Methods in Enzymology Vol 284: Lipases, Part A

Preface The pace of lipase research has been accelerating. The powerful tools of molecular biology have been brought to bear, more new lipase amino acid sequences and three-dimensional structures are appearing, and new approaches for handling their complicated interracial kinetics are being reported. In addition, more ways are being discovered and used to control lipase activity and for harnessing their catalytic prowess to pull greater efficiency into older chemical processes. Indeed, studies of heterogeneous lipase catalysis, long passed over by many academic researchers in favor of more experimentally tractable homogeneous, single-phase enzyme systems, are moving closer to the level of depth previously reserved for proteases, their hydrolytic cousins. To the usual problems of abundance and purity that enzymologists and structural biologists generally face, lipases present the additional difficulty associated with multiphase systems. Unlike proteases, the substrates that are hydrolyzed by lipases are most efficiently presented to the enzyme in a separate, lipid phase. The presence of a suitable second phase appears to bring about an increase in lipase activity and, in some cases, effect a change in their three-dimensional structures. Part of the expanding interest in lipases derives from the increasing applications for these enzymes and from the success of new techniques for studying them. Previous volumes of Methods in Enzymology have dealt specifically with phospholipids, their degradation (Volume 197, Phospholipases), and their biosynthesis (Volume 209, Phospholipid Biosynthesis). The recent explosion of interest in lipases led us to develop Volumes 284 and 286. The first, Biotechnology (Volume 284), includes sequencing, cloning, and structural studies of lipases and, the second, Enzyme Characterization and Utilization (Volume 286), includes the purification of novel lipases, kinetics and assay issues, aspects of lipid metabolism, and the use of lipases in organic synthesis. Research in the lipase field has been dominated by European scientists and stimulated by the European Community Bridge Program. In addition, there has been a great deal of research emphasis on this field in industrial laboratories. Thus, the authorship of this volume is truly international and includes a diverse mixture of academic and industrial scientists. Expert secretarial assistance from Mary Kincaid, Ophelia Chiu, and Vina Wong helped enormously with the development of this volume. Editorial assistance from Shirley Light is greatly appreciated. BYRON RUBIN EDWARD A . DENNIS xiii

[ 1]

UNDERSTANDING

[1] S t r u c t u r e

INTERFACIAL PROPERTIES OF LIPASES

as Basis for Understanding Properties of Lipases

3

Interfacial

By M~ROSLAWCYGLER and JOSEPH D. SCHRAG* Introduction Lipases are water-soluble enzymes that catalyze the hydrolysis of ester bonds in triacylglycerols. In 1958, Sarda and Desnuelle 1 investigated the lipolytic activity of pancreatic lipase and carefully documented the "interracial activation" phenomenon--the enhancement of activity by the presence of micelles. They made a suggestion that pancreatic lipase works most efficiently on the two-dimensional surface of the micelle. In this and in a subsequent article, 2 Desnuelle and co-workers postulated that the lipase becomes activated when adsorbed at the water-lipid interface and that this activation process is associated with a conformational change in the enzyme. This interfacial activation phenomenon was subsequently observed for many other lipases of various origins and became a characteristic feature used to distinguish "true" lipases from esterases (e.g., see Ref. 3; reviewed in Ref. 4). The more recent discovery of lipolytic enzymes that are not interfacially activatable despite clear sequence homology to other lipases 5-7 showed that the classification based solely on the interracial activatability is too simplistic. Ransac et al. 4 discussed this question thoroughly and proposed to define lipase as "a carboxylesterase able to catalyze hydrolysis of long chain acylglycerols (p. 270)." Nevertheless, the activity of the majority of the known lipases increases in the presence of a water-lipid interface, and it is the molecular nature of this activation that has fascinated research* The authors of this chapter carried out research and contributed to the chapter on behalf of the National Research Council of Canada, and therefore "an interest in the copyright of the chapter belongs to the Crown in right of Canada, that is, to the Government of Canada. t L. Sarda and P. Desnuelle, Biochim. Biophys. Acta 30, 513 (1958). 2 p. Desnuelle, L. Sarda, and G. Ailhard, Biochim. Biphys. Acta 37, 570 (1960). 3 B. Borgstrrm and H. L. Brockman, "Lipases." Elsevier, Amsterdam, 1984. 4 S. Ransac, F. Carri~re, E. Rogalska, R. Verger, F. Marguet, G. Buono, E. P. Melo, J. M. S. Cabral, M. P. Egloff, H. van Tilbeurgh, and C. Cambillau, "Molecular Dynamics of Biomembranes" (J. A. F. Op den Kamp, ed.), Vol. H96, pp. 265-304. NATO ASI series. Springer-Verlag, Berlin, 1996. 5 C. Martinez, P. De Geus, M. Lauwereys, G. Matthyssens, and C. Cambillau, Nature (London) 356, 615 (1992). 6 E. Lesuisse, K. Schanck, and C. Colson, Eur. J. Biochem. 216, 155 (1993). 7 A. Hjorth, F. Carri~re, C. Cudrey, H. WSldike, E. Boel, D. M. Lawson, F. Ferrato, C. Cambillau, G. G. Dodson, L. Thim, and R. Verger, Biochemistry 32, 4702 (1993). METHODS IN ENZYMOLOGY, VOL. 284

Copyright © 1997 Government of Canada.

4

PRIMARY AND TERTIARY STRUCTURE

[ 1]

ers. Various biophysical and biochemical methods have been developed to study the activation phenomena and many of them, and their results, are described in other chapters of this volume. Although these investigations provided much information on the macroscopic properties of lipases, their stereoselectivity, the influence of the environment (solvent, interface properties, and parameters) on the reaction outcome, etc., the first direct evidence that a conformational rearrangement does, indeed, occur at the water-lipid interface came from crystallography. The three-dimensional structures of the first three, seemingly different, lipases were determined in short succession: the 27-kDa Rhizomucor miehei lipase, s the 46-kDa human pancreatic lipase, 9 and the 60-kDa Geotrichum candidum lipase. 1° Two surprising findings emanated from these studies. The first finding, recognized immediately in the original papers, was that the active site, composed of a Ser-His-Asp/Glu catalytic triad reminiscent of the serine proteases, 11 is not exposed on the protein surface but rather is covered by surface loops and is not accessible to the substrate. Yet there was no doubt about the enzymatic competence of the enzymes used for crystallization. The second surprising finding was the commonality of the basic fold of these lipases within their catalytic domains, lz,13 This fold, which has also been found in many esterases and other hydrolytic enzymes, has been termed the aJfl-hydrolase fold (see [4] in this volume13a). That three different lipases crystallized from aqueous solutions in enzymatically incompetent conformations suggested that the observed conformations are not an artifact of the crystallization conditions, but instead represent the biologically relevant inactive forms that are prevalent in solution. On the basis of these crystal structures it was obvious that at least some of the surface loops would have to change their conformations or positions significantly for the substrate to be able to access the active site. 8-~° This prediction was quickly confirmed experimentally. First, the strucs L. Brady, A. M. Brzozowski, Z. S. Derewenda, E. Dodson, G. Dodson, S. Tolley, J. P. Turkenburg, L. Christiansen, B. Huge-Jensen, L. Norskov, L. Thim, and U. Menge, Nature (London) 343, 767 (1990). 9 F. K. Winkler, A. D'Arcy, and W. Hunziker, Nature (London) 343, 771 (1990). 10j. D. Schrag, Y. G. Li, S. Wu, and M. Cygler, Nature (London) 351, 761 (1991). 11 D. M. Blow, Nature (London) 343, 694 (1990). 12D. L. Ollis, E. Cheah, M. Cygler, B. Dijkstra, F. Frolow, S. M. Franken, M. Harel, S. J. Remington, I. Silman, J. Schrag, J. L. Sussman, K. H. G. Verschueren, and A. Goldman, Protein Eng. 5, 197 (1992). 13 M. Cygler, J. D. Schrag, and F. Ergan, Biotechnol. Genet. Eng. Rev. 10, 143 (1992). 13aj. D. Schrag and M. Cygler, Methods Enzymol. 284, [4], 1997 (this volume).

[ 1]

UNDERSTANDING INTER.FACIAL PROPERTIES OF LIPASES

5

tures of the Rhizomucor miehei lipase complexed with two different inhibitors were reported I4,15) and showed a large rearrangement of a single loop (lid or flap) relative to the uncomplexed structure. This movement opened access to the active site and exposed the nucleophilic serine. A similar effect was caused by a phospholipid molecule bound in the active site of the pancreatic lipase-colipase complex. 16 In this case, the conformational change was larger and involved two loops. Another direct confirmation of the conformational rearrangement came from the determination of the structure of Candida rugosa lipase, a homolog of G. candidum lipase, crystallized under two different conditions and displaying two different conformations for a large surface loop: one that covers the active site and another in which the loop is moved to the side, exposing the active site. I7'18 This was further corroborated by the structures of C. rugosa lipase complexed with phosphonyl and sulfonyl inhibitors. 19 Another example of a conformational rearrangement of the lid has been observed in Pseudomonas lipases. 2° To date, the 3-dimensional structures of more than 10 lipases determined by X-ray crystallography have been described in the literature and work on more is near completion. Apart from those mentioned above they are Pseudomonas glumae lipase,21 Chromobacterium viscosum lipase (nearly identical to P. glumae lipase,e2 Candida antarctica lipase B, 23 Humicola lanuginosa lipase,24 Fusarium solani cutinase,5 Rhizopus delemar lipase,24 14 A. M. Brzozowski, U. Derewenda, Z. S. Derewenda, G. G. Dodson, D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. A. Patkar, and L. Thim, Nature (London) 351, 491 (1991). 15 U. Derewenda, A. M. Brzozowski, D. M. Lawson, and Z. S. Derewenda, Biochemistry 31, 1532 (1992). 16H. van Tilbeurgh, M. P. Egloff, C. Martinez, N. Rugani, R. Verger, and C. Cambillan, Nature (London) 362, 814 (1993). x7 p. Grochulski, Y. Li, J. D. Schrag, F. Bouthillier, P. Smith, D. Harrison, B. Rubin, and M. Cygler, J. Biol. Chem. 268, 12843 (1993). 18 p. Grochulski, Y. Li, J. D. Schrag, and M. Cygler, Protein Sci. 3, 82 (1994). 19p. Grochulski, F. Bouthillier, R. J. Kazlauskas, A. N. Serreqi, J. D. Schrag, E. Ziomek, and M. Cygler, Biochemistry 33, 3494 (1994). 20 j. D. Schrag, Y. Li, M. Cygler, D. Lang, T. Burgdoff, H.-J. Hecht, D. Schomburg, T. Rydel, J. Oliver, L. Strickland, M. Dunaway, S. Larson, and A. McPherson, Structure 5, 187 (1997). 21 M. E. Noble, A. Cleasby, L. N. Johnson, M. R. Egmond, and L. G. Frenken, FEBS Lett. 331, 123 (1993). z2 D. Lang, B. Hoffmann, L. Haalck, H.-J. Hecht, F. Spener, R. D. Schmid, and D. Schomburg, J. Mol. Biol. 259, 704 (1996). 23j. Uppenberg, M. T. Hansen, S. Patkar, and T. A. Jones, Structure 2, 293 (1994). 24 U. Derewenda, L. Swenson, R. Green, Y. Wei, G. G. Dodson, S. Yamaguchi, M. J. Haas, and Z. S. Derewenda, Nat. Struct. Biol. 1, 36 (1994).

6

PRIMARY AND TERTIARY STRUCTURE

[1]

a second isoform of C. rugosa lipase (referred to as a cholesterol esterase25), Penicillium camembertii lipase, 24 P s e u d o m o n a s cepacia lipase, 2° Penicillium sp. U Z L M 4 lipase (J. D. Schrag and M. Cygler, unpublished, 1997), and a P s e u d o m o n a s mendaccino lipase. 26 H e r e we briefly describe common features of the lipase fold, discuss the characteristics of the "closed" vs " o p e n " conformation, and explore the differences between lipases. Last, we describe substrate-binding sites and discuss possible interactions of lipases with the lipid layer.

Lipase Fold Lipases with known three-dimensional structures span a wide range of molecular weights, from - 1 9 kDa (cutinase) to - 6 0 kDa (G. c a n d i d u m lipase). All of them, with the exception of pancreatic lipases, contain only one domain. These catalytic domains belong to the a,/3 doubly wound protein fold 27 and are formed from a parallel ]3 sheet and a number of helices that flank the sheet on both sides. The minimal fragment of this fold common to all lipases is a subset of the a//3-hydrolase fold as described by Ollis et al.12 and contains a five-stranded 13 sheet and two a helices (B and C in Fig. 1). In addition, helix A exists in all lipases except for those of the R. miehei family, and helix D usually exists in the form of a short, distorted turn that is positioned in space between that of helix D and E of the a//3-hydrolase fold. T h e / 3 strands correspond to strands/33 to/37 of the a//3-hydrolase fold (see [4] in this volume13a). The positions of the catalytic residues in this nomenclature are as follows: serine after strand /35, histidine after strand/38, and acid after strand/37. Pancreatic lipase is an exception to this arrangement, with the acid after strand/36. 9'28Although the order of triad residues, their location, and the lipase fold have been conserved in evolution, the topological location of the lid-forming loops differs between lipases (see p. 14). In some lipases the lid is located N terminal to the nucleophile, whereas in others it is C terminal to the nucleophile. In addition, whereas in some lipases only one loop undergoes rearrangement, in others one or even two additional loops move together 25D. Ghosh, Z. Wawrzak, V. Z. Pletnev, N. Li, R. Kaiser, W. Pangborn, H. JOrnvall, M. Erman, and W. L. Duax, Structure 3, 279 (1995). z6R. Sarma, J. Dauberman, A. J. Poulose, J. Van Beilen, S. Power, B. Shew, G. Gray, S. Norton, and R. Bott, in "Lipases: Structure, Mechanism and Genetic Engineering." (L. Alberghina, R. D. Schmid, and R. Verger, eds.) Vol. 16, pp. 71-76. GBF Monographs, CEC-GBF International Workshop, Braunschweig, September 13-15. VCH Verlagsgesellschafl mbH, Weinheim, Federal Republic of Germany, 1991. 27j. S. Richardson, Adv. Protein Chem. 34, 167 (1981). 28j. D. Schrag, F. K. Winkler, and M. Cygler, J. Biol. Chem. 267, 4300 (1992).

[ 11

U N D E R S T A N D I N G I N T E R F A C I A L P R O P E R T I E S OF LIPASES

kc

~

7

A

[. io 132

t6

L

]7

FIG. 1. The common lipase fold. Arrows indicate /3 strands and rectangles indicate a helices. /3 Strands are numbered according to the nomenclature of the a//3-hydrolase fold (Ollis et al)2). Secondary structural elements shown in black or white (strands/33-/37 and helices B and C) occur in all lipases; those shown in gray (strand/32 and helices A, D, and F) occur in most. Helices A and F, shown with dashed border lines, are on the concave side of the/3 sheet; the other helices are on the convex side. Helix D is often composed of only one (distorted) turn. with the lid loop o v e r significant distances to create the active conformation. Cutinase is an example of a lipase without a lid, and in this structure all of the loops near the active site are relatively short. 5

Two Classes of Conformers: Closed and Open A c o m p a r i s o n of three-dimensional structures of related molecules obtained f r o m X - r a y diffraction experiments can provide a wealth o f information on the flexibility a n d / o r mobility of various parts of the proteins, going b e y o n d what can be learned f r o m a static view of a single molecule. T o that effect, the crystallization process m a y be used to investigate the structural features of a protein fold that are sensitive to external factors and differentiate t h e m f r o m those features that are i n d e p e n d e n t of the protein environment. O n e of the i m p o r t a n t features of an e n z y m e is the shape of the substratebinding site and its accessibility to the substrate. Structures of the majority of e n z y m e s show that their active sites are o n the surface of the molecule, accessible to the solvent. This is not always the case for lipases. T h e various lipase structures d e t e r m i n e d to date can be divided into two categories:

8

PRIMARY AND TERTIARY STRUCTURE

[1]

those with the active site accessible from the solvent (open form) and those with an inaccessible active site (closed form). For some lipases both forms have been experimentally observed (see Table I), which immediately raises the question of the relevance of these forms for the enzymatic activity of lipases. The fact that the open and closed conformations are observed not only within one homologous family of lipases but across families suggests that these two states are important for the function of these enzymes. Within homologous lipases all the open forms are quantitatively similar, as are the closed forms. When the structures of the native lipases (uncomplexed) are analyzed, we find that there is some correlation between the crystallization conditions and the conformational state of the lipase. This is summarized in Table I. The polyethylene glycol conditions in general promote the crystallization of the closed form, whereas a substantial content of alcohol (e.g., 2-methyl-2,4-pentanediol) or detergent in the crystallization solution is conducive to the crystallization of lipases in an open state. The polyethylene glycol is believed to affect protein solubility during crystallization by partial exclusion of water from around the protein, 29 but the local environment of the protein remains water based. The addition of alcohol or detergent, however, changes significantly the microscopic properties of the solution and influences protein environment. Another factor influencing the equilibrium between the closed and open forms is the presence of substrate-like (inhibitor) molecules. When they are bound in the active site region, either covalently or noncovalently, only the open conformation is observed.

Closed (Inactive) Conformation The closed conformations are presumed to predominate in aqueous media as indicated by the lower lipolytic activity of most lipases in the absence of an interface. At the same time, the persistence of a low level of activity shows that other conformations allowing access of the substrate to the active site are also at least transiently present in solution. The closed conformations are characterized by an unoccupied active site, which is shielded from the solvent by one or more loops forming the lid. The contacting surfaces of the lid and the remaining part of the protein are complementary, with the exception of an area near the nucleophile, where a cavity under the lid is frequently found. This cavity usually contains some water molecules. Overall, the external surfaces of the dosed forms of lipases are reasonably hydrophilic, typical for water-soluble proteins. A closer examination of the distribution of polar atoms (nitrogen and oxygen) on 29 T. Arakawa and S. Timasheff,

Biochemistry 24, 6756 (1985).

[ 1]

UNDERSTANDING INTERFACIAL PROPERTIES OF LIPASES

9

the molecular surface shows that in some lipases, but not all, the surface has a more hydrophobic character on the lid-containing face. This is shown in Fig. 2 for C. rugosa and H. lanuginosa lipases. Open Conformation The features of the lipase surface in the open conformation are quite different from those of the closed conformation. The movement of the lid not only opens access to the active site, but at the same time exposes a large hydrophobic surface (Fig. 3a). In R. miehei lipase the activation rearrangement is associated with ~700 ,~2 of hydrophobic area being exposed. 15 At the same time, some of the hydrophilic surface previously exposed becomes buried. The decrease in the hydrophilic surface is ~450 ,~2. An even larger area, more than 1000 A2, of newly exposed hydrophobic surface has been observed for the C. rugosa lipase. TM Similarly, a significant increase in hydrophobic surface around the active site was reported for pancreatic lipase. 16 In all open conformations of lipases, the nucleophile is at the bottom of a depression created by the lid movement. In the observed conformation of C. antarctica lipase B, the path leading to the active site is rather narrow (Fig. 3 b ) . 23 The more hydrophobic character of the face containing the active site can also be noticed in cutinase, an enzyme that does not have a lid (Fig. 3c) and whose structure represents the active state of this enzyme. In particular, loop 175-190 on one side of the nucleophile, Ser-120, contains many hydrophobic residues.

Oxyanion Hole An important component of the catalytic machinery of hydrolases, first postulated for serine proteases, 3°'31 is the oxyanion hole, a constellation of properly located hydrogen bond donors (usually main-chain NH groups) that helps to stabilize the intermediate state arising during the reaction, in which the carbonyl oxygen bears a partial negative charge. In serine proteases, this oxyanion hole exists in a competent form in the substrate-free enzyme. This is not the case with many lipases. In most of them at least one of the residues that contribute to the oxyanion hole is a part of one of the moving loops and attains its proper position and orientation only in the open conformation of the lipase. Among the presently known structures, the C. rugosa lipase is somewhat of an exception. Its oxyanion hole is already preformed in the closed conformation and does not change on lid 30 D. M. Blow, J. J. Birktoft, and B. S. Hartley, Nature (London) 221, 337 (1969). 31 j. D. Robertus, J. Kraut, R. A. Alden, and J. J. Birktoft, Biochemistry 11, 4293 (1972).

