11, 381-408 fluorescence spectroscopy, 11, 418430 geometry and quaternary structure, 11, 440-442 infrared spectroscopy, 11, 374-379 ionizable groups, 11, 430440 secondary and tertiary structures, 11, 373-374 ultraviolet absorption of peptide groups, 11, 379-380 ultraviolet difference spectroscopy, 11, 408-417 regulation of concentration balance between synthesis and degradation, I, 402-403 catabolite repression, I, 400401 feedback repression of synthesis, I, 401402 substrate induction of synthesis, I, 399-400 Enzyme crystallography, state of the art, I, 86-87 Enzyme crystals differences from other crystals, I, 5-7 Fourier description structure analysis and phase problem, I, 29-32 as sum of waves, I, 26-29 growth of, batch, I, 19 equilibrium dialysis, I, 19-22 vapor diffusion, I, 19


mounting and radiation damage, I, 22-23 structure and symmetry, I, 7-13 Enzyme regulation diagnostic tests, I, 356358 allosteric protein evaluation, I , 372375 cooperativity index, I, 358 double reciprocal plots, I , 356-357 equations of state, I, 386388 fitting saturation curves, I, 361365 frozen conformational states, I, 370-371 Hill plot, I, 358-359 minimal substrate technique, I, 372 Scatchard or Klotz plots, I, 359-361 Leelocity curves, I, 368369 Y X or N x versus log (X) plots, I, 357 gloasary, I, 395-396 molecular models, qualitative features, I, 344-348 quantitative molecular parameters derivation of general equation, I, 34a353 simple models, I, 353-355 Epimerase(s) definition and history, VI, 365-357 keto-enol rearrangement and, VI, 373-374 noncarbohydrate, VI, 378 oxidation-reduction and, VI, 369-371 proton shifts and, 11, 295-298 Epinephrine, glycogen synthetase and, IX, 338, 341 Epoxidase(s) general considerations, VII, 199-200 metabolic roles, VII, 200-201 Equations of state, enzyme regulation and, I, 365-368 Equilibria chemical, metabolic regulation and, I, 418-419 Erthrocytes, phosphofructokinase of, 257 Eecherichia coli acetyl coenzyme A carboxylase subunits, VI, 60-64 N-acetylglutamate-5-phosphotransferase of, IX, 513 acid phosphatase of, IV, 498

adenosine diphosphorylglucose pyrophosphorylase energy charge and, VIII, 104-107 manganese effect, VII, 102-104 mutants, VIII, 109-117 alkaline phosphatase, I, 251-254, IV, 373-415 asparaginase, IV, 107-116 aspartate metabolism, regulation of, I , 457-459 aspartate transcarbamylase of, IX, 225497 aspartokinases of, VIII, 513-544 adenylylation of, VIII, 44-45 cytochrome b, of, XI, 579, 580, 581 cytochrome b-562, properties and structure, X I , 584-585 deoxyribonuclease, ATP-dependent, IV, 259 deoxyribonucleic acid ligase isolation and physical properties, X, 239-243 mechanism, X , 244-252 role in vivo, X, 254-259 deoxyribonucleic acid polymerase I, physiological role, X, 139-141 deoxyribonucleic acid polymerase 11, physiological role, X, 142 deoxyribonucleic acid polymerase, 111, physiological role, X, 142-143 deoxyribonucleic acid polymerase, exonucleases and, IV, 255-258 endonuclease I, IV, 259-280 endonuclease 11, IV, 204-285 exonucleases I and 111, IV, 253 exonuclease I V of, IV, 254-255 3' + 5' exonuclease, IV, 256 5' + 3' exonuclease, IV, 256-258 fatty acid synthetase, 3decynoyl-Nacetyl-cysteamine and, V, 461-463 fructose-l,6-diphosphatase of, IV, 63% 639 P-galactosidase, VII, 624-625 8-galastosidase, mutations, I, 255-266 glutaminase, IV, 80-93 glutamine synthetase of, VIII, 40-44, X, 755-807 inorganic pyrophosphatase catalytic properties, IV, 518-526 molecular properties, IV, 501418

491


membrane adenosine triphosphatase, X , 416-421 methyltransferase of, IX, 154-160 phage lytic enzymes, V, 355-361 F series polysaccharide depolymerase, V, 392-393 X endolysin, V, 385-392 N20F’ lytic enzyme, V, 382-385 T 2 lysozyme, V, 379-382 T4 lysozyme, V, 361-379 phosphofructokinase of, VIII, 256-257 polynucleotide phosphorylase of, VII, 548-570 mutant, VII, 571-572 restriction endonucleases, IV, 263-264 ribonucleases of, IV, 241-243 succinyl coenzyme A synthetase of, X, 582591 thioredoxin reductase, general properties, XIII, 144-145 tryptophan synthetase catalytic properties, VII, 22-30 molecular properties, VII, 8-21 vitamin BIZmethyltransferase of, IX, 122-154 Ester(s), subtilisin and, 111, 592-593 Esterase(s) classification and distribution, V, 43-45 modification of, I, 124-127 other, V, 67-89 Estrone sulfatase, V, 6-7 Ethanolamine deaminase activation by monovalent cations, VI, 545 assays, VI, 542 cobamide binding sites, VI, 543-544 inhibitors, cobamide, VI, 544-545 isolation, VI, 541-542 occurrence, VI, 540-541 other properties, VI, 546-547 physical properties, VI, 542-543 specificity of coenzyme requirement, VI, 544 substrate specificity and binding, VI, 545-546 a-Ethylmalate, synthesis, VII, 426 Etiocholanolone sulfatase, V, 9-10 Euglena gracilis, chloroplast adenosine triphosphatase, X, 394

Eukaryotes deoxyribonucleic acid polymerases of, X , 173-209 polypeptide chain initiation inhibitors, X, 43 initiation factors, X, 2943 initiator aminoacyl-transfer ribonucleic acid and, X, 28-29 messenger ribonucleic acid translation, X,43-44 ribonucleic acid polymerases, X,261331 Evolution allosteric proteins, I, 390393 principles, cytochrome c and, I, 274285 structure-function relationships in proteins, I, 267-274 Evolutionary factors expression in protein structure convergence, I, 328-329 development of novel properties, I, 334-337 divergence, I, 314-328 loss of function and survival value, I, 332-334 parallelism, I, 329-332 Exonuclease(s), bacterial, IV, 252-259 F Fast reaction techniques application of conformational changes, 11, 112-114 enzyme-substrate reactions, 11, 108112 Fatty acid(s) activation, VIII, 6-11 biosynthesis, acyl carrier protein and, VIII, 164-165 unsaturated, cis-trans isomerization, VI, 390-394 Fatty acid: coenzyme A ligases catalytic properties, general considerations, X, 475-477 Fatty acid synthetase(s), I, 226-228 3-decynoyl-N-acetylcysteamine and, V, 461-463 Fatty acyl coenzyme A synthetases distribution and isolation


acetate : coenzyme A ligase, X, 470 long chain fatty acid: coenzyme A ligase, X, 471-472 medium chain fatty acid :I coenzyme A ligase, X, 470-471 medium-long chain fatty acid: coenzyme A ligase, x, 472-473 other related acid: coenzyme A ligases, X, 473-474 scope of chapter, X, 469-470 Feedback inhibition metabolic regulation and, I, 403404 allosteric concept, I, 404406 cooperative effect, I, 406408 terminology, I, 408 Feedback regulation multifunctional pathways, I, 444445 aspartate metabolism, I, 457-459 concerted inhibition, I, 449-450 cumulative inhibition, I, 452454 enzyme multiplicity, I, 445-447 heterogeneous metabolic pool inhibition, I, 454-455 multivalent repression, I, 455-457 sequential controls, I, 447-449 synergistic inhibition, I, 450452 Fermentation, reductive monocarboxylic acid cycle and, VI, 213-214 Ferredoxin( s) bacterial background, XII, 37-39 chemical properties, XII, 4 4 4 6 physical properties, XII, 39-44 chemical properties apoproteins and reconstitution, XII, 25-28

chemical modification, XII, 28-29 electron transfer reactions, XII, 29 exopeptidases and, XII, 28-31 two irons per center background, XII, 15-20 chemical properties, XII, 25-31 perturbants of EPR spectra, XII, 23-25

physical properties, XII, 20-23 Ferredoxin-linked carboxylations general considerations, VI, 193-196, 214-216

a-ketobutyrate synthase, VI, 203-204 a-ketoglutarate synthase, VI, 201-203

a-ketoisovalerate synthase, VI, 205 phenylpyruvate synthase, VI, 205-207 pyruvate synthase, VI, 197-201 reductive carboxylic acid cycle of bacterial photosynthesis, VI, 207213

reductive monocarboxylic acid cycle of fermentative metabolism, VI, 213-214

Fibrinogen derivatives, thrombin and, 111,298-299 thrombin and, 111, 295-298 Ficin, 111, 538-542 Fish, glycogen synthetase of, IX, 358 Flavin coenzyme(s), structure and chemistry, XII, 423-425 Flavodoxins composition, molecular weight and purification, XII, 59-60 discovery, nomenclature and distribution, XII, 58-59 flavin mononucleotide in, XII, 65-66 flavin-protein interactions apoprotein preparation and properties, XII, 82-83 modified flavins and, XII, 85-87 protein modification and, XII, 87-88 thermodynamics and kinetics, XII, 83-85

function, XII, 60-63 oxidation-reduction potentials, XII, 98-102

reactivity comproportionation, XII, 102-103 dithionite, XII, 103-104 oxygen and ferricyanide, XII, 105108

redox proteins and, XII, 108-109 regulation by iron, XII, 63-65 spectroscopic properties circular dichroism, XII, 94-95 fluorescence, XII, 93-94 magnetic resonance, XII, 95-98 optical absorption spectra, XII, 88-91, 99

single crystal absorbance, XII, 91-93 structure, XII, 66-67 determination and comparison of chemical sequences, XII, 67-70 three-dimensional, XII, 70-82


Flavoprotein monooxygenase(s) external, XII, 204-206 bacterial luciferase, XII, 226-229 m-hydroxybenzoate-4-hydroxylase, XII, 225 m-hydroxybenzoate-6-hydroxylase, XII, 224-225 p-hydroxybenzoate hydroxylase, XII, 211-216 imidazoleacetate monooxygenase, XII, 225-226 kynurenine-3-hydroxylase,XII, 230-231 melilotate hydroxylase, XII, 21722I microsomal amine oxidase, XII, 229-230 orcinol hydroxylase, XII, 223-224 phenol hydroxylase, XII, 221-223 salicylate hydroxylase, XII, 206-211 internal, XII, 193-194 arginine, XII, 203-204 lactate, XII, 194-199 lysine, XII, 199-203 Flavoprotein oxidase ( s ) chemical mechanism flavin reduction, XII, 474-503 oxidation by oxygen, XII, 503-505 definition of, XII, 421-423 kinetics computer simulation, XII, 442-443 strategy, XII, 425-426 summary, XII, 443-445 mechanism confirmation, XII, 437442 correlation of steady-state and transient kinetics, XII, 435-437 molecular properties and kinetic mechanism n-amino acid oxidase, XII, 445-466 L-amino acid oxidase, XII, 456461 glucose oxidase, XII, 461-466 monoamine oxidase, XII, 466-471 old yellow enzyme, XII, 471-473 steady-state kinetics rate equation, XII, 429-432 velocity measurements, XII, 426429 transient-state kinetics oxidative half-reaction, XII, 435

493 reductive half-reaction, XII, 432431 Fluorescence, ribonuclease, IV, 718-719 Fluorescence spectroscopy protein structure, 11, 418421,429-430 typical cases, 11, 421-429 Fluorocitrate, aconitase and, V, 428-430 Formaldehyde (and congeners) transfer deoxycytidylate hydroxymethyltransferase, IX, 209-210 deoxyuridylate hydroxymethyltransferase, IX, 210 glycine decarboxylase, IX, 221-223 thymidylate synthetase, IX, 210-215 serine hydroxymethyltransferase, IX, 215-221 Formate (and congeners) transfer 5-amino-4-imidazole carboxamide ribonucleotide transformylase, IX, 204-205 formiminoglutamate formiminotransferase, IX, 206-207 formiminoglycine formiminotransferase, IX, 206 5-formiminotetrahydrofolate cyclodeaminase, IX, 202-203 N-formylglutamate transformylase, IX, 207-208 5-formyltetrahydrofolate cyclodehydrase, IX, 207-208 10-formyltetrahydrofolate deacylase, IX, 200 10-formyltetrahydrofolate synthetase, IX, 198-200 glycinamide ribonucleotide transformylase, IX, 203-204 5,lO-methenyltetrahydrofolate cyclohydrolase, IX, 201 methionyltransfer ribonucleic acid transformylase, IX, 208-209 Formiminoglutamate formiminotransferase, properties, IX, 206-207 Formiminoglycine formiminotransferase, properties, IX, 206 5-Formiminotetrahydrofolate cyclodeaminase, properties, IX, 202-203 N-Formylglutamate transformylase, properties, IX, 207-208

494 5-Formyltetrahydrofolate cyclodehydrase, properties, IX, 201-202 10-Formyltetrahydrofolate deacylase, properties, IX, 200 10-Formyltetrahydrofolate synthetase, properties, IX, 198-200 Frog muscle, glycogen synthetase of, IX, 357 Fructose-1,Bdiphosphatase(8) activation by disulfide exchange coenzyme A and acyl carrier protein, IV, 623-624 cystamine and, IV, 622-623 homocystine, IV, 624-625 as regulatory mechanism, IV, 625626 assay methods and mechanism of action, IV, 615-616 Candida utilis inhibition by AMP, IV, 636-637 purification and properties, IV, 635836 regulation, IV, 640 relation to SDPase, IV, 638 structure, IV, 637438 comparative properties, IV, 645646 Eschen'chia coli, IV, 638439 higher plants and blue-green algae physiological role, IV, 642-643 purification and properties, IV, 640642 regulation, IV, 643 historical review, IV, 612413 kidney purification and properties, IV, 629630 regulation, IV, 630431 molecular structure binding sites for divalent cation, IV, 628-629 binding sites for F D P and AMP, IV, 627-628 induced conformational changes, IV, 629 molecular weight and subunit structure, IV, 826-627 muscle evidence for presence. IV. 632 physiological ;ole, IV; 634-835


purification and properties, IV, 632633 structure and relation to other enzymes, IV, 633-634 physiological role, IV, 613-615, 644645 proteolysis, changes induced, IV, 618 purification and properties, IV, 629630, 632-633 optimum pH and cation effects, IV, 617418 purification procedures, IV, 616-617 substrate specificity, IV, 618 regulation, I, 439-441,IV, 613-615, 630431 modification of tyrosine residues and, IV, 619-620 papain and, IV, 619 pH and, IV, 618419 pyridoxal phosphate and, IV, 620 Saccharomyces cerevisiae, IV, 640 slime molds, IV, 640 sulfhydryl groups, activation and, IV, 621422 L-Fucose isomerase, properties, VI, 346348 L-Fuculose 1-phosphate aldolase catalytic reaction assay, VII, 314 equilibrium constant, VII, 314 substrate specificity, VII, 314 properties, VII, 313-314 Fumarase catalytic properties active site affinity labeling, P,563564

catalytic site number, V, 562-563 kinetics, V, 552-557 substrate specificity, V, 557-562 general considerations, V, 539-540 historical development, V, 540-541 isotope exchange and, 11, 304-308 mechanism of action, V, 564-568 modification of, I, 140-141 molecular properties amino acid composition, end groups and peptide maps, V, 544-545 dissociation and recombination of subunits, V, 546-549 physical properties, V, 542-544


subunit structure, V, 545-546 thiol groups, V, 549-552 preparation and assay, V, 541-542 stereospecificity, 11, 171-176 Fungi, see also Molds aldolase of, VII, 215-216 aspartate transcarbamylases of, IX, 302-306 carboxypeptidases of, 111, 790-791 glutamate dehydrogenases Neurospora crassa, XI, 323-329 others, XI, 329-332 nuclear ribonucleic acid polymerase, X, 310-311 ribonucleases of, IV, 208-239 G

n-Galactarate dehydrase, properties, V, 580-581 Galactonate dehydrase, properties, V, 578-579 Galactose oxidase chemical and physical properties, XII, 528-529 copper of, XII, 529-530 discovery and purification, XII, 527528 inhibitors, XII, 531 mechanism, XII, 532-533 molecular properties and kinetic mechanism, XII. 461-466 optical properties, XII, 530-531 specificity, XII, 531 p-Galac tosidase assay and standardization, VII, 620623 bacterial suspensions and animal material, VII, 624 standard assays and units, VII, 623 transferase assay, VII, 624 chemical properties, VII, 636-639 enzymic properties active site, VII, 657-658 condensation reactions, VII, 660 inhibition studies, VII, 660-661 kinetics, VII, 648-4351 mechanism, VII, 651-657 metal activation, VII, 645-648

other sources, VII, 661-663 pH dependence, VII, 644-645 specificity, VII, 641-644 transfer reaction, VII, 658-660 general background, VII, 618 immunological properties, VII, 839641 mutations and, I, 255-256 occurrence, VII, 619-620 physiochemical properties associated forms, VII, 633-634 denaturation and renaturation, VII, 634-636 size, shape and quaternary structure, VII, 627-633 purification animal tissues, VII, 626-627 Escherichia coli, VII, 624-625 other microorganisms, VII, 625426 Galactosyl transferase biological significance, IX, 377 catalytic properties kinetics, IX, 371-376 reaction catalyzed, IX, 369 substrate specificity, IX, 369-371 purification and properties, IX, 367369 Gastricin, gene duplication and, I, 308 Gastric lipase, properties, VII, 605-606 Gaucher’s disease, acid phosphatase and, IV, 496 Genes cistron and types of mutations, I, 243-245 duplication, I, 3 W 3 0 3 carboxypeptidases, I, 308-309 ethanol dehydrogenaaes, I, 309-311 gastricin, I, 308 a-lactalbumin, I, 311-314 lysozyme, I, 311-314 pancreatic proteinases, I, 303-307 pepsin, I, 308 rennin, I, 308 sulfhydryl proteinases, I, 307-308 Genetic drift, speciation of homologous proteins and, I, 321428 Glucagon, glycogen synthetase and, IX, 341 a-Glucan phosphorylases allosteric transitions, VII, 473-474

496


desensitization, VII, 480-482 kinetic and structural basis, VII, 474-480 catalytic reaction nucleotide activation, VII, 439-441 substrate specificity, VII, 437-438 chemical properties amino acid analysis and end groups, VII, 446-447 primary structure, VII, 448-451 enzyme structure, physical properties, VII, 441-446 general considerations, VII, 435-437 mechanism of catalysis kinetics, VII, 460-463 protein functional groups, VII, 463466 vitamin Ba coenzyme, VII, 451-459 regulation associationdissociation phenomena, VII, 469-473 interconversion of a and b forms, VII, 466469 mechanism of allosteric transitions, VII, 473-482 o-Glucarate dehydrase, properties, V, 580 Gluconate dehydratase, properties, V, 578 Glucosamine-6-phosphate isomerase, properties, VI, 314-318 Glucosaminate dehydrase, properties, V, 586