10

PRIMARY

AND

TERTIARY

STRUCTURE

[1]

0

0

0

--~

,-~

-n

~

~

-n 0

0

L)

o e~

0

~oo

o

0

o 100, sequence length >400, not annotated as a fragment, and not represented in the structural alignment were retrieved (in total, 23 entries). Adding New Sequences to Initial Alignment A new HMM was built from the structural alignment and the sequences retrieved during the first library search, by forcing the hmmt program to leave the structural alignment undisturbed. We tried first to do the HMM training by using the initial HMM (from the structural alignment) as a "hint" (which will allow it to change during the model-building process), based on the assumption that the Stamp alignment outside SCRs will be suboptimal, at least compared to standard multiple alignment, and that 42 M. Gribskov, A. D. McLachlan, and D. Eisenberg, Proc. Natl. Acad. Sci. U.S.A. 84, 4355 (1987). 43 A. Krogh, M. Brown, I. S. Mian, K. Sjolander, and D. Haussler, J. Mol. Biol. 235,1501 (1994). 44 S. R. Eddy, Curr. Opin. Struct. Biol. 6, 361 (1996). 42 S. R. Eddy, G. Mitchison, and R. Durbin, J. Comput. Biol. 2, 9 (1995). 46 S. R. Eddy, Intelligent Systems for Molecular Biology (ISMB) 3, 114 (1995). 47 T. Smith and M. Waterman, J. Mol. Biol. 147, 195 (1981).

[2]

41

IDENTIFICATION OF CONSERVED RESIDUES

1400

1200

1000 ""..

\\

.=

o 0 =E =E

800-

"..... ,....., .\o.~ k ".,.

\

600-

400"

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. . . . . . . .

.

Sequences

FIG. 4. Distribution of HMMer score values collected by searching the SwissProt database with three different models. ( - - ) The initial model, based on the 3D structural alignment; ( - - - ) the second model, in which closely related sequences have been included; ( . . - ) the final model, in which all significantly similar sequences have been included, with some minor exceptions. A full description of these data can be found in text.

allowing HMMer to modify the alignment in these regions would make them better suited for searching. However, it turned out that HMMer was not able to retain a reasonable alignment inside several SCRs, probably because of low sequence similarity, and forcing HMMer to retain the structural alignment seemed to generate better models.

Iteration of Search Process The library search of the SwissProt database was then repeated using the model from the extended data set. This search generated score values (Fig. 4, second model) with a clear distinction between the sequences included in the model or sequences with high homology to these, sequences related to sequences in the model, and unrelated sequences. Again a library of sequences was generated from the output, this time all entries with score values >16 (which is close to the limit for significant hits), sequence lengh >100, not annotated as a fragment, and not represented in the structural alignment. A total of 46 entries was retrieved. Some initial alignments showed that four sequences lacked the GxSxG motif known to be typical

42

PRIMARY AND TERTIARY STRUCTURE

[2]

TABLE II SELECTED LIPASE AND ESTERASE PROTEIN SEQUENCES a

Score

SwissProt code

Description

Source

PDB

1029.97 1025.18 1014.95 990.90 960.78 959.68 941.46 934.79 930.47 899.80 897,15 896,05 872,55 857,65 854.67 853.32 847.56 843.19 831.81 824.71 800.77 794.13 789.87 788.62 777.32 766.28 749.83

ACES_MOUSE ACES_RAT ACES_HUMAN ACES_BOVIN CHLE_HUMAN CHLE_MOUSE CHLE_RABIT ACES_TORCA ACES_TORMA EST6_DROMA EST6_DROSI EST6_DROME ESTB_DROPS ESTI_HUMAN ACES_CHICK ESTP_RAT ESTC_DROPS ESTI_RAT EST_MOUSE ESTA_DROPS LIP3_CANRU ESTP_DROME LIP4_CANRU LIPI_CANRU LIP2_CANRU ESTI_RABIT LIP5_CANRU

Acetylcholinesterase Acetylcholinesterase Acetylcholinesterase Acetylcholinesterase Cholinesterase Cholinesterase Cholinesterase Acetylcholinesterase Acetylcholinesterase Esterase 6 Esterase 6 Esterase 6 Esterase 5b Liver carboxylesterase Acetylcholinesterase Pi 6.1 esterase Esterase 5c Liver 60-kDa esterase E1 Carboxylesterase Esterase 5a Lipase 3 Esterase P Lipase 4 Lipase 1 Lipase 2 Liver 60-kDa carboxylesterase 1 Lipase 5

Mus musculus Rattus norvegicus Homo sapiens Bos taurus Homo sapiens Mus musculus Oryctolagus cuniculus Torpedo californica Torpedo marmorata Drosophila mauritiana Drosophila simulans Drosophila melanogaster Drosophila pseudoobscura Homo sapiens Gallus gallus Rattus norvegicus Drosophila pseudoobscura Rattus norvegicus Mus musculus Drosophila pseudoobscura Candida rugosa Drosophila melanogaster Candida rugosa Candida rugosa Candida rugosa Oryctolagus cuniculus Candida rugosa

1MAH

1ACE

1CLE

1CRL

of most lipases 48'49 ( G L T _ D R O M E , N R T _ D R O M E , T H Y G _ B O V I N , T H Y G _ H U M A N ) . These sequences were removed from the library, and a final H M M was generated, as described above. The SwissProt databank was searched with this final model (Fig. 4, third model; Table II). We see that the model now seems to describe all entries included in the model, as they generally have a high score value (although the score value for the most closely related sequences has dropped). A small number of sequences that are not included in the model do have a significant score; these are 48 D. L. Ollis, E. Cheah, M. Cygler, B. Dijkstra, F. Frolow, S. M. Franken, M. Harel, S. J. Remington, I. Silman, J. Schrag, J. L. Sussman, K. H. G. Verschueren, and A. Goldman, Protein Eng. 5, 197 (1992). 49 H. W. Anthonsen, A. Baptista, F. DrablOs, P. Martel, S. B. Petersen, M. Sebasti~o, and L. Vaz, in "Biotechnology Annual Review" (M. R. E1-Gewely, ed.), p. 315. Elsevier Science, Amsterdam, 1995.

[2]

43

IDENTIFICATIONOF CONSERVED RESIDUES TABLE II (continued)

Score

SwissProt code

Description

Source

739.53 717.68 702.53 701.36 700.36 691.98 679.25 671.26 659.17 650.08 647.60 633.36 628.03 553.66 551.74 511.82 491.92 489.16 488.29 435.70

ACEI_CAEEL SASB_ANAPL ESTF_MYZPE ESTE_MYZPE EST2_RABIT BAL_HUMAN BAL_RAT ACES_DROME LIP1-GEOCN LIP2_GEOCN ESTI_CAEBR ESTS_DROVI ESTI_CAEEL D2_DICDI CRYS_DICDI PNBA_BACSU EST2_CAEEL ESTI_CULPI ESTJ_HELVI PCD_ARTOX

Acetylcholinesterase 1 Fatty acyl-CoA hydrolase Esterase FE4 Esterase E4 Liver 60-kDa carboxylesterase 2 Bile salt-activated lipase Bile salt-activated lipase Acetylcholinesterase Lipase 1 Lipase 2 Gut esterase Esterase S Gut esterase cAMP-regulated D2 protein Crystal protein p-Nitrobenzyl esterase Esterase CM06B1 Esterase B1 precursor Juvenile hormone esterase Phenmedipham hydrolase

Caenorhabditis elegans Anas platyrhynchos Myzus persicae Myzus persicae Oryctolagus cuniculus Homo sapiens Rattus norvegicus Drosophila melanogaster Geotrichum candidum Geotrichum candidum Caenorhabditis briggsae Drosophila virilis Caenorhabditis elegans Dictyostelium discoideum Dictyostelium discoideum Bacillus subtilis Caenorhabditis elegans Culex pipiens Heliothis virescens Arthrobacter oxidans

303.33 271.59 190.02 140.68

THYG_BOVIN THYG_HUMAN GLT_DROME NRT_DROME

Thyroglobulin Thyroglobulin Glutactin Neurotactin

Bos taurus Homo sapiens Drosophila melanogaster Drosophila melanogaster

PDB

1THG

a Protein sequences retrieved by the final hidden Markov model, sorted according to HMMer score. Only sequences with score values >16 and not annotated as fragments are shown. The last four sequences lack the GxSxG motif and are not included in the final alignment.

s e q u e n c e s s h o w i n g a significant s i m i l a r i t y to lipases a n d e s t e r a s e s , b u t t h e y l a c k t h e G x S x G motif. T h e y a r e p r o b a b l y of lipase o r e s t e r a s e origin, b u t h a v e a c q u i r e d n e w o r m o d i f i e d functionality. It was d e c i d e d to l e a v e t h e s e s e q u e n c e s o u t o f t h e a l i g n m e n t to a v o i d p o t e n t i a l noise p r o b l e m s , w h i c h can b e i n t r o d u c e d b y t h e inclusion of n o n t y p i c a l s e q u e n c e s . T h e final m o d e l also r e c o g n i z e s a small n u m b e r o f n e w s e q u e n c e s n o t i n c l u d e d in p r e v i o u s m o d e l s , in p a r t i c u l a r t h e p a l m i t o y l - C o A h y d r o l a s e (PMCH_RAT). However, these sequences were only fragments and not r e l e v a n t for inclusion in t h e final a l i g n m e n t .

Making Multiple Sequence Alignment T h e H M M t h a t w e h a v e g e n e r a t e d can b e c o n v e r t e d d i r e c t l y i n t o an a l i g n m e n t b y using t h e H M M e r t o o l kit. This was d o n e for t h e final m o d e l ,

44

PRIMARY AND TERTIARYSTRUCTURE

[2]

using the h m m a program. A n u m b e r of conserved positions could be identified. However, in particular for the first part of the alignment, large sections did not seem to be consistent with the structural alignment. Therefore, an alternative approach was used to m a k e the final multiple alignment. The initial structural alignment was used as a profile in ClustalW, 5°,51 and then all sequences in the final library were aligned on the basis of information in this profile. Visual inspection showed that this generated a reasonable multiple alignment for most of the sequence space, and this alignment was used without further refinement. This alignment includes both closely related sequences and more distant homologs. T o generate an even m o r e robust alignment with respect to structural information another alignment based on the library for the second H M M was also generated. This includes only sequences that are similar to the sequences for which we have 3D structural information.

Identification of C o n s e r v e d R e s i d u e s After having defined the multiple alignments we want to identify "imp o r t a n t " positions in the sequences on the basis of the information found in these alignments. Several different approaches are available for doing this, such as the K a b a t score, 52 conservation index, 53 or entropy measures like the one used in hssp. 54 For this analysis we have chosen to use a simple criterion, focusing on conserved positions. A position is classified as conserved if all residues found at that position in the alignment belong to the same class. We then need to define reasonable classes of residues. The simplest approach m a y be to treat each residue type as an individual class, i.e., look only for total conservation. However, this is normally too restrictive to be useful; for example, it is known that both aspartate and glutamate are used as the acidic residue of the typical lipase/esterase catalytic triad. T h e r e are several ways that we can group residues, based on codon usage, physical data, mutation data, etc. This is reflected, e.g., in the n u m b e r of different mutation matrices that have been developed. Useful discussions can be found in Johnson and Overington s5 and in Vogt et al. 56 It is important 5oD. G. Higgins and P. M. Sharp, Gene 73, 237 (1988). 51D. G. Higgins, A. J. Bleasby, and R. Fuchs, Comput. Appl. Biosci. 8, 189 (1992). 52E. Kabat, T. Wu, H. Bilofsky, M. Reid-Miller, and H. Perry, "Sequences of Proteins of Immunological Interest." National Institute of Health, Bethesda, Maryland, 1983. 53W. A. Hide, L. Chan, and W. H. Li, J. Lipid Res. 33, 167 (1992). 54C. Sander and R. Schneider, Proteins 9, 56 (1991). 55M. S. Johnson and J. P. Overington, Z Mol. Biol. 233, 716 (1993). 56G. Vogt, T. Etzold, and P. Argos, J. Mol. Biol. 249, 816 (1995).

[2]

IDENTIFICATION OF CONSERVED RESIDUES

45

to realize that it may be relevant to assign individual residues to several classes, depending on how these are defined. For the current analysis we have used the classification ((IVLM), (GAST), (FYW), (NEDQ), (KR), (P), (C), (H)), which means that, e.g., isoleucine, valine, leucine, and methionine are accepted as equivalent residues. This classification is close to what we obtain if we do a principal coordinates analysis on the Blosum 62 score matrix, 57it is somewhat related to the ClustalW classification scheme, and it is inspired by the work of Johnson and Overington. 55 The classification also reflects similarities in standard volume of residues, in particular for the nonpolar residues. This is consistent with the study of Gerstein et aL 58 showing that there are significant constraints on how much the volume of residues at buried positions can vary during evolution, reflecting the fact that most proteins have a tightly packed core. However, it is important to remember that this classification is only one of several possible choices, and the choice of classification scheme (or scoring scheme in general) may be influenced by the type of information being searched for. To give some room for sequencing errors and other, more atypical deviations, at most one residue at each position of the small alignment and two residues for the large alignment can belong to a different class than the majority of the residues without breaking the conservation at that position. Definition of Conserved Positions

Both alignments were analyzed by the approach described above, and the result for the large alignment is shown in Table 111,59 together with possible explanations (see below). A surprisingly high number of positions seem to be conserved (66 in total), given the high number of quite dissimilar sequences included in the alignment. There also seems to be a difference in the distribution of conserved positions, with more conserved positions in the first part of the alignment. To study this observation in more detail we tried to identify domains in the relevant structures. It is not evident how to split these structures into domains. We have used the Protein Domain Assignment server 6°'61 to analyze lcrl, lace, and lthg. This classifies lthg as consisting of two domains, splitting it just before the active site serine; lcrl is also classified as a two-domain fold, with a 57 S. Henikoff and J. G. Henikoff, Proc. Natl. Acad. ScL U.S.A. 89, 10915 (1992). 58 M. Gerstein, E. L. Sonnhammer, and C. Chothia, J. MoL Biol. 236, 1067 (1994). 59 R. J. Kazlauskas, Trends Biotechnol. 12, 464 (1994). 60 S. A. Islam, J. Luo, and M. J. Sternberg, Protein Eng. 8, 513 (1995). 61 M. J. Sternberg, H. Hegyi, S. A. Islam, J. Luo, and R. B. Russell, Intelligent Systems for Molecular Biology (ISMB) 3, 376 (1995).

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[11]

Two NOVELLIPASESFROMB. therrnocatenulatus

205

lytic triad and that the oxyanion hole is formed by the residues around His-14. However, no significant homology apart from the region of the nucleophilic serine was found with most other lipases and esterases in the EMBL and Swiss-Prot databases. But lipase BTL-2 shows a significant homology of 30-35% with the mature lipases of Staphylococcus hyicus, S. aureus, and S. epidermidis, 34-37which also have a similar molecular weight of around 45,000. An alignment of the mature lipase BTL-2 amino acid sequence with that of the three Staphylococcus lipases is given in Fig. 4. Like the lipases from BaciUuspumilus and B. subtilis, 23'24 an alanine replaces the first glycine in the pentapeptide GIy-X-Ser-X-Gly, which is conserved among microbial and mammalian lipases. This replacement might be a common feature of Bacillus lipases. The lipase production obtained under the control of the native promoter was rather low (600 U/g cells). Nevertheless, we tried to purify this lipase and investigate some of its properties. The isolation of the lipase started with the disruption of the ceils by sonication followed by the incubation of the suspension for 25 min at 65° (heat precipitation). Precipitated protein and cell debris were then removed by centrifugation and the clear supernatant was applied to coupled Q- and S-Sepharose columns that had been previously equilibrated with Tris buffer, pH 7.5. Under these conditions the lipase did not bind, but many other proteins did. Unbound lipase was collected and loaded on a phenyl-Sepharose column that had been equilibrated with 0.5 M (NH4)2804 in Tris buffer, pH 7.5. A final elution of bound lipase with 2% (w/v) octyl glucoside in distilled water resulted in a pure enzyme with a specific activity of 10,225 U/mg, a total yield of 49%, and a purification factor of 329. SDS-PAGE showed a single protein band corresponding to a molecular weight of 32,000 for the purified lipase, even though the deduced sequence indicates a molecular weight of 43,000 for the mature lipase. But N-terminal sequencing of the purified lipase showed that during expression the signal peptide is correctly cleaved at the position previously assumed and that 34 C. Y. Lee and J. J. Iandolo, J. Bacteriol. 166, 385 (1986). 35 K. Nikoleit, R. Rosenstein, H. M. Verheij, and F. G6tz, Eur. J. Biochern. 228, 221 (1995). 36 A. M. Farrell, T. J. Foster, and K. T. Holland, J. Gen. Microbiol. 139, 267 (1993). a7 F. GOtz, F. Popp, E. Korn, and K. H. Schleifer, Nucleic Acids Res. 13, 5895 (1985).

FIG. 4. Alignment of the mature lipase BTL-2 from B. thermocatenulatus with unprocessed lipases from Staphylococcus aureus 34,35 [(1), strain PS54; (2), strain NCTC8530], S. epidermidis, ~ and S. hyicus. 37 Residues of BTL-2 homologous to at least one of the Staphyloccus lipases are highlighted in light gray and those found in all five lipases are highlighted in dark gray. The region around the oxyanion hole and the amino acids of the catalytic triad are boxed.

206

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[ 1 1]

the 32,000 band on the SDS gel is the lipase. Abnormal migration during S D S - P A G E might be responsible for this discrepancy in molecular weight, which has also been reported for other proteins. 38,39A first brief characterization of this purified recombinant lipase BTL-2 showed that the properties of lipase BTL-2 differ significantly from those of the lipase BTL-1 and most other Bacillus lipases (Table Ill). For example, BTL-2 has a molecular weight of 43,000, whereas the molecular weight of BTL-1 and most other Bacillus lipases ranges from 16,000 t o 22,000. 21-24 In addition, BTL-2 shows maximum activity at alkaline pH, but BTL-1 and most other lipases zl,28'4°-42 show maximum activity in a lower pH range of pH 5.6-8.0. The lipase BTL-2 was remarkably stable in the presence of most detergents and organic solvents. Many other lipases would be quickly denatured under these conditions. 42 A more detailed characterization of lipase BTL-2 is given in the following sections. Overexpression of BTL-2 in Escherichia coli The low level of BTL-2 expression under the control of its native promoter in pLIP2 made it necessary to develop an efficient expression system that would allow us to produce large amounts of this promising lipase for crystallization and further characterization, including its potential in biotechnological applications. For this purpose, we subcloned the BTL-2 gene in the E. coli expression vector pCYTEXP1, 43 which contains the strong A promoter PL together with the temperature-sensitive ~ repressor ci857. Expression of the gene is induced by shifting the cultivation temperature from 30 to 42 ° (heat shock) for 3-4 hr after reaching an optical density (OD) of 0.8-0.9 at 578 nm. The vector pCYTEXPI(pT1) was generated from the expression vector pTI-ompAROL, 44 which also contains the lipase gene from Rhizopus oryzae and the presequence of the outer membrane protein OmpA of E. coli. Two general expression strategies were developed45: (1) expression of mature BTL-2 lipase in the cytoplasm of E. coli and (2) secretion of BTL-2 38 R. E. Blankenship, Antonie van Leeuwenhoek 65, 311 (1994). 39 R. E. Daley and W. Wickner, J. Biol. Chem. 260, 15925 (1985). 40 T. Iizumi, K. Nakamura, and F. Fukase, Agric. Biol. Chem. 54, 1253 (1990). 41 H. Sztajer, H. Ltinsdorf, H. Erdmann, U. Menge, and R. D. Schmid, Biochim. Biophys. Acta 1124, 253 (1992). 42 W. StOcklein, H. Sztajer, U. Menge, and R. D. Schmid, Biochim. Biophys. Acta 1168, 181 (1993). 43 T. N. Belev, M. Singh, and J. E. G. McCarthy, Plasmid 26, 147 (1991). 44 H. D. Beer, Ph.D. thesis. University of Hannover, Hannover, Germany, 1994. 45 M. L. Rtia, H. Atomi, C. Schmidt-Dannert, and R. D. Schmid, submitted (1997).

[11]

TWONOVELLIPASESFROMB. thermocatenulatus

207

lipase in the periplasm of E. coli by the use of either the original leader sequence of BTL-2 or the OmpA signal sequence. Toward this end three different expression vectors were constructed and introduced into E. coli BL321: (1) pT1-BTL2, containing the mature lipase gene, (2) pTl-preBTL2, containing the prelipase gene, and (3) pT1-OmpABTL2, containing the mature lipase gene fused to an OmpA leader sequence. Figure 5 outlines the cloning strategies for the construction of these three expression vectors. Appropriate restriction sites for the ligation of vector and insert were created by polymerase chain reaction (PCR) using oligonucleotide primers containing the desired sites. In all three cases, the initiation codon ATG of the lipase gene (with or without leader sequence) is located at the NdeI site of pT1. Transformants were checked for lipase expression on LB-tributyrin plates. Plasmids of transformants exhibiting lipase activity of LB-tributyrin plates were isolated and the correct insertion of the lipase gene was checked by restriction analysis and sequencing of the insert. One plasmid of each construction (pT1-BTL2, pTl-preBTL2, and pT1OmpABTL2) with the correct insertion was chosen for further experiments. To determine the amount of active lipase expressed by E. coli BL321 containing plasmid pT1-BTL2, pTl-preBTL2, or pT1-OmpABTL2, cells from 200-ml cultures were harvested by centrifugation after 3 hr of heat shock and disrupted by sonication. After the removal of cell debris by centrifugation, the lipase activity in the supernatant was determined using tributyrin as a substrate. After 30 min of heat shock, the production of lipase started and reached a maximum after 3 hr. A comparison of total lipase activity obtained with the three expression vectors after 3 hr of heat shock is shown in Table IV. Escherichia coli BL321 cells harboring the plasmid pTI-ompABTL2 produced larger amounts of lipase activity (30,000 U/g cells) than cells harboring pT1-BTL2 (7000 U/g cells) or pTl-preBTL2 (9000 U/g cells). These expression levels represent a 12- to 50-fold improvement compared to pLIP2 (600 U/g ceils) containing the BTL-2 gene under the control of its native promoter. The expression vector pT1-OmpABTL2 was also introduced into two more E. coli host cells, JM105 and DH5a. The expression level (Table IV) obtained with DH5a was no different than that obtained with BL321, but a nearly 25-fold increase in the production of soluble lipase was reached when JM105 was used as the host cell (660,000 U/g cells). Total cellular protein obtained before and after the induction of lipase expression by heat shock for 3 hr was analyzed by SDS-PAGE. The proteins were stained with Coomassie Brilliant Blue (Fig. 6A). To determine the activity of the recombinant lipase directly on SDS gels (Fig. 6B), we developed a method to remove the SDS and at the same time renature the lipase

208

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[11]

TWO NOVELLIPASESFROM B. thermocatenulatus

209

TABLE IV EXPRESSIONLEVELOFBTL-2 LIPASEIN VARIOUS Escherichia coli EXVRESSlONVECTORSANDHOSTCELLSa Plasmid

E. coli host

Expression level (U/g of cells)b

pT1-BTL2 pT1-preBTL2 pT1-OmpABTL2

BL321 BL321 BL321 DH5a JM105

7,000 9,000 30,000 30,000 660,000

"From Biochem. Soc. Trans. 25, 178 (1997), with permission. b pH-stat assay with tributyrin as substrate (60° and pH 7.5). by incubating the gel for 20 min at room temperature in a 20 m M Tfis buffer, p H 8.5, containing 0.5% (w/v) Triton X-100. 45 After renaturation the gel was stained for lipolytic activity with 1-napthyl acetate as the substrate. 01 After 3 hr of heat shock E. coli BL321 p T l - p r e B T L 2 produced a new protein band with a molecular weight of 43,000 (Fig. 6A, lane 5). However, despite the fact that the soluble lipase activities produced by both plasmids pT1-BTL1 and p T l - p r e B T L are similar (Table IV), E. coli BL321 pT-BTL2 failed to produce a band that was clearly visible after staining for protein. Activity staining indicated that cells harboring pT1-BTL2 produced a band of similar intensity and the same molecular weight 41,000, as those with p T l - p r e B T L 2 (Fig. 6B, lanes 4 and 5, respectively). However, plasmid pT1preBTL2 expressed an additional band of 43 kDa that was also visible after Coomassie staining (Fig. 6A, lane 5). This protein band was always retained in the pellet of the cell lysate. N-Terminal sequencing of both the 41-kDa lipase produced by plasmids pT1-BTL2 and p T l - p r e B T L 2 and the insoluble 43-kDa lipase band expressed by plasmid p T l - p r e B T L 2 revealed that the insoluble 43-kDa lipase represents the unprocessed lipase BTL2 still containing its hydrophobic signal peptide. The N-terminal sequence of the 41-kDa soluble lipase produced by both plasmids pT1-BTL2 and pT1preBTL2 was identical with that of the mature lipase BTL-2. Thus, E. coli was unable to process the preenzyme fully. Plasmid p T 1 - O m p A B T L 2 genFIG.5. Constructionsof plasmids pTl-preBTL2, pT1-BTL2,and pT1-OmpABTL2.Plasmid pLIP2 contains a 1300-bpDNA fragment from B. therrnocatenulatusin pUC18, whichincludes the coding sequence for the mature BTL-2 lipase (black arrow) and the N-terminal signal peptide (m). Plasmid pT1-OmpAROL is a secretion cloning vector, wherein the expression of a foreign gene, cloned in the multiple cloningsite, is under the control of the heat-inducible APLpromoter. (ll) Coding region for the OrnpA signal peptide.

210

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING 1

2

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FIG, 6. SDS-polyacrylamide gel electrophoresis of total cellular proteins stained for protein (A) and activity (B): E. coli BL321 harboring pT1-BTL2 (lanes 1 and 4), pTl-preBTL2 (lanes 2 and 5), and pT1-OmpA BTL2 (lanes 3 and 6) were grown at 30° in LB medium containing ampicillin (100/zg/ml) to an optical density of 0.8-0.9 at 578 nm. The temperature was then raised to 42 ° to induce the expression of the lipase and the culture was incubated for another 3 hr. The cells were solubilized as described in text and subjected to SDS-polyacrylamide electrophoresis. The gels were stained with Coomassie Brilliant Blue for protein (A) and with 1-naphthyl acetate for activity (B). The molecular weights of the standards ( x 10 -3) are indicated on the left.

erated a new and, as expected from the produced lipase activity, broad protein band of 44 kDa on an SDS gel after staining for protein and activity (Fig. 6A and B, lanes 6). N-Terminal sequencing of the produced lipase showed that, similar to pTl-preBTL2, the OmpA signal sequence was not cleaved at all by E. coli. With both pTI-preBTL2 and pT1-OmpABTL2 we tried to increase the amount of mature lipase BTL-2 by decreasing the heat-shock temperature from 42 to 37°, conditions that have been described to reduce the rate at which newly synthesized proteins are released by the protein synthesis machinery into the cell c y t o p l a s m . 46'47 In the case of pTl-preBTL2, the amount of precursor produced was significantly decreased, but the increase in expressed mature lipase was only small, even after 24 hr of heat shock. A decrease in the cultivation temperature for lipase expression had no influence on the processing of OmpA-BTL2, however. In the case of pT1OmpABTL2 it is likely that the cell machinery responsible for the processing of the protein was simply overloaded by such a high level of lipase 46 C. H. Schein and M. H. M. Notebom, Bio/Technology 6, 291 (1988). 47 R. C. Hockney, Trends Biotechnol. 12, 456 (1994).

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Two NOVELLIPASESFROMB. thermocatenulatus

211

production, as has also been reported for staphylococcal nuclease A . 48 When expressed in E. coli JM105, pT1-OmpABTL2 produced 80 times more soluble lipase than in either E. coli BL321 or DH5a (Table IV). But with SDS-PAGE no difference could be detected in the expression levels of the three strains. Because a large amount of OmpA-lipase activity is still associated with the precipitate of the cell lysate from all three strains, it is possible that in E. coli JM105 a larger amount of soluble OmpA-lipase is released after cell disruption. To utilize the large amounts of lipase expressed by E. coli JM105 pT1OmpABTL2, it was necessary to cleave the OmpA leader sequence from the lipase BTL-2. Daley and Wickner 39 reported the successful cleavage of the leader sequence from the OmpA protein using proteinase K. We followed their example and used proteinase K to cleave the OmpA signal peptide. First, the insoluble lipase was extracted from the precipitate of the cell lysate with 1% (w/v) cholate in 50 mM Tris buffer (pH 8.0) for 16 hr at 4°. In this way, 2.2 million units of lipase (using tributyrin as substrate) was solubilized from 2 g of E. coli JM105 cells in a relatively pure state with a specific activity of 16,208 U/mg. Next, the digestion of the OmpA lipase with proteinase K in a final concentration of 0.7 U/ml for 15 min at 0° (the reaction was stopped by addition of 10 mM phenylmethylsulfonyl fluoride [PMSF]) yielded 82% of the lipolytic activity before digestion. Small peptide fragments produced by the proteinase digestion were removed by ultrafiltration through a 100-kDa membrane. A 16-fold concentrated enzyme solution with 1.152 million units and a specific activity of 36,000 U/mg could be obtained with this simple purification procedure. SDS-PAGE of the digested lipase showed a protein band corresponding to a molecular weight about 2,000, smaller than the lipase before digestion (Fig. 7). The OmpA signal sequence consists of 21 residues and has a molecular weight of 2,400, which is consistent with the smaller size of the digested lipase. Large-Scale Production and Purification of BTL-2

For crystallization it must be ensured that only the mature lipase form is present in the protein solution. Hence, E. coli BL321 pT1-BTL2 was chosen for cultivation in a 100-liter fermenter. The medium consisted of LB supplemented with glucose (10 g/liter) and ampicillin (100 mg/liter) and adjusted to pH 7.3. The cells were grown at 30° for 5 hr until the end of the exponential growth phase (OD578nm 1.65). Lipase expression was induced by raising the temperature to 42° (heat shock) and reached a 48 M. Takahara, D. W. H!bler, P. J. Barr, J. A. Gerlt, and M. Inouye, J. Biol. Chem. 260, 2670 (1985).

212

ISOLATION, CLONING,EXPRESSION,AND ENGINEERING

[1 11

94 67 43 30 20 14

1

2

3

FIG.7. SDS-polyacrylamidegel electrophoresis:Digestionof OmpA lipasewithproteinase K after cholate extraction from insoluble precipitate of the cell lysate. Lane 1, molecular weight standard (× 10-3); lane 2, sample after cholate extraction of OmpA lipase; lane 3, sample of proteinase K-digestedlipase. The gel is stained with silver for protein. maximum after 4 hr of induction, at which time the cells were harvested. The final cell mass was 4.8 g/liter and the lipase yield was 53,700 U/g cells. 49 Lipase, which is located in the cytoplasm of E. coli cells, was isolated by disrupting the cells in a high-pressure homogenizer followed by centrifugation. The crude enzyme solution was then loaded on a butyl-Sepharose column that had been equilibrated with a Tris buffer, pH 8.0, containing 0.1 M NaC1. After washing the column with equilibration buffer, bound lipase was eluted with 1% (w/v) cholate in Tris buffer, pH 8.0. Fractions with activity were pooled and concentrated by ultrafiltration through a 50-kDa membrane. Final purification of the concentrated lipase solution was achieved by gel filtration using a TSK G300 column (Tosohaas, Tokyo, Japan). The purification procedure resulted in a highly active and pure lipase with a yield of 32% and a specific activity of 54,887 U/mg. 49 Table V summarizes the results of the purification. The key purification step was the hydrophobic interaction chromatography with butyl-Sepharose, which resulted in a 28-fold increase in specific activity. On S D S - P A G E (Fig. 8) a single band with a molecular weight of 40,000 could already be detected by silver staining after this purification step. After ultrafiltration and gel filtration, the same major band was visualized together with three minor 49M. L. Rtla, C. Schmidt-Dannert,S. Wahl, A. Sprauer, and R. D. Schmid,J. Biotechnol., in press (1997).

TWO NOVELLIPASESFROM B. thermocatenulatus

[11]

213

TABLE V SUMMARYOF PURIFICATIONOF RECOMBINANTLIPASEBTL-2 PRODUCEDWITH Escherichia coli BL321 pT-BTL2a

Step

Total units (U)

Total protein (rag)

Specificactivity (U/mg) b

Cell breakage Butyl-Sepharose Ultrafiltration Gel filtration

942,270 481,815 403,875 296,391

2,151 38 15.8 5.4

438 12,354 25,562 54,887

Purification factor 1 28 58 125

Yield (%) 100 51 42 32

From Biochem. Soc. Trans. 25, 178 (1997), with permission. b pH-stat assay with tributyrin as substrate (60° and pH 7.5).

a

bands by both protein and activity staining (activity staining not shown). The fact that all four bands showed lipase activity means that the minor bands might possibly represent aggregate forms of the lipase. N-Terminal sequence analysis showed the identity of the purified protein with the mature lipase BTL-2 previously expressed in only small quantities and with the amino acid sequence predicted f r o m the nucleotide sequence.

Physicochemical Properties o f B T L - 2 The pure and highly active enzyme was characterized and its physicochemical properties were c o m p a r e d to those of the BTL-2 lipase previously expressed only weakly in E. coli under the control of its native p r o m o t e r (Table III). 49 Both lipases have the same N-terminal sequence and a p I of 7.2. A difference of 8,000 was found in the molecular weight, as estimated !~iili

67 43 30 20 14. 1

2

3

4

5

F~G. 8. SDS-polyacrylamide gel elecrophoresis of BTL-2 lipase at different purification steps: Lane 1, molecular weight standards (× 10-3); lane 2, sample after cell breakage; lane 3, sample after butyl-Sepharose; lane 4, after sample ultrafiltration; lane 5, sample after TSK G3000 column. The gel is stained with silver for protein.

214

I S O L A T I O NCLONING, , EXPRESSION, AND ENGINEERING

l111

by SDS-PAGE, between the weakly expressed lipase (32,000) and that of the overexpressed lipase (40,000). The molecular weight predicted on the basis of the amino acid sequence was 43,000. The reason for this discrepancy might be attributed to an abnormal migration of the weakly expressed lipase during SDS-PAGE, as previously explained. The major difference between the two enzymes was found in their specific activity toward tributyrin. That of the overexpressed enzyme (54,788 U/mg) was five times higher than that of the weakly expressed lipase (10,225 U/mg). The reason for this discrepancy is not clear at present, but might be due to conformational differences. Also, the presence of inactive enzyme in the enzyme preparation of the weakly expressed lipase might contribute to this difference. Similar results have also been reported for native and overexpressed recombinant rhodanase from bovine liver. 5° The specific activity of the overexpressed lipase BTL-2 is 320 to 1675 times higher than that of BTL-1 and other small Bacillus lipases. 21-24 In addition, most lipases have specific activities below 3000 U/mg. 4°-42 As expected, the lipase BTL-2, like BTL-1, reaches maximum activity between 55 and 75 °. With triolein as substrate maximal activity was found at 75 ° and with tributyrin at 60°. Published thermostable lipases from mesophiles exhibit only low activities at temperatures above 60°. Also, BTL-2 is thermostable, like most lipases from Bacillus and P s e u d o m o nas. 9'1538'21,28'51 The overexpressed lipase BTL-2 was stable up to 50° when incubated for 30 min at pH 9.0. Both lipases, BTL-1 and the weakly expressed lipase BTL-2, were slightly less stable below 40 °. The pH activity of BTL-2 at 60 ° depends on the substrate. With triolein maximum activity was found at pH 9.0, decreasing rapidly so that no activity could be detected any longer at pH 6.0. With tributyrin, however, a maximum was reached at pH 8.0 and, in contrast to triolein, 70% residual activity was detected at pH 6.0. After 14 hr of incubation at 30 °, BTL-2 was stable within the pH range of 9.0-11.0. Thus, unlike BTL-1 and most other lipases, 21'2s'40-42'51 BTL-2 is a thermoalkalophilic lipase. Most detergents enhanced the activity, with the increase ranging from 50% {3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (CHAPS) and octyl-/3-D-glucopyranoside (OGP)} to 80% (Triton X-100 and cholate) when added to the enzyme solution of overexpressed lipase. After 1.5 hr of incubation at 30° in the presence of these detergents, no significant loss of pNPP-hydrolyzing activity was observed. But the addition of SDS, Lubrol PX, Tween 20, or Tween 80 immediately inactivated the 50 D. M. Miller, G. P. Kurzban, J. A. Mendoza, J. M. Chirgwin, S. C. Hardies, and P. M. Horowiz, Biochim. Biophys. Acta 1121, 286 (1992). 51 H.-K. Kim, M.-H. Sung, H.-M. Kim, and T.-K. Oh, Biosci. Biotech. Biochem. $8, 961 (1994).

[11]

Two NOVELLIPASESFROMB. thermocatenulatus

215

enzyme. The weakly expressed lipase showed a similar behavior, but Triton X-100 inhibited this lipase, whereas Tween 20 did not. Lipases from both preparations showed only about a 20% decrease in activity when incubated in the presence of 30% (v/v) methanol, propanol, or acetone for 1 hr at 30°. After prolonged incubation for 4 days at room temperature in the presence of 30% (v/v) propanol no further decrease of activity was detected, indicating that the lipase BTL-2 is exceptionally stable in the presence of organic solvents. With respect to substrate specificity, BTL-2 exhibited the highest activity with tributyrin, both at pH 7.5 and 8.5 (Fig. 9). The lipase hydrolyzed triacylglycerols up to Cls with nearly the same efficiency at both pH values. The activity on triacylglycerols was between 40 (C6 or C8) and 20% (C12 or C16 ) that of the activity on tributyrin. Triacetin (Ca) was a poor substrate, especially at pH 8.5, where no activity was detected. However, the correla-

100

-

80,

.~

60,

;> ..~

~

4o

20

2

4

6

8

10

12

14

16

18

Chain length of acyl group FIG. 9. Activity of the purified lipase on triacylglycerols with various chain lengths of the acyl group was determined by the pH-stat assay at 60° and pH 7.5 (open columns) or pH 8.5 (filled columns).

A/280 nm 0.11-

A/280 nm

>500 kDa

A

0.08-

0.100.070.09-

0.06-

0.08-

0.05-

0.07-

0.04-

0.060.03 0.05.

0.02 -

235 kDa

0.04-

0.01-

0.03" 0

-

%

0+02-

' 4() ' 8'0 ' 120' 160' 2(~0 '24"0

0.01-

J

0"

Ve (ml)

4 kDa A/280 nm

' 4o ' A/280 nm

8'0 '

i~,o' i;o' 2oo' 2;.o v,+(++,.,,)

235 kDa

0.07 -

l 0.030.02.

C

~72kD

0.05 0.04-

0 . - -

0.03 -

' ,+~) ' 8;~ ' I';O' 11o ' 2+0'

0+07.

0.06-

a

0.01.

A/280 nm

D

0.08-

2;o VeI.'.>

0.02-

0.01-

44 kDa

E

0-

0.06. 40 0.05. 0.04.. 0.03. 0.02. 0.01.. O.

' 4;~ ' 8~ ' 1~0' 1~0 ' 2~0' 2;0 v~500 kDa) is observed when a low ionic strength buffer (10 mM Tris) is used. Under the conditions used for purification [50 mM Tris buffer (pH 7.5), 52 M. G. Van Oort, A. M. Th. J. Deever, R. Dijkman, M. L. Tjeenk, H. M. Verheij. G. H. De Haas, E. Wenzig, and F. G6tz, Biochemistry 28, 9278 (1989). 53 G. Pablo, A. Hammon, S. Bradley, and J. E. Fulton, J. Invest. Dermatol. 63, 231 (1974). 54 M. A. Taipa, M. R. Aires-Barros, and J. M. S. Cabral, J. Biotechnol. 26, 111 (1992).

FIo. 10. Elution behavior of BTL-2 lipase on a TSK G3000 column under different running conditions: (A) 10 mM Tris-HC1 (pH 7.5), (B) 50 mM Tris-HC1 (pH 7.5) containing 0.1 M NaC1, (C) 50 mM Tris-HC1 (pH 7.5) containing 0.1% (w/v) cholate, (D) 50 mM Tris-HC1 (pH 7.5) containing 1% (w/v) cholate, and (E) 50 mM Tris-HC1 (pH 7.5) containing 30% (v/v) propanol. In all experiments, 1 ml of sample was loaded on the column and eluted at room temperature at 4 ml/min, except in (E), in which the flow rate was 3 ml/min.