D-c%ICOSeisomerase nature of, VI, 341-344 properties, VI, 349-354 Glucose8phosphatae.e distribution, IV, 600-610 catalytic properties, IV, 565-588 assay methods, IV, 566-567 control of phosphotransferase activity, IV, 592-595 kinetics and reaction mechanism, IV, 572-592 reactions catalyzed, IV, 567-571 thermodynamic considerations, IV, 571-572 detergents and, IV, 556-557 detergent-like effects, IV, 559-560 direct effects, IV, 557-559 modifying effects, IV, 560-562

historical phosphohydrolase activity, IV, 545546 phosphotransferase activity, IV, 546-547 kinetic studies, IV, 572-574 activators and inhibitors, IV, 578582 mechanism, IV, 582-592 pH and, IV, 574-576 substrate concentration, IV, 576-577 temperature, IV, 577-578 membranous nature, possible significance, IV, 562-564 metabolic roles and regulation, IV, 596-597 control in vivo, IV, 597-599 speculation, IV, 599 phospholipids and, IV, 554-556 reactions catalyzed multifunctional nature, IV, 567-568 substrate specificity, IV, 568-571 relation to other enzymes, IV, 552 solubilization and attempted purification, IV, 553-554 Glucose-6-phosphate distribution intracellular, IV, 548-551 phylogenetic and tissue, IV, 547-548 glycogen synthetase and, IX, 341-344 Glucose-6-phosphate isomerase catalytic properties active form of substrate, VI, 293-294 anomerase activity, VI, 294-296 assay, VI, 278-279 kinetic parameters, VI, 289-293 mechanism of action, VI, 296-301 molecular architecture active site amino acids, VI, 285-287 amino acid composition, VI, 278-279 conformational states, VI, 284-285 molecular weight and related properties, VI, 276-278 multiple forms, VI, 281-284 subunit structure, VI, 279-28E occurrence and function, VI, 272-276 Glucosidase-transferase assay of, V, 211-212 glycogen storage disease and, V, 221222


Glucosulfatase, V, 11 Glutamate pyrrolidone carboxylate formation from, enzymic, IV, 133-139 nonenzymic, IV, 130-133 D-Glutamate cyclo transferase, pyrrolidone carboxylate formation and, IV, 133-136 L-Glutamate cyclotransferase, pyrrolidone carboxylate formation and, IV, 138 Glutamate dehydrogenase(s), cellular location, XI, 305-306 chemical modification, coenzyme site and specificity, XI, 352-354 cysteine residues, XI, 347-348 histidine residues, XI, 346-347 lysine residues, XI, 343-346 other modifications, XI, 348-349 spectrophotometric studies, XI, 349 substrate site, XI, 349-352 composition, XI, 320-322 distribution and coenzyme specificity, XI, 296-297 animals, XI, 300-305 bacteria, XI, 297-299 fungi, XI, 297 plants, XI, 299-300 electrophoretic and spectrophotometric properties, XI, 322-323 function and equilibrium, XI, 294-295 genetics and regulation of enzyme synthesis, bacteria, XI, 332-334 fungi, XI, 323-332 plants, XI, 334-335 historical background, XI, 295-296 kinetic studies, XI, 354-357 metamorphosis and, XI, 306 modification of, I, 137 oligomer structure, hexameric model, XI, 315-317 immunochemistry, XI, 317-318 molecular weight, XI, 314-315 trimer formation, XI, 317 polymerization, mechanism, XI, 308, 310-312 nucleotide effects, XI, 312-314 significance, XI, 307-308 primary structures, XI, 335-343

purification of, XI, 307 reaction mechanism, XI, 357-360 regulation of, I, 443-444, XI, 360-366 stability, denaturation and dissociation, XI, 319-320 tertiary structure, XI, 318-319 Glutamate mutase assay, VI, 524-525 catalytic properties conditions affecting activity, VI, 531-532 equilibrium, VI, 532 interaction of components and coenzyme, VI, 528-530 mechanism, VI, 532-534 substrate and coenzyme specificity, VI, 530-531 purification and molecular properties component E, VI, 525-526 component S, VI, 526-528 stereochemistry, 11, 206-210 Glutaminase(s) Azotobacter agik's, IV, 97-98 Escheiichia coli, IV, 80-81 acyl transferase reactions, IV, 84-85 assay, IV, 81 deuterium oxide and, IV, 90 6-diazo-5-oxonorleucine and, IV, 85-87 mechanism of action, IV, 90, 92-93 occurrence, IV, 81 other inhibitors, IV, 87 pH effects on kinetic parameters, IV, 88,89 purification, IV, 82 relationship to other acylases, IV, 90, 91 specificity, IV, 82-84 temperature and, IV, 88 survey, IV, 93-95 Glutamine occurrence and function of, X, 699704, 755-757 pyrrolidone carboxylate formation from enzymic, IV, 139-141 nonenzymic, IV, 130-133 L-Glutamine cyclotransferase, pyrrolidone carboxylate formation and, IV, 139-141

498


Glutamine synthetase, X, 757-759 active site mapping, X, 720-733 adenylylation and deadenylylation, VIII, 4044 historical development of problem, X, 784-786 regulatory protein, X, 786-787 uridylylation and deuridylation of regulatory protein, X, 787-789 adenylylation effect biosynthetic activity, X, 794-796 y-glutamyltransferase activity, X, 796-800

adenylylation state determination enayme assays, X, 802-803 spectral analysis, X, 802 cascade control, X, 789-792 covalent modification of, I, 409-413 cumulative feedback inhibition, X, 776 mechanism, X, 777-780 divalent cation control adenosine triphosphate: cation ratio,

x,783-784

relaxation phenomena, X, 781 specificity, X, 781-783 general catalytic properties, X, 708-709 hybrid forms, X, 804-807 intermediate formation of y-glutamyl phosphate, X, 716-720 mechanism, X, 733-743, 759-763 multiple molecular forms, X, 800-801 determination of state of adenylylation, X, 802-803 heterologous interaction, X, 804-807 preparation of partially adenylylated form, X, 801-802 partially adenylylated direct isolation, X, 801 in vitro preparation, X, 801 mixing of forms, X, 802 subunit dissociation and reassociation, X, 802 partial reactions adenosine triphosphate formation,

X, 714-715 arsenolysis of glutamine, X, 713 cycloglutamyl phosphate synthesis, X, 715 exchange reactions, X, 709-710

methionine sulfoxime phosphate formation, X, 714 boxoproline formation, X, 713-714 physical and chemical properties, X, 704-708

physicochemical properties, X, 792-793 adenylylation site, X, 793-794 pyrrolidone carboxylate formation by, IV, 136-137 regulation of, X, 743-754, 775-792 repression of formation, X, 780-781 structure chemical composition, X, 769-771 physical characteristics, X, 763-769 taut, relaxed and tightened forms, X, 771-775

Glutaminyl peptides, pyrrolidone carboxylate formation from, IV, 139-141 y-Glutamyl amino acids, pyrrolidone carboxylate formation from, IV, 142-146

y-Glutamyl cyclotransferase, pyrrolidone carboxylate formation and, IV, 141-146

y-Glutamyl-cysteine synthetase general catalytic properties, X, 676-678 mechanism, X, 683-687 partial reactions adenosine triphosphate hydrolysis,

X, 678-679 enayme4activated glutamatelcomplex formation, X, 683 exchange reactions, X, 680-681 5-oxoproline formation, X, 679-680 phosphorylation of methionine sulfoxime, X, 681-683 pyrophosphate synthesis, X, 679 physical properties, X, 675-676 pyrrolidone carboxylate formation and, IV, 136-137 sources, purification and assay, X, 674-675

y-Glutamyl lactamase, see y-L-Glutamyl cyclotransf erase y-Glutamyltransferase agaricaceae, IV, 95-96 survey, IV, 93-95 y-Glutamyl transpeptidase kidney, IV, 96-97

499


pyrrolidone carboxylate formation and, IV, 141 Glutathione S-epoxidetransferase, VII, 205-206

catalytic properties factors influencing, VII, 209-210 kinetics and specificity, VII, 209 measurement of reaction, VII, 208 notes on mechanism, VII, 210 products of reaction, VII, 208 molecular properties molecular weight, VII, 207 multiple forms, VII, 206-207 purification, VII, 206 stability, VII, 208 Glutathione reductase kinetic studies, XIII, 138-141 metabolic functions, XIII, 129-133 thiol groups, XIII, 141-142 two-electron-reduced enzyme, properties, XIII, 133-138 Glutathione synthetase acyl phosphate intermediate, X, 690-692

general catalytic properties, X, 689-690 historical background, X, 671-674 mechanism, X, 693-695 partial reactions, X, 693 physical properties, X, 688-689 Glyceraldehyde-&phosphate dehydrogenase, historical background, XIII, 2-3 isolation, XIII, 3-4 mechanism of action other activities, XIII, 44-49 physiological activity, XIII, 38-44 metabolic role, XIII, 45-49 pyridine nucleotide binding, XIII, 28-30

cooperativity of, XIII, 30-35 preexisting asymmetry model, XIII, 35-38

reaction catalyzed, XIII, 1 structure apoenzyme, XIII, 19-20 chemical modification, XIII, 20-24 dissociation and hybridization, XIII, 24-27 holoenzyme X-ray structure, XIII, 9-19

primary, XIII, 5-9 Glycerate kinases assay and distribution, VIII, 504-505 catalytic properties, VIII, 507-508 metabolic role, VIII, 505-506 molecular properties purification and state of purity, VIII, 506-507 stability, VIII, 507 Glycerol kinases assay and distribution, VIII, 488492 catalytic properties product inhibition, VIII, 501-502 substrate specificity and kinetics, VIII, 497-501 thermodynamics, VIII, 502 metabolic role, VIII, 492-493 molecular properties chemical modification, VIII, 496-497 composition, VIII, 494 purification and state of purity, VIII, 493494 size and subunit structure, VIII, 494-495

stability, VIII, 495-496 regulation mammals, VIII, 504 microorganisms, VIII, 502-503 L-Glycerol-3-phosphate dehydrogenase, properties, XIII, 256-260 Glycidol phosphate, enolase and, V, 534 Glycinamide ribonucleotide transformylase, properties, IX, 203-204 Glycine, oxidative decarboxylation of, IX, 221-223 Glycine amidotransferase biological distribution, IX, 498499 catalytic properties reaction mechanism, IX, 500-503 substrate specificity, IX, 499-500 regulation, IX, 503305 Glycogen structure determination, debranching enzymes and, V, 228-234 Glycogen phosphorylase, covalent modification of, I, 413415 regulation of, I, 441442 Glycogen storage disease, glucosidasetransferase and, V, 221-222

500


Glycogen synthetase adrenal, IX, 353 assay, IX, 317418 association with glycogen, IX, 311-312 blood erythrocytes, IX, 355 leukocytes, IX, 354-355 platelets, IX, 355 brain, IX, 353-354 control of activity epinephrine effect, IX, 338, 341, 351-352

glucagon and, IX, 341, 351-352 glucose and, IX, 347-348 glycogen and, IX, 336, 340 insulin activation, IX, 336-338, 340-341, 348-351

muscle contraction events, IX, 338-340

donor specificity, IX, 318 general properties of a and b forms, IX, 324-325 glucosyl acceptor de novo synthesis, IX, 320-321 oligosaccharides, IX, 320 polysaccharides, IX, 319-320 heart, IX, 340-341 historical background, IX, 310-311 inactive form, IX, 330-331 kinase, IX, 326-327 liver, IX, 341-353 muscle, IX, 332340 nomenclature, IX, 322-324 nonmammalian organisms, IX, 357-361 phosphatase liver, IX, 328-330 muscle, IX, 327-328 physicochemical properties muscle, IX, 313-315 other tissues, IX, 316-316 properties of the two forms activity of, IX, 335-336 glucose 6-phosphate and, IX, 332-334, 341-344

inorganic phosphate and, IX, 334, 344-345

magnesium and other ligands, IX, 335, 345

nucleotides, IX, 334-335, 345 pH and, IX, 335, 346

physiological conditions and, IX, 348347

proteolytic inactivation, IX, 331 purification, IX, 312-313 reaction catalyzed, IX, 316-317 reaction mechanism, IX, 321-322 regulation of, I, 441-442 spleen, IX, 354 system of interconversion, IX, 325-326 tumors, IX, 356-357 tissues sensitive to sex hormones, IX, 355-356

Glycosidases, modification of, I, 128-129 Glycosulfatase (s) ccllulose polysulfatase, V, 11-12 cerebroside sulfatase, V, 13-14 chondrosulfatase, V, 12 glucosulfatase, V, 11 Glyoxylate condensing enzymes, general remarks, VII, 411412 Gout, molybdenum hydroxylase genetics and, XII, 400-402 Gram-negative bacteria phage lytic enzymes Aerobacter cloacae, V, 398 Azotobacter, V, 397498 Klebsiella pneumoniae, V, 394-395 Pseudomonas, V, 395-397 Salmonella, V, 398 Gram-positive bacteria phage lytic enzymes Bacillus megaterium, V, 408410 Bacillus stearothermophilus, V, 410-411

Micrococcus lysodeikticus, V, 401402 staphylococci, V, 398401 streptococci, V, 402-408 Guanidine hydrochloride, inorganic pyrophosphatase and, IV, 508-510 Guanidino kinase(s) bivalent metal ions and, VIII, 471473 catalytic reaction, VIII, 459 chemical stop assays forward reaction, VIII, 464 reverse reaction, VIII, 465 continuous recording assays potentiometric, VIII, 466 spectrophotometric, VIII, 465406 discovery and isolation, VIII, 459461 distribution and function, VIII, 461-464


equilibrium, VIII, 485-486 function of amino acid residues cysteine, VIII, 477-480 others, VIII, 480-482 historical background, VIII, 457-459 isotopic assays, VIII, 465 mechanism, VIII, 482-485 molecular properties amino acid composition, VIII, 469470

immunological reactions, VIII, 470-471

molecular weight and subunit composition, VIII, 466-468 stability, VIII, 468-469 pH optimum, VIII, 476-477 substrate specificity general comments, VIII, 473 guanidines, VIII, 474-476 nucleotides, VIII, 476 Guanine aminohydrolaw, IV, 76-77 Guanosine aminohydrolase, IV, 77-78 Guanosine diphosphate-n-mannose oxidoreductase properties, V, 479 Guanosine kinase, see Inosine-guanosine kinase Guanosine monophosphate, synthesis, VIII, 39-40 Guanosine monophosphate kinase assay, IX, 84 distribution and purification, IX, 82-84 kinetic and molecular properties, IX, 84-85

substrate specificity; reaction mechanism, IX, 85-86 Guanosine 5'-phosphate reductase, modification of, I, 137 Guinea pig serum, asparaginase of, IV, 105-107 H

Heart aspartate transaminase of, IX, 398-462 glycogen synthetase of, IX, 340-341 mitochondria1 adenosine triphosphatase catalytic properties, X, 382-383 cold inactivation, X, 380 inhibitors and activators, X, 385-386 molecular properties, X, 378-380,381

501 nature of active site, X, 383 purification and stability, X, 377-378 5'-nucleotidase of, IV, 347-348 succinyl coenzyme A synthetase of, X, 591-594 Heavy atom derivatives globular macromolecules factors in derivative formation, I, 73-86

preparation and classification, I, 70-73

useful heavy atom compounds, I, 73 Helicorubin, properties, XI, 592 Heme proteins, modification of, I, 142 Hemoglobin allosteric properties, I, 388-390 mutations and, I, 257-259 Hemoprotein(s), cytochro,me M i k e , XI, 576-577 Hexarate dehydrase(s), properties, V, 579-581

Hexokinase (s) aggregation phenomenon, IX, 40-41 amino acid composition and essential groups, IX, 41 chemical studies comparison of native isoenzymes, IX, 10-12 nature of proteolytic modification, IX, 12-13 comparative aspects, IX, 46-48 isoelectric point, IX, 41 isoenzymes and modification by endogenous proteases, IX, 2-6 mechanism adenosine triphosphatase reaction, IX, 18-19 conformational changes, IX, 27-28 enzyme-substrate interaction and, IX, 43-44 equilibrium measurements, IX, 19-22 kinetic studies, IX, 13-17, 41-43 phosphoenzyme intermediate, IX, 23-24, 44

sulfhydryl groups and, IX, 25-27 modification by added proteases, IX, 6-7

molecular weight and subunit structure, IX, 39-40


molecular weight in nondenaturing solvents, IX, 7-8 multiple forms, IX, 31-33 purification procedures, IX, 37-38 regulation, IX, 29-31, 4446 relation of soluble to insoluble forms, IX, 33-37 subunit size, IX, 8-10 Hill plots, enzyme regulation and, I, 358-359

Hirudin, thrombin and, 111, 304405 Histidine ammonia-lyase catalytic process function of prosthetic group, VII, 159-162

metal ion activation, VII, 162-163 prosthetic group, VII, 154-159 rate limiting step, VII, 164-166 reaction sequence, VII, 148-154 distribution, purification and kinetic properties, VII, 137-142 mechanism of action, VII, 195-196 size and constitution, VII, 146-148 Histidine deaminase, role of metals, 11, 509-510

Histidine decarboxylase, Schiff base and, 11, 356-357 Histidine residues aspartate transcarbamylase, IX, 267 chemical modification, I, 173 chymotrypsin catalytic reaction and, 111, 234 chemical modification, 111, 234 pH and, 111, 231-233 creatine kinase, VIII, 434 phosphofructokinase, VIII, 272 ribonuclease, IV, 685-689 subtilisin, 111, 580-584 Histone kinases, properties, VIII, 579-580 Homoarginine residues, proteinase inhibitors and, 111, 461-452 Homocitrate synthase, properties, VII, 425-426

Homocysteine synthetase(s1, properties, VII, 61-62 Homoserine dehydratase, properties, VII, 67-59 Homoserine dehydrogenase, see Aspartokinase

Hormones, phosphofructokinase and, VIII, 277-278 Hyaluronate-endo-p-glucuronidase, V, 313 Hyaluronate lyase, V, 313-314 Hyaluronidase (s) bacterial, V, 309 biological effects, V, 319-320 historical background, V, 307-308 kinetics, V, 318-319 leech, V, 313 lysosomal, V, 312 microbial, V, 313-314 preparation and assay, V, 315-317 properties, V, 318 submandibular, V, 312-313 substrates additional, V, 311 chondroitin, V, 310 chondroitin 4- and B-sulfate, V, 310 dermatan sulfate, V, 310-311 hyaluronate, V, 309 testicular, V, 311-312 testicular type, V, 308,311-313 Hydrogenase, proton transfer and, 11, 317-318