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[11]

0.1 M NaC1], an overall lipase aggregate size of 235 kDa was observed. The addition of 0.1% (w/v) cholate to the running buffer led to a partial disaggregation of the lipase (lipase peaks at 235 and 72 kDa), whereas with 1% (v/v) cholate one sharp peak corresponding to a molecular weight of 64,000 was observed. In the presence of 30% (v/v) propanol during gel filtration, a sharp peak corresponding to a molecular weight of 44,000 for the lipase monomer was eluted, which is very close to the molecular weight of 43,000 estimated from the amino acid sequence. The deaggregation of lipases in the presence of propanol was also reported for the lipases of Penicillium sp. 41 and Pseudomonas cepacia. 26 These observations clearly show that lipase BTL-2 is aggregated in the absence of detergents or organic solvents. Broad peaks during gel filtration indicate the existence of a heterogeneous mixture of aggregate sizes. In the presence of cholate, where lipase peaks of 64 and 72 kDa eluted that do not correspond to the molecular weight of the monomer, it is likely that the lipase is associated with cholate molecules. Other detergents, such as 1% (w/v) Brij, failed to dissociate completely the molecular aggregates of the lipase. At present it is not clear whether lipase BTL-2 interacts with bile salts in a specific way, although only bile salts (cholate, taurocholate, deoxycholate, or CHAPS) were able to elute the lipase from the butylSepharose column during purification. Other detergents (octyl glucoside, Brij, and Triton X-100), organic solvents (propanol, acetone, or methanol) or guanidine, all compounds that are effective in weakening hydrophobic interactions, were ineffective. With the lipase from P. cepacia a strong negative correlation between aggregation and activity has been described, possibly because the aggregation involves the active sites of the lipase molecule. 26 This was also found in the case of the lipase BTL-2. For this investigation, lipase fractions with different aggregation states obtained after gel filtration were incubated for 5 rain at room temperature in the presence or absence of 1% (w/v) cholate, after which the activity was checked at 30° using triolein as the substrate (Table VI). Under those conditions, in which monomers of the lipase existed in solution [30% (v/v) propanol], the activity did not change on incubation with the detergent. For this reason, the ratio between the specific activities measured before and after incubation with detergent was one. In contrast, the activity of the lipase with the highest molecular weight (>500,000) after gel filtration increased by a factor of 6.8 after incubation with 1% (w/v) cholate. Lipase forms eluted from the gel-filtration column with a molecular weight of 235,000 and 72,000 exhibited intermediate increases in activity after incubation with the bile salt (by a factor of 4.l and 1.9, respectively). Nevertheless, the effect of aggregation was strongly dependent on the substrate employed for the activity assay (tributyrin or

[11]

TWONOVELLIPASESFROMB. thermocatenulatus

219

TABLE VI COMPARISON OF ESTIMATED MOLECULAR MASS OF BTL-2 LIPASE OBTAINED AFTER GEL-FILTRATION CHROMATOGRAPHY UNDER SEVERAL RUNNING CONDITIONS AND SPECIFIC ACTIVITY RATIOSa'b

Running conditions Tris Tris Tris Tris

buffer buffer buffer buffer

(pH (pH (pH (pH

7.5, 7.5, 7.5, 7.5,

10 50 50 50

mM) mM), 0.1% (w/v) cholate mM), 1% (w/v) cholate mM), 30% (v/v) propanol

MW (estimated)

(U/mg)+/(U/mg) -c

>500,000 235,000 72,000 44,000

6.8 4.1 1.9 1.0

From Biochem. Soc. Trans. 25, 178 (1997), with permission. b Ratios in units per milligram, measured before ( - ) and after (+) 5 rain of incubation with 1% (w/v) cholate. c pH-stat assay with triolein as substrate (30° and pH 8.5).

triolein) and the temperature during the assay (30 or 60°). Whereas the tributyrin-hydrolyzing activity of aggregated BTL-2 (molecular weight > 500,000) was unchanged at 30 and 60° by incubation with 1% (w/v) cholate, a strong enhancement of the triolein-hydrolyzing activity was produced, especially at 30°. One explanation for this could be that during tributyrin hydrolysis, mono- and dibutyrin are produced. Monoacylglycerides are nonionic surfactants that are widely used as emulsifiersY It might be possible that, because of its water solubility (in contrast to the long-chain monoolein), monobutyrin is itself capable of breaking down the lipase aggregates, so that lipase monomers are always present in the assay. A more specific effect of tributyrin cannot be excluded. Conclusion We have found a strain of the thermophile B. thermocatenulatus that has at least two different lipases. One of these lipases, BTL-1, exhibited similarities to other Bacillus lipases previously published. But the second lipase, BTL-2, which was not detected during the cultivation of B. therrnocatenulatus, is different from most other lipases of Bacillus. Moreover, this lipase BTL-2 shows homology to lipases from Staphylococcus strains, which are also gram positive. Until now no structure of such a large prokaryotic lipase has been available. Hence, we developed an efficient expression system and a simple purification procedure that allow the production of the large amounts of pure enzyme needed for crystallization. Crystallization and X-ray diffraction analysis of the lipase BTL-2 are in progress. Lipase 55 K. Holmberg and E. Osterberg, J. Am. Oil Chem. Soc. 65, 1544 (1988).

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

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BTL-2 proved to be a stable enzyme, with optimal activity at elevated temperatures and at alkaline pH, which are promising properties for biotechnological applications. The discovery of two different lipases in B. thermocatenulatus prompted us to ask why this strain needs two lipolytic enzymes, what their physiological function is, and why only BTL-1 was detected in the culture broth of B. thermocatenulatus on induction with olive oil. It would be worthwhile investigating other Bacillus strains from which either a small lipase 21-24 or a higher molecular weight lipase 51is known to determine if a second lipolytic enzyme also exists in these strains.

[12] V e r n o n i a L i p a s e : A P l a n t L i p a s e w i t h S t r o n g F a t t y Acid Selectivity B y PATRICK A D L E R C R E U T Z , THOMAS GITLESEN, IGNATIOUS N C U B E , and JOHN S. R E A D

Introduction Lipases are useful tools in lipid processing. One important advantage of lipase-catalyzed processing compared to chemical methods is the inherent selectivity of the enzymatic reactions. The 1,3-regioselectivity of several lipases has been used extensively. A typical example is the production of cocoa butter substitutes, which is carried out on an industrial scale. Furthermore, there is great interest in the use of lipases for the preparation of chiral building blocks in the production of pharmaceuticals, agrochemicals, etc. In these applications, the often broad substrate specificity and high stereoselectivity of the lipases are utilized. The fatty acid selectivity of lipases has great potential for the enrichment of different fatty acids from natural fats and oils. Several promising reports have appeared in the literature. The fatty acid selectivity of Geotrichum candidum lipase was observed long ago. 1 The lipase was selective for fatty acids having a cis-9 double bond, for example, oleic acid. Although the originally described strain is no longer available, lipases from other G. candidum strains express similar selectivity.2

1 A. A. Alford and D. A. Pierce, J. Food Sci. 26, 518 (1961). 2 M. W. Baillargeon, R. G. Bristline, and P. E. Sonnet, Appl. Microbiol. Biotechnol. 30, 92 (1989).

METHODS IN ENZYMOLOGY,VOL. 284

Copyright© 1997by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/9/$25.00

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[ 12]

BTL-2 proved to be a stable enzyme, with optimal activity at elevated temperatures and at alkaline pH, which are promising properties for biotechnological applications. The discovery of two different lipases in B. thermocatenulatus prompted us to ask why this strain needs two lipolytic enzymes, what their physiological function is, and why only BTL-1 was detected in the culture broth of B. thermocatenulatus on induction with olive oil. It would be worthwhile investigating other Bacillus strains from which either a small lipase 21-24 or a higher molecular weight lipase 51is known to determine if a second lipolytic enzyme also exists in these strains.

[12] V e r n o n i a L i p a s e : A P l a n t L i p a s e w i t h S t r o n g F a t t y Acid Selectivity B y PATRICK A D L E R C R E U T Z , THOMAS GITLESEN, IGNATIOUS N C U B E , and JOHN S. R E A D

Introduction Lipases are useful tools in lipid processing. One important advantage of lipase-catalyzed processing compared to chemical methods is the inherent selectivity of the enzymatic reactions. The 1,3-regioselectivity of several lipases has been used extensively. A typical example is the production of cocoa butter substitutes, which is carried out on an industrial scale. Furthermore, there is great interest in the use of lipases for the preparation of chiral building blocks in the production of pharmaceuticals, agrochemicals, etc. In these applications, the often broad substrate specificity and high stereoselectivity of the lipases are utilized. The fatty acid selectivity of lipases has great potential for the enrichment of different fatty acids from natural fats and oils. Several promising reports have appeared in the literature. The fatty acid selectivity of Geotrichum candidum lipase was observed long ago. 1 The lipase was selective for fatty acids having a cis-9 double bond, for example, oleic acid. Although the originally described strain is no longer available, lipases from other G. candidum strains express similar selectivity.2

1 A. A. Alford and D. A. Pierce, J. Food Sci. 26, 518 (1961). 2 M. W. Baillargeon, R. G. Bristline, and P. E. Sonnet, Appl. Microbiol. Biotechnol. 30, 92 (1989).

METHODS IN ENZYMOLOGY,VOL. 284

Copyright© 1997by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/9/$25.00

[ 121

Vernonia LIPASESELECTIVITY

221

Several different lipases have been used to enrich y-linolenic acid. 3-6 In all cases the lipase was less active on ~/-linolenic acid than on most other fatty acids. Either esterification of a mixture of free fatty acids was carried out so that ~/-linolenic acid was enriched in the free fatty acid fractions, 3-5 or the triglyceride was partially hydrolyzed so that the ~/-linolenic acid was enriched in the glyceride fraction. 5 In a similar way erucic acid has been enriched from rapeseed oil,7,8 and C20 cis-5 polyunsaturated fatty acids have been enriched from Biota orientalis oil. 9 Furthermore, lipase from rape has been shown to discriminate against cis-4 and cis-6 unsaturated fatty acids, 1° and Rhizopus lipase has been shown useful in the enrichment of docosahexaenoic acid in fish oil fractions as well. 11 Lipase fatty acid chain length profiles are not as well investigated as the effect of unsaturation on lipase activity. Studies have been performed on hydrolysis of triacylglycerols in aqueous emulsions 12-14 and hydrolysis of fatty acid alkyl esters in organic solvent. ~5 Unfortunately, artifacts can be introduced by the physical properties of the substrate or the reaction system. Investigations on hydrolysis of triglycerides in organic solvent by five different lipases showed that the effect of chain length on hydrolytic activity is in general much smaller than the effect of unsaturation. 16 It has been shown that some plant seeds contain lipases that show selectivity for the most common fatty acids in that particular kind of seed. 17 It can thus be of interest to study lipases from plant seeds that contain unusual fatty acids. In this chapter, we describe a lipase from seed of Vernonia galamensis. These seeds contain about 40% oil and the oil contains up to 80% vernolic acid (cis-12,13-epoxy-cis-9-0ctadecenoic acid). Much of

3 M. J. Hills, I. Kiewitt, and K. D. Mukherjee, Biotechnol. Lett. 11, 629 (1989). 4 T. m. Foglia and P. E. Sonnet, J. Am. Oil Chem. Soc. 72, 417 (1995). 5 M. S. K. Syed Rahmatullah, V. S. K. Shukla, and K. D. Mukherjee, Z Am. Oil Chem. Soc. 71, 563 (1994). 6 Deleted in proof. 7 p. E. Sonnet, T. A. Foglia, and S. H. Feairheller, J. Am. Oil Chem. Soc. 70, 378 (1993). 8 G. P. McNeill and P. E. Sonnet, J. Am. Oil Chem. Soc. 72, 1301 (1995). 9 M. S. F. Lie Ken Jie and M. S. K. Syed Rahmatullah, J. Am. Oil Chem. Soc. 72, 245 (1995). 10 M. J. Hills, I. Kiewitt, and K. D. Mukherjee, J. Am. Oil Chem. Soc. 67, 561 (1990). 11 V. B. Yadwad and L. C. Noronha, Biotechnol. Bioeng. 38, 956 (1991). 12 M. Iwai, S. Okumura, and Y. Tsujisaka, Agric. Biol. Chem. 39, 1063 (1975). 13 M. Sugiara and M. Isobe, Chem. Pharm. Bull. 23, 1226 (1975). 14M. Iwai, in "Lipases" (B. Borgstr6m and H. L. Brockman, eds.), p. 416. Elsevier Science, New York, 1984. 15 M. S. Rangheard, G. Langrand, C. Triantaphylides, and J. Baratti, Biochim. Biophys. Acta 1004, 20 (1989). 16M. Berger and M. P. Schneider, Biotechnol. Lett. 13, 641 (1991). 17y. H. Lin, C. Yu, and A. H. C. Huang, Arch. Biochem. Biophys. 244, 346 (1986).

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

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the vernolic acid is present in the form of trivernolin. In the following sections, we describe the purification and properties of the lipase as well as purification and properties of trivernolin and vernolic acid, which are used in the studies of the fatty acid selectivity of the lipase. Purification of Vernonia galamensis Seed Lipase

Lipase Assay Method Principle. The Vernonia lipase is assayed by measuring the release of resorufin from 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufin ester. Reagents 1,2-O-Dilauryl-rac-glycero-3-glutaric acid-resorufin ester Dioxane (analytical reagent) Procedure. The assay mixture contains 1.35 ml of enzyme solution and 0.15 ml of 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufin ester (1 mg/ ml) in dioxan. The mixture is incubated at 37°. Samples of 250 /zl are withdrawn after 0.5 and 10 min and added to 1 ml of dioxane. The mixture is centrifuged at 3000 rpm for 5 min at room temperature. The amount of resorufin released is determined by measuring absorbance at 572 nm. Definition of Unit and Specificity. One unit of enzyme is the amount that catalyzes the release of 1/zmol of resorufin per minute at 37°. Specific activity is defined as units per milligram of protein.

Purification Procedure The extraction and all purification steps are carried out at room temperature and all centrifugation steps are done at 7°.

Step 1: Extraction of Lipase from Vernonia Seeds and Differential Centrifugation. Vernonia galamensis seeds are washed several times in distilled water and soaked in distilled water for about 15 hr. The seeds are then washed several times with distilled water and 20 g of seed is gently homogenized using a pestle and mortar in 80 ml of buffer [150 mM Tris-HCl (pH 7.5), 2 mM dithiothreitol (DTT), 0.5 mM EDTA, and 0.4 M sucrose]. The homogenate is filtered through cheesecloth and centrifuged at 1000 g for 5 min. The lipid layer is carefully removed. The resulting supernatant is centrifuged at 20,000 g for 30 min. About three-quarters of the activity is recovered in the pellet. The pellet is washed three times by resuspending in the homogenizing buffer and centrifuging at 20,000 g for 20 min. The lipase in the pellet is solubilized by suspending the pellet in 10 ml of homogenizing buffer containing 1% (w/v) deoxycholic acid but without

Vernonia LIPASESELECTIVITY

[ 12]

223

sucrose. Material that is not solubilized is r e m o v e d by centrifugation at 20,000 g for 15 min, Step 2: Sephacryl S-300 Gel Filtration. The solubilized lipase (3 ml) is loaded onto a Sephacryl S-300 (60 x 2.5 cm) column and eluted with elution buffer [50 m M Tris-HCl ( p H 7.5), 100 m M NaC1, 2 m M D T T , 0.5 m M E D T A , and 1% (w/v) deoxycholic acid] at a flow rate of 25 ml/hr. Fractions of 4.2 ml are collected. The lipase activity is eluted within the void volume fractions (Fig 1). The purification is summarized in Table I.

Properties of Lipase Solubility, Molecular Weight, Stability, p H Optimum. The lipase is highly insoluble in nondetergent buffers but becomes soluble in 1% (w/v) deoxycholate. As a result of this p o o r solubility the lipase activity is eluted at an apparent aggregate molecular weight of i x 106. A molecular weight of 116,000 is obtained for the lipase from sodium dodecyl sulfate0.8

3000

0.6-

- 2000 t"q

8 0.4o

- 1000

2~

0.2

0 j 10

20

30

40

-0 50

Fraction no. FIG. 1. Elution profile for the elution of Vernoniagalarnensislipase. Absorbance at 280 nm (--) and lipase activity (O). The solubilized lipase (3 ml) was loaded onto a Sephacryl S-300 column (60 × 2.5 cm) and eluted at a flow rate of 25 ml/hr with fractions of 4.2 ml being collected. [Reprinted from Biochim. Biophys. Acta 1257, I. Ncube, T. Gitlesen, P. Adlercreutz, J. S. Read, and B. Mattiasson, p. 149 (1995). With kind permission of Elsevier Science-NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.]

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[1 2]

TABLE I PURIFICATION OF Vernonia galamensis LIeASEa

Purification state

Purification (fold)

Specific activity (U/mg protein)

Activity yield (%)

1000 g supernatant 20,000 g pellet Gel-filtration eluate

1 34 73

163 5,556 11,905

100 60 28

"Data from Ncube et al. (1995). 22

polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 2). The lipase shows maximum activity at pH values between pH 8.0 and 9.0. Immobilization

Lipase fractions from the Sephacryl S-300 gel filtration are pooled and 40 ml of the pool is added to 1 g of porous polypropene that has been wetted with 3 ml of ethanol. The mixture is shaken overnight on a reciprocal shaker. The polypropene with the adsorbed lipase is collected by filtration and washed with several volumes of distilled water followed by 100 ml of 10 mM sodium phosphate buffer, pH 8.0. The preparation is dried overnight under reduced pressure. The lipase is stable for weeks if stored in a desiccator at room temperature. Extraction of Vernonia Oil and Purification of Trivernolin Oil from Vernonia seeds (50 g) is extracted by homogenizing in 200 ml of cold acetone (-20°). Solid material is removed by vacuum filtration and the acetone is evaporated under nitrogen gas at 50 °. The oil obtained (15 g) is dissolved in 50 ml of hexane and centrifuged at 3000 rpm for 10 min to remove the precipitated material. Fatty acids are removed from the oil by washing with an equal volume of methanol-bicarbonate buffer (pH 10) (1 : 1, v/v). Hexane is removed from the oil by evaporation under nitrogen gas. Oil (2 g) is then loaded onto a silica gel 60 (230-400 mesh) column (25 × 3 cm) that has been equilibrated with hexane. The oil is washed into the column with hexane. Nonpolar material is eluted with 200 ml of hexane-ether (95 : 5, v/v). The triglycerides are eluted with hexane-ether (70: 30, v/v). To obtain an elution profile of the triglycerides small fractions of about 1 ml are collected every 5 min for monitoring of total lipid content and fatty acid composition of the fractions. Bulk fractions of about 25 ml are collected and aliquots of 100/zl withdrawn from the fractions for gas chromatography (GC) determination of the fatty acid composition. Frac-

[ 12]

Vernonia LIPASESELECTIVITY

225

Lane number 5

4

3

2

|

205 97 69

46

~

30 ~

O

21

FIG. 2. SDS-polyacrylamide gel electrophoresis of Vernonia lipase on 12.5% (w/v) acrylamide gels. Lanes 1 and 7, molecular weight markers; lane 2, crude extract; lane 3, 20,000 g supernatant; lane 4, resuspended pellet; lane 5, solubilized pellet; lane 6, gel-filtration lipase active pool. [Reprinted from Biochim. Biophys. Acta 1257, I. Ncube, T. Gitlesen, P. Adlercreutz, J. S. Read, and B. Mattiasson, p. 149 (1995). With kind permission of Elsevier ScienceNL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.] tions that fall within the pure trivernolin p e a k are pooled and the solvent evaporated under nitrogen gas to obtain pure trivernolin. P r e p a r a t i o n of Vernolic Acid b y E n z y m a t i c H y d r o l y s i s of Trivernolin Trivernolin (300 mg) is dissolved in 1 ml of water-saturated isooctane and 100 mg of Lipozyme (Novo Nordisk, Bagsvaerd, D e n m a r k ) is added. T h e mixture is incubated in a shaking water bath set at 45 ° and at a shaking speed of 150 rpm. The extent of hydrolysis is followed by silica gel thinlayer c h r o m a t o g r a p h y (TLC), using a mobile phase of h e x a n e - e t h e r - a c e t i c acid (80:20:1, by volume). After complete hydrolysis of trivernolin the solution is r e m o v e d from the Lipozyme and added to 2 ml of m e t h a n o l chloroform (1.4 : 1.2, v/v). The vernolic acid is extracted into aqueous meth-

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[ 12]

anol by addition of 1 ml of 200 mM bicarbonate buffer, pH 10. The aqueous methanol phase is removed and the vernolic acid extracted by adding 1 ml of n-hexane followed by 100/zl of 6 M HC1. The hexane layer is withdrawn and the hexane evaporated in a stream of nitrogen gas to obtain pure vernolic acid. Properties of Trivernolin and Vernolic Acid

Physical Appearance and Stability Trivernolin is a clear, light oil that is stable for weeks if stored at - 2 0 °. Vernolic acid is a white solid at room temperature and remains stable at - 2 0 °. General Considerations Concerning Determination of Fatty Acid Selectivity Lipases are active on many types of substrates. The natural substrates are triglycerides, but partial glycerides and many other esters can also be hydrolyzed by lipases. Furthermore, ester synthesis and transesterification reactions can be carried out, especially if organic reaction media are used. Lipases can express fatty acid selectivity in all these types of reactions and the selectivity is not necessarily identical in all reactions. It is not straightforward to determine fatty acid selectivity using a triglyceride substrate. If the fatty acids are unevenly distributed in the three positions in the triglyceride molecules, which they indeed are in most natural fats and oils, the apparent fatty acid selectivity can be biased by the regioselectivity of the lipase. 18 It is also difficult to obtain a synthetic triglyceride substrate with completely randomly distributed fatty acids, and thus other substrates must be used. The approach taken here is to use a mixture of synthetic triglycerides containing the same fatty acid in all three positions. The fatty acid specificity can also be measured using free fatty acids as substrates. Here, this kind of fatty acid specificity was studied in an acidolysis reaction: The fatty acids were incorporated into the triglyceride substrate, which originally consisted of tricaprylin. Some important parameters in the analyses are discussed in the following sections.

Solvent The properties of solvents and especially their ability to dissolve water and substrate will affect both reaction rate and equilibrium. Simple triglycer18 p. Villeneuve, M. Pina, D. Montet, and J. Graille, J. A m . Oil Chem. Soc. 72, 753 (1995).

[ 121

Vernonia LIPASESELECTIVITY

227

ides containing fatty acids with carbon chain lengths longer than 10 cannot be mixed at room temperature. An organic solvent must be used to obtain homogeneous substrate mixture. Lipases operate in vivo at the interface between a polar water phase and an apolar substrate phase. Apolar solvents such as hexane methyl tert-butyl ether or diisopropyl ether can be used to mimic the apolar substrate phase. The substrate solubility in these solvents is in the range of 10 mM. In the present study isooctane was used as solvent.

Water Water plays an important role in lipase-catalyzed reactions both as a reactant and an enzyme activator. In the case of hydrolysis there is a net consumption of water during the reaction whereas for acidolysis there is no change in water content of the reaction mixture, but water is needed as an intermediate substrate. Water is also needed by the enzyme as an activator to sustain enzyme activity. To study effects of reaction conditions on enzymatic reaction rates, it is necessary to keep the hydration of the enzyme constant. Otherwise the effects observed are often due only to redistribution of water in the system. Even small changes such as changing the substrate concentration can change the solubility of water in the reaction medium and thereby influence enzyme hydration. To keep enzyme hydration constant, control the thermodynamic water activity in the reactor. This can easily be done by equilibration with saturated salt solutions. 19 The enzyme preparation and the substrate solution can be separately equilibrated to the same water activity before the reaction is started. If the reaction is to be studied over a long period of time, it is advisable to control the water activity continuously.2° In the present study preequilibration of enzyme preparations and substrate solutions was carried out and initial reaction rates were measured.

Enzyme Preparation Lipases are not soluble in pure organic solvents and will be present as a separate phase. The lipase preparation can either be used directly as a powder or be immobilized in several ways onto carriers. The latter approach makes the reaction system easier to handle and reduces the mass transfer limitations in the system.

19 H. L. Goderis, G. Ampe, M. P. Feyten, B. L. Fouwr, W. M. Guffens, S. M. Van Cauwenbergh, and P. P. Tobback, Biotechnol. Bioeng. 30, 258 (1987), 20 E. Wehtje, I. Svensson, P. Adlercreutz, and B. Mattiasson, Biotechnol. Tech. 7, 873 (1993).

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[12]

Methods for Determination of Fatty Acid Selectivity

Hydrolysis of Triglyceride Mixtures in Isooctane Method. Hydrolysis reactions are done at a water activity of 0.97 obtained by preequilibrating both enzyme preparation and substrate solution with a saturated solution of K2SO419). The standard substrate solution is a mixture of simple triglycerides of each of the fatty acids C4 : 0, C6 : 0, C8 : 0, C10: 0, C12 : 0, C14 : 0, C16 : 0, C18 : 1, and trivernolin dissolved in isooctane, each to a concentration of 10 mM. The reaction is started by adding 100 mg of lipase preparation to 2 ml of substrate solution. The reactions are carried out at 40 ° in a shaking water bath that is set at a shaking speed of 150 rpm. Samples of 100/zl are withdrawn for high-performance liquid chromatography (HPLC) analysis of the released fatty acids. High-Performance Liquid Chromatography Analysis. The H P L C analysis of fatty acids is carried out in an H P L C system equipped with an ultraviolet (UV) detector using a Licrospher RP-C18(5/xm) column (250 × 4 mm; Merck, Darmstadt, Germany). Samples from the enzyme reaction are diluted in 400/zl of acetonitrile. Tridecanoic acid is added as an internal standard. Ten microliters of phenacylation reagent consisting of phenacyl bromide and 18-crown-6 ether (10 mg/ml of molar ratio 10 : 1 in acetonitrile) is added. The reagent is added in slight deficit to avoid artifact peaks from the reagent overlapping the fatty acid peaks of the short fatty acid at the beginning of the chromatograms. About 20 mg of KCO3 (s) is added to each reaction vial to convert the free fatty acids to their reactive potassium salts. The samples are heated to 80 ° for 1 hr, cooled to room temperature, and filtered through filter paper to remove remaining triglycerides. 21Elution is done at room temperature using an acetonitrile-water gradient. Elution is started with an initial concentration of acetonitrile of 60%, which is raised to 99% over 10 min and held at 99% for 2 rain. Fatty acid phenacyl esters are monitored by determining UV adsorption at 254 nm. Results. Among the saturated fatty acids, the Vernonia lipase showed rather pronounced selectivity for caprylic acid (C8 : 0, Fig. 3). The reaction rate obtained with triolein was quite low, while trivemolin was hydrolyzed rapidly, more than four times faster than C8:0. When Candida rugosa lipase was investigated in the same way, trivernolin was hydrolyzed more rapidly than the other triglycerides but the differences were much smaller. 22 21 A. W. Reed, H. C. Deeth, and D. E. Clegg, J. Assoc. Anal. Chem. 67, 718 (1984). ~2 I. Ncube, T. Gitlesen, P. Adlercreutz, J. S. Read, and B. Mattiasson, Biochim. Biophys. Acta 1257, 149 (1995).

Vernonia LIPASESELECqTV1TY

[ 12]

229

2.5-

2

"~ ~

~

1.5

1

0.5

0

Triglyceride

FIG. 3. Fatty acid specificity of Vernonia galamensis lipase in triacylglycerol hydrolysis in isooctane. Equimolar mixtures of monoacid-triacylglycerols (10 mM each) were hydrolyzed using lipases adsorbed on polypropene, [Data from Ncube et al. (1995). 22]

It is thus clear that the Vernonia lipase expresses a strong selectivity for vernolic acid in the form of trivernolin.

Transesterification of Tricaprylin with Fatty Acid Mixtures Method. The substrate mixture is 100 m M tricaprylin and 30 m M in each of the fatty acids C7 : 0, C10: 0, C12 : 0, C14 : 0, C16 : 0, C18 : 1, and vernolic acid dissolved in isooctane. Shorter fatty acids cause inhibition of the enzyme and are therefore not included in the test. The reaction is carried out in 4-ml vials and the reaction mixture consists of 2 ml of substrate solution and 100 mg of lipase preparation. The reactions are carried out at a water activity of 0.43 (attained by preequilibration with a saturated solution of K2CO3) on a water bath at a temperature of 40 ° and shaking speed of 150 rpm. Samples (100/~1) are withdrawn for G C analysis of the esterified fatty acids. Gas-Liquid Chromatographic Analysis. The fatty acids in glycerides are converted to the corresponding methyl esters using sodium methoxide 23 23 I. Svensson, P. Adlercreutz, and B. Mattiason, Appl. Microbiol. BiotechnoL 33, 255 (1990).

230

ISOLATION,

CLONING,

EXPRESSION,

[12]

AND ENGINEERING

with tridecanoin as an internal standard. Analysis of the methyl esters is done on a Shimadzu (Tokyo, Japan) GC14A gas chromatograph equipped with a flame ionization detector (FID) using an SP2380 column (Supclco, Bellefonte, PA) (30 m, 0.32-mm i.d., and 0.2-/zm film thickness). In the analysis of the fatty acids of Vernonia oil, the initial column temperature is 170° for 2 min and is raised to 190° at 5°/rain and held at this temperature for 8 rain. In the analysis of fatty acids from the transesterification reaction samples the initial temperature is 40° and is raised to 190° at 15°/min and held at this temperature for 15 min. Helium is used as the carrier gas. In both cases the vernolic acid is eluted well after the other fatty acids. Results. The Vernonia lipase incorporated vernolic acid into the triglyceride fraction more rapidly than any of the other fatty acids (Fig. 4). Among the other fatty acids, heptanoic acid was incorporated faster than the others. The specificity of the Vernonia lipase for vernolic acid is thus not limited to the natural substrate, the triglyceride, but is also expressed for the free fatty acids.

Michaelis-Menten Kinetics of Hydrolysis of Triolein and Trivernolin Method. Candida rugosa lipase was immobilized by a method similar to that used with the Vernonia lipase. In the kinetic study, the reaction 0.3

0.25 t .o .~

0.2J

't:2 e~

.~, ~ 0.15

~

0.1 o.05

0

i

i

~

i

i

i

I oo

U Fatty acid

X 0

Fxc. 4. Fatty acid specificity of Vernonia galamensis lipase in acidolysis of tricaprylin in isooctane. Equimolar mixtures (30 mM each) of the fatty acids and 10 mM tricaprylin were used as substrates and the lipase was adsorbed on polypropene. [Data from Ncube et al. (1995).22]

[ 121

Vernonia LIPASESELECTIVITY

231

T A B L E II KINETIC CONSTANTS FOR HYDROLYSIS OF TRIVERNOLIN AND TRIOLEIN BY Vernonia LIPASE AND Candida LIPASEa

Km (raM)

Vm= (/zmol • hr -1. mg protein -1)

Lipase

Trivernolin

Triolein

Trivernolin

Triolein

Candida Vernonia

39 -+ 12 68 - 10

28 -4- 3 58 +- 10

1 2 1 - 13 18 _+ 1

130 _-_ 4 3.8 -+ 3

a Data from Ncube et al. (1995). 22

mixtures are 50 mg of lipase preparation in 1.0 ml of substrate solution for Vernonia lipase and 25 mg in 500 /xl of substrate solution for Candida lipase. The substrate solutions consist of either trivernolin or triolein at concentrations ranging from 0 to 200 mM in isooctane. The initial water activity in all solutions is kept the same (0.97) by preequilibration of both enzyme preparation and substrate solution over saturated solutions of K2804. To determine the initial rates of hydrolysis, samples of 100/zl are withdrawn for HPLC analysis of the released oleic acid or vernolic acid. Results. The hydrolysis of triglycerides in isooctane catalyzed by the lipases from V. galarnensis seeds and from C. rugosa could be described by the Michaelis-Menten equation. Because vernolic and oleic acid differ only in that the former acid contains an epoxy group, it was of interest to compare the hydrolysis of triolein and trivernolin. It was found for both enzymes that the Km values were slightly higher for trivernolin compared to triolein (Table II). For the Candida lipase, the Vmaxvalues for the two

T A B L E III FATTY ACID SELECTIVITYOF SOME PLANT SEED LIPASES Lipase source

Best substrate

Ref.

Corn Castor bean Rapeseed Elm seed Oat seed Vernonia seed

Trilinolein, triolein Triricinolein Trierucin Tricaprin Triolein Trivernolin

a a a a b This work

a y . H. Lin, C. Yu, and A. H. C. Huang, Arch. Biochem. Biophys. 244, 346 (1986). b G. J. Piazza, A. Bilyk, D. P. Brower, and M. J. Haas, J. Am. Oil Chem. Soc. 69, 978 (1992).

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substrates were similar. However, the V m a x value for trivernolin was about five times higher than that for triolein for the Vernonia lipase. It is thus this difference in V m a x that causes the selectivity for trivernolin. It would be interesting to investigate the mechanism behind this large rate enhancement caused by the epoxy groups in the fatty acids. An important step in the elucidation of this mechanism would be to determine the three-dimensional structure of the Vernonia lipase. Discussion It is quite natural that the lipases present in plant seeds are effective in hydrolyzing the triglycerides that are predominant in the seeds. This kind of specificity has been observed previously in lipases from a few plant seeds (Table III). However, this work is the first to describe selectivity for epoxide-containing fatty acids. In looking for lipases with novel specificities it can thus be worthwhile to investigate lipases from plant seeds containing triglycerides built up of unusual fatty acids.

[13] H e p a t i c L i p a s e : H i g h - L e v e l E x p r e s s i o n Structure Determination

and Subunit

B y JOHN S. HILL, RICHARD Co DAVIS, DAWN YANG, MICHAEL C. SCHOTZ, a n d HOWARD WONG

Introduction Through its ability to catalyze the hydrolysis of triglycerides and phospholipids, hepatic lipase (HL) influences the metabolism of chylomicron remnants, intermediate-density lipoproteins (IDLs), and high-density lipoproteins (HDLs). 1 Hepatic lipase is synthesized and secreted by hepatocytes, and binds to the lumenal surface of the endothelium in liver sinusoids through an ionic interaction with heparan sulfate proteoglycans.2 The detection of HL catalytic activity but not protein synthesis in the ovary and adrenal gland suggested that HL is transported and binds specifically to these tissues as well.3,4In addition, there is evidence to suggest that, indepen1 T. Olivecrona and G. Bengtsson-Olivecrona, Curr. Opin. Lipidol. 4, 187 (1993). 2 T. Kuusi, E. A. Nikkila, I. Virtanen, and P. K. J. Kinnunen, Biochem. J. 181, 245 (1979). 3 M. H. Doolittle, H. Wong, R. C. Davis, and M. C. Schotz, Z Lipid Res. 28, 1326 (1987). 4 E. A. Hixenbaugh, T. R. Sullivan, J. F. Strauss III, E. A. Laposata, M. Komaromy, and L. G. Paavola, J. BioL Chem. 266, 4222 (1989).

METHODS IN ENZYMOLOGY, VOL. 284

Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25.00

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I S O L A T I O N , CLONING, EXPRESSION, A N D E N G I N E E R I N G

[13]

substrates were similar. However, the V m a x value for trivernolin was about five times higher than that for triolein for the Vernonia lipase. It is thus this difference in V m a x that causes the selectivity for trivernolin. It would be interesting to investigate the mechanism behind this large rate enhancement caused by the epoxy groups in the fatty acids. An important step in the elucidation of this mechanism would be to determine the three-dimensional structure of the Vernonia lipase. Discussion It is quite natural that the lipases present in plant seeds are effective in hydrolyzing the triglycerides that are predominant in the seeds. This kind of specificity has been observed previously in lipases from a few plant seeds (Table III). However, this work is the first to describe selectivity for epoxide-containing fatty acids. In looking for lipases with novel specificities it can thus be worthwhile to investigate lipases from plant seeds containing triglycerides built up of unusual fatty acids.

[13] H e p a t i c L i p a s e : H i g h - L e v e l E x p r e s s i o n Structure Determination

and Subunit

B y JOHN S. HILL, RICHARD Co DAVIS, DAWN YANG, MICHAEL C. SCHOTZ, a n d HOWARD WONG

Introduction Through its ability to catalyze the hydrolysis of triglycerides and phospholipids, hepatic lipase (HL) influences the metabolism of chylomicron remnants, intermediate-density lipoproteins (IDLs), and high-density lipoproteins (HDLs). 1 Hepatic lipase is synthesized and secreted by hepatocytes, and binds to the lumenal surface of the endothelium in liver sinusoids through an ionic interaction with heparan sulfate proteoglycans.2 The detection of HL catalytic activity but not protein synthesis in the ovary and adrenal gland suggested that HL is transported and binds specifically to these tissues as well.3,4In addition, there is evidence to suggest that, indepen1 T. Olivecrona and G. Bengtsson-Olivecrona, Curr. Opin. Lipidol. 4, 187 (1993). 2 T. Kuusi, E. A. Nikkila, I. Virtanen, and P. K. J. Kinnunen, Biochem. J. 181, 245 (1979). 3 M. H. Doolittle, H. Wong, R. C. Davis, and M. C. Schotz, Z Lipid Res. 28, 1326 (1987). 4 E. A. Hixenbaugh, T. R. Sullivan, J. F. Strauss III, E. A. Laposata, M. Komaromy, and L. G. Paavola, J. BioL Chem. 266, 4222 (1989).

METHODS IN ENZYMOLOGY, VOL. 284

Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25.00

[13]

SUaUNITSTRUCTUREOF HEPATICLIPASE

233

dent of its catalytic activity, HL can act as a ligand to facilitate the uptake of remnant lipoproteins through its interaction with cell surface proteoglycans and the low-density lipoprotein receptor-related protein (LRP). 5-8 Although HL appears to have a number of roles in lipoprotein metabolism, it is evident in human HL deficiency that remnant metabolism is primarily affected. 9 This observation is consistent with animal studies in which the inhibition of HL activity by administration of HL antibodies decreased the rate of chylomicron remnant uptake by the liver. 1°'11 However, there are several in vitro studies that demonstrate that HL also participates in the remodeling of HDL. 12-14 This has been clearly demonstrated in gene-targeted and transgenic HL animals in which the metabolism of the predominant plasma lipoprotein species, HDL, is significantly affected. 15-17 Mature human HL is a 476-amino acid glycoprotein with a calculated polypeptide molecular weight of 53,431, whereas the purified denatured enzyme has an apparent molecular weight of 65,000, reflecting the contribution of N-linked carbohydrate. ~8-2° Hepatic lipase is a member of a lipase family that also includes lipoprotein lipase (LPL) and pancreatic lipase

5 p. Diard, M.-I. Malewiak, D. Lagrange, and S. Griglio, Biochem. J. 299, 889 (1994). 6 A. Nykjaer, M. Nielson, A. Lookene, N. Meyer, H. R¢igaard, M. Etzerodt, U. Beisiegel, G. Olivecrona, and J. Gliemann, J. Biol. Chem. 269, 31747 (1994). 7 M. Z. Kounnas, D. A. Chappell, H. Wong, W. S. Argraves,and D. K. Stricldand, J. Biol. Chem. 270, 9307 (1995). 8 A. Krapp, S. Ahle, S. Kersting, Y. Hua, K. Kneser, M. Nielsen, J. Gliemann, and U. Beisiegel, J. Lipid Res. 37, 926 (1996). 9 R. A. Hegele, J. A. Little, C. Vezina, G. F. Maguire, L. Tu, T. S. Wolever, D. J. A. Jenkins, and P. W. Connelly, Arterioscler. Thromb. 13, 720 (1993). 10F. Sultan, D. Lagrange, H. Jansen, and S. Griglio, Biochim. Biophys. Acta 1042, 150 (1990). 11 S. Shaft, S. E. Brady, A. Bensadoun, and R. J. Havel, J. Lipid Res. 35, 709 (1994). 12j. R. Patsch, S. Prasad, A. M. Gotto, and W. Patsch, J. Clin. Invest. 80, 341 (1987). 13M. A. Clay, H. H. Newnham, and P. J. Barter, Arterioscler. Thromb. 11, 415 (1991). 14 A. Barrans, X. Collet, R. Barbaras, B. Jaspard, J. Manent, C. Vieu, H. Chap, and B. Perret, J. Biol. Chem. 269, 11572 (1994). 15 S. J. Busch, R. L. Barnhart, G. A. Martin, M. C. Fitzgerald, M. T. Yates, S. J, T. Mao, C. E. Thomas, and R. L. Jackson, J. Biol. Chem. 269, 16376 (1994). 16j. Fan, J. Wang, A. Bensadoun, S. J. Lauer, Q. Dang, R. W. Mahley, and J. M. Taylor, Proc. Natl. Aead. Sci. U.S.A. 91, 8724 (1994). 17 G. E. Homanics, H. V. de Silva, J. Osada, S. H. Zhang, H. Wong, J. Borensztajn, and N. Maeda, J. Biol. Chem. 270, 2974 (1995). 18 G. Stahnke, R. Sprengel, J. Augustin, and H. Will, Differentiation 35, 45 (1987). 19 S. Datta, C.-C. Luo, W.-H. Li, P. Vantuinen, D. H. Ledbetter, M. A. Brown, S.-H. Chen, S.-W. Lui, and L. Chan, J. Biol. Chem. 263, 1107 (1988). 20 G. A. Martin, S. J. Busch, G. D. Merideth, A. D. Cardin, D. T. Blakenship, S. J. T. Mao, A. E. Rechtin, C. W. Woods, M. M. Racke, M. P. Schafer, M. C. Fitzgerald, D. M. Burke, M. A. Flanagan, and R. L. Jackson, J. Biol. Chem. 263, 10907 (1987).

234

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

I 13]

(PL). 21-e3 Amino acid homology, including the conservation of disulfide bridges among all three enzymes, suggests that they share similar threedimensional structures. 24 However, on comparison of their phylogenetic relationships, it is apparent that HL and LPL are more closely related to each other than either enzyme is to PL. 22 The low protein yields associated with the purification of HL from human postheparin plasma have not permitted a more detailed physical analysis of this enzyme. For example, the only previous estimations of the functional molecular weight of HL have relied on gel-filtration techniques. The primary disadvantage of conventional size-exclusion chromatography methods is that the elution position depends not only on the molecular weight of the molecule, but also on its shape. In addition, if the protein has a tendency to adhere to the column matrix, the molecular weight may be underestimated. In this chapter we describe procedures developed for a large-scale production and purification of recombinant HL. The purification of sufficient quantities of this enzyme has enabled the analysis of its subunit structure by three independent means: intensity light scattering, sedimentation equilibrium, and radiation inactivation.