Hydrogen bonds, propinquity effects and, 11, 254-264 Hydrolase(s), role of metals in mechanism, 11, 519-525 D-2-Hydroxy acid dehydrogenase, properties, XIII, 272-273 erythro-p-Hydroxyaspartate dehydratase, properties, VII, 47-48 m-Hydroxybenzoa te-4-hydroxylase, properties, XII, 225 m-Hydroxybenroa te-6-hydroxylase, properties, XII, 224-225 p-Hydroxybenzoate hydroxylase, properties, XII, 211-216 8-Hydroxydecanoyl thioester dehydrase distribution, V, 464 function of, V, 441443 general considerations, V, 441443 inhibition by substrate analogs, V, 455-456

acetylenic inhibitor reaction site, V, 456-458

3-decynoyl-N-ace tylcysteamine and fatty acid synthetase, V, 461-463


inhibitor specificity, V, 458459 isomeriration of 3-decynoyl-Nacetylcysteamine, V, 459-461 modification of, I, 141 mutants, V, 463 protein structure active site, V, 453-455 disulfide bridges, V, 452-453 nitration, V, 455 reaction mechanism isotope effects and labeled substrates, V, 449-450 kinetic studies, V, 446-449 trapping of intermediates, V, 451-452 substrate specificity, V, 444-445 optical and geometric, V, 446 thioester, V, 445-446 4-Hydroxy-2-ketoglutarate aldolase, properties, VII, 299-301 5-Hydroxymethyl deoxycytidine monophosphate kinase, properties, IX, 94-95 8-Hydroxy-8-methylglutarylcoenzyme A cleavage general, VII, 432-433 properties of beef liver enzyme, VII, 433-434 ,8-Hydroxy-8-methylglutaryl coenzyme A synthase distribution, VII, 429 general considerations, VII, 427428 properties of yeast enzyme, VII, 429-431 stereochemistry of product, VII, 428 p-Hydroxyphenylpyruvate hydroxylase, XII, 179-180 catalytic properties, XII, 180-183 purification and molecular properties, XII, 180 I

Imidazole rings, trypsin, 111, 269-270 Imidazolylacetate monooxygenase, properties, XII, 225-226 Imido adenylate derivatives synthesis, VIII, 37 argininosuccinate, VIII, 38-39 guanosine monophosphate, VIII, 39-40

503 Immunochemistry, chemical modification and, I, 199-200 Inclusion compounds, propinquity effects and, 11, 274-279 Indoleamine 2,3-dioxygenase historical, XII, 130-131 molecular and catalytic properties, XII, 131-132 Influenza virus neuraminidase isolation and purification, V, 328 purification of virus particles, V, 327-328 Infrared spectroscopy, enzyme structure and, 11, 374-379 Inhibition studies nomenclature and basics, 11, 18-21 prediction of patterns, 11, 21-25 types of experiments, 11, 25-43 Initial velocity studies bireactant, 11, 7-10 prediction of patterns, 11, 10-13 terreactant, 11, 13-18 Initiation factors discovery and function, X, 6-12 eukaryote miscellaneous, X, 42-43 ribosome associated, X, 29-33 supernatant, X, 33-42 initiation cycle and, X, 12-13 properties factor-1, X, 26-28 factor-2, X, 13-17 factor-3, X, 7-26 Inorganic ions metabolic regulation and divalent cations, I, 420-423 monovalent cations, I, 419-420 Inorganic pyrophosphatase catalytic properties assay, IV, 534 interaction with inhibitors, IV, 525-526 ion and inhibitor effects, IV, 518-519 kinetics, IV, 535-538 mechanism, IV, 538-539 nature and binding of active substrate and role of magnesium, IV, 522-525 pH effects, IV, 518

504


reversal of reaction, IV, 519-520 substrate specificity and stoichiometry, IV, 520-522,534-535 chemical composition amino acid content, IV, 512 cyanogen bromide cleavage products, IV, 514 N- and C-terminal amino acids, IV, 512-514

tryptic digest maps, IV, 514 chemical modification, IV, 514-515 cyanate and diazonium-1H-tetrazole, IV, 516-518 2,4&trinitrobenzene sulfonate, IV, 515-516

molecular properties electron microscopy, IV, 506-508 homogeneity, IV, 502-503 metalloenzyme, IV, 532-534 physicochemical parameters, IV, 530-531

purification, IV, 501-502, 530 reconstitution from subunits, IV, 510-612

reversible divalent cation binding, IV, 531-532 size, IV, 504 other, IV, 539-541 physical properties optical properties, IV, 505-506 sedimentation and diffusion coefficients, IV, 504 viscosity, IV, 505 subunits in quanidine hydrochloride optical properties, IV, 510 sedimentation and diffusion coefficients and intrinsic viscosity, IV, 509

size, IV, 509 Inosamine phosphate amidinotransferase biological distribution, IX, 505-506 catalytic properties reaction mechanism, IX, 507-508 substrate specificity, IX, 506-507 regulation, IX, 508-509 Inosine-guanosine kinase, properties, IX, 54-56 Inosinic acid dehydrogenase, modification of, I, 137-138

Insects, alcohol dehydrogenase of, XI, 189-190

Insulin, glycogen synthetase and, IX, 336338, 340-341

Intestine monoglyceride lipase of, VII, 603-605 5'-nucleotidase of, IV, 345 Intramolecular reactions, miscellaneous, 11, 238-250 Invertase, V, 292-293 biosynthesis, V, 294-295 catalytic properties, V, 305 active site, V, 300-301 inhibitors, V, 301-302 kinetics, V, 302 mechanism, V, 302-303 determination, V, 292 localization and multiple forms, V, 293-294

properties, V, 298-300 purification, V, 295-298, 304-305 Invertebrates, cytochrome b-like pigments of, XI, 564 Ionizable groups, protein structure and, 11, 430-440 Iron-sulfur proteins, categories conjugated, XII, 3-4 ferredoxin, XII, 2 high potential, XII, 2-3 eight irons background, XII, 37-39 chemical properties, XII, 4 4 4 6 physical properties, XII, 3944 four irons per center background, XII, 31-33 high potential proteins, XII, 35-37 low potential proteins, XII, 34 Iron-sulfur enzymes, XII, 47-50 mitochondrial, XII, 56 model compounds, XII, 48-47 nitrogenase system, XII, 60-56 nomenclature, XII, 2 xanthine oxidase, XII, 56 Isoamylase (8) plant, V, 208 pseudomonad and Cytophaga, V, 204-206

yeast, V, 206-208


Isocitrate dehydrogenase, stereospecificity, 11, 168-171 Isocitrate lyase, VII, 382-383 assay and isolation, VII, 383 molecular properties cofactors, VII, 384 inhibitors, VII, 385 molecular weight and subunits, VII,

proton transferase and, 11, 283-285 rate equations, 11, 63-65

Jack bean ureaae of, IV, 2-5

384

sulfhydryl groups, VII, 385 Isoleucine residues chymotrypsin chemical modification, 111, 238-239 conformation and, 111, 239-243 pH and, 111, 236-237 chymotrypsinogen, 111, 175-176 Isomerase(s) aldo-keto, proton shifts and, 11,290-295 Isopentylpyrophosphate isomerase assays, evaluation, VI, 567-568 function, VI, 566-567 mechanism, VI, 570-572 occurrence, VI, 565-566 properties inhibitors, VI, 569 metal ion and, 569 Michaelis constant, VI, 569 pH optimum, VI, 568 reversibility and position of equilibrium, VI, 569-570 synthesis of substrate and product, VI, 567

dsopropylmalate synthase properties, VII, 423 effect of leucine, VII, 424-425 Isotope effects primary, 11, 284-285 examples, 11, 325-329 origin and magnitude, 11, 322-324 pepsin and, 111, 180 secondary, 11, 285-287 examples, 11, 331-333 origin and magnitude, 11, 329-331 Isotopic exchange kinetics basic considerations, 11, 4 3 4 4 ping pong mechanism, 11, 48-52 sequential mechanism, 11, 44-48 lack of, 11, 287

K

Kallikreins, 111, 482-483 Keratinase, microbial, 111,763-765 p-Keto acid decarboxylase(s), Schiff bases and, 11, 359 p-Ketoacyl acyl carrier protein synthetase catalytic properties assays, VIII, 190 mechanism, VIII, 194-199 pH optimum, substrate specificity and kinetics, VIII, 190-194 historical background, distribution and metabolic significance, VIII, 188-189

molecular properties, VIII, 189-190 a-Ketobutyrate synthase, properties, VI, 203-204

2-Keto-3-deoxy-~-arabonate aldolase, properties, VII, 296 2-Keto3-deoxy-~-arabonate dehydratase, properties, V, 583-585 2-Keto-3-deoxy-o-fuconate aldolase, properties, VII, 296 2-Keto-3-deoxy-D-glucarate aldolase properties, VII, 297 5-Keto-4deoxyglucarate dehydratase, properties, V, 585-586 2-Keto3-deoxy-manno-octosonate aldolase, properties, VII, 298 2-Keto-3-deoxy-6-phosphogalactonic aldolase, properties, VII, 295-296 2-Keto3deoxy-6-phosphogluconic aldolase general properties Pseudomonas putida, VII, 283-285 Pseudomonas saccharophila, VII, 285 mechanism isotope exchanges, VII, 290-291 oxalacetate decarboxylation, VII, 291-292


role of an additional base, VII, 292-295 role of Schiff base formation, VII, 289-290 modification of, I, 139 structure other characteristics, VII, 287-288 subunit composition and arrangements, VII, 285-287 3-Keto-dihydrosphingosine, formation of, VII, 355-356 a-Ketoglutarate dehydrogenase complexes, I, 224-225 a-Ketoglutarate synthase, properties, VI, 201-203 a-Ketoisovalerate synthase, properties, VI, 205 A'3-Ketosteroid isomerase animal tissue catalytic mechanism, VI, 617-618 distribution and properties, VI, 615-617 catalytic mechanism hydrogen isotopes and, VI, 605-607 proposal for, VI, 612-615 ultraviolet and fluorescence studies, VI, 607-612 catalytic properties active-sitedirected inhibitor, VI, 601 competitive inhibitors, VI, 6oo-gO1 kinetic studies, VI, 604 metal ions, chelators and urea, VI, 800 pH effects, VI, 602 reversibility, VI, 603-604 substrate specificity, VI, 59WOo thermodynamic parameters, VI, 602-603 historical, VI, 591-592 molecular properties chemical modification, VI, 595 induction, purification and crystallization, VI, 592-593 structure, VI, 598-599 ultracentrifugation, VI, 593-594 ultraviolet and fluorescence spectra, VI, 594-595 Kidney argininosuccinase of, VII, 178-179

fructose-1,6-diphosphatase purification and properties, IV, 629-630 regulation, IV, 630-631 7-glutamyl transpeptidase of, IV, 96-97 vitamin Ba methyltransferase of, IX, 164-165 Kineticist tools of, 11, 6-7 inhibition studies, 11, 18-43 initial velocity studies, 11, 7-18 isotope exchange, 11, 43-52 miscellaneous, 11, 52-61 Kinetics, basic theory, 11, 6 nomenclature and notation, 11, 2-5 Kinetic systems far from equilibrium computer solutions to rate equations, 11, 74-83 integrated rate expressions, 11, 71-74 single substrate-single product Michaelis-Menten mechanism, 11, 74-79 near equilibrium relaxation amplitudes, 11, 99-108 relaxation spectra, 11, 83-99 Kinin-destroying enzymes, 111, 483 Kinin-forming enzymes catheptic kininogenases, 111, 483 kallikreins, 111, 482483 Klebsiellu pneumoniae phage polysaccharide depolymerase enzyme properties and role, V, 394-395 preparation and assay, V, 394 Klotz plots, enzyme regulation and, I, 359-361 Kynurenine3hydroxylase, properties, XII, 230-231

1

a-Lactalbumin bovine, VII, 681-682 gene duplication and, I, 311-314 lactose synthetase and, IX, 366 8-Lactamase, see Penicillinase Lactate dehydrogenase, away, XI, 199-200

507

TOPICAL SUWECT INDEX

coenzyme binding oxidized, XI, 280-281 reduced, XI, 277-280, 281 fluorescence and spectroscopy coenzyme, XI, 268 protein, XI, 264-268 histidine-195, state of protonation, XI, 289 historical, XI, 192-195 isolation, XI, 200-202 isozymes, evolution of genes, XI, 198-199 general, XI, 195 known genes, XI, 195-198 D-lactate specific enzymes, XI, 199 kinetic studies, transient phase, XI, 57-59 mechanism, XI, 289-292 modification, I, 136 crystals, XI, 261 mild proteolysis, XI, 257-258 side chains, XI, 258-261 physical properties, XI, 261-264 reactions catalyzed cyanide addition, XI, 270 glyoxylate and XI, 269-270 ketopyruvate reduction, XI, 268-269 reviews, XI, 192 steady-state kinetics noninhibiting substrate concentrations, XI, 270-273 steady-state inhibitors, XI, 273-274 structure, amino acid sequence, XI, 202-205 composition, XI, 202, 203 oligomer, XI, 250-257 threedimensional structure, XI, 205-250

substrate binding, XI, 290-291 absence of enzyme-substrate compounds, XI, 281-282 active ternary complex, XI, 284-286 enzyme-anion complexes, XI, 282 enzyme :coenzyme :inhibitor complex, XI, 282-284 ternary complex transient kinetics, XI, 286-289 substrate inhibition lactate. XI. 275-276 pyruvate, XI, 274-275

significance of abortive complex, XI, 276-277

D(-)-Lactate

dehydrogenase, XIII,

269-270

enzymic properties, XIII, 270-272 physical properties, XIII, 270 L( +)-Lactate dehydrogenase cytochrome b, core, XIII, 266-267 enzymic properties, XIII, 267-269 historical background, XIII, 263-264 physical properties, XIII, 264-266 Lactate oxidase, properties, XII, 194-199 Lactic acid racemase, properties, VI, 380 Lactobacillus, histidine decarboxylase of, 11, 356-357 Lactose synthetase historical background, IX, 364-365 requirement for two proteins identification of a-lactalbumin, IX, 366

relationship to lysozyme, IX, 366-367 resolution of, IX, 365466 Leaves, adenosine diphosphoryl glucose pyrophosphorylase of, VIII, 86-90 Leech, hyaluronidase of, V, 313 Leucine aminopeptidase assay, 111, 83-84 chemical properties, 111, 88-89 enzymic properties inhibitors, 111, 96-98 mechanism of action, 111, 98-100 role of metal ion, 111, 89-91 substrate specificity and kinetics, 111, 91-96 historical background, 111, 82-83 physical properties, 111, 86-88 purification, 111, 84-86 use in sequence studies, 111, 101-102 Lipase(s1 activity, determination of, VII, 578680

castor bean, VII, 613-614 catalytic properties active site, VII, 593-595 chemical structure of substrate, VIZ, 595-599

colipase and, VII, 595 effectors and, VII, 594601 ester bond synthesis and, VII, 601602

508


phosphoglycerides and, VII, 602-603 positional specificity, VII, 591-593 substrate physical state, VII, 586591

definition of, VII, 575-577 distribution animal, VII, 577 microorganisms, VII, 578 plant, VII, 577-578 gastrointestinal gastric, VII, 605-606 intestinal, VII, 603-605 microorganisms, VII, 614-616 milk, VII, 611-613 pancreatic, VII, 580-581 catalytic properties, VII, 586-603 molecular properties, VII, 583-586 purification, VII, 581-583 tiasue adipose tissue hormone sensitive, VII, 609-610 lipoprotein lipase, VII, 606-609 liver lipases, VII, 611 Lipoamide dehydrogenase distribution, XIII, 106-107 kinetic studies, VIII, 115-117 mechanism, XIII, 126-129 mechanism of Maasey and Veeger, review of, XIII, 111 metabolic functions, XIII, 107-110 role of NAD' as modifier, XIII, 117120

structural studies, XIII, 120-126 two-electron-reduced enzyme, properties, XIII, 111-115 Lipoate, activation of, 17-18 Lipoprotein lipase, properties, VII, 606609

Liver acetyl coenzyme A carboxylase of, VI, 78-79

acid phosphatases, IV, 484-493 alcohol dehydrogenase of, XI, 20-22, 56-57, 107-109, 111-117

aldolase, VII, 241-244 argininosuccinase of, VII, 171-178, 179 fructose-1,6-diphosphatase, IV, 618-633 glycogen synthetase of, IX, 341-353 lipases, VII, 611 mitochondria1 adenosine triphosphatase

catalytic properties, X, 388-389 molecular properties, X, 388 purification, X, 387-388 5'-nucleotidase of, IV, 343-345 sulfatase A of, V, 27-36 sulfatase B of, V, 37-38 sulfite oxidase of, XII, 414-419 vitamin Bn methyltransferase of, IX, 164-165

London forces, propinquity effects and, 11, 254-264 Long chain fatty acid:coenayme A ligase catalytic properties partial and exchange reactions, X, 486-487

substrates and inhibitors, X, 485-486 Luciferase, bacterial, properties, XII, 226-229

Luciferin, adenylylation of, VIII, 19-20 Lyase(s1, role of metals in mechanism, 11, 508-519 Lysine, biosynthesis, VI, 504 Lysine monooxygenase, properties, XII, 199-203, 293-294

n-cu-Lysine mutase, VI, 551-552 cofactor requirements, VI, 552 comparison to L-plysine mutase, VI, 554-555

cofactor requirements, VI, 555-559 2,5diaminohexanoate and, VI, 554 inhibitors, VI, 553 partial reactions, VI, 553-554 purification of complex, VI, 552 L-p-Lysine mutase assay, VI, 548-549 cofactor requirements, VI, 550 comparison to n-a-lysine mutase, VI, 554-555

cofactor requirements, VI, 555-559 inhibitors, VI, 551 occurrence, VI, 548 purification and physical properties, VI, 549-550 reversibility, VI, 551 Lysine residues acylation biotin activating enzyme, VIII, 18 lipoate activating enzyme, VIII, 1718

creatine kinase, VIII, 432-434


ribonuclease, IV, 801 subtilisin, modification of, 111, 596-598 Lysosomes, hyaluronidase of, V, 312 Lysozyme, see abo Phage lysozyme activity biological role, VII, 669-670 muramidase and chitinase, VII, 667669 analysis of rate enhancement, VII, 864-865, 868 general acid catalysis, VII, 885 ion pairing and, VII, 867 substrate distortion, VII, 865-867 association with hydrogen ions, VII, 745-746 apparent ionization constants, VII, 736-739 enthalpy and volume changes, VII, 735-736 environment of ionizable groups, VII, 739-745 isionic and isoelectric pH, VII, 732 potentiometric titrations, VII, 732735 association with other ions and molecules, VII, 746-755 avian, VII, 677-4380 bacteriophage T2 catalytic properties, V, 381-382 chemical properties, V, 380-381 physicochemical properties, V, 380 purification, V, 379 bacteriophage T4 catalytic properties, V, 369-374 chemical properties, V, 366-369 enzyme assays, V, 361464 physicochemical properties, V, 366 purification, V, 364-366 role in life cycle, V, 375-379 bond rearrangement mechanism, VII, 855-856 chemical modification, I, 207-211 chemical modifications amino groups, VII, 780-785 arginine, VII, 785-786 carboxyl groups, VII, 786-788 cystine, VII, 789-790 histidine, VII, 791 methionine, VIJ, 791-792 other reactions, VII, 797-798

509 tryptophan, VII, 792-796 tyrosine, VII, 796-797 chromophore properties, VII, 799-802 absorbance, VII, 802-803 chromophore exposure, VII, 809 circular dichroism, VII, 807-809 fluorescence, VII, 803-806 solvent and saccharide perturbation, VII, 809 conformation of egg-white model main polypeptide chain, VII, 692699 side chains, VII, 699-707 water structure, VII, 707 crystallographic studiea of inhibitor complexes, VII, 707-708 a- and p-N-acetylglucosamine, VII, 708 enzyme-substrate complex structure, VII, 712-714 supplementary binding studies, VII, 714-717 tri-N-acetylchitotriose, VII, 708-711 denaturation, VII, 760-766 guanidine hydrochloride, VII, 774776 nonaqueous solvents, VII, 776-777 nuclear magnetic resonance, VII, 777-780 properties, VII, 770-772 stability, VII, 766-770 thermal, VII, 772-774 urea, VII, 776 early history, VII, 666-667 fluorescence spectroscopy, 11, 426-429 gene duplication and, I, 311-314 hen egg-white amino acid sequence, VII, 672-676 composition, VII, 672 preparation and purification, VII, 670-672 synthesis, VIT, 67M77 human, VII, 680-881 hydrodynamic measurements, shape and solvation, VII, 717-725 hydrogen exchange, VII, 730-732 large substrates bacterial cells and cell walls, VII, 836-841 chitin and derivatives, VII, 841-843


low molecular weight substrates, VII, 846-847

bond cleaved, VII, 847 chain length and, VII, 847-848 character of substrate, VII, 848-849 Hammett constant, VII, 851-852 isotope effect, VII, 850 kinetic constants, VII, 850-851 kinetics of saccharide binding, VII, 852-855

pH and, VII, 849-850 rate enhancement, VII, 850 temperature and, VII, 849 mechanism, VII, 856-857 acetamido participation, VII, 863864

saccharide binding, VII, 808-835 substrates, kinetics and mechanism, VII, 836-868 X-ray studies, VII, 682-717 X-ray studies, I, 67-69 analysis of structure, VII, 6824392 conformation of egg-white model, VII, 692-707 crystallography of inhibitor complexes, VII, 707-717 Lysyl hydroxylase catalytic properties, XII, 166-167 purification and molecular properties, XII, 165 o-Lyxose isomerase, properties, VI, 344345

environment, VII, 863 general acid catalysis, VII, 857-860 glycosyl carbonium ion and, VII, 862-863

location of catalytic center, VII, 857 substrate distortion, VII, 860-862 optical properties, VII, 725-730 relationship to a-lactalbumin, IX, 366367

saccharide binding, VII, 809-810 acceptor and substrate reactivity, VII, 822-825 free energies of association, VII, 810-820

multiple modes of binding, VII, 833835 pH dependence, VII, 832-833 separation of group contributions, VII, 829-832 separation of site contributions, VII, 825-829 thermodynamics of association, VII, 820-822

self-association, VII, 756-757 structure in crystal and solution, VII, 757-760

transfer reactions, VII, 843-846 vertebrate chemical modifications, VII, 780-809 denaturation, VII, 760-780 general considerations, VII, 666-670 physical properties, VII, 717-760 preparation, composition and sequence, VII, 670-682

M

Macromolecular complexes, metabolic regulation and, I, 426-428 Maanesium, enolase and, V, 524526 Malate, cleavage of, VII, 431 Malate dehydrogenase, catalytic properties, active site structure, X I , 390-395 kinetic analyses, XI, 385-390 distribution and preparation, X I , 370373

mitochondrial, environment of, XI, 395-396

molecular properties, amino acid composition, XI, 375376, 377

molecular weight, XI, 373-374 nature of subforms, XI, 376, 378 subunit structure, XI, 374375 structure of NAD'dependent cytoplasmic interaction with coenzyme, X I , 382385

pig heart crystal structure, X I , 379382

types of, XI, 369-370 Malate synthase condensation mechanism, VII, 420-422 occurrence, VII, 412-414 proton transfer and, 11, 316-317 purification and properties, VII, 415 reversibility, VII, 417

511


specificity, VII, 415-417 stereochemistry, VII, 417419 Maleate isomerase, properties, VI, 38% 385

Maleic anhydride, pyruvate carboxylase and, VI, 22-23 Maleyl isomerase(s), properties, VI, 385390

Malonyl coenzyme A-acyl carrier protein transacy lase catalytic properties assays, VIII, 179-180 mechanism, VIII, 180-185 pH optimum, substrate specificity and kinetics, VIII, 180 historical background, distribution and metabolic significance, VIII, 176178

molecular properties, VIII, 178-179 Mammals amino acid decarboxylases of, VI, 221224

glutamine synthetase of, X, 699-754 hexokinase mechanism, IX, 4144 occurrence of multiple forms, IX, 31-33

purification and molecular properties, IX, 37-41 regulation, IX, 44-46 relation of solubie to insoluble forms, IX, 33-37 neuraminidase of, V, 325-327 Mandelic acid racemase, properties, VI. 379-380

D-Mannonate dehydrase, properties, V, 579

D-Mannose isomerase, properties, VI, 344-345

L-Mannose isomerase, properties, VI, 345-346

Mannosed-phosphate isomerase catalytic properties general kinetic parameters, VI, 307309 mechanism of action, VI, 310-314 zinc effect on nonemymic isomerization, VI, 309-310 characterization as zinc metalloenzyme, VI, 305-307

history, occurrence and function, VI, 302-304

molecular properties of yeast enzyme, VI, 304-305 Medium chain fatty acid:coenzyme A ligases catalytic properties formation of butyryl adenylate, X, 484

steady state kinetics and reaction mechanism, X, 484-485 substrates and inhibitors, X , 483 Melilotate hydroxylase, properties, XII, 217-221

Metabolic functions regulation of balance allosteric determination of catalytic function, I, 442444 compensatory control mechanisms, I, 434-439

metabolite interconversion systems. I, 43@-434 oppositely directed exergonic reactions, I, 439442 Metabolic regulation adenylate energy charge and, I, 470476

covalently bonded modifiers and, I, 484

operational response curves, I, 482-483 principles chemical equilibria and, I, 418-419 compartmentalization and, I, 423426 covalent enzyme modification, I, 409-415

enzyme concentration, I, 399403 feedback inhibition, I, 403-408 inorganic ions, I, 419-423 macromolecular complexes, I, 426428

proteolysis, I, 416-417 unidirection of reversible reactions, I, 428-430 Metabolic systems, analogy with electronic systems, I, 463-465 Metal ( s ) catalysis by, VI, 401406 kinetics and, 11, 59-61 microbial proteinases and, 111, 772-773

512


Metal bridge complexes binary complex formation, 11, 494495 development of concept, 11, 485-489 enzyme-metal linkage, 11, 490494 reactions within coordination sphere, 11, 497-498 ternary complex formation, 11, 495497

Metal complexes reaction chelation mechanisms, 11, 453-455 coordinated ligand reactions, 11, 455463

ligand substitution mechanisms, 11, 450-453

Metal ions properties relevant to catalysis general, 11, 448-450 reactions of metal complexes, 11, 450-463

Metalloenzymes, enzymic properties, 111, 76-77

5,lO-Methenyltetrahydrofolate cyclohydrolase, properties, IX, 201 Methionine adenosyltransferase catalytic properties activators and pH effects, VIII, 133135

assay, VIII, 129-130 energetics, VIII, 139-141 kinetics, VIII, 137-139 reversibility, partial reactions and mechanistic considerations, VIII, 130-133

substrate specificities and inhibition by substrate analogs, VIII, 135-137 net reaction, VIII, 125-127 purification and physical properties, VIII, 127-129 regulation and genetics mammals, VIII, 142-143 microorganisms, VIII, 141-142 significance and distribution, VIII, 123-125

Methionine residues chemical modification, I, 173 chymotrypsinogen, 111, 179 ribonuclease, IV, 682483 subtilisin, modification of, 111, 598-599

Methionyl transfer ribonucleic acid transformylase, properties, IX, 208209

4-Methoxybenzoate O-demethyl-monooxygenase, properties, XII, 285-287 3-Methylaspartate ammonia-lyase catalytic procesa evidence for carbanion mechanism, VII, 127-135 substrate and activator binding, VII, 121-127

distribution, purification and kinetic properties, VII, 118-119 size and constitution, VII, 119-121 p-Methylcrotonyl coenzyme A carboxylase distribution, VI, 4 1 historical background and metabolic significance, VI, 39-40 mechanism of action, VI, 4 2 4 5 molecular characteristics, VI, 42 reaction catalyzed, VI, 38-39 substrate specificity, VI, 4 1 4 2 a-Methyleneglutarak mutase, VI, 534535

assay, VI, 535 catalytic properties coenzymes, W,536 equilibrium, VI, 536-537 mechanism, VI, 537 substrates and inhibitors, VI, 536 purification and molecular properties, IV, 535-536 Methyl groups asymmetrical, stereospecificity of malate synthesis, 11, 157-164 7-Methyl-y-hydroxy-a-ketoglutaric aldolase, properties, VII, 301-302 Methylmalonyl coenzyme A epimerase, stereochemistry, 11, 206-210 Methylmalonyl coenzyme A mutase, VI, 511-512

assays, VI, 51%513 catalytic properties coenzymes, VI, 517-519 equilibrium, VI, 519 mechanism, VI, 519-524 pH and, VI, 519 substrates, VI, 517 distribution, VI, 513


purification and molecular properties animal tissue, VI, 515-517 Propionibacterium shermanii, VI, 514-515 Methylmalonyl coenzyme A racemase, properties, VI, 378-379 N5-Methyltetrahydrofolate-homocysteine methyltransferases, see Methyltransferase, Vitamin Bn methyltransferase Methyltransferase assay and purification, IX, 154-155 catalytic properties and folate binding, IX, 156-158 folate substrates, IX, 161-162 occurrence, IX, 160-161 physical properties, IX, 155-156 reactions catalyzed, IX, 121-122 repression of synthesis, IX, 158-160 Mevaldate reductase, stereospecificity, 11, 186-189 Mevalonate labeled, preparation and use, 11, 192204 Mevalonate kinase, stereospecificity, 11, 186-189 Micelles, propinquity effects and, 11, 264-274 Michaelis-Menten mechanism single substrate-single product, 11, 7475 early phase solutions, 11, 77 general solutions to one intermediate mechanism, 11, 75-77 steady state solutions, 11, 77-79 Microbial proteinase(s) chemical properties, 111, 728-730, 742743, 749-754, 770-773 distribution and isolation, 111, 724-728, 741-742, 745-749, 767-770 enzymic properties, 111, 734-740, 742743, 758-763,776-786 inhibitors, 111, 762-763, 777-778 kinetics and mechanism, 111, 763, 778-786 optimum pH, 111, 758-759, 776-777 substrate specificity, 111, 759-762, 778-786 physical properties, 111, 731-733, 742743

513 conformation studies, 111, 755-758, 775-776 molecular weight, physical constants and isoelectric point, 111, 754-755, 773 stability, 111, 755, 773-775 Micrococcus luteus endonuclease, ATPdependent, IV, 261-262 polynucleotide phosphorylase of, VII, 548-570 ultraviolet repair enzymes, IV, 269-270 Micrococcus lysodeikticus adenosine triphosphatase of, X, 421-425 phage-induced lytic enzyme catalytic properties, V, 401-402 purification, V, 401 stability, V, 402 Microorganisms, see also Bacteria elc. aldolases of, VII, 255-256 amylase of, V, 239-271 lipases of, VII, 614-616 3’-nucleotidases of, IV, 354 ribonucleoside 2’,3’-cyclic phosphate diesterase of, IV, 356-363 succinate dehydrogenase of, XIII, 254256 type b cytochromes in, XI, 577-584 Microsomes electron transport, XIII, 148-149 cytochrome b, reductase system, XIII, 150-151 cytochrome P-450 reductase system, XIII, 149-150 mixed function amine oxide, XIII, 153-154 synergism between systems, XIII, 151-153 Milk, lipases, VII, 611-613 Milk xanthine oxidase catalytic properties mechanism of action, XII, 365-388 reactions catalyzed, XII, 344-365 historical background, XII, 303-304 molecular properties chemical modification, XII, 317-326 composition and physical properties, XII, 310-317 magnetic interactions of redox groups, XII, 342-344

514


purification, XII, 304-310 redox group magnetic and optical properties, XII, 326-342 Mitochondria adenosine triphosphatase assay, X, 377 beef heart, X, 377-386 rat liver, X, 387-389 yeast, X, 386-387 nicotinamide adenine dinucleotide dehydrogenases, XIII, 177-178 energy conservation and, XIII, 214216

high molecular weight, XIII, 187-189 inhibitors of, XIII, 203-207 low molecular weight, XIII, 189-198 relevance of low and high molecular weight dehydrogenases, XIII, 198203

transhydrogenation and, XIII, 207214

ubiquinone reductase (Complex I), XIII, 178-187 ribonucleic acid polymerase of, X, 318-328

type b cytochromes in, XI, 564-565 Molds, see abo Fungi amylases catalytic properties, V, 263-271 molecular properties, V, 236-263 glycogen synthetase of, IX, 361 proteases acid, 111, 723-744 diisopropylfluorophosphate sensitive, 111, 744-765 metal-chelator-sensitive, 111, 765-786 other, 111. 786-795 Mblybdenum iron-sulfurflavin hydroxylase catalytic properties mechanism of reduction, XII, 394397

oxidizing substrates, XII, 397400 distribution and biological importance, XII, 301-303 enzymes to be considered, XII, 300-301 genetic studies Aspergillus nidulans, XII, 412414 Drosophila melanogaster, XII, 406412

nitrate reductase and, XII; 402406 xanthinuria and gout in man, XII, 400-402

molecular properties, XII, 389-394 other, XII, 388-389 Monoamine oxidase, molecular properties, and kinetic mechanism, XII, 466-471

Monooxygenase (s) copper-containing dopamine, XII, 294-295 phenol, XII, 295-297 flavin and pterin-linked, background, XII, 191-193 heme containing flavoproteins and, XII, 269-280 general mechanisms, XII, 280-285 iron-sulfur proteins as electron donors, XII, 259-269 iron and copper-containing functions, XII, 257-258 historical aspects, XII, 253-255 nomenclature, XII, 256-257 role in oxygen activation, XII, 256 iron-containing, heme, XII, 258-285 nonheme, XII, 285-294 model studies and possible mechanisms, XII, 241-262 nonheme iron-containing lysine, XII, 293-294 4methoxybenzoate 0-demethyl, XII, 285-287 phenylalanine, XII, 287-289 proline, XII, 292-293 tryptophan, XII, 291-292 tyrosine, XII, 290-291 pterin-linked, XII, 231 Multienzyme complexes biological significance, I, 237-240 biosynthesis of aromatic amino acids, I, 228-237 fatty acid synthetases, I, 226-228 a-ketoglutarate dehydrogenase, I, 224225

pyruvate dehydrogenase, composition and organization, I, 215220

regulatory features, I, 220-224

"OhCAL SUBJECT INDEX

Multiple isomorphous replacement how heavy an atom, I, 37-39 location of heavy atoms, I, 32-35 outline of method, I, 32 phase determination and error assessment, I, 35-37 Mung bean, 3'-nucleotidase of, IV, 353 Muscle aldolase, VII, 224-241, 258 creatine kinases of, VIII, 395-401 enolase, glycidol phosphate and, V, 534 fructose-l,6-diphosphatase evidence for presence, IV, 632 physiological role, IV, 634-635 purification and properties, IV, 632633

structure and relation to other enzymes, IV, 633-634 glycogen synthetase of, IX, 332-340 phosphofructokinase of, VIII, 254-256 phosphorylase kinase of, VIII, 557-564 Mutants advantages and limitations of, I, 265266

isolation of, I, 245-249 Mutations alkaline phosphatase and, I, 251-254 amino acid analogs and, I, 262-265 p-galactosidase and, I, 255-256 hemoglobin and,, I, 257-259 other enzymes and proteins, I, 259-262 reversion, suppression and complementation, I, 249-251 tryptophan synthetase, I, 256-257 types, cistron and, I, 243-245 Myelin, ribonucleoside 2',3'-cyclic phosphate diesterase in, IV, 364-365 Myokinase, see Adenylate kinase Myrosulfatase, V, 15-17 Myxobacter, protease of, 111, 786-788 N

Negative charge, proteinase inhibitors and, 111, 422-423 Nervous tissue 5'-nucleotidase of, IV, 346-347 ribonucleoside 2',3'-cyclic phosphate diesterase of, IV, 363-364

intracellular localization, IV, 364-365 physiological role, IV, 365 properties and substrate specificity, IV, 364 Neuraminidase (s) assay method, V, 339-341 biological significance, V, 341-342 historical background, V, 321-323 inhibitors, V, 339 kinetic data, IV, 337-339 occurrence, V, 323 bacterial, V, 324-325 mammalian, V, 325-327 viral, V, 324 properties, 329-331 purification influenza virus, V, 327-328 Vibrio cholerae, V, 328-329 substrate specificity configurational, V, 331-332 esterification of neuraminic acid, V, 336-337

N-substitution of neuraminic acid,

v, 335

0-substitution of N-acetylneuraminic acid, V, 336 position of glycoside linkage, V, 334335

steric hindrance in natural substrates, V, 332-333 Neurospora adenylosuccinase of, VII, 191-193 argininosuccinase of, VII, 180-181 Neurospora crassa acid phosphatase, IV, 497 aspartate transcarbamylase, IX, 302306

argininosuccinase of, VII, 180-181 invertase, V, 303-304 catalytic properties, V, 305 purified preparations, V, 304-305 Neutral proteinase(s) metal-chelator-sensitive, 111, 765-766 chemical properties, 111, 770-773 distribution and isolation, 111,767770

enzymic properties, 111, 776-786 physical properties, 111, 773-776 Nicotinamide adenine dinucleotide, pyruvate carboxylase and, VI, 33-34

516


Nicotinamide adenine dinucleotide dehydrogenase Azotobacter vinelandii, XIII, 221 mammalian mitochondria, XIII, 177178

energy conservation and, XIII, 214216

high molecular weight, XIII, 187-189 inhibitors of, XIII, 203-207 low molecular weight, XIII, 189-198 relevance of low and high molecular weight, XIII, 198-203 transhydrogenation and, XIII, 207214

ubiquinone reductase (Complex I), 178-187

yeast, XIII, 216-221 Nicotinamide adenine dinucleotide kinase assay, IX, 77-78 distribution, purification and stability, IX, 76-77 kinetic and molecular properties, IX, 78-79

reaction mechanism, IX, 79-80 substrate specificity, IX, 80-82 Nicotinamide nucleotide transhydrogenase