DNA Transfection and Production of Recombinant H u m a n Hepatic Lipase Full-length human H L cDNA TM is cloned into the HindIII and NotI sites of the expression vector pcDNAI/Neo (Invitrogen, San Diego, CA). Chinese hamster ovary (CHO)-Pro5 cells are maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with sodium pyrufate, glutamine, nonessential amino acids, 10% fetal bovine serum (FBS), and antibiotics. To mediate the transfection of CHO cells, coprecipitates of plasmid D N A and CaPO4 are prepared. 2s The calcium phosphate-DNA mixture is incubated at room temperature for 30 rain before it is added to a 50% confluent CHO monolayer. Stably transfected ceils are selected by growth in the presence of geneticin (G418 sulfate, 400/xg/ml) and surviving 21 O. Ben-Zeev, C. M. Ben-Avram, H. Wong, J. Nikazy, J. E. Shively, and M. C. Schotz, Biochim. Biophys. Acta 919, 13 (1987). 22 T. G. Kirchgessner, J.-C. Chant, C. Heinzmann, J. Etienne, S. Gnilhot, K. Svenson, D. Ameis, C. Pilon, L. D'Auriol, A. Andalibi, M. C. Schotz, F. Galibert, and A. J. Lusis, Proc. Natl. Acad. Sci. U.S.A. 86, 9647 (1989). 23 W. A. Hide, L. Chan, and W.-H. Li, Z Lipid Res. 33, 167 (1992). 24 Z. S. Derewenda and C. Cambillau, J. Biol. Chem. 2 ~ , 23112 (1991). 25 C. Chen and H. Okayama, Mol. Cell. Biol. 7, 2745 (1987).

[ 13]

SUBUNIT STRUCTURE OF HEPATIC LIPASE

235

colonies are selected and expanded. Cell clones expression maximal quantities of HL are identified by enzyme activity analysis. After growth to confluency in sixteen 225-cm2 flasks, cells are washed with DMEM and fresh DMEM supplemented with 1% (v/v) Nutridoma (serum substitute to minimize contaminating proteins, Boehringer-Mannheim, Indianapolis, IN) and heparin (10 units/ml) are added to each flask. The medium is harvested and replaced every 24 hr for a 10-day period. After centrifugation at 1000 g for 10 min at 4° to remove cellular debris, the harvested medium is stored at - 8 0 °. Purification of Recombinant H u m a n Hepatic Lipase Three liters of thawed medium is mixed with NaC1 and benzamidine to a final concentration of 0.5 M and 0.5 mM, respectively. A four-step procedure is utilized, consisting of octyl-Sepharose, heparin-Sepharose, hydroxylapatite, and dextran sulfate-Sepharose. Hepatic lipase activity is monitored as described previously26 using radiolabeled triolein substrate. The apparent Km and Vm~xvalues for a triolein substrate are determined from a linear regression analysis of a double-reciprocal plot of lipolytic activity (/zmol/min/mg) versus triolein concentration (raM). Protein concentration is determined by the Bradford method. 27 All purification steps are carried out at 4 °.

Step 1: Octyl-Sepharose Chromatography. The medium (3 liters) is loaded at a flow rate of 150 ml/hr onto an octyl-Sepharose column (2,5 × 25 cm) previously equilibrated with 5 mM (1.03 g/liter) barbital buffer, pH 7.2, containing 0.5 M (29.22 g/liter) NaC1 and 20% (v/v) glycerol (buffer A). Following a wash with 800 ml of buffer A, the lipase is eluted with 700 ml of 5 mM barbital buffer, pH 7.2, containing 0.35 M NaC1 (20.45 g/liter), 20% (v/v) glycerol, and 1.2% (w/v) Triton N-101. Step 2: Heparin-Sepharose Chromatography. The eluate from the octylSepharose column is applied to a heparin-Sepharose column (2.5 x 20 cm), which is then washed with 800 ml of buffer A prior to elution with 500 ml of 1 M (58.44 g/liter) NaCI, 20% (v/v) glycerol, 5 mM barbital (pH 7.2). Step 3. Hydroxylapatite Chromatography. The eluted lipase is passed through a 2-ml hydroxylapatite column to remove contaminating proteins and then diluted with an equal volume of 20% (v/v) glycerol, 5 mM barbital (pH 7.2) before it is loaded onto a dextran sulfate-Sepharose column (1 × 5 cm). 26 M. H. Doolittle, H. Wong, R. C. Davis, and M. C. Schotz, Z Lipid Res. 28, 1326 (1987). 27 M. D. Bradford, Anal Biochem. 72, 248 (1976).

236

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[13]

TABLE I PURIFICATION OF HUMAN HEPATIC LIPASE SECRETED BY HEPATIC LIPASE-TRANSFECTED CHO

Culture medium Hydroxylapatite flowthrough Dextran sulfateSepharose eluent HL concentrate

CELLS a'b

Volume (ml)

Protein (mg)

Specific activity

Purification

(~mol/min/mg)

(fold)

Recovery (%)

3300 500

131 1.10

1.5 109

1 73

-61

50

0.62

152

101

48

1

0.55

146

97

41

Reproduced with permission from J. S. Hill, R. C. Davis, D. Yang, J. Wen, J. S. Philo, P. H. Poon, M. L. Phillips, E. S. Kempner, and H. Wong, J. Biol. Chem. 271, 22931 (1996). b As described in text, HL was purified using sequential chromatography consisting of four steps: octyl-Sepharose, heparin-Sepharose, hydroxylapatite, and dextran sulfateSepharose. The data presented are representative of several experimental purifications. The presence of Triton N-101 did not permit the measurement of activity present in the octyl- and heparin-Sepharose eluant fractions. Specific activity is expressed as nanomoles of free fatty acid released per minute per microgram of protein.

a

Step 4. Dextran Sulfate-Sepharose Chromatography. The dextran sulfate-Sepharose column is washed with 50 ml of 0.5 M NaC1, 5 mM barbital (pH 7.2) and then eluted with 50 ml of 1 M NaCI, 5 mM barbital (pH 7.2). The collected eluant is concentrated in an Amicon (Danvers, MA) filtration unit using a YM30 membrane to a final volume of 1-2 ml. To preserve enzyme activity, freeze-thaw cycles should be avoided and the purified enzyme should be stored in the presence of 20% (v/v) glycerol. The results of a typical purification from 3300 ml of medium from transfected CHO cells are shown in Table I. Following concentration of the final eluant, approximately 0.5 mg of protein was obtained with an overall yield based on activity of about 40% with a calculated HL specific activity of 146/xmol/min/mg. This specific activity is similar to previous reports of HL purified from human postheparin plasma 28'29and recombinant HL made by stably transfected rat hepatoma McARH7777 cells. 3° Kinetic analysis of the purified enzyme with a triolein emulsion substrate indicated an apparent Vn~, of 1.4 (-+ 0.3) mmol/min/mg and an apparent Km of 16 28 y. Ikeda, A. Takagi, and A. Yamamoto, Biochim. Biophys. Acta 1003, 254 (1989). 29 C. F. Cheng, A. Bensadoun, T. Bersot, J. S. T. Hsu, and K. H. Melford, J. Biol. Chem. 260, 10720 (1985). 30 Z.-S.Ji, S. J. Lauer, S. Fazio, A. Bensadoun, J. M. Taylor, and R. W. Mahley, J. Biol. Chem. 269, 13429 (1994).

[ 131

SUBUNIT STRUCTURE OF HEPATIC LIPASE

237

(+- 2) mM (n = 3). The purified HL bound to heparin-Sepharose and eluted as a single peak with maximum elution at 0.7 M NaC1 (data not shown). This affinity for heparin is consistent with previous reports for human HE. 29,31 Thus, the properties of the recombinant enzyme were the functional equivalent of the native enzyme. Determination of Subunit Structure of Hepatic Lipase

Intensity Light Scattering Size-exclusion chromatography is performed with a Superose 12 (Pharmacia, Piscataway, NJ) column (30 × 0.6 cm) equilibrated with 0.1 M sodium phosphate buffer containing 0.5 M NaC1, using a 500-/A sample loop operated at a flow rate of 0.5 ml/min. The eluant is monitored using three online detectors in series: a light-scattering detector (Wyatt Minidawn, Santa Barbara, CA), a refractive index detector (Polymer Laboratories PLRI, Vienna, VA), and a UV absorbance monitor at 280 nm. The polypeptide molecular weight of a glycoprotein can be calculated using Eq. (1): MWp =

K(UV)(LS)/ep(RI) 2]

(1)

where UV, LS, and RI are the signals from the absorbance, 90° light scattering, and differential refractive index detectors, respectively, 8p is the extinction coefficient (the absorbance of a solution containing 1 mg of polypeptide/ml for a 1-cm pathlength), MWp is the polypeptide molecular weight, and K is a calibration constant that depends on instrument conditions. The calibration constant, K, is measured by running bovine serum albumin (Sigma, St. Louis, MO) and ovalbumin (Sigma) molecular weight standards. 32,33The extinction coefficient of human HL is calculated as 1.22 using the Gill and von Hippel method. 34 The elution characteristics of purified HL are analyzed using a Superose 12 gel-filtration column in the presence of 0.1 M sodium phosphate and various NaC1 concentrations. A single protein peak is obtained with the salt concentration ranging from 0.15 to 1 M NaC1 (data not shown). Following calibration of the light-scattering/size-exclusion chromatography system using molecular weight standards, a 500-/,1 sample of purified enzyme (150 /,g) is injected to assess the subunit structure of HL. The chromatograms 31 H. L. Dichek, C. Parrott, R. Ronan, J. D. Brunzell, H. B. Brewer, Jr., and S. SantamarinaFojo, J. Lipid Res, 34, 1393 (1993). 32T. Takagi, J. Chromatogr. 280, 124 (1983). 33 T. Arakawa, K. E. Langley, K. Kameyama, and T. Takagi, Anal. Biochem. 203, 53 (1992). 34 S. C. Gill and P. H. von Hippel, Anal. Biochem. 182, 319 (1989).

238

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[13]

from all three online detectors and corresponding activity assay of each fraction are shown in Fig. 1. The first peak, eluting at about 6.4 ml, has considerable light scattering but lacks absorbance, indicating that there is a small amount of aggregates (Fig. 1A). The second peak at about 9.6 ml also corresponds to the ultraviolet absorbance and refractive index measurements and has a calculated molecular weight [Eq. (1)] of 107,000, in agreement with the expected value for a dimer of hepatic lipase (the monomer polypeptide formula weight of HL is 53,431). Also, this second light-scattering peak corresponds to fractions determined to be lipolytically

A

O~

B

r

,

i

,

i

i

I

,

i

,

l

,

l

,

i

.

l

i

300

25o 200

~E150 .g" loo

E ~ > > >

~ b > H b ~ . . . .

o~ >~

. . . . . . . .

< c a < <


. . . . . . . .

~

. . . .

. . . . . .

. . . . . .

H b H H H H ~

~

.

.

N
3GalNAc structure of the pancreatic BSDL could be sialylated and fucosylated. It is worth noting that fucose prevails over sialic acid in FAP, which correlates with data suggesting that the epitope recognized by MAb J28 is an TABLE I CARBOHYDRATE COMPOSITION OF NORMALAND PATHOLOGICALVARIANTS OF BILE SALTDEPENDENT LIPASEa

Pathological variant Normal variant J28 (mol/mol protein)

Man GlcNAc

Gal GalNAc

Fuc NeuAc

5.0 3.5 12.7 11.7 10.4 10.6

PCC J28PCC J28FAP J28 + (mol/mol protein) (mol/mol protein) (mol/mol protein) 7.2 2.2 13.2 11.8 11.5 10.3

6.6 4.4 11.0 11.7 12.8 6.2

8.0 2.6 6.7 7.9 11.8 3.1

Bile salt-dependent lipase was purified from pancreatic juices from either a normal donor (normal variant) or patients with chronic pancreatitis (pathological variants, PCC), according to the method described by D. Lombardo, O. Guy, and C. Figarella, Biochim. Biophys. Acta 5277 142 (1978). All these preparations were devoid of reactivity with MAb J28 (J28-). The oncofetal variant of BSDL (FAP) reactive with MAb J28 (J28 +) was isolated according to E. Mas, N. Abouakil, S. Roudani, F. Miralles, O. Guy-Crotte, C. Figarella, M. J. Escribano, and D. Lombardo, Biochem. J. 2897 609 (1993). Data were compiled from this last reference and from E. Mas, N. Abouakil, S. Roudani, J. L. Franc, J. Montreuil, and D. Lombardo, Eur. J. Biochem. 216, 807 (1993).

348

I S O L A T I O NCLONING, , EXPRESSION, AND ENGINEERING

[20]

O-linked structure involving fucose residues (our unpublished observation). Although differences between BSDL and FAP do not seem to be restricted to sugars,47modifications of the glycosylation of BSDL can affect its activity, as can be suspected from the low specific activity of the oncofetal glycovariant measured either on nonspecific substrates (4-nitrophenyl acetate and hexanoate) or cholesteryl esters. 47This fact is of importance when considering the production of recombinant BSDL in either eukaryotic or prokaryotic cells. The fundamental difference between the DNA, RNA, and protein synthesis, on the one hand, and the glycan synthesis, on the other, is that the first depend on the reproduction of a template, whereas the second is dependent on the control and specificity of a series of glycosidases and glycosyltransferases executing concerted or ordered reactions without the need for a template. Therefore, the glycosylation of any recombinant protein is dependent on the expression system. In this respect, information concerning the role of glycans linked to a protein and in particular to BSDL may be valuable. Role of N-Glycosylation of Bile Salt-Dependent Lipase Four experimental approaches are accessible for assessing the role of protein glycosylation. Enzymes that cut glycans to the innermost residue of either N-linked structures (Endo H, PNGase F) or O-linked structures (O-glycanase) are provided by some manufacturers and can be used on purified glycoproteins. Some of these enzymes need detergent to catalyze the lysis of glycan structures; under these conditions the denaturation of the protein by the detergent can preclude effects induced by deglycosylation. Chemical methods using, for example, trifluoromethane sulfonic anhydride are sometimes too drastic to be useful. Drugs that affect either the transfer "en bloc'of the oligomannoside structure to the nascent polypeptide or the maturation of N-linked structures during the protein transport can be successfully used when performing studies on cell models expressing the protein constitutively. Drugs that affect O-linked glycosylation (aryl-Nacetyl-ct-galactosaminides48) may have secondary effects because they possibly affect the expression of such structures on plasma membranes and consequently cell-to-cell recognition (our unpublished observation). Mutagenesis directed to the sites of glycosylation and expression of the unglycosylated protein in any kind of cell are also ways to gain information on the role of glycosylation. For BSDL all of these experimental approaches were taken. The two first methods were of little value with BSDL (our unpublished observation), whereas the two last methods were very informative. 48S. F. Kuan,J. C. Byrd,C. Bashaum,and Y. S. Kim,J. Biol. Chem. 264, 19271 (1989).

[20]

GLYCOSYLATION OF BILE SALT-DEPENDENT LIPASE

349

The rat pancreatoma AR4-2J cell line is a unique line capable of synthesis and secretion of B S D L . 49 Tunicamycin, a structural analog of UDPGlcNAc and the most widely used inhibitor of N-linked glycosylation,5° blocks the transfer of GlcNAc-l-phosphate to dolichol phosphate and thus the formation of Glc3Man9GlcNAc2-pyrophosphoryl dolichol. This drug had no effect on the synthesis of BSDL by AR4-2J cells, but the enzyme was isolated with a lower molecular weight and did not incorporate radiolabeled mannose compatible with the expression of a BSDL lacking its N-linked structure. 51 The nonglycosylated BSDL, although still active, was less efficient than the native enzyme; the former protein was also less stable to temperature changes. It was also shown that the rate of secretion of BSDL by AR4-2J cells exposed to tunicamycin was about two times slower than that of cells grown in the absence of the drug. Nevertheless, drugs that affect the processing of the oligomannose structure, such as castanospermine, 1deoxymannojirimycin, or swainsonine, had no effect either on the enzyme activity or on its secretion. 51 This result corroborates the observation that the glycosylation of human BSDL may vary with pancreatic pathology without affecting its secretion. 44 Therefore, the transfer of the oligosaccharide precursor to the nascent BSDL is essential for the folding of the fully active BSDL and is required for enzyme secretion. The same effect of tunicamycin on both rate of secretion and activity of BSDL was observed with Chinese hamster ovary (CHO) cells transfected with the complete cDNA encoding the rat BSDL. 52 Nevertheless, controversial studies report on the lack of importance of N-glycosylation for functional properties of BSDL expressed in C127 cells transfected with a cDNA variant with altered N-glycosylation site (Asn-187 to Gin). This variant was fully active, secreted, and displayed the same stability to pH, heat, and proteolysis by trypsinJ 3 Whether these differences came from intrinsic properties of the transfected material (e.g., human versus rat) or from cell lines used for the BSDL expression is not yet known. Role of O-Glycosylation of Bile Salt-Dependent Lipase On the basis of the sequence of cloned BSDL studied to date, two questions need to be answered: The first concerns the role of the tandem 49 y. Huang and D. Y. Hui, J. Biol. Chem. 266, 6720 (1991). 50 j. A. Pizzey, F. A. Bennett, and G. E. Jones, Nature (London) 305, 315 (1983). 51 N. Abouakil, E. Mas, N. Bruneau, A. Benajiba, and D. Lombardo, J. Biol. Chem. 268, 25755 (1993). 52 K. R. Morlock-Fitzpatrick and E. A. Fisher, Proc. Soc. Exp. Biol. Med. 208, 186 (1995). 53 L. Hansson, L. Bltickberg, M. Edlund, L. Lundberg, M. Str6mqvist, and O. Hernell, J. Biol. Chem. 268, 26692 (1993).

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[20]

repeated sequences encoded by exon 11, the number of which varies by species, and the second concerns the importance of the O-linked glycosylation present on these sequences. Each repeated sequence is rich in proline (P), glutamic acid (E), serine (S), and threonine (T). Proteins that are rapidly degraded within eukaryotic cells frequently contain such sequences, referred to as the PEST (for Pro, Glu, Ser, and Thr) region. 54 On BSDL, PEST regions coexist with the C-terminal cluster of O-linked oligosaccharides 21 and are absence in sequences of other known pancreatic enzymes. The function of this domain of repeated identical sequences remains unknown, but it may function to dictate intracellular processing and transport of BSDLY When this domain was deleted by recombinant DNA techniques, the truncated BSDL was normally expressed and displayed full activity and functional properties such as activation by bile salts and heparin binding. 53'55-58 In contrast to the results of Bl~ickberg et al., 57 Loomes56 and Di Persio et aL, 55 suggested that the C-terminal repeats may regulate proteolytic degradation of the protein and substrate delivery to the enzyme. Although truncated BSDL was expressed in various cell models, e.g., prokaryotic cells such as E s c h e r i c h i a coli 58 or eukaryotic cells such as monkey kidney fibroblast type 1 (COS-I) cellsy murine C127 cells, 53'57 or baby hamster kidney (BHK) cells, 56 this cannot be argued to explain the discrepancy in results. However, the size of the recombinant BSDL appeared to be of importance. All truncated forms of BSDL that displayed full activity and functional properties included residues Ala-1 to residue His-536. 55,57,58 Those that were neither catalytically active nor insensitive to proteolysis were shorter and included residues Ala-1 to Tyr-48955 or residues Ala-1 to Leu-519. 56 Interestingly, BSDL from which the sequence from residue Pro490 to the end was deleted was not secreted owing to its retention within a particulate fraction of transfected cells. 55 Therefore, it seems likely that the C-terminal tail of BSDL and particularly the sequence between residues Pro-490 and Glu-534 is required for normal intracellular processing and secretion of the enzymeY This domain, encoded by exon 11, has a highly variable sequence between species and precedes the tandemly repeated sequences. Therefore, the intracellular processing of BSDL can be different between species and could be related to the glycosylation of this domain. Interestingly, the rat and human enzymes, which are both O-glycosylated, are associated with intracellular membranes by means of a 94-kDa protein 54 S. Rogers, R. Wells, and M. Rechsteiner, Science 234, 364 (1986). 55 L. P. Di Persio, C. P. Carter, and D. Y. Hui, Biochemistry 33, 3442 (1994). 56 K. M. Loomes, Eur. J. Biochem. 230, 607 (1995). 57 L. Bl~ickberg, M. Str6rnqvist, M. Edlund, K. Juneblad, L. Lundberg, L. Hansson, and O. HerneU, Eur. J. Biochem. 228, 817 (1995). 58 D. Downs, Y. Y. Xu, J. Tang, and C. S. Wang, Biochemistry 33, 7979 (1994).