AB-specific historical, XIII, 62-64 kinetics and reaction mechanism, XIII, 75-78 molecular properties, XIII, 69-71 occurrence, XIII, 64-66 preparation and assay, XIII, 66-69 reconstitution, XIII, 78-79 relationship to energy-coupling system, XIII, 71-75 BB-specific historical, XIII, 52-53 molecular properties, XIII, 57-59 occurrence, XIII, 53-54 purification and assay, XIII, 54-57 reaction mechanism and regulation, XIII, 59-62 definition, XIII, 51-52 physiological roles, XIII, 79-81 fatty acid synthesis, XIII, 88 mitochondrial glutamate and isocitrate metabolism, XIII, 85-88

mitochondrial monooxygenase reactions, XIII, 83-85 redox state of mitochondrial nicotinamide nucleotides, XIII, 81-82 Nitrate reductase, molybdenum hydroxlase “common cofactor,” XII, 402406

Nitrite reductase(s), properties of, XIII, 273-279

Nitrogenase, properties of, XII, 50-56 Nonheme iron proteinb), metal complexes and, 11, 531-533 Nuclear magnetic resonance, ribonuclease, IV, 723-725 Nuclear preparations rat liver, pyrrolidone carboxylate formation by, IV, 138-139 Nucleases, see abo Deoxyribo- and Ribonucleases modification of, I, 130 X-ray diffraction studies, I, 67-69 Nucleophilic catalysis, isomerization and, VI, 397-401 Nucleoside 3’,5’-cyclic phosphate diesterase, IV, 365-366 distribution, IV, 366 inhibitors and activators, IV, 368-370 intracellular localBation, IV, 367-368 metal ions, pH and substrate affinity, IV, 368 physiological function, IV, 370-371 possibility of other diesterases, IV, 370 substrate specificity, IV, 366-367 Nucleoside diphosphokinase(s) assay methods, coupled, VIII, 321-325 isotopic, VIII, 325 staining procedure, VIII, 325 catalytic properties conformational changes, VIII, 331 metal requirements, VIII, 329-330 reaction catalyzed, VIII, 320 specificity, VIII, 320-321 sulfhydryl groups, VIII, 330-331, distribution, VIII, 309-313 function in the cell, VIII, 331-333 historical development, VIII, 307-309 kinetics and catalytic mechanism pH and temperature effects, VIII, 328329

517


substrate concentration effect, VIII, 326-328

molecular properties occurrence of isozymes, VIII, 313314

phosphorylated enzyme, VIII, 315320

physical properties, VIII, 315 purification procedures, VIII, 314315

Nucleotide kinases, reaction catalyzed, I X , 49-50 3’-Nucleotidase microorganisms, IV, 354 mung bean, IV, 353 rye grass, IV, 353 wheat seedling, IV, 353-354 5’-Nucleotidase (s) bacterial, IV, 338-340 bull seminal plasma, IV, 342-343 cardiac tissue, IV, 347-348 comparison of, IV, 349-352 Ehrlich ascites cells, IV, 348-349 intestinal, IV, 345 liver, IV, 343-345 nervous tissue, IV, 346-347 other vertebrate tissues, IV, 348 pituitary gland, IV, 346 potatoes, IV, 349 snake venom, IV, 342 yeast, IV, 341342 Nucleotide kinases, reaction catalyzed, I X , 50 Nucleotidyl transferring enzymes, role of metals in mechanism, 11, 502-504 Nucleus ribonucleic acid polymerases animal, X, 262-300 higher plant, X, 311-318 yeast and fungi, X, 300-311

0 Old yellow enzyme, molecular properties and kinetic mechanism, XII, 471-473 Oleate formation from stearate, 11, 179-184 hydration, stereospecificity, 11, 184-186 Oligonucleotides, polynucleotide phosphorylase and, XII, 549-552

Optical rotatory dispersion protein structure and, 11, 381-382, 408 secondary, 11, 382-386 tertiary, 11, 386-391 typical cases, 11, 391-407 ribonuclease, IV, 719-723 Orcinol hydroxylase, properties, XII, 223-224

Ornithine mutase, properties, VI, 559 Oxalacetate formation from phosphoenolpyruvate, general considerations, VI, 117119, 165-168

Oxidation-reduction reactions between metal complexes, 11,529-530 cytochrome c, 11,534-538 mechanistic principles, 11, 530-531 nonheme iron proteins, 11, 531-533 xanthine and aldehyde oxidases, 11, 533-534

within metal complexes mechanistic principles, 11, 525 xficotinamide adenine dinucleotidelinked dehydrogenases, 11, 525-528 vitamin BIZmechanisms, 11, 628-529 Oxidoreductase (8) stereospecificity absolute coenzyme configuration, 11, 144-147

historical background, 11, 134-137 hydrogen transfer and, 11, 137-144 significance of A- and B-side specificity, 11, 154-157 substrates and, 11, 147-151 without hydrogen transfer, 11, 151154

Oxygenases, see Dioxygenases, Monooxygenases Oxygen binding proteins, X-ray diffraction studies, I, 53-63 P

Pancreas pig, phospholipase A, of, V, 76-77 Pancreatic elastase, see Elastase Papain active site chemical modification, 111, 515-516 comparison with other proteinases, 111, 498-499


geometry of, 111, 496498 location of thiol group, 111, 514 amino acid composition and sequence, 111, 507-509 assay methods, 111, 518-519 chemical modifications, I, 205-207 active site, 111, 515-516 other reactions, 111, 516-518 crystallization of, 111, 486-488 fructose-1,6-diphosphatase and, IV, 619 heavy atom derivatives, 111, 488-490 kinetic studies acyl enzyme intermediate, 111, 525532 deacylation, 111, 533-635 kinetic constants and pH, 111, 535537 pH, ionic strength and temperature effects, 111, 525 partial reduction, 111, 617 physical properties hydrodynamic properties, 111, 503 immunochemical studies, 111, 606 spectrophotometric and fluorescence properties, 111, 503-505 stability, 111, 505-506 preparation and crystallization, 111, 502-503 specificity esterase and thiolesterase activity, 111, 523-524 nucleophile binding, 111, 524-525 peptide and amide bond hydrolysis, 111, 519-523 transamidation and transesterification, 111, 524 three-dimensional structure description, 111, 491-496 electron density map, 111,490-491 water-insoluble derivatives, 111, 117518 Papaya latex, other proteolytic enzymes, 111, 537-538 Papaya peptidase A, 111,538 Parallelism, protein evolution and, I, 329332 Pasteur effect, phosphofructakinase and, VIII, 274-276 Pea seeds, argininosuccinase of, VII, 181182

Pentacovalency, pseudorotation and, VIII, 214-219 Penicillinase assay methods, IV, 35-39 background, IV, 23-25 catalytic reaction, IV, 27 conformation and function nonspecific transitions, IV, 44-15 specific transitions, IV, 45-46 definitions and specificity, IV, 25-26 factors affecting activity activators and inhibitors, IV, 43-44 pH and temperature, IV, 42-43 immunological studies, IV, 46 kinetics and substrate specificity, IV, 39-40 molecular properties composition and sequence analysis, IV, 31-35 purification and physical properties, IV, 2731 occurrence, IV, 26 structural modification of, IV, 41-42 substrate structural modifications, IV, 40-41 Pepsin action esterase activity, 111, 151 organic sulfite cleavage, 111, 151-152 Specificity, 111, 142-151 theories of, 111, 160-164 amino acids composition, 111, 128-130 sequence, 111, 130-133 assay, 111, 124-125 autolysis of, 111, 139-140 chemical modification, 111, 133-137 condensation reactions, 111, 156 denaturation of, 111, 137-138 electrophoretic mobility, 111, 127 formation from pepsinogen, 111, 138139 gene duplication and, I, 308 historical background, 111, 120 inhibition of, 111, 154-156 isotope effects, 111, 160 molecular weight and shape, 111, 126127 occurrence

519


classification and nomenclature, 111, 121-123

other pepsinlike enzymes, 111, 123 optical properties, 111, 127-128 pH dependence, 111, 152-154 protein cleavage by, 111, 140-142 purification of, 111, 124 ribonuclease and, IV, 673 side chain specificity, 111, 145-147 stereochemical specificity, 111, 147-151 transpeptidation reactions, 111, 157-160 Pepsinogen, included with Pepsin Peptide(s) synthesis, nucleic acid independent, VIII, 11-17 Peptide bonds cleavage, proteinase inhibitors and, 111, 452-454 Peptide chain elongation, see also Elongation scheme of, X , 54 Peptide groups, ultraviolet absorption, 11, 379-380 PH chymotrypsin and, 111, 231-233, 236237

p-galactosidase and, VII, 635, 644-645 guanidino kinases and, VIII, 476-477 kinetic parameters and, 11, 52-56 methylmalonyl coenzyme A mutase and, VI, 519 phosphoglucomutase and, VI, 455 pyruvate carboxylase and, VI, 21 Phage, see also Bacteriophage Phage lysozyme, see also Lysozyme application to other biologically important problems, V, 350-352 early history, V, 344-345 recent developments, V, 345-349 Phenol o-monooxygenase, properties of, XII, 221-223, 296-297 Phenylalanine ammonia-lyase catalytic process function of prosthetic group, VII, 159-162

metal ion activation, VII, 162-163 prosthetic group, VII, 154-159 rate limiting step, VII, 164-166 reaction sequence, VII, 148-154

distribution, purification and kinetic properties, VII, 142-146 mechanism of action, VII, 195-196, size and constitution, VII, 146-148 Phenylalanine hydroxylase, properties, XII, 232-238 Phenylalanine 4-monooxygenase, properties, XII, 287-289 Phenylpyruvate synthase, properties, VI, 205-207 Phosphagen kinase, see Guanidino kinase(s) Phosphate, adenosine diphosphoryl glucose pyrophosphorylase and, VIII, 91-92 Phosphate esters hydrolysis of acyclic di- and triesters, VIII, 207-208 metaphosphate mechanism for monoesters, VIII, 202-206 Phosphoenolpyruvate, enzymatic synthesis, X, 631-633 Phosphoenolpyruvate carboxykinase discovery, distribution and physiological role, VI, 136-138 physical properties, VI, 143-148 reactions catalyzed and their properties metal requirement, VI, 141-142 nucleoside phosphate specificity, VI, 140-141

oxalacetate or phosphoenolpyruvate formation, VI, 139 pH optimum, VI, 142-143 pyruvate formation, VI, 139-140 thiol reagents and, VI, 143 regulation activity, VI, 151-154 concentration, VI, 150-151 intracellular location, VI, 148-150 Phosphoenolpyruvate carboxylase discovery, distribution and physiological role, VI, 119-122 kinetic and regulatory properties, VI, 126-133

mechanism of action, VI, 133-136 nature of reaction catalyzed, VI, 122124

role of metals in mechanism, 11, 507508

520


structural studies, VI, 124-126 Phosphoenolpyruvate carboxytransphosphorylase characteristics and mechanism of catalyzed reaction, VI, 157-161 different forms, structure and catalytic activities, VI, 163-164 discovery, distribution and physiological role, VI, 154-157 kinetic parameters, effectors and inhibitors, VI, 162 Phosphoenolpyruvate synthetase catalytic properties kinetic studies, X, 643-645 mechanism, X, 638441 metal ion requirements, X, 645 pH and equilibrium, X, 645-646 regulation, X, 648-649 specificity, X, 646-647 stoichiometry, X, 637-638 molecular properties bound divalent metal ion, X, 637 molecular interconversions, X, 635636

phosphoryl and pyrophosphoryl enzyme, X, 636-637 purification, X, 633-634 stability, X, 634-635 sulfhydryl groups, X, 635 Phosphofructokinase assay of, VIII, 243-244 catalytic properties cation requirement, VIII, 247-248 isotopic exchange, VIII, 252 kinetic studies, VIII, 248-252 phosphoryl acceptor specificity, VIII, 244-245

phosphoryl donor specificity, VIII, 24&247

control of glycolysis hormones and, VIII, 277-278 Pasteur effect, VIII, 274-276 pyridine nucleotide oscillations, VIII, 276 purification, VIII, 241-243 reaction catalyzed, VIII, 240-241 regulation of, I, 439-441, VIII, 261-269 role of specific groups histidine, VIII, 272 other functional groups, VIII, 272-274

thiols, VIII, 269-272 structural properties Clostridium pasteurianum, VIII, 256 dilution effects, VIII, 259-260 Escherichia coli, VIII, 255257 isozymes, VIII, 257 molecular weight, VIII, 253-254 phosphorylation, VIII, 260-261 rabbit erythrocyte, VIII, 257-258 rabbit muscle, VIII, 254-256 Phosphoglucomutase activation, VI, 439442 assay, VI, 417-418 all-or-none, VI, 420421 colorimetric, VI, 418419 coupled, VI, 419 radiometric, VI, 419-420 catalytic reaction central complex structural differences, VI, 432 isomeric forms of phosphoenzyme, VI, 430-432 isotope exchange reactions, VI, 429430

kinetics, VI, 426-429 reaction sequence, VI, 421-424 roles of glucose diphosphate, VI, 424426

inhibition anions, VI, 442444 cations, VI, 444-446 chemical modification, VI, 446-447 miscellaneous, VI, 447 poor substrates and analogs, VI, 444 metal ion effects activation, VI, 448-449 addition and release of magnesium, VI, 449-451 addition and release of other metals, VI, 451-452 complexes in vivo, VI, 454 metal-binding site, VI, 453454 structural changes, VI, 452-453 pH and temperature effecta, VI, 455 physical and chemical properties, VI, 413-415

polymorphism, VI, 416-417 purity, VI, 412-413 stability, VI, 416

TOPICAL SUFLJECT INDEX

preparation chromatography, VI, 409410 dephospho-enzyme, VI, 411412 isolation, VI, 408-409 phosphate-labeled, VI, 410-411 specificity, VI, 436-439 structural studies active site phosphate group, VI, 455-456 active site phosphopeptide, VI, 456-457 conformational studies, VI, 457-458 thermodynamics hydrolysiv of phospho-enzyme, VI, 434-436 overall reaction, VI, 433 phosphate transfer to glucose phosphate, VI, 433-434 6-Phosphogluconate dehydrase distribution, V, 573-574 properties, V, 575-578 3-Phosphoglycerate, adenosine diphosphoryl glucose pyrophosphorylase and, VIII, 91-92 3-Phosphoglycerate kinase biological behavior occurrence, VIII, 337-338 reaction catalyzed, VIII, 338-337 species variation and genetics, VIII, 338-340 historical background, VIII, 335-336 molecular properties molecular weight, VIII, 342 primary structure, VIII, 342343 secondary structure, VIII, 344 tertiary structure, VIII, 344-346 purification procedures, VIII, 340-341 reaction kinetics backward and forward reactions, VIII, 346-348 metal ion specificity, VIII, 349 nucleotide specificity, VIII, 348-349 postulated mechanism, VIII, 349-351 Phosphoglycerate mutase assay, VI, 462-464 inorganic and simple organic compounds, effects, VI, 474-475 kinetics, VI, 469-470 pH and temperature effects, VI, 475

521 phosphate transfer to water, VI, 472474 physical and chemical properties, VI, 460-462 preparation and purity, VI, 459-460 reaction sequence, VI, 464-468 Specificity, VI, 471 stability and storage, VI, 462 thermodynamics, VI, 470-471 Phosphoglycolate, triosephosphate isomerase and, VI, 335-336 Phospholipase(s), general, V, 71-73 Phospholipase A,, V, 73 isolation and purification, V, 74-75 Crotalus adamanteus venom, V, 75-76 Crotalus atrox venom, V, 77-78 pig pancreas, V, 76-77 physical and chemical characteristics amino acid content, V, 80-81 amino acid sequence, V, 81-82 molecular weight, V, 78-80 sources, V, 74 substrates and mode of attack, V, 74 Phospholipase C isolation and purification Bacillus cereus, V, 83-84 Clostridium perfringens, V, 84-85 sources, V, 82-83 Phospholipids, glucose-6-phosphatase and, IV, 554-556 Phosphomutase(s), other sugars, VI, 458-459 Phosphoribosylpyrophosphate synthetase catalytic properties conditions affecting activity, X, 617 equilibrium constant, X, 617 mechanism, X, 618-621 substrates and activators, X, 614-617 inhibition by metabolites bacterial enzyme, X, 621-622 mammalian enzyme, X, 622-623 physiological significance, A, 623 occurrence and purification, X, 611-612 other regulatory aspects, X, 623-624 physical and chemical properties, X, 612-614 reaction catalyzed and assay methods, X, 608-611 related enzymes, X, 607-608


Phosphorus acyclic, nucleophilic reactions and, VIII, 208-214 Phosphorylase kinase heart muscle, VIII, 564 liver and other mammalian tissues, VIII, 565 nonmammalian sources, VIII, 565 skeletal muscle, VIII, 557-564 0-Phosphorylethanolamine phospholyase, properties, VII, 5 2 5 3 Phosphoryl transfer catalysis intramolecular, VIII, 219-227 metal ion, VIII, 227-231 enzymic mechanisms, VIII, 232-233 bimolecular or aeaociative, VIII, 235-238

metaphosphate, VIII, 233-235 Phosphotransferase, glucose-bphosphatase and, IV, 592-595 Phosvitin kinase, properties, VIII, 5& 581

Photoisomerization, related problems and, VI, 395-397 Photosynthesis bacterial, XI, 509-510 green sulfur, XI, 514-516 purple nonsulfur, XI, 610-512 purple sulfur, XI, 512-514 reductive carboxylic acid cycle of, VI, 207-213 Physarum polycephalum, ribonuclease PPI of, IV, 241 Physiological function design requirements, I, 465-467, 484488

adenylate energy charge, I, 470-476 interactions between input signals, I, 476-484 kinetic properties, I, 467470 Pituitary gland, 5’-nncleotidase of, IV, 346 Plants acid phosphatase of, IV, 497 adenosine diphosphoryl glucose pyrophosphorylase algae, VIII, 90 leaves, VIII, 86-90 nonchlorophyllous tissue, VIII, 93-

94

alcohol dehydrogenase of, XI, 188-189 amino acid decarboxylases of, VI, 221-224

aspartate transcarbamylase of, IX, 307-308

cytochromes b, microsomes, XI, 591 mitochondria, XI, 589-591 photosynthetic systems, XI, 587-589 fructose-l,6diphosphatase

physiological role, IV, 642-643 purification and properties, IV, 640-642

regulation, IV, 643 isoamylases of, V, 208 nuclear ribonucleic acid polymerase, X, 311-318 pullulanase of, V, 202-204 viral ribonucleic acids, terminal sequences, X , 85-86 Pneumococci, endonucleases, IV, 260-261 Point mutations, protein structure and, I, 286-292 Polymerization statistics of, X , 157-158 oligomer synthesis, X, 159-180 polymer synthesis, X, 158-159 Polynucleotide ( 8 ) enzymic methylation, general considerations, IX, 167-168 polynucleotide phosphorylase and, VII, 552-557 staphylococcal nuclease and kinetic measurements, IV, 186-187 specificity, IV, 185-186 Polynucleotide phosphorylase catalytic reactions, VII, 545-546 Clostridium and mutant Escherichia enzymes, VII, 571-572 enzyme-polynucleotide complex, VII, 570 exchange reactions, VII, 570-571 other practical uses, VII, 572-574 phosphorolysis, VII, 548-557 polymerization, VII, 557-570 specificity, VII, 546-548 degradation of, VII, 540-542 general background, VII, 533-535 in vivo


control of synthesis and activity, VII, 538-539 distribution and localization, VII, 536-538

physiological role, VII, 538 molecular weight and subunits, VII, 543-545

polymerization by kinetics, VII, 557-566 mechanism, VII, 566-570 purification, VII, 539-540 thermal stability, VII, 542-543 Polypeptide chain initiation eukaryotes inhibitors, X, 43 initiation factors, X, 29-43 initiator aminoacyl-transfer ribonucleic acid and, X, 28-29 messenger ribonucleic acid transla: tion, X, 43-44 prokaryotes general, X, 2-5 initiation factors, X,6-28 initiator aminoacyl-transfer ribonucleic acid and, X, 5-6 regulation interference factors, X, 44-45 messenger recognition, X, 46-51 Polypeptide chain termination events of protein synthesis and, X, 114-117

mechanism interaction of release factors with ribosomes, X, 101-108 in vitro assay, X, 100-101 peptidyl-transfer ribonucleic acid hydrolysis, X, 108-114 role of guanine nucleotides, X, 106108

requirements soluble protein factors, X, 95-100 terminator codons, X, 88-95 Polyprenyl biosynthesis stereospecificity 3-hydroxy3-methylglutaryl coenzyme A synthesis, 11, 186-189 preparation and use of labeled mevalonates, 11, 192-204 squalene biosynthesis, 11, 190-192

Polysaccharide depolymerase Aerobacter phages, V, 398 Azotobacter phage, V, 397-398 bacteriophage F series catalytic properties and biological significance, V, 393 enzyme assay, V, 392 partial purification, V, 392-393 stability, V, 393 Klebsiella phage enzyme properties and role, V, 394-395

preparation and assay, V, 394 Pseudomanas phages, V, 395-397 Porphobilinogen synthesis, mechanism of, VII, 333-337 Potatoes, 5’-nucleotidase of, IV, 349 Procarboxypeptidase B, physical and chemical properties, 111, 67-68 Proelastase, activation of, 111, 331-332 Prokaryotes polypeptide chain initiation general, X, 2-5 initiation factors, X, 6-28 initiator aminoacyl-transfer ribonucleic acid and, X, 5-6 Proline iminopeptidase, 111, 115 Proline 4-monooxygenase, properties, XII, 292-293 Proline reductase, Schiff base and, 11, 358

Proline residues, proteinase inhibitors and, 111, 423 Prolyl hydroxylase, XII, 152-154 assays, XII, 161 catalytic properties, XII, 156-160 nonvertebrate, XII, 163-165 purification and molecular properties, XII, 154-156 regulation, XII, 161-163 Propane-l,2-diol dehydrase, stereochemistry, 11, 210-214 Propinquity effects activation parameters and kinetic order, 11,250-254 approximation through noncovalent forces charge-transfer, 11, 254-264 Debye, 11, 254-264 hydrogen bonds, 11, 254-264

524 inclusion compounds, 11, 274-279 London, 11, 254-264 micelles, 11, 264-274 evaluation of concepts, 11, 220-226 Propionyl coenzyme A carboxylase distribution, VI, 48 historical background and metabolic significance, VI, 46-48 mechanism of action, VI, 51-53 molecular characteristics, biotin binding site, VI, 50-51 reaction catalyzed, VI, 46 stereochemistry, 11, 208-210 substrate specificity, VI, 49 a-(n-Propyl)malate, synthesis, VII, 426-427 Prostate gland acid phosphatase assay, IV, 457 electrophoresis, IV, 468-469 functional groups, IV, 469-472 general, IV, 455-457 kinetics, IV, 457466 physical properties, IV, 476 preparation, IV, 466-468 transphosphorylation, IV, 472-473 use as a reagant, IV, 473-476 Protease(s), see also .Proteinases activation, proteolysis and, I, 416 gene duplication and pancreatic, I, 303-307 dfhydryl, I, 307-308 hexokinases and, IX, 2-7 modification of, I, 118-124 serine, sequence homologies, 111, 343 352 X-ray diffraction studies, 1, 63-67 Protein (8) allosteric alternative approaches, I, 385-388 evaluation of, I, 372-375 evolutionary considerations, I, 390393 hemoglobin, I, 388-390 molecular basis of cooperativity, I, 375-379 protein design, I, 379-381 status of simple models, I, 381-385 chain termination and eukaryotic, X, 99-100


prokaryotic, X, 95-99 cleavage by pepsin, 111, 140-142 functional group adenylylation, VIII, 40-49 genetic phenomena in chain shortening, I, 292-293 deletion, addition, chain extension, I, 293-300 gene duplication, I, 3W314 point mutations, I, 286-292 homologous, speciation of, I, 321-328 structure, common characteristics, I, 87-89 structure-function relationships, evolution and, I, 267-274 Proteinase(s), see also Protease(s) acid, see Acid proteinases alkaline, see also Alkaline proteinases bacterial, 111, 605-606 inhibitor association with, 111, 428-433 inhibitor complexes, dissociation of, 111, 434-436 microbial, see Microbial proteinases neutral, see Neutral proteinases thiol, microorganisms and, 111, 791-795 Proteinase inhibitors assays active site titrants, 111,393-396 competitive enzyme assays, 111, 396 errors in, 111, 396-400 association constants enzyme titration methods, 111, 402403 physicochemical methods, 111,400402 potentiometric method, 111, 403-406 competitive inhibition and, 111, 391393 complex formation, changes in conformation-sensitive parameters, 111, 406-410 crystalline inhibitors and complexes, 111, 383-384 definition, 111, 378 disulfide loop, reactive site and, 111, 422-423 enzyme-susceptible bond in reactive site, 111, 419 historical background, 111, 376-378


identities and nomenclature, 111, 378379

kallikrein inhibitor and pancreatic inhibitors, 111, 379-380 serum inhibitors, 111, 382 soybean inhibitors, 111, 380-382 inhibition a t same reactive site, 111, 470-473

molecular differences analogies and homologies, 111,457463

specificity toward other enzymes,

111, 463473 nonoverlapping reactive sites, 111, 466-468

overlapping independent reactive sites, 111, 468470 physical properties, 111, 384-388 reactive site model chemical model of inhibitor, 111, 443-447

control dissociation of complex, 111, 437439

detection of reactive sites, 111, 412-418

equilibria of hydrolysis, 111, 423428 general properties, 111, 418-423 kinetics of interaction, 111,428437 nature of stable complex, 111, 450451

objections to, 111, 451457 overshoot of complex, 111,410-412 residue replacement a t reactive site,

111, 439-441 sites for other enzymes, 111, 441443 temporary inhibition, 111, 447-449 special purification techniques, 111, 389-391

N-terminal residue in reactive site,

111, 419-420 virgin and modified, interconversion,

111, 436437 Protein kinase(s) cyclic nucleotide-regulated, VIII, 566-578

historical background, VIII, 555-557 nonclassified, VIII, 578379 acidic nuclear protein kinases, VIII, 580

histone kinases, VIII, 579-580

phosvitin kinases, VIII, 580-581 substrate-specific phosphorylase kinase, VIII, 557-565 pyruvic dehydrogenase kinase, VIII, 565-566

Pro teolysis metabolic regulation and blood coagulation, I, 416417 proteolytic enzyme activation, I, 416 polynucleotide phosphorylase and,

VII, 557 Prothrombin activating enzyme, 111, 315-317 amino acid and carbohydrate composition, 111,313 aasays of, 111, 308 disulfide bridges, 111, 313 isolation and purity, 111, 308-311 metabolism biosynthesis, 111, 320-321 turnover rate, 111,320 number of polypeptide chains, 111, 314-315

physical properties, 111, 311412 structural aspects models, 111, 318-319 proteolysis, 111, 317-319 secondary proteolysis, 111, 319 N- and C-terminal analysis, 111,313314

Proton dissociation-replacement reactions, 11, 312-313 citrate synthase, 11, 316-317 hydrogenase, 11, 317-318 malate synthase, 11, 316-317 stereochemistry, 11, 313-315 1,l-Proton shifts, epimerases and, 11, 295-298

l,2-Proton shifts, aldo-keto isomerases,

11, 290-295 l,3-Proton shifts, allylases and, 11,299302

Protozoa, glycogen synthetase of, IX, 359 Pseudomonads aspartokinases of, VIII, 551-552 isoamylase of, V, 204-206 Pseudomonus, proteinases, of, 111, 769770

Pseudomonas aeruginosa, phage polysaccharide depolymerase, V, 395-397


Pseudomonas putida, phage polysaccharide depolymerase, V, 397 Pseudomonas testosteroni As-3-ketosteroid isomerase catalytic properties, VI, 5 9 W mechanism, VI, 605-615 molecular properties, VI, 592699 Pseudouridine kinase, properties, IX, 62 Pullulanase Aerobacter aerogenes preparation and physical properties, V, 195-197 reversion reactions, V, 201 substrate specificity and action pattern, V, 197-201 plant, V, 202204 Purine aminohydrolase (s) assay methods, IV, 51 distribution, IV, 49-51 historical background, IV, 48 Purine nucleoside phosphorylase assays direct spectrophotometry, VII, 505 inorganic orthophosphate estimation, VII, 504 isotopic assays, VII, 505 pentose estimation, VII, 503-504 spectrophotometry coupled with xanthine oxidase, VII, 504-505 catalytic mechanism equilibrium studies, VII, 511 reaction mechanism, VII, 512 reaction sequence, VII, 511-512 distribution in nature, VII, 485-490 historical development, VII, 483485 kinetics substrate concentration and, VII, 505-510

temperature and pH effects, VII, 510-511

metabolic functions bacterial metabolism, VII, 494 chemotherapy, VII, 493-494 erythrocyte metabolism, VII, 4 9 2 493

fish skin, VII, 494495 nucleoside metabolism, VII, 490492 properties, VII, 495-496 purification, VII, 495 reactions catalyzed, VII, 496-500

specificity carbohydrates, VII, 502-503 purines, VII, 500-502 subunit structure, VII, 514 sulfhydryl groups, VII, 513-514 Pyridine nucleotide ( s ) deoxythymidine diphosphate-D-glucose oxidoreductase and, V, 474478 phosphofructokinase and, VIII, 276 Pyridine nucleotide-disulfide oxidoreductases mechanism, similarities and contrasts, XIII, 94-99 reaction catalyzed-chemical similarities and crom-reactivity, XIII, 92-94 structure, similarities and contrasts, XIII, 99-105 Pyridoxal, reactions with amino acids, 11, 339-345 Pyridoxal-linked reactions absorption spectra and, VII, 62-65 amino acids and, VII, 33-39 mechanisms, VII, 65-66 @-elimination and, VII, 66-72 7-elimination and replacement, VII, 72-73

stereochemistry, VII, 73-74 Pyridoxal phosphate, fructose-1,6diphosphatase and, IV, 620 Pyridoxal phosphate enzymes apoenzymes and, 11, 346-348 classification, 11, 367-368 comparative characteristics, 11, 363-364 mechanism of reaction, 11, 349-356 peptide sequences, 11, 366 structural and spectral properties, 11, 348-349

Pyrimidines, dioxygenase reactions of, XII, 169-179 Pyrimidine deoxyribonucleoside 2'-hydroxylase, catalytic properties, XII, 176178

Pyrimidine nucleoside monophosphokinases, IX, 87-88 Pyrocatechase structure, physical probes, 11, 406-407 Pyrophosphokinase(s), other, X, 624-628 Pyrrolidone carboxylate derivatives, enzymic formation of, IV, 146147

TOPICAL SUBJECT INDEX detection and determination, IV, 125127

enzymic formation from glutamate D-glUtaInate cyclotransferase, IV, 133-136

L-glutamate cyclotransferase, IV, 138 glutamine synthetase, IV, 136-137 yglutamylcysteine synthetase, IV, 136-137

rat liver nuclear preparations, IV, 138-139

enzymic formation from glutamine and glutaminyl peptides L-glutamine cyclotransferase, IV, 139-141

y-ghtamyl cyclotransferase, IV, 141 y-glutamyl transpeptidase, IV, 141 enzymic formation from y-glutamyl amino acids y-L-glutamyl cyclotransferase and, IV, 142-146 historical background, IV, 124-125 metabolism, IV, 149-151 natural occurrence, IV, 127-130 nonenzymic formation, IV, 130-133 Pyrrolidone carboxylyl peptidase, IV, 147-149

Pyrrolidonyl peptidase, 111, 113-114 Pyruvate carboxylase acyl coenzyme A derivatives and general properties, VI, 24-27 parameters reflecting enzyme conformation, VI, 29-31 specificity of activation, VI, 27-29 aspartate and, VI, 31-33 first partial reaction, VI, 9-10 immunochemical studies, VI, 23 general properties, VI, 2-3 generalized minimal mechanism, VI, 6-7

monovalent cation effects, VI, 6 partial reactions, VI, 3-5 presence of bound biotin, VI, 3 requirements, VI, 5-6 historical background, VI, 1-2 mild denaturation and chemical modification maleic anhydride, VI, 22-23 other reagents, VI, 23 sulfhydryl reagents, VI, 21-22

temperature, VI, 19-20 urea and pH, VI, 20-21 molecular parameters and quaternary structure, VI, 16-18 nicotinamide adenine dinucleotide and, VI, 33-34 phosphoenolpyruvate and, VI, 34 product inhibition-two-site mechanism, VI, 7-8 regulation of synthesis, VI, 34 role of metals, 11, 511-515 second partial reaction nature of bound metal ion, VI, 10-11 role of bound metal ion, VI, 11-15 Pyruvate dehydrogenase complexes composition and organization, I, 215220

regulatory features, I, 220-224 Pyruvate kinase assay, VIII, 371 catalytic mechanism, VIII, 379-382 control, VIII, 378-379 historical background, VIII, 353-355 kinetics inhibitors, VIII, 375-377 substrates and activators, VIII, 372-375

molecular properties chemical modification, VIII, 360-361 composition, VIII, 358 ronformationnl change, VIII, 361364

purification, VIII, 355-358 structure, VIII, 358-359 muscle, role of metal in mechanism, 11, 504-506 stoichiometry and specificity cofactors, VIII, 366-370 number of active sites, VIII, 370371

reaction catalyzed, VIII, 364 substrate specificity, VIII, 364-366 thermodynamics, VIII, 371-372 yeast, role of metals in mechanism, 11, 506-507

Pyruvate, phosphate dikinase catalytic properties kinetic studies, X, 643, 644-645 mechanism, X, 641-642 metal ion requirements, X, 645


regulation, X, 648-649 specificity, X,646 stoichiometry, X,638 molecular interconversions, X, 635636 phosphoryl and pyrophosphoryl enzyme, X, 636-637 purification, X, 633-634 stability, X, 634 sulfhydryl groups, X, 635 Pyruvate synthase, properties, VI, 197201 Pyruvic dehydrogenase kinase, properties, VIII, 565-566

Q Qj3 replicase, elongation factors and, X,

83-85

R Rana cateebiana collagenase, 111, 689-690 catalytic properties, 111, 691-693 preparation, 111, 69M91 Rate equations derivation chemical reaction, 11, 61-63 isotopic exchange, 11, 63-65 Reactions, reversible, unidirection of, 428-430 Red cell acid phosphatase general properties, IV, 477 purification and separation of genetic types, IV, 477-484 Reduced nicotinamide adenine dinucleotide kinase, properties, IX, 82 Reduviin, thrombin and, 111, 304-305 Relaxation amplitudes kinetic studies near equilibrium calculation of amplitudes, 11, 10510s thermodynamic effects of chemical reactions, 11, 101-105 transformation to normal concentration variables, 11, 99-101 Relaxation spectra kinetic studies near equilibrium

alternative treatment of multistep mechanisms, 11, 91-93 analysis and interpretation, 11,95-99 multistep mechanisms, 11, 89-91 one-step mechanisms, 11, 83-84 thermodynamically dependent reactions, 11, 93-95 transient and stationary solutions of rate equations, 11, 84-87 two-step mechanisms, 11, 87-89 Rennin, gene duplication and, I, 308 R-enzyme, see Pullulanase j3-Replacement reactions, pyridoxallinked, VII, 54-57 Retinal isomerase, pigment regeneration and, VI, 587-589 Reverse transcriptase biological role noninfectious murine sarcoma virus, X, 232 other inhibitors, X, 233 rifamycins, X, 233 Rous sarcoma virus a, X, 231-232 temperature sensitive Rous sarcoma virus, X, 232-233 comparison to other polymerases, X, 233-235 inhibitors rifamycins, X, 231 sulfhydryl reagents, X, 230-231 nuclease activity deoxyribonuclease, X, 220-221 ribonuclease, X,221 ribonuclease H,X, 221-222 primer and direction of synthesis, X, 225-226 problems, X, 229-230 properties, X, 218 sire, X, 219-220 storage and stability, X, 219 properties of catalytic reaction deoxyribonucleoside triphosphate, X,224 divalent cations, X, 224-225 other conditions, X, 225 purification, X, 216-218 serological relationships avian leukosis viruses, X, 223 general considerations, X,222


mammalian C-type viruses, X, 223224 other viruses, X,224 solubilization, X, 215 template fidelity of synthesis, X, 228-229 preferences, X,226-228 requirements, X, 226 size, X,229 virus purification and, X, 214-215 Reversions, mutations and, I, 249-251 L-Rhamnose isomerase, properties, VI, 345-346 L-Rhamnulose 1-phosphate aldolaae catalytic reaction assay, VII, 308-309 equilibrium constant, VII, 310-311 metal ions and, VII, 309 pH optimum, VII, 309 substrate binding and reaction sequence, VII, 311-313 turnover number, VII, 309 historical background, VII, 304 metabolic significance, VII, 305 molecular properties isolation, VII, 305 physical properties, VII, 306 structure, VII, 306-308 occurrence, VII, 304305 Rhodopseudomonas capsulatus, aspartokinase of, VIII, 544-545 R hodopseudomonua spheroides 8-aminolevulinate synthetase of, VII, 344-345 aspartokinase of, VIII, 552-553 membrane adenosine triphosphatase,

x,429

Rhodospirillum rubrum, adenosine diphosphoryl glucose pyrophosphorylase of, VIII, 81-86 Riboflavin kinase, properties, IX, 74-75 Ribonuclease (s) aggregation of, IV, 744-746 assays, IV, 747-750 rhain conformation and solvent-induced changes, IV, 725-726 added electrolytes, IV, 735-737 organic solvents, IV, 733-735 thermal and acid transitions, IV,