[9.0]

GLYCOSYLATION OF BILE SALT-DEPENDENT LIPASE

351

immunologically related to the glucose-regulated protein of 94 kDa (Grp94). 59'60 This association takes place in the endoplasmic reticulum, and BSDL is released from the Grp-94-related 94-kDa protein once fully glycosylated in or after leaving the trans-Golgi compartment. 59'6°With this background, it seems difficult to understand the role of the O-linked glycosylation in either the intracellular processing or the enzyme properties using truncated recombinants of BSDL. The use of cells with defects in O-glycosylation, such as the CHO l d l D cell line, appears more appropriate. 61 This cell line transfected with the full-length cDNA of the rat BSDL has been used in one study. 52Results indicated that, contrary to the N-linked glycosylation, the O-linked glycosylation did not affect enzyme secretion or the stimulation of its activity by bile salts. We have extended this study to the intracellular processing of BSDL and were unable to detect any degradation of the enzyme in the absence of O-linked glycosylation (our unpublished observation). Consequently, the PEST sequences do not seem to divert non-O-glycosylated BSDL to a degradation cell compartment. A way to reconcile the presence of PEST sequences and the secretion of BSDL instead of its degradation is to propose that the association of BSDL with membranes from the endoplasmic reticulum to the trans-Golgi compartment is sufficient to protect the enzyme from degradation independent of the O-glycosylation of PEST sequences. Conclusion Despite enormous advances due to recombinant D N A techniques, the understanding of the complex function of protein glycosylation remains primitive. The role of glycans on glycoproteins may be threefold. First, the glycan may participate in the folding of the protein and influence its stability. Second, the glycan may not be directly associated with the function of the glycoprotein but may modulate its half-life by, for example, favoring or inhibiting hepatic clearance. Finally, the glycan may have biological function through its interaction with target molecules such as the homing of lymphocytes via selectins. As discussed here, the N-linked glycosylation of BSDL seems important for the folding and secretion of BSDL, but further processing of the glycan structure did not affect the secretion and activity of the enzyme. Consequently the expression of recombinant BSDL in baculovirus-infected Sf9 i n s e c t cells 62 appeared appropriate for high-level production of BSDL for 59 N. 60 N. 61 D. 62 L.

Bruneau and D. Lombardo, J. Biol. Chem. 270, 13524 (1995). Bruneau, P. Lech~ne de la Porte, and D. Lombardo, Eur. J. Biochem. 233, 209 (1995). M. Kingsley, K. F. Kozarsky, L. Hobbie, and M. Krieger, Cell 44, 749 (1986). P. Di Persio, J. A. Kissel, and D. Y. Hui, Protein Expr. Purif. 3, 114 (1992).

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[20]

further crystallization of the enzyme. With regard to the C-terminal mucinlike tail, we should be more cautious. Transfer of the enzyme from the intestinal lumen to the bloodstream may necessitate the O-linked glycosylation of BSDL, which under those conditions may not only be species specific but may also depend on the cell line used as the model for captation. If through the production of recombinant BSDL in cells that glycosylate BSDL differently, behavior different from the native protein 63 is observed, then a new hypothesis will be required. Overall, results obtained until now indicate, as hypothesized by us 10 years ago, that "the enzyme could be called to assume a function behind the mucosa" (cited from Ref. 20). This function still remains to be characterized, but in the light of more recent data [the ability of circulating BSDL to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of oxidized LDL, and the presence of sialyl Lewisx and sialyl Lewisa antigen on e i t h e r m i l k 41 and pancreatic BSDL (our unpublished results)], one wonders whether the C-terminal O-glycosylated tail of the protein may be involved in the interaction of BSDL with apolipoprotein and/or contribute to an adhesive activity of the protein. Although it has been shown that apoB100 peptides bind to proteoglycans,64 how BSDL interacts with LDL and what the putative role of BSDL is in adhesive processes must be further analyzed. There is certainly a reason why human eosinophils express a 74kDa form of BSDL, which can be either the unglycosylated protein or a variant possessing fewer tandem repeats than the 100-kDa human enzyme. This variant must be further characterized before drawing any conclusion. Nevertheless, the role of the eosinophil enzyme, which is localized in granules, is probably to destroy parasites, TM and likely not to interfere with lipoprotein metabolism. Beside the role of its glycosylation, the intracellular role of BSDL or of its oncofetal glycovariant FAP needs to be further analyzed. Thus it has been suggested that the lipolytic activity of BSDL would be essential for the metabolism of lipids during growth and differentiation of fetal c e l l s 19 and for exocytosis in acinar cells. 65 Acknowledgments The research in this laboratory is made possible by institutional funding from the Institut National de la Sant6 et de la Recherche M6dicale (Paris, France) and by grants awarded from the Association pour la Recherche contre le Cancer (Villejuif, France), the Conseil G6n6ral des Bouches-du-Rh6ne (Marseille, France), and the Conseil R6gional of Provence-Alpes63 M. StrOmqvist, K. Lindgren, L. Hansson, and K. Juneblad, J. Chromatogr. A718, 53 (1995). 64 t . Chan, J. Biol. Chem. 267, 25621 (1992). 65 M. Withiam-Leitch, R. P. Rubin, S. E. Koshlukova, and J. M. Aletta, J. Biol. Chem. 270, 3780 (1995).

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C6te-d'Azur (Marseille, France). Eric Mas was a recipient of a fellowship from the Ligue Nationale contre le Cancer (Paris, France). Authors address their special thanks to Mrs. V. Sbarra, Mr. D. Lecestre, and C. Crotte for their generous help. This work is dedicated to the students who were working or are still working in our group.

[21] S t e r e o s e l e c t i v i t y o f L i p a s e f r o m R h i z o p u s o r y z a e toward Triacylglycerols and Analogs: Computer-Aided Modeling and Experimental Validation By

LUTZ HAALCK, FRITZ PALTAUF, JURGEN PLEISS, ROLF

D.

SCHMID,

FRITZ SPENER, a n d PETER STADLER

Introduction The genus Rhizopus has long been known to produce extracellular lipases. In fact, many Rhizopus species such as R. delemar, R. microsporus, R. niveus, and R. arrhizus were found to be efficient producers of various lipases that could be purified and characterized.1 All Rhizopus lipases studied so far have a molecular mass between 29 and 39 kDa, hydrolyze triacylglycerols preferentially at the sn-1 and sn-3 positions, and show little selectivity for hydrolysis of triacylglycerols with different fatty acid moieties. As this behavior is similar to the selectivities of human pancreatic lipase, Rhizopus lipases have been proposed as substitution products for patients suffering from pancreatic insufficiency.2 Cloning of the genes encoding the lipases from R. delemar 3"4and Rhizopus oryzae 5 has revealed that their nucleotide sequences are nearly identical, comprising a signal sequence (26 amino acids), a prosequence (97 amino acids), and a region encoding the mature lipase (269 amino acids). The proenzyme (39 kDa), which is fully active, is cleaved by proteolysis in vivo to provide the mature form of the lipase (29 kDa). Evidence has been obtained that the prosequence is involved in the correct folding and thus may have a chaperone-like function5 in this lipase, which contains three disulfide bridges and one free cysteine in the prosequence. On expression

1 M. Iwai and Y. Tsujisaka, in "Lipases" (B. Borgstrrm and H. L. Brockman, eds.), p. 443. Elsevier, Amsterdam, 1984. 2 A. Noma and B. Borgstr6m, Scand. J. Gastroenterol. 6, 217 (1971). 3 M. J. Haas, J. Allen, and T. R. Berka, Gene 109, 107 (1991). 4 R. D. Joerger and M. J. Haas, Lipids 28, 81 (1993). 5 D. Beer, G. Wohlfahrt, R. D. Schmid, and J. E. G. McCarthy, Biochem. J. 319, 351 (1997).

METHODSIN ENZYMOLOGY,VOL.284

Copyright© 1997by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/97 $25.00

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C6te-d'Azur (Marseille, France). Eric Mas was a recipient of a fellowship from the Ligue Nationale contre le Cancer (Paris, France). Authors address their special thanks to Mrs. V. Sbarra, Mr. D. Lecestre, and C. Crotte for their generous help. This work is dedicated to the students who were working or are still working in our group.

[21] S t e r e o s e l e c t i v i t y o f L i p a s e f r o m R h i z o p u s o r y z a e toward Triacylglycerols and Analogs: Computer-Aided Modeling and Experimental Validation By

LUTZ HAALCK, FRITZ PALTAUF, JURGEN PLEISS, ROLF

D.

SCHMID,

FRITZ SPENER, a n d PETER STADLER

Introduction The genus Rhizopus has long been known to produce extracellular lipases. In fact, many Rhizopus species such as R. delemar, R. microsporus, R. niveus, and R. arrhizus were found to be efficient producers of various lipases that could be purified and characterized.1 All Rhizopus lipases studied so far have a molecular mass between 29 and 39 kDa, hydrolyze triacylglycerols preferentially at the sn-1 and sn-3 positions, and show little selectivity for hydrolysis of triacylglycerols with different fatty acid moieties. As this behavior is similar to the selectivities of human pancreatic lipase, Rhizopus lipases have been proposed as substitution products for patients suffering from pancreatic insufficiency.2 Cloning of the genes encoding the lipases from R. delemar 3"4and Rhizopus oryzae 5 has revealed that their nucleotide sequences are nearly identical, comprising a signal sequence (26 amino acids), a prosequence (97 amino acids), and a region encoding the mature lipase (269 amino acids). The proenzyme (39 kDa), which is fully active, is cleaved by proteolysis in vivo to provide the mature form of the lipase (29 kDa). Evidence has been obtained that the prosequence is involved in the correct folding and thus may have a chaperone-like function5 in this lipase, which contains three disulfide bridges and one free cysteine in the prosequence. On expression

1 M. Iwai and Y. Tsujisaka, in "Lipases" (B. Borgstrrm and H. L. Brockman, eds.), p. 443. Elsevier, Amsterdam, 1984. 2 A. Noma and B. Borgstr6m, Scand. J. Gastroenterol. 6, 217 (1971). 3 M. J. Haas, J. Allen, and T. R. Berka, Gene 109, 107 (1991). 4 R. D. Joerger and M. J. Haas, Lipids 28, 81 (1993). 5 D. Beer, G. Wohlfahrt, R. D. Schmid, and J. E. G. McCarthy, Biochem. J. 319, 351 (1997).

METHODSIN ENZYMOLOGY,VOL.284

Copyright© 1997by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/97 $25.00

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ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[2 1]

in Escherichia coli, inclusion bodies are f o r m e d that can be reconstituted to fully active lipase. A s Rhizopus lipase shows about 60% sequence h o m o l o g y to the lipase f r o m Rhizomucor miehei, whose structure has b e e n solved at a resolution of 1.9 ~,6 a structural m o d e l o f R . oryzae lipase based on the X - r a y structure of R. miehei lipase can be built and has b e e n c o r r o b o r a t e d by site-directed mutagenesis. 7 T h e structure of R. delemar lipase was d e t e r m i n e d by X - r a y crystallography at a resolution of 2.6 ~ . s G e n e t i c experiments on the substrate specificity of R. oryzae lipase s h o w e d that substitution of a m i n o acids in the predicted acyl chain-binding groove, in particular P h e 9 5 A s p 9 and Val209Trp, 9,1° resulted in e n h a n c e d p r e f e r e n c e for short- and m e d i u m - c h a i n triacylglycerols. T h e s t e r e o p r e f e r e n c e of pure R. arrhizus lipase (reclassified to R. oryzae u ) has b e e n t h o r o u g h l y studied using m o n o m o l e c u l a r films of pure dicaprin. 12 A m o d e r a t e preference for hydrolysis of the sn-1 acyl chain is o b s e r v e d in these experiments. If triacylglycerols and analogs were used in emulsion, s t e r e o p r e f e r e n c e shifts f r o m sn-1 to sn-3 d e p e n d i n g on the steric requirements of the sn-2 substituent of the substrate. 13 In this chapter, we describe the p r e p a r a t i o n of R. oryzae lipase f r o m r e c o m b i n a n t E. coli, the analysis of stereoselectivity of this lipase t o w a r d triacylglycerols and analogs, and c o m p u t e r modeling to gain insight into the molecular basis of its stereopreference. P r o d u c t i o n of Rhizopus oryzae L i p a s e

Cloning and Expression o f Rhizopus oryzae Lipase in Escherichia coli D N A Isolation. T h e fungus Rhizopus oryzae D S M 853 is g r o w n on C z a p e k - D o x m e d i u m 14 s u p p l e m e n t e d with 2% (w/v) malt extract. Total 6 A. M. Brzozowski, U. Derewenda, Z. S. Derewenda, G. G. Dodson, D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. A. Patkar, and L. Thim, Nature (London) 351, 491 (1991). v H. D. Beer, G. Wohlfahrt, J. E. G. McCarthy, D. Schomburg, and R. D. Schmid, Protein Eng. 9, 507 (1997). 8 U. Derewenda, L. Swenson, Y. Wei, R. Green, P. M. Kobos, R. Joerger, M. J. Haas, and Z. S. Derewenda, J. Lipid Res. 35, 524 (1994). 9 R. D. Joerger and M. J. Haas, Lipids 29, 377 (1994). a0H. Atomi, U. Bornscheuer, M. M. Soumanou, H. D. Beer, G. Wohlfahrt, and R. D. Schmid, P. J. Barnes & Associates, High Wycombe, Proc. 21st World Cong. Int. Soc. Fat Res., Vol. 1, 49, 1996. u M. J. Haas and R. D. Joerger, in "Food Biotechnology Microorganisms" (Y. H. Hui and G. G. Khachatourians, eds.), p. 564. VCH, New York, 1995. 12E. Rogalska, S. Nury, I. Douchet, and R. Verger, Chirality 7, 505 (1995). 13po Stadler, A. Kovac, L. Haalck, F. Spener, and F. Paltauf, Eur. J. Biochem. 227, 335 (1995). 14K. Tsuchiya, S. Tada, K. Gomi, K. Kitamoto, C. Kumagai, Y. Jigami, and G. Tamura, Appl. Microbiol. Biotechnol. 38, 109 (1992).

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STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

355

D N A is isolated according to a modified procedure of Raeder and Broda. 15 Southern and colony hybridization are carried out according to standard procedures 16using two oligonucleotides corresponding to conserved N- and C-terminal regions of the homologous lipases from Rhizomucor miehei, 17 R. niveus, 18 and R. delemar 4 as described by Beer et al. 5 Transformation and Expression Experiments. The cDNA encoding the mature R. oryzae lipase is excised from plasmid pCYTEXP15'19 as an N d e I B a m H I fragment and ligated to the pCRII vector (Invihogen, Leek) for amplification and sequencing of the lipase gene. For plasmid propagation E. coli strain InvaF' is used. The N d e I - B a m H I fragment is subcloned into the expression vector pET3a, giving pET3-ROL. Transformation with plasmid D N A is accomplished using CaCl2-treated E. coli cells as described by Sambrook et aL 16 Escherichia coli strain BL21(DE3) harboring the pET3-ROL recombinant plasmid is grown on solid Luria-Bertani (LB) agar medium containing ampicillin (100/zg/ml). Cells from a single colony are inoculated into LB broth containing the same concentration of ampicillin and grown overnight at 37 °. Five milliliters are taken to inoculate 2 liters of LB broth (five 400-ml portions). The cultures are shaken vigorously at 37 ° until the optical density at 600 nm reaches 0.6 to 1. Isopropyl-/3-thiogalactopyranoside (IPTG) is added to a final concentration of 1 mM. The cells are harvested 3 hr after induction. Refolding o f Recombinant Rhizopus oryzae Lipase. The recombinant mature R. oryzae lipase (ROL) is produced as insoluble inclusion bodies in the cytoplasm. The renaturation procedure is adopted from Joerger and Haas. 4The cells obtained from a 2-liter culture (about 12 g) are resuspended in 100 ml of 50 mM Tris-HCl (pH 8.0), 5 mM ethylendiaminetetraacetic acid (EDTA), 10% (w/v) sucrose. Lysozyme is added to a final concentration of 0.8 mg/ml and the mixture is incubated for 30 min at 30°. On addition of 400 ml of buffer [10 mM Tris-HC1 (pH 8.0), 1 mM EDTA, 0.5% (v/v) Triton X-100] the lysate is suspended, sonicated three times for 20 sec with a tip-type sonicator (15 W), and centrifuged at 15,000 g for 30 min. The pellet is resuspended in 10 mM Tris-HC1 (pH 8.0), 1 mM EDTA and incubated for 1 hr at 37° with benzonase (90 U/ml) for D N A degradation. After centrifugation at 15,000 g for 30 min, the inclusion bodies are dissolved in 200-800 ml of solubilization buffer [8 M urea, 25 mM phosphate buffer (pH 7.0), 1 mM EDTA, and 5 mM dithiothreitol (DTT)] at room tempera15 U. Raeder and P. Broda, Lett. Microbiol. L 17 (1985). 16j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning," 2nd Ed., p. A.1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. 17 E. Boel, B. Huge-Jensen, M. Christensen, L. Thim, and N. Fiil, Lipids 23, 701 (1988). 18 W. Kugimiya, Y. Otani, and M. Hashimoto, Japanese Patent 64-80290 (1989). 19T. N. Belev, M. Singh, and J. E. G. McCarthy, Plasmid 26, 147 (1991).

356

ISOLATION, CLONING,EXPRESSION,AND ENGINEERING

[9.1]

ture for 1 hr. The protein content is estimated by densitometry after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The solution is centrifuged at 15,000 g for 30 min and cystine is added to the supernatant to a final concentration of 15 mM. The pH is adjusted for 10 min between pH 8.5 and 9.0. The solution is added dropwise to 10 to 20 vol of cold (4°) 50 mM phosphate buffer (pH 8.0), 1 mM EDTA, and 5 mM cysteine. The final volume depends on the protein content of inclusion bodies. Best results are achieved at a final protein concentration of 10/xg/ml and 48 hr of renaturation. As judged from densitometry after S D S - P A G E the yield of active mature lipase is 60%.

Purification of Rhizopus oryzae Lipase Native Rhizopus oryzae Lipase. Rhizopus oryzae lipase (Lot No. 80,000 ST) formerly denoted as R. arrhizus lipase, 11 was purchased from Gist Brocades (Delft, The Netherlands). Twenty grams of the crude fermentation product is dissolved in 200 ml of starting buffer (10 mM Tris-HCL, pH 6.8). Using fast protein liquid chromatography (FPLC; Pharmacia, Uppsala, Sweden) cation-exchange chromatography is performed on a Fractogel EMD TMAE column (5 x 20 cm; E. Merck AG, Darmstadt, Germany) equilibrated with the same buffer at a flow rate of 4 ml/min. After sample injection the column is washed with 400 ml of starting buffer. Under these conditions, most of the lipase does not bind to the column and elutes directly with the void volume. For regeneration the column is washed with 200 ml of 0.7 M NaC1 in starting buffer, giving rise to a fraction containing protein contaminants, colorants, and about 20% of the lipase, which is discarded. The protein profile is monitored spectrophotometrically at 280 nm and lipase activity is determined using p-nitrophenyl palmitate as the substrate as described by Winkler and Stuckmann. 2° The pH of the void volume containing the lipase is adjusted to pH 7.2, NaC1 is added to a final concentration of 2 M, and the sample is subjected to hydrophobic interaction chromatography on a phenyl-Sepharose Fast Flow column (5 x 20 cm; Pharmacia). This low substitution grade material ensures weak binding of the hydrophobic protein and allows for elution without detergents. The lipase retained is eluted with concentration gradients of decreasing NaCI (2-0 M) and concomitantly increasing ethylene glycol (0-80%, v/v) at a flow rate of 2 ml/min. The lipase-containing fractions obtained at 65% ethylene glycol are pooled, and salt and ethylene glycol are removed by passing the protein at a flow rate of 50 ml/min over a Sephadex G-25 column (10 x 20 cm; Pharmacia) equilibrated with 5 mM 20U. K. Winkler and M. Stuckmann, J. Bacteriol. 138, 663 (1979).

[21]

STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

357

calcium acetate, pH 4.7. Subsequently, the solution is applied at a flow rate of 2 ml/min to cation-exchange chromatography on a SP-Sepharose HP column (XK 16/20; Pharmacia). Elution with a linear concentration gradient increasing from 0 to 0.7 M NaC1 in 5 mM calcium acetate, pH 4.7, gives rise to two lipolytically active fractions at 0.47 and 0.54 M NaC1, corresponding to a 32-kDa prolipase and the mature 29-kDa lipase, respectively. The yields are 10 and 43 mg for the prolipase and for the mature lipase, respectively. It is well established that Rhizopus as well as Rhizomucor lipases are expressed as preproenzymes, which are secreted into the periplasm as proenzymes and further processed to the mature lipase. 4 N-terminal sequencing revealed that the N terminus of the purified 32-kDa prolipase corresponds to the N terminus reported for the R. niveus lipase. TM This observation supports the idea that the full-length prolipase (39 kDa) is sequentially cleaved by at least two proteases. Recombinant Mature Rhizopus oryzae Lipase. The solution containing renatured lipase (10-20 liters) is adjusted to pH 5.5 and applied to cationexchange chromatography on Fractogel EMD-SO3- 650(M) column (5 x 20 cm; E. Merck AG) at a flow rate of 30 ml/min, equilibrated with 50 mM sodium phosphate buffer, pH 5.5. The lipase is eluted using a linear concentration gradient increasing from 0 to 1.5 M NaCI in 50 mM sodium phosphate buffer, pH 5.5. The lipase-containing fractions eluting at 0.25 M NaC1 are pooled, concentrated, and applied in aliquots of 15 ml at a flow rate of 3 ml/min to size-exclusion chromatography on a Fractogel BioSEC650(S) Superformance column (2.6 x 60 cm; E. Merck AG) equilibrated with 10 mM sodium phosphate buffer, pH 5.5, and 100 mM NaC1 for final polishing (Rt 68.7 min). Starting from the wet cell paste this procedure yields 150 mg of lipase (estimated yield, 40%). Enzymatic activity of the purified lipases is determined titrimetrically using a pH-stat apparatus with olive oil 21 or tributyrin as substrate) 3 One unit is defined as the lipase activity that liberates 1/zmol of fatty acids per minute at 37 °. The native mature R O L shows a specific activity of 6700 and 2200 U/mg using triolein and tributyrin, respectively, as substrates. These specific activities found for the recombinant mature R O L are essentially the same as those for the native mature lipase as tested for both substrates. Preparation of Substrates Synthesis of substrates is described starting from commercially available material (Fig. i). The purity of substrates and intermediates is routinely 21R. Ruyssen and A. Lauwers, "Pharmaceutial Enzymes: Properties and Assay Methods." E. Story-Scientia,Gent, Belgium, 1978.