726-731

thermodynamics, IV, 740-744 transitions in derivatives, IV, 738740 urea and, IV, 731-733 chemical modification of functional groups amino groups, IV, 677-682 arginine, IV, 689-690 carboxyl groups, IV, 675-677 cystine-disulfide groups, IV, 690-696 histidine, IV, 685-689 intramolecular crosslinks, IV, 696697 methionine, IV, 682-683 other reagents, IV, 697 chemical modification of functional groups serine and threonine, IV, 696 tyrosine, IV, 684-685 chemical synthesis and S-peptide summary, IV, 697-705 classification of, IV, 205-207 discussion of mechanism and stability lysine 41 and, IV, 801 opposite vs. adjacent attack, IV, 791-794 pH and, IV, 801-806 proton transfer and rate-limiting step, IV, 795-796 role of oxygen, IV, 79-01 stabilization of intermediates, IV, 794-795 structure, IV, 785-788 transphosphorylation and hydrolysis, IV, 788-791 enzymic cleavage of main chain chymotrypsin, IV, 674 elastase, IV, 672-673 pepsin, IV, 673 subtilisin, IV, 669-672 trypsin, IV, 673-674 fungal, general survey, IV, 208-211 historical background, IV, 647-649 isolation and chromatography, IV, 649-653 macromolecular inhibitors, IV, 758-759 mechanism of catalysis Mathias and Rabin et al., IV, 780781 Roberts et al., IV, 784


Usher, IV, 783-784 Wang, IV, 782-783 Witzel, IV, 781-782 microbial, of interest, IV, 239-243 list of, IV, 243-249 physical parameters diffusion coefficient, IV, 708-709 electrophoretic mobility, IV, 710-711 fluorescence, IV, 718-719 hydration and axial ratio, IV, 709710

hydrogen ion equilibrium, IV, 711712

hydrogen exchange, IV, 712-714 molecular weight, IV, 709 physical parameters nuclear magnetic resonance and electron paramagnetic resonance, IV, 723-725 optical rotatory dispersion and circular dichroism, IV, 719-723 partial specific volume, IV, 705-707 radius of gyration, IV, 707-708 refractive index increment, IV, 707 sedimentation behavior, IV, 709 ultraviolet absorption spectra, IV, 714-717

viscosity, IV, 710 reaction catalyzed, IV, 746-747 small molecule effectors, IV, 759-772 specificity, IV, 750-751 base, IV, 754-758 phosphate, IV, 758 sugar, IV, 752-754 steady state kinetic data ionic strength and, IV, 777-778 Michaelis constants and turnover number, IV, 772-777 organic solvents and, IV, 779-780 structure amino acid sequence, IV, 653-654 physical probes, 11, 396-401 three-dimensional, IV, 654-669 Ribonuclease N,, IV, 230-231 applications, IV, 232-234 preparation, IV, 231 properties, IV, 231-232 R.ibonuclease T, applications, IV, 222-223

preparation, IV, 212-213 properties, IV, 213-214 specificity and mode of action, IV, 215-218

structure and function, IV, 218-222 Ribonuclease Tz applications, IV, 229-230 preparation, IV, 223-224 properties, IV, 224-225 specificity and mode of action, IV, 225-229

Ribonuclease Uz applications, IV, 237-239 preparation, IV, 234-235 properties, IV, 235 specificity, IV, 235-237 Ribonucleic acid synthesis, VIII, 20-21 polynucleotide adenylyltransferases, VIII, 24-26 polynucleotide phosphorylase, VIII, 23-24

ribonucleic acid polymerase, VIII, 21-23

Ribonucleic acid polymerase(s) animal general properties, X, 280-283 historical, X, 262-264 inhibitors, X, 295-299 intracellular localization, X, 279-280 nomenclature, X, 264-266 physiological role, X, 299-300 purification, X, 266-269 regulation in vivo, X, 300 stirnulatory factors, X, 293-294 structure, X, 269-279 template specificity, X, 284-293 bacterial assay, X, 338-339 chain elongation, X, 359-366 chain initiation, X, 353-359 chain termination, X, 366-370 inhibitors, X, 370-374 outline of reaction, X, 346-348 purification, X, 335-338 template binding, X, 348-353 variety of reactions catalyzed, X, 344-346

chain elongation kinetics, X, 364-366


nondissociable ternary complex, X, 359-361 specificity, X, 361-363 chloroplast, X, 329-330 covalent modification diphosphopyridine nucleotidedependent, VIII, 48-49 possible artifact, VIII, 49 inhibitors, X, 370 agents affecting template, X, 373374 agents interacting with enzyme, X, 371-373 fungal, X, 310-311 higher plant enzyme properties, X, 314-316 factors, X, 317-318 function, X, 318 molecular properties, X, 314 solubilization and purification, X, 311-314 mi tochondrial enzyme properties, X, 325-328 molecular properties, X, 324-325 solubilization and purification, X, 318-324 synthesis, X, 328 nuclear animal, X, 262300 higher plant, X, 311-318 yeast and fungi, X , 300-311 structure of bacterial enzyme dissociation and reconstitution, X, 342-343 missing subunit problem, X, 343-344 molecular weight, X, 341-342 subunits and, X, 340-341 yeast, X, 300-301 enzyme properties, X, 306-309 function, X, 310 molecular properties, X, 304-306 solubilization and purification, X, 301-304 stimulatory factors, 309310 Ribonucleoside 2’,3’-cyclic phosphate diesterase nervous ‘tissue, IV, 363-364 intracellular localization, IV, 364-356 physiological rale, IV, 365 properties and substrate specificity, IV, 364

x,

531 Ribonuceloside 2’,3’-cyclic phosphate diesterase with 3’-nucleotidase activity cellular localization, IV, 361-362 a metalloenzyme, IV, 362-363 physiological function, IV, 363 properties kinetics and mechanism of action, IV, 358-361 physical and chemical, IV, 358 substrate specificity, IV, 357458 Ribonucleotides reduction, regulation of, I, 442-443 Ribose-5-phosphate isomerase general, VI, 318-320 catalytic properties assay methods, VI, 321-322 mechanism, VI, 323-324 Michaelis constants, VI, 322-323 molecular properties, VI, 320-321 Ribosomal ribonucleic acid methyltransferase biological significance, IX, 189-190 isoIation and properties, IX, 187-189 occurrence, IX, 187 Ribosome(s) elongation and, X, 67, 77-78 sites involved, X, 09-77 structure and, X, 68-69 elongation factors and, X, 62-03, 66-67 release factor interaction bacterial, X , 101-103 mammalian, X, 103-106 sites involved in elongation guanosine triphosphatase and factor binding sites, X, 73-76 peptidyltransferase center, X, 69-72 role of 30 S proteins, X, 76-77 Ribulose-1,d-diphosphate carboxylase general considerations, VI, 169-173 kinetics and specificity, VI, 181-183 “carbon dioxide,” VI, 183-184 divalent metal ion, VI, 185-186 inhibitors, VI, 186-187 ribulose diphosphate, VI, 184-185 light activating factors, VI, 191-192 mechanistic considerations, VI, 187-191 molecular properties composite quaternary structure, VI, 179-180

532


native enzyme, VI, 173-178 subunits, VI, 178-179 reaction, VI, 180-181 o-Ribulose-5-phosphate 3’-epimerase, properties, VI, 374375 ~Ribulose-5-phosphate 4’-epimerase, properties, VI, 372-373 Rubredoxin, chemical properties, XII, 12-15 historical background, XII, 4-6 physical properties, XII, 6-12 Rye grass, 3’-nucleotidase of, IV, 353

s Saccharomyces, see also Yeast acid phosphatase, IV, 497 Saccharomyces cerevisiae aspartate transcarbamylase of, IX, 302-306

aspartokinase of, VIII, 553 fructose-l$-diphosphatase, regulation, IV, 640 Salicylate hydroxylase, properties, XII, 206211

Salmonella phage lytic enzyme, V, 398 Sarcoplasmic membrane(s) calcium binding by, X, 450451 calciumdependent adenosine triphosphatase ion requirements, X, 446-447 lipid depletion and, X, 449 membrane permeability and, X, 445446

temperature and pH, X, 447 thiol reagents and, X, 449-450 calcium-independent adenosine triphosphate, X, 444445 calcium transport by absence of precipitating anions, X, 451 adenosine triphosphate extra splitting, X, 453-455 presence of precipitating anions, X, 451453

release from preloaded vesicles, X, 455456

composition lipids, X, 442 proteins, X, 440-442 in situ, X, 434-435

iso1ated contaminants, X, 437 procedures, X, 435-437 shape and size of vesicles, X, 437-439 lipid functions enzyme properties, X, 465467 membrane permeability, X, 465 lipoprotein structure, physical properties, X, 442-444 Saturation curves, fitting of, I, 361365 Scatchard plots, enzyme regulation and, I, 359-361 Schiff base(s) formation general characteristics, 11, 336-339 nonenzymic catalytic effects, 11, 339-346

other than pyridoxal, TI, 345-346 Schiff base enzyme(s) noncarbonyl, 11, 358-359 aldolases and transaldolases, 11, 359-360

8-aminolevulinate dehydratase, 11, 361-362

comparative properties, 11, 369 p-keto-acid decarboxylases, 11, 359 Seminal plasma bull, 5’-nucleotidase of, IV, 342-343 Serine hydroxymethyltransferase, properties, IX, 215-221 Serine peptides, microbial proteinases and, 111, 751-752 Serine residues, chemical modification, I, 173 chymotrypsin amide hydrolysis and, 111, 224231 ester hydrolysis and, 111, 218-224 ribonuclease, IV, 696 subtilisin, 111, 575-576 conversion to cysteine, 111,577-580 sequence in other proteinases, 111, 576-577

Serratia marcescens, adenosine diphosphorylglucose pyrophosphorylase of, VIII, 107-108 Serum, acid phosphatase in, IV, 495-496 Slime molds, fructose-l,6-diphosphatases of, IV, 640 Solvation, physical organic models, 11, 226-238


Sorangium, proteinases of, 111, 747, 752-754, 789-790

Spinach adenosine diphosphoryl glucose pyrophosphorylase of, VIII, 86-89 chloroplast adenosine triphosphatase assay, X , 389 catalytic properties, X, 391393 cold inactivation, X, 390-391 molecular properties, X, 390, 391 nucleotide binding, X, 39-94 purification, X, 389-390 Spleen acid deoxyribonuclease catalytic properties, IV, 276-285 distribution, intracellular localization and biological role, IV, 285-287

physical and chemical properties, IV, 272276

acid exonuclease, IV, 330-336 acid phosphatase, IV, 493-495 deoxyribonuclease of, IV, 272-287 glycogen synthetase of, IX, 354 Squalene, epoxidase and, VII, 211 Staphylococcal nuclease active site, stereochemical probes,

IV, 195-196 behavior in solution, IV, 183-184 covalent structure, IV, 180-183 crystallographic studies, introduction,

IV, 156-159 fluorescence spectroscopy, 11, 429 fragments, complementation of, IV, 196-199

general, IV, 153-156 historical background, IV, 177-178 isolation, IV, 178-179 mechanism, IV, 174-175 peptide chain conformation, IV, 159-163

proposed future studies, IV, 175 studies in solution, IV, 17%174 substrate specificity and catalytic mechanisms polynucleotide substrates, IV, 185-187 size and specificity of active site,

IV, 191-195 synthetic substrates and inhibitors,

IV, 187-191

533 synthetic analogs and, IV, 199-204 synthetic substrates and inhibitors kinetic measurements, IV, 190-195 specificity, IV, 187-189 thymidine-3',5'-diphosphate and calcium ion binding, IV, 163-171 ultraviolet difference spectroscopy, 11, 413-414

a warning, IV, 174 Staphylococci, lytic phage enzymes, V, 398-399

Staphylococcus, acid phosphatase of, IV, 498

Staphylococcus aurews phage lytic enzyme, V, 400-401 virolysin, V, 399-400 Starch structure determination, debranching enzymes and, V, 228-234 Stearate, conversion to oleate, 11, 179-184 Stereochemistry a,j3 elimination reactions, 11, 309-312 proton transfer, 11, 313-315 Stereospecificity, nicotinamide adenine dinucleotide-dependent oxidoreductases, 11, 134-157 Steroid sulfatase(s), V, 4-6 androstenolone sulfatase, V, 7-9 cortisone sulfatase, V, 10 estrone sulfatase, V, 6-7 etiocholanolone sulfatase, V, 9-10 Strain, physical organic models, 11, 226-238

Streptococcal proteinase activation, 111, 627-628 amino acids active site, 111, 626-627 composition, 111, 624-625 N- and C-terminal, 111, 625-626 assay methods, 111, 627 immunological properties, 111, 615-619 kinetics of hydrolysis dielectric constant and, 111, 636-637 esterase vs. peptidase activity, 111, 638-639

p H and, 111, 633-636 structural inhibitors, 111, 637-638 mechanism of action, 111, 639-647 physical properties electrophoresis, 111, 614

534 gel filtration and chromatography, 111, 613-614 molecular weight, 111, 614 stability, 111, 614-615 preparation and crystallization proteinase, 111, 611413 zymogen, 111, 610-611 specificity esterase activity, 111, 631-632 peptide and amide bonds, 111, 628-631 transferase activity, 111, 632633 sulfhydryl group nature, 111, 622-623 reactivity, 111, 623-624 zymogen-to-enzyme transformation autocatalytic, 111, 621 bacterial cell walls, 111,621-622 preformed streptococcal proteinase, 111, 621 subtilisin, 111, 620-621 trypsin, 111, 619-620 Streptococci endonucleases, IV, 260-261 phage lytic enzyme, V, 402-403 enzyme assay, V,403-404 group C phages and, V,404-406 other phages, V, 406-408 Streptococcus faecalis, membrane adenosine triphosphatase, X,400-416 Streptomyces, proteinases of, 111, 746-747, 752-754, 768-769 Structural information other methods of obtaining Fourier difference maps and salt difference maps, I, 4145 noncrystallographic symmetry, I, 45-46 single isomorphous replacement and variations, I, 39-11 Submandibular gland, hyaluronidase of, V, 312-313 Substrate bridge complexes electronic structure of, ATP, 11, 478-481 formation mechanism, 11,48143 reaction mechanism, 11, 483-485 Subtilisin (s) active site studies histidine and, 111, 580-584


serine and, 111, 575-580 chemical modification lysine residues, 111, 596-598 methionine residues, 111, 598-599 tyrosine residues, 111, 599-602 historical background and development, 111, 562-563 inhibitors, dye binding and, 111, 602-605 physical, chemical and stability properties subtilisin Amylosacchariticus, 111, 566-567 subtilisin BPN’, 111, 565-566 subtilisin Carlsberg, 111, 564-565 subtilisin Novo, 111, 566 practical uses, 111, 606-607 detergents and, 111, 608 protein sequencing, 111, 807 primary structure comparison of sequences, 111, 571-575 general comparison, 111, 567 subtilisin Amylosacchariticus, 111, 569-571 subtilisin BPN’, 111,567-569 subtilisin Carlsberg, 111, 569 subtilisin Novo, 111, 509 ribonuclease and, IV, 669-672 substrate specificity and enzymic properties mechanism of action, 111, 593-596 protein and peptide substrates, 111, 584-586 synthetic substrates, 111, 586-593 transesterification and transpeptidation, 111, 593 X-ray structure background, 111, 547452 catalytic site, 111, 553-560 comparison with subtilisin Carlsberg, 111, 560 general description, 111, 552-553 Subtilisin BPN’, chemical modification, I, 203-205 Subunits acetyl coenzyme A carboxylase, VI, 60-79 adenylosuccinase, VII, 192-193 aldolases, VII, 221-224

!K)PICAL SUBJECT INDEX

amino acid decarboxylases, VI, 248-253 argininosuccinase, VII, 176-177 aspartate transcarbamylase, IX, 230-243 aspartokinases, VIII, 518-519, 541 carbamate kinase, IX, 103-104 creatine kinase, VIII, 394-395 fumarase, V, 545-549 glucose-6-phosphate isomerase, VI, 279-281 glycerol kinase, VIII, 494-495 guanidino kinases, VIII, 4-88 hexokinase, IX, 8-10, 39-40 2-keto-3-deoxy-6-phosphogluconic aldolase, VII, 285-287 phosphofructokinase, VIII, 254-257 polynucleotide phosphorylase, VII, 543-546 purine nucleoside phosphorylase, VII, 514 thiolase, VII, 396-397 transcarboxylase, 95-101 triosephosphate isomerase, VI, 327-329 uridine diphosphate-n-glucose 4’epimerase and, VI, 366-368 Succinate dehydrogenase mammaliam, XIII, 222-223 enzymic properties, XIII, 236-245 inhibitors and modifiers, XIII, 245-247 mechanism, XIII, 251-254 molecular properties, XIII, 223-236 regulatory properties, XIII, 247-251 microorganisms and, XIII, 254-256 stereospecificity, 11, 176-179 Succinyl coenzyme A mutase, stereochemistry, 11, 206-210 Succinyl coenzyme A synthetase anomalous reactions, X, 605-606 catalytic intermediates others, X, 602603 phosphoensyme, X, 596-600 succinyl phosphate, X, 600-602 Escherichia coli active site, X, 590 characterization of phosphoenzyme, x, 583-584 purification, X, 582483 quaternary structure, X, 584-589 reactivity and stability, X, 689

535 size, X, 584 sulfhydryl groups, X, 590-591 other sources, X, 594 pig heart phosphoenzyme formation, X, 592-593 polymorphism, X, 593-594 quaternary structure, X, 591-592 size, X, 591 possible regulatory properties, X, 606 reaction catalyzed, X, 581-582 steady state kinetics, X, 603-605 substrate specificity coenzyme A, X, 595 nucleoside di- and triphosphates, X, 595-596 succinate, X, 594-595 Sucrose phosphorylase covalent glucose-enzyme configuration of bond, VII, 524 isolation and properties, VII, 521423 kinetics, VII, 510-521 mechanism of catalysis, VII, 524-526 purification and properties, VII, 518-519 water and alcohols as acceptors, VII, 528632 Sulfarnatases, V, 18-19 Sulfatase A other sources, V, 36-37 ox liver, V, 27-36 Sulfatase B other sources, V, 38-39 ox liver, V, 37-38 Sulfatophosphate sulfohydrolases, V, 17-18 Sulfate, activation of, VIII, 35-37 Sulfate-activating enzymes, regulation and control, X, 665-669 Sulfhydryl groups, see also Cysteine, Thiol groups amylases, V, 244-245 argininosuccinase and, VII, 177-178 aspartate transcarbamylase, IX, 263-265 streptococcal proteinase, 111,622-624 Sulfhydryl reagents, pyruvate carboxylase and, VI, 21-22 Sulfites organic, pepsin and, 151-152


Sulfite oxidases liver catalytic properties, XII, 417-419 molecular properties, XII, 414-417 Sullite reductase(s), XIII, 286-287 NADPHdependent, properties, XIII, 287-295

reduced methyl viologendependent, XIII, 295 Sulfonamide, carbonic anhydrase and, V, 652-658, 661 Superoxide dismutase away methods, XII, 539-540 catalytic mechanism enzymic dismutation of 02-,XII, 552-556

inhibition, XII, 556 model complexes, XII, 657 crystallization, XII, 542 determination of purity and concentration, XII, 540 historical, XII, 533 molecular properties apoenzyme, XII, 548-549 native enzyme, XII, 542-548 reconstitution, XII, 549-551 redox properties, XII, 551-552 physiological role, XII, 533 prokaryotic and mitochondrial, XII, 537-538

purification methods, XII, 538-539 sources, XII, 538 suppression, mutations and, I, 24925 1

Synovial fluid, collagenases, of, 111, 693-696

T

Tabanin, thrombin and, 111, 304-305 Tadpole liver, glycogen synthetase of, IX, 357-358 Tartrate epoxidase, VII, 201 catalytic properties factors influencing, VII, 205 kinetics, VII, 204-205 measurement of reaction, VII, 203-204

specificity and products, VII, 204

molecular properties cysteine and amino acid composition, VII, 202 molecular weight, VII, 202-203 purification, VII, 202 Temperature creatine kinase and, VIII, 420-422 p-galactosidase and, VII, 634-635 phosphoglucomutase and, VI, 455 pyruvate carboxylase and, VI, 19-20 Terminal deoxynucleotidyl transferase biological directions, X, 169-171 historical background, X, 145-147 mechanism, X, 160-161 metal ligand inhibitors, X, 161-163 other inhibitors, X, 163-165 unititiated synthesis, X, 165-168 nature of the reaction enzyme, X, 154-157 initiators, X, 149-152 metal ions, X, 152-153 nucleoside triphosphates, X, 147-149 pyrophosphate, X, 153-154 practical applications oligomeric additions, X, 167-188 polymeric additions, X, 166-167 random copolymers, X, 168-169 N-Terminal exopeptidases, background, 111, 81-82 Terminator codons biochemical identification, X, 93-95 genetic aspects, X, 88-93 Testicle, hyaluronidase of, V, 311-312 Tetranitromethane, p-hydroxydecanoyl thioester dehydrase and, V, 455 Thiamine pyrophosphokinase, X, 624427 Thiolase active site, amino acid sequence, VII, 404-405

biological function control of enzymic activity, VII, 400 metabolic significance, VII, 399 regulation of synthesis, VII, 401 catalytic properties equilibrium, VII, 397 8-ketoacyl groups and, VII, 398-399 thiol groups and, VII, 397 inhibitors, VII, 401-402 historical background, VII, 391-392 isolation and stability, VII, 393-394


mechanism, VII, 402-404 molecular properties, VII, 394-395 occurrence, VII, 392-393 subunit structure and reversible dissociation, VII, 395-397 Thiol groups, see also Cysteine, Sulfhydryl groups fumarase, V, 549-552 papain location of, 111, 514 modification of, 111, 515-516 phosphofructokinase, VIII, 269-272 Thiolsubtilisin, displacement reactions, I, 116117 Thioredoxin reductase general properties, XIII, 144-145 light-activated reduction-neutral scmiquinone, XIII, 147-148 metabolic functions, XIII, 142-144 reduced states, mechanism, XIII, 145-147 specificity of, XIII, 144 Threonine residues chemical modification, I, 173 ribonuclease, IV, 696 Threonine, serine dehydratase(s) inhibition by serine, VII, 47 mammalian liver, VII, 39-42 microbial, VII, 4 2 4 5 other sources, VII, 4647 Threonine synthetase, properties, VII, 59-60 Thrombin amino acid and carbohydrate composition, 111, 285-286 assay of, 111, 282-283 catalytic properties general, 111, 292-293 inhibitors, 111, 302306 mechanism, 111, 306-307 protein and polypeptide substrates, 111, 295-300 purported substrates, 111, 300-302 synthetic substrates, 111, 293-295 A and B chain amino acid sequences, 111, 287-290 disulfide bridges, 111, 290-291 general, 111, 278 historical background, 111, 278-280

537 homology and tertiary structure, 111, 291-292 isolation and purity, 111, 283-284 occurrence, 111, 280-282 physical properties and molecular weight, 111, 284-285 inhibitors polypeptide, 111, 304-305 protein, 111, 305-306 synthetic, 111, 302-304 sequence homology, 111, 290 N- and C-terminal 'amino acids, 111, 287 Thymidine 3',5'-diphosphate binding, staphylococcal nuclease and, IV, 163-171 Thymidine diphosphate-L-rhamnose synthetase, properties, VI, 375-376 Thymidylate synthetase, properties, IX, 210-215 Thymine 7-hydroxylase catalytic properties, XII, 174-176 Tosyl elastase tertiary structure comparison with hypothetical model, 111, 364-365 electron density map, 111, 357-358 model of, 111, 358-363 Tosyllysine chloromethyl ketone, results obtained, I, 112-113 Tosylphenylalanine chloromethyl ketone chymotrypsin and, I, 94-96 results obtained, I, 112-113 Transaldolase distribution and purification, VII, 262-265 enzymic properties, VII, 265-268 function of, VII, 259-260 historical background, VII, 261-262 mechanism of action, VII, 271-277 metabolic role, VII, 277-280 molecular properties, VII, 269-271 Schiff bases and, 11, 359-360 Transamination, see Amino group transfer Transcarboxylase catalytic properties assay and general properties, VI, ioaiog


equilibria and free energy, VI, 109-111

kinetics and reaction mechanism, VI, 111-1 15

role of cobalt and zinc, VI, 111 electron microscopy, VI, 101-108 historical background, VI, 84-89 molecular weight, metal content, and biotin content and linkage, VI, 92-95

purification, VI, 91-92 role in propionic acid fermentation, VI, 89-91 subunits dissociation, VI, 95-99 reconstitution, VI, 99-101 Transfer ribonucleic acid aminoacylation assays, X, 509 reaction product; X, 508-509 enzyme complexes, formation and detection, X, 521-522 “recogniiion” problem, X, 522-523 chemical modification and, X, 524-525

conclusions, X, 528 heterologous aminoacylation, X, 525-526

isoacceptor ribonucleic acid sequencing, X, 523-524 method of “dissected molecules,” X, 525

mutant ribonucleic acid analysis, X, 524

topology of synthetase complexes, X, 526-528 structure, X, 518-521 Transfer ribonucleic acid methyltransferase biological significance, IX, 184-186 occurrence, IX, 168-169 properties ionic stimulation, IX, 174-175 substrate specificity, IX, 172-174 purification, IX, 169-172 regulation bacteriophage infection, IX, 182 hormonal, IX, 179-182 inhibition, IX, 176-179

tumor tissue, IX, 183-184 virus infection, IX. 182-183 2,4,6Trinitrobensene sulfonate, inorganic pyrophosphatase and, IV, 515-516

Triosephosphate isomerase catalytic properties function in vivo, VI, 337-338 kinetic parameters, VI, 333335 mechanism, VI, 338-340 phosphoglycolate and, VI, 335-336 substrate active states, VI, 336-337 history and general, VI, 326 modification of, I, 138 molecular properties, VI, 326-327 active site structure, VI, 330333 isoenzymes, VI, 329-330 molecular weight, VI, 327 subunit structure, VI, 327-329 Trypsin activation, 111, 244-245 active-site-directed reagents and, I, 103-112

chemical modification activation and, 111, 271-272 amino groups and imidazole rings, 111, 269-270 disulfide bridges, 111, 271 tyrosine and tryptophan residues, 111, 270-271 water-insoluble derivatives, 111, 272-273

chemical structure, 111, 255-260 crystalline, heterogeneity of, 111, 254 fragment, inhibitor complex with, 111, 454

inhibitors low molecular weight, 111, 273-274 naturally occurring polypeptides, 111, 274-275

inorganic pyrophosphatase and, IV, 514

mechanism and active site, 111, 260-261 catalytic site, 111, 261-262 specificity and binding sites, llI, 262-263

physicochemical properties and stability, 111, 254-255 ribonuclease and, IV, 673-674 substrates and specificity


assays, 111, 267-269 modified substrates, 111, 267 role of side chain, 111, 263-265 role of substrate structure, 111, 266 substitution of amino and carboxyl groups, 111, 265-266 Trypsin inhibitor pancreatic, chemical modification of, 111, 443-444 Trypsinogen activation of, 111, 261-254 preparation of, 111, 251 Tryptophanase, properties, VII, 4849 L-Tryptophan 2,3-dioxygenase catalytic properties, XII, 129-130 historical, XII, 127-128 molecular properties, XII, 128-129 Tryptophan hydroxylase, properties, XII, 240-241 Tryptophan 5-monooxygenase, properties, XII, 291-292 Tryptophan residues, chemical modification, I, 173 trypsin, 111, 270-271 Tryptophan synthetase catalytic properties individual reactions, VII, 27-30 reaction mechanism, VII, 24-26 reactions catalyzed, VII, 22-24 historical background, VII, 1-4 molecular properties a-p2 affinity and equilibrium, VII, 21 a-chain purification and properties, VII, 8-12 p-component purification and properties, VII, 16-20 mutant a chain, VII, 13-16 mutant p subunits, VII, 20-21 mutations and, I, 256-257 occurrence, VII, 4-5 other organisms and, VII, 21-22, 30 tryptophanase and, VII, 6-8, 81 use in studies on protein synthesis and regulation, VII, 5-6 Tumor viruses, deoxyribonucleic acid polymerases of, X , 211-235, see ako Reverse transcriptase p-Tyrosinase, properties, VII, 49-51 Tyrosine hydroxylase, properties, XII, 238-240

Tyrosine 3-monooxygenase, properties, XII, 290-291 Tyrosine residues, chemical modification, I, 174 creatine kinase, VIII, 434-436 fructose-l,6diphosphatase, IV, 619-620 ribonuclease, IV, 684-685 subtilisin, modification of, 111, 599-602 trypsin, 111, 270-271

U Ultraviolet absorption peptide groups, 11, 379-380 ribonuclease, IV, 714-717 Ultraviolet difference spectroscopy protein structure, 11, 408409 solvent perturbation and, 11, 410-413 typical cases, 11, 413417 Urea a-galactosidase and, VII, 635 pyruvate carboxylase and, VI, 20-21 Urease(s) catalytic properties active site studies, IV, 20-21 kinetics, IV, 18-20 mechanism, IV, 15-16 substrate specificity, IV, 16-18 jack bean enzymic activity measurement, IV, 4-5 isolation and purification, IV, 2-4 molecular properties, IV, 5-8 chemical composition and behavior, IV, 11-12 derivatives, IV, 12-13 immunological behavior, IV, 13 molecular weight, IV, 8-10 other, IV, 10-11 other sources, IV, 13-15 Uridine-cytidine kinase assay, IX, 57-58 distribution and purification, IX, 5657 kinetic and molecular properties, IX, 59-60 reaction mechanism, IX, 60-61 regulatory properties, IX, 61-62 substrate specificity, IX, 58-59

540


Uridine diphosphate-N-acetyl-n-glucosamine 2'-epimerase, properties, VI, 371-372

Uridine diphosphate-&glucose 4'-epimerase, VI, 357-358 activators, VI, 359362 bound pyridine nucleotide protein conformation and, VI, 368369

subunit association and, VI, 365368 kinetics and specificity, VI, 358-359 mechanism of catalysis, VI, 362-366 Uridine diphosphoryl glucose pyrophosphorylase analytical and synthetic applications, VIII, 54-55 measurement of activity, VIII, 52-53 metabolic function cytology, VIII, 55-56 metabolism, VIII, 5559 regulation, VIII, 59-62 properties kinetics, VIII, 65-68 mechanism, VIII, 69-71 optima, VIII, 62 specificity, VIII, 68-69 structure, VIII, 6 2 6 5 purification, VIII, 53-54 Uridine monophosphate kinase, properties, IX, 90-91 V Velocity curves, enzyme regulation and, I, 368-369 Venom enzymes hydrolyzing phosphate esters, IV, 328 5'-nucleotidase of, IV, 342 Venom exonuclease chemical nature, IV, 317-319 general, IV, 313-317 structural determination identification of (I and u terminals, IV, 326-328 ribooligonucleotide sequences, IV, 324-326

substrate structural characteristics conformation, IV, 319-320 monophosphoryl group and, IV, 322324

nature of bases, IV, 320-321 nature of sugar, IV, 320 Vibrio cholerae, neuraminidase of, V, 328-329

Viruses neuraminidases of, V, 324 virion deoxyribonucleic acid polymerase others, X, 214 tumor viruses, X, 213-214 Visual pigments(s) light interaction early intermediates, VI, 584-585 later intermediates, VI, 585-586 overall reaction, VI, 583-584 molecular properties criteria of purity, VI, 576 linkage between retinal and protein, VI, 580-582 lipids, VI, 579-580 preparation, VI, 575-576 protein, VI, 576-577 retinal chromophore, VI, 577-579 structure and color, VI, 582-583 nature of, VI, 573-574 regeneration following illumination, VI, 587489 sites in photoreceptor, VI, 575 Vitamin B,, mechanisms, metal complexes and, 11, 528-529

Vitamin BIZcoenzyme amino group migrations and, VI, 539540

mutases and, VI, 509-511 Vitamin BIZcoenzyme-requiring dehydrases apoenzyme properties, V, 496-497 coenzyme analogs and, V, 493-496 enzyme-coenzyme interaction, V, 492493

general considerations, V, 481-482 nature of hydrogen transfer, V, 485492

substrate to product interconversion, V, 482-485 Vitamin BIZmethyltransferase alkylation studies and light stability, IX, 137-143 assay, Ix, 122-123

541


catalytic properties methyl transfers catalyzed, IX, 129135

propyl iodide inhibition, IX, 127-129 radioactive folate binding, IX, 1 3 6 137

mechanism, IX, 151-154 occurrence, IX, 162-164 physical properties absorption spectrum, IX, 124-125 resolution-reconstitution and molecular weight, IX, 125-127 purification, IX, 123-124 role of S-adenosyl methionine, IX, 143-151

Vitamin B,. adenosyltransferase catalytic properties activators and inhibitors, VIII, 151152

assay, VIII, 148-149 kinetics and substrate specificity, VIII, 150-151 reversibility, partial reactions and mechanistic considerations, VIII, 149-150

net reaction, VIII, 145-147 purification and physical properties, VIII, 147-148 significance and distribution, VIII, 144-145

W

activation refolding, 111: 182-183 arginine 145,111, 176179 catalytic site, 111, 179-182 isoleucine 16, 111, 175-176 methionine 192, 111, 179 clastase, 111, 353-356 glossary of symbols, I, 89 molecular symmetry determination and, I, 15-18 molecular weight determination and, I, 13-15 power and limitations, I, 3-5 sub tilisin background, 111, 547-552 catalytic site, 111, 553-560 comparison with subtilisin Carlsberg, 111, 560 general description, 111, 552-553 X-ray diffraction globular macromolecules heavy atom derivatives, I, 69-86 molecules studied, I, 52-69 lysozyme analysis of structure, VII, 682-692 conformation of egg-white model, VII, 692-707 crystallography of inhibitor complexes, VII, 707-717 origin of, I, 23-25 o-Xylonate dehydrase, properties, V, 582 D-Xylose isomerase properties, VI, 349-354 role of metals, 11, 511

Wheat acetyl coenzyme A carboxylase of, VI,

Y

78-79

3’-nucleotidase of, IV, 353-354 X

Yeast adenylosuccinase of, VII, 185-191 alcohol dehydrogenase of, XI, 2223, 171-186

Xanthine oxidase, see Milk xanthine oxidase metal complexes and, 11, 533-534 properties of, XII, 56 Xanthinuria, human, molybdenum hydroxylase genetics and, XII, 400402 X-ray crystallography carboxypeptidase A, 111, 1 7 4 6 chemical modification and, I, 201-202 chymotrypsinogen, 111, 169-175

aldolase of, 11, 515-516, VII, 258 enolase carboxymethylation, V, 533 photooxidation, V, 533-534 glycogen synthetase of, IX, 359-361 hex0kinases chemical studies, IX, 10-13 mechanism, IX, 13-28 modification by added proteases, IX, 6-7

542 molecular weight and subunit structure, IX, 7-10 regulation, IX, 29-31 two isozymes and endogenous proteases, IX, 2-6 p-hydrory-p-methylglutaryl coenzyme A synthase, VII, 429-431 inorganic! pyrophosphatase catalytic properties, IV, 534-539 molecular properties, IV, 530-539 invertase, V, 292-293 biosynthesis, V, 294-295 catalytic properties, V, 300303 localization and multiple forms, V, 293-294 properties, V, 298-300 purification, V, 295-298 isoamylase of, V, 206-208 mannose-6-phosphate isomerase of, VI, 304-305


methyltransferase of, IX, 161 mitochondria1 adenosine triphosphatase properties, X, 386 purification, X, 386-387 nicotinamide adenine dinucleotide dehydrogenase of, XIII, 216221 nuclear ribonucleic acid polymerase, X, 301-310 5'-nucleotidase of, IV, 341442 proteases acid, 111, 723-744 diisopropylfluorophosphate-sensitive, 111, 744-765 metal-chelator sensitive, 111, 765-786 other, 111, 786-795 2

Zinc, carbonic anhydrase and, V, 641-642

A 8 C D E

6 7 0 9 O

F G H 1

1 2 3 4

1 5

The Enzymes. Volume XIII: Oxidation-Reduction. Part C: Dehydrogenases (II), Oxidases (II), Hydrogen Peroxide Cleavage. Third Edition

The Enzymes. Volume XIII: Oxidation-Reduction. Part C: Dehydrogenases (II), Oxidases (II), Hydrogen Peroxide Cleavage. Third Edition

The Enzymes, Vol XII: Oxidation-Reduction, Part B (Electron Transfer (II), Oxygenases, Oxidases (I)), 3rd Edition

Hydrogen Peroxide: Medical Miracle

Hydrogen Peroxide: Medical Miracle

Hierachies [Part Ii Of Ii]

The Crossing part II

The Black Book volume III-part II

The Real Book - Volume II (C Instruments)

Volume II

Proserpina, Volume II (Illustrated Edition)

Volume II

Volume II

Volume II

Volume II

NETWORKING 2011, Part II

Material Science Part II

Grundgesetze part ii

TEXTILE MATHEMATICS - PART II

Wolf's Obsession Part II

Serat Paramayoga Part II

The Fourth Stall Part II

Grundgesetze part ii

Guide Dog, Part II

Index, part II

Environmental photochemistry part II

Dynamics, Part II

The Fourth Stall Part II

The Enzymes, Vol II: Kinetics and Mechanism, 3rd Edition

The Enzymes, Vol II: Kinetics and Mechanism, 3rd Edition

Chromatin and Chromatin Remodeling Enzymes Part C, Volume 377

The Enzymes. Volume XIII: Oxidation-Reduction. Part C: Dehydrogenases (II), Oxidases (II), Hydrogen Peroxide Cleavage. Third Edition