358

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING i.

O A

x

.o

o

~

.

B

(X = C6H5); UA~H4,Et20

ii~ (X= H): 1) R2Br,EtONa,EtOH 2) u~.., E,O~ prochiral sn-2-monoacylglyce rol or analog

t...

prochiral tdacylglycerol or analog

sn-1

O

N 1) ROSO2CH3,KOH,benzene

~

RCOCI, CHCH2/ pyridine

_CH2-OH

2) HCI/MeOH

o

_CH2-O--C--RI

~=

tY-;. 1) RCOCI,CH2CI2/pyddfne 2) H2/ Pd, EtOH

.o

C

[21]

H2N~

f

sn-2

CH2-OH

sn-2

sn-3

esterification in organic solvents

xz RCCCI,H20/THF

CH2-O--C--R 3 sr~3

hydrolysis in aqueous buffer enzyme catalyzed reaction

I

~HO

enantiomeric diacylglycerols or analogs 1:12: type of moiety i phenyt ii alloA JJ alkyloxy iv acyloxy X acylamino

type of bonding C-C phenyl C-C anoA

O .

II I CH2-O--C--R

ester amide

. _CH2-OH and

ether

CH2-OH sn-1,2-isomer

(~','~'~H

0

R,=~

(~H2-O--C--R3 sn-2,3-isomer

(N)-I-phenyle~yU~¢yanate,toluene, 1,4-diazabicyclo[2.2.2]octa~e

lipase stareoselectivity: relative retie of stereoisomers (enantiomeric excess) reflecting sn-1 or sn-3 preference in esterification and hydrolyis reactions

diastereornedc carbamate derivatives, HPLC separation on silica gel column

FIG. 1. Scheme for synthesis of substrates and procedure for determination of lipase stereoselectivity in hydrolysisand esterificationexperiments.

checked by thin-layer chromatography (TLC) on silica gel plates (0.2 mm; E. Merck AG). Spots are visualized by charring at 120 ° after spraying with 50% (v/v) sulfuric acid. Final products are characterized by their nuclear magnetic resonance (NMR) spectra obtained on a Varian (Palo Alto, CA) Gemini 200 using tetramethylsilane as an internal standard.

1,3-O-Benzylideneglycerol An equimolar mixture of water-free glycerol (92 g, 1 mol) and freshly distilled benzaldehyde (106 g, 1 mol) in 600 ml of toluene is stirred under reflux in the presence of 1.8 g of p-toluenesulfonic acid (2% of glycerol,

[2 1]

STEREOSELECTIVITY OF LIPASE FROM

Rhizopus oryzae

359

w/w). 12 The water that is produced during the formation of the benzylidene acetal is removed azeotropically using a water separation funnel. When water evolution has stopped, the cooled reaction mixture is seeded with 1,3O-benzylideneglycerol crystals and stored at 0° overnight. The crystalline product is isolated by filtration and suspended in cold toluene containing sufficient sodium hydroxide (0.5 N in methanol) to neutralize the acid catalyst. The product is dissolved by warming and the toluene solution is washed with 1% aqueous sodium dihydrogenphosphate, dried over anhydrous sodium sulfate, and crystallized at 0 °. Recrystallization from benzenehexane (1 : 1) results in 41 g of 1,3-O-benzylideneglycerol, mp 58-60 ° (literature 22 mp 63.5-64.5 ° for the cis-isomer).

Hexadecyl Methane Sulfonate The alkylmethane sulfonate is obtained in 90% yield by reacting hexadecanol with methane sulfochloride in methylene chloride in the presence of triethylamine as described by Paltauf and Hermetter. 23 The product has a m p of 52-53 ° (literature 24 mp 54-55°).

2-O-Hexadecyl-sn-glycerol The 2-O-hexadecyl-sn-glycerol is prepared in analogy to 1-O-alkylglycerols as described by Baumann and Mangold. 24 To a solution of 4.3 g of 1,3-O-benzylideneglycerol (23.9 mmol) in 100 ml of benzene 3.6 g of powdered potassium hydroxide (64.3 mmol) is added. The suspension is refluxed for 1 hr using a water separation funnel to remove water by azeotropic distillation. Hexadecyl methanesulfonate (5 g, 15.6 mmol) dissolved in 20 ml of water-free benzene is then added dropwise, and refluxing is continued for 4 hr. After cooling, 100 ml of water is added and the product is extracted with diethyl ether (twice, 100 ml each). The combined organic phases are washed twice with 100 ml of water until neutral and dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure to yield 1,3-O-benzylidene-2-O-hexadecylglycerol as a light yellow, waxlike product. On TLC this substance shows an Rf of 0.76 with chloroform-acetone-acetic acid (96:4:1, by volume) as the developing solvent. The crude 1,3-O-benzylidene-2-O-hexadecylglycerol (7.9 g) is hydrolyzed by stirring for 2 hr at room temperature in a solution consisting of 8 ml of concentrated hydrochloric acid, 33 ml of water, and 100 ml of methanol. After removal of the organic solvent under reduced pressure, 22 F. H. Mattson and R. A. Volpenhein, J. Lipid Res. 3, 281 (1962). 23 F. Paltauf and A. Hermetter, Methods Enzymol. 197, 135 (1991). 24 W. J. Baumann and H. K. Mangold, Z Org. Chem. 29, 3055 (1964).

360

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[2 1]

the residue is extracted with 100 ml of diethyl ether. The ether extract is washed with water until neutral (twice, 100 ml each) and dried over anhydrous sodium sulfate. The sovlent is evaporated and 4.4 g (89% yield based on hexadecylmethane sulfonate) of 2-O-hexadecylglycerol is isolated after recrystallization from 100 ml of n-heptane. The product shows a single spot on TLC (Rf 0.10) with chloroform-acetone-acetic acid (96 : 4 : 1, by volume) and has a m p of 64-65 °. Thin-layer chromatography analysis on boric acidimpregnated plates using the solvent system chloroform-methanol (98:2, v/v) confirms the isomeric purity of the 2-O-hexadecylglycerol (Rf 0.27); no 1-O-hexadecylglycerol is detected (gf 0.13). AH NMR (CDCI3): ~, 0.90 (t, 3H, CH3); 1.20-1.74 (m, 28H, alkyl); 2.03 (m, 3H,/3-CH2); 3.40-3.50 (m, 1H, CH); 3.53-3.59 (t, a-CH2); 3.62-3.82 (m, 4H, CH20). 2 - Octadecanoyl-sn -glycerol

To a solution of 500 mg (2.78 mmol) of 1,3-benzylideneglycerol in 5 ml of methylene chloride-pyridine (3 : 1, v/v) 842 mg (2.78 mmol) of octadecanoyl chloride in 3 ml of methylene chloride is added dropwise at 0°. The reaction mixture is then stirred for 3 hr at room temperature. After the addition of 20 ml of methylene chloride the mixture is extracted with 20 ml of ice-cold 2% (v/v) hydrochloric acid. The organic phase is washed subsequently with two 20-ml portions of water and dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure to yield crude 1,3-benzylidene2-octadecanoylglycerol. The solid material shows an Re of 0.59 with chloroform-acetone-acetic acid (96 : 4 : 1, by volume) and is used without further purification. The crude intermediate (1.2 g) in 20 ml of ethanol is hydrogenated at room temperature for 2 hr in the presence of 250 mg of 10% palladium-carbon (Pd-C). The catalyst is removed by centrifugation and the solvent is evaporated under reduced pressure. Recrystallization from 20 ml of light petroleum gives 643 mg (65% yield based on 1,3-benzylideneglycerol) of pure sn-2-octadecanoylglycerol, mp 70-71 °. A TLC analysis of the product shows a single spot with an Rf of 0.09 with the solvent system chloroform-acetone-acetic acid (96 : 4 : 1, by volume). Using boric acid-impregnated plates and the solvent system chloroform-methanol (98 •2, v/v) the appearance of a single spot (Rf 0.26) indicates the absence of detectable amounts of the corresponding 1-acylglycerol isomer (Rf 0.11). 1H NMR (CDCI3): t~, 0.90 (t, 3H, CH3); 1.20-1.80 (m, 30 H, alkyl); 2.39 (t, 3H, a-CH2); 3.85 (d, 4H, CH20); 4.87-5.00 (m, 1H, CH). 2-Deoxy-2-hexadecanoylamino-sn-glycerol

Serinol (2 g, 22.0 mmol) dissolved in 20 ml of water and 6.1 g of triethylamine (60.6 mmol) is monoacylated by addition of 5 g (18 mmol)

[21]

STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

361

of hexadecanoyl chloride dissolved in 20 ml of tetrahydrofuran in analogy to the procedure described by Dijkman et al. 25 The reaction mixture is stirred for 24 hr at 40°. After evaporation of the solvents under reduced pressure the residue is dissolved in 40 ml of chloroform. On washing with 40 ml of 5% hydrochloric acid the crude product precipitates and is isolated by filtration. The pure product (3.25 g, 55% yield) is obtained by recrystallization from hot chloroform. The product (mp 124-126 °) has an Rf of 0.29 using the solvent system chloroform-methanol (9 : 1, v/v). 1H NMR (CDC13C D 3 O D , 1 : 1): & 0.90 (t, 3H, C H 3 ) ; 1.10-1.70 (m, 26 H, alkyl); 2.17 (t, 2H, a-CH2); 3.52-3.69 (m, 4H, C H 2 0 ) ; 3.78-3.88 (m, 1H CH). 2-Deoxy-2-hexadecyl-sn-glycerol

To a stirred sodium ethanolate solution [3.2 g of sodium (139 milliatom) in 80 ml of absolute ethanol] 31.6 g (197 mmol) of diethylmalonate is added dropwise. Then 42.6 g (139 mmol) of hexadecyl bromide is added and the reaction mixture is refluxed for 4 hr. After 40 ml of the solvent is removed by distillation the mixture is cooled to room temperature and the precipitated sodium bromide is disoslved by addition of 300 ml of ice-cold water. The organic phase is separated and the aqueous layer is extracted with two 300-ml portions of diethyl ether. The combined organic phases are dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure to give 63 g of the crude alkylated diethylmalonate. Thin-layer chromatography analysis using light petroleum-ethyl acetate (5:1, v/v) shows the alkylation product (Rf 0.74) and unreacted diethylmalonate (R~ 0.42). The crude material is dissolved in 100 ml of diethyl ether and is added dropwise to 8.3 g of lithium alanate (219 mmol) suspended in 220 ml of diethyl ether. The reaction mixture is stirred under reflux for 2.5 hr. After cooling the excess lithium alanate is destroyed by careful dropwise addition of ice-cold water. Precipitated aluminum hydroxides are dissolved with 300 ml of 10% sulfuric acid and the product is extracted with 500 ml of diethyl ether. The organic phase is washed with saturated aqueous sodium chloride and dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure and the crude product (approximatley 30 g) is purified by column chromatography on silica gel. Impurities are eluted using a stepwise gradient of light petroleum-ethyl acetate (80: 20 to 50 : 50, v/v); pure 2-deoxy-2-hexadecylglycerol(22 g, 53% yield based on hexadecyl bromide) is eluted with ethyl acetate as a solvent. The product (mp 79-83°; literature26 82-84 °) shows a single spot on TLC (R~ 0.15) using heptane25 R. Dijkman, N. Dekker, and G. H. de Haas, Biochim. Biophys. Acta 1043, 67 (1990). 26 p. L. Anelli, F. Montanari, S. Quici, G. Ciani, and A. Sironi, J. Org. Chem. 53, 5292 (1988).

362

ISOLATION, CLONING,EXPRESSION,ANDENGINEERING

[21]

ethyl acetate (7:3, v/v). 1H NMR (CDC13-CD3OD, 1:1): ~, 0.90 (t, 3H, CH3); 1.10-1.70 (m, 31 H, alkyl); 3.47-3.69 (m, 4H, CH20).

2-Deoxy-2-phenyl-sn-glycerol Reduction of commercially available 2-phenyldiethylmalonate (Fluka, Buchs, Switzerland) with lithium alanate is accomplished in analogy to the procedure described above for the 2-alkyl analog. The pure product is obtained with 78% yield by vacuum distillation (bp. 120°, 10 -4 bar) and shows a single spot on TLC (Rf 0.09) using chloroform-acetone-acetic acid (96 : 4: 1, by volume).

Acylation of Monoacylglycerol Analogs Acylation of monoacylglycerol analogs is performed by a procedure exemplified below for the preparation of 1,3-dioleoyl-2-O-hexadecylglycerol. To a solution of 1.07 g (3.38 mmol) of 2-O-hexadecylglycerol in 20 ml of methylene chloride-pyridine (3 : 1, v/v) 2.15 g of oleoyl chloride (7.14 mmol) in 3 ml of methylene chloride is added at 0°. The reaction mixture is then stirred for 2 hr at room temperature. The reaction mixture is washed with 20 ml of ice-cold 2% (v/v) hydrochloric acid and then with water (twice, 20 ml each time) until neutral. The organic phase is dried over anhydrous sodium sulfate and the solvent is removed under reduced pressure to give 2.82 g of the crude diacyl derivative. Purification by medium-pressure liquid chromatography (MPLC) on silica gel with light petroleum-diethyl ether (95 : 5, v/v) gives 2.65 g (92% yield) of the pure product. The oily product shows an Rf of 0.80 using the solvent system chloroformacetone-acetic acid (96 : 4 : 1, by volume). Assay of Lipase Stereoselectivity

Principle The procedure described below for the determination of lipase stereoselectivity is based on the method described by Rogalska et al. 27 (Fig. 1). The enantiomeric diacylglycerols or analogs (sn-l,2 and sn-2,3 isomers) resulting from lipase-catalyzed hydrolysis of prochiral triacylglycerols or analogs, or from lipase-catalyzed esterification of prochiral 2-acylglycerols or analogs, are derivatized with (R)-phenylethylisocyanate (PEIC) to form the corresponding diastereomeric carbamates, which are separated by highperformance liquid chromatography (HPLC) on a silica gel columnJ 3 The relative ratio of sn-l,2 and sn-2,3 stereoisomers reflects the preference of 27E. Rogalska, S. Ransac, and R. Verger,Z BioL Chem.265, 20271 (1990).

[21]

STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

363

the lipase in hydrolysis or esterification reactions. For example, formation of excess sn-2,3 isomer reflects preference for the sn-1 position during hydrolysis of a triacylglycerol or analog, but preference for the sn-3 position in esterification of a 2-monoacylglycerol or analog. Care must be taken to avoid further hydrolysis of diacylglycerols or analogs during action of the lipase on triacylglycerols or analogs. Similarly, further acylation of diacylglycerols or analogs during esterification of 2-acylglycerols or analogs must be avoided by choosing the appropriate reaction conditions (amount of lipase and reaction time to yield 99%; Fluka) 1,4-Diazabicyclo[2.2.2]octane (DABCO) TLC solvent A: Chloroform-acetone-acetic acid (96 : 4 : 1, by volume) TLC solvent B: n-Heptane-ethyl acetate (70:30, v/v) HPLC: Eurospher 100 5/zm (250 x 4 mm; Knauer, Berlin, Germany) Mobile phase A: n-Heptane-2-propanol (99.6:0.4, v/v)

[21]

STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

365

Mobile phase B: n-Heptane-methanol-2-propanol (98.7:0.7:0.6, by volume) Procedure. The crude lipid mixture (approximately 1 mg) obtained after hydrolysis of triacylglycerols and analogs or after esterification of 2monoacylglycerols or analogs is dissolved in 100 /~1 of dry toluene and reacted with 3/zl of (R)-phenylethylisocyanate (PEIC) in the presence of catalytic amounts (approximately 0.1 mg) of 1,4-diazabicyclo[2.2.2]octane. The derivatization is carried out under an argon atmosphere in a sealed Wheaton vial. To ensure complete carbamoylation the mixture is stirred at room temperature for 12 hr. The solvent is removed under a gentle stream of nitrogen and excess isocyanate is evaporated under reduced pressure (1 mtorr, 6 hr) at room temperature. Prior to HPLC analysis the crude carbamate derivatives are purified by preparative TLC on silica gel plates with a fluorescence indicator (0.2 mm, F254; E. Merck AG) using solvent system A or B (Table I). The corresponding bands are visualized under ultraviolet light. The plates are sprayed with water and the respective zone is scraped into a 10-ml glass tube. The product is eluted with two 2-ml portions of n-heptane-2-propanol (3:2, v/v) under vortexing. After centrifugation (2500 rpm, 3 min) the organic solvent is withdrawn and evaporated under a stream of nitrogen. The residue is dissolved in 200/zl of n-heptane for HPLC analysis. TABLE I THIN-LAYER CHROMATOGRAPHIC SEPARATION OF PRODUCTS OBTAINED AFTER LIPASE-CATALYZED HYDROLYSIS OF 1,3-DIOLEOYL-2-X-GLYCEROL OR LIPASE-CATALYZED ESTERIFICATION OF 2-X-GLYCEROL WITH OLEIC ACID AND CORRESPONDING DIASTEREOMERIC I(3)-OLEOYL-2-X-GLYCEROL (R) -PHENYLETHYLISOCYANATE DERIVATIVESa

Rf values sn-2 Glycerol moiety (X)

tri b

di b

mono b

di-PEIC

2-Acyl (e.g., oleoyl)

0.79

0.09

0.42 d

2-O-Hexadecyl 2-Deoxy-2-hexadecyl 2-Deoxy-2-phenyl 2-Deoxy-2-aminopalmitoyl

0.80 0.82 0.78 0.64

0.36 0.47 c 0.39 0.45 0.38 0.16

0.10 0.12 0.10 0.02

0.47 d 0.49 d 0.39 d 0.41 b

a tri, Substrate triacylglycerol or analog; di, diacylglycerol or analog; mono, monoacylglycerol or analog; di-PEIC, (R)-phenylethylisocyanate derivative of diacylglycerol or analog. b Solvent system A: Chloroform-acetone-acetic acid (96:4:1, by volume), oleic acid Rf 0.22. c 1,3-Dioleoylglycerol. a Solvent system B: n - H e p t a n e - e t h y l acetate (7 : 3, v/v).

366

[2 ] ]

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

High-Performance Liquid Chromatography Analysis of Diastereomeric R-(+ )-Phenylethylisocyanate Derivatives of Diacylglycerols and Analogs. The resolution of diastereomeric PEIC derivatives of sn-l,2- and sn-2,3diacylglycerols and analogs (Fig. 2) is performed on a Hewlett-Packard (Palo Alto, CA) 1050 HPLC system equipped with a silica gel column. Peaks are monitored with a variable ultraviolet (UV) detector set at 210 nm. The mobile phase is n-heptane-2-propanol (99.6:0.4, v/v) at a flow rate of 0.8 ml/min for the separation of all derivatives except the 1(3)-oleoyl2-deoxy-2-palmitoylaminoglycerol carbamates. The latter compounds are separated using n-heptane-methanol-2-propanol (98.7:0.7:0.6, by volume) as eluent at a flow rate of 0.8 ml/min. The distribution of sn-l,2 and sn-2,3 stereoisomers is calculated after automatic integration of the corresponding HPLC peak areas. Determination of Absolute Configuration. The elution order of the diasteromeric diacylglycerol carbamate derivatives is determined with 1,2dioleoyl-sn-glycerol (Sigma, Munich, Germany) as a standard. The corresponding 1-oleoyl-2-O-hexadecyl-sn-glycerol is synthesized as described by Stadler et al.i3 1-Oleoyl-2-deoxy-2-palmitoylamino-sn-glycerol is prepared from L-serine methyl ester in analogy to Dijkman et al. z5 Each of the former optically pure sn-l,2 isomers is converted into the respective carbamate derivative and analyzed by HPLC. Retention times are compared to those of the respective derivatives obtained from rac-l,2 isomers. The sn-l,2 isomers eluted as second peak ( R t sn-2,3 < R t sn-l,2). The same retention behavior is observed for l(3)-acyl-2-deoxy-2-alkylglycerols. The steric configuration of 1-acyl-2-deoxy-2-alkyl-sn-glycerols is confirmed by determination of the optical rotation in comparison to literature data as described. I3 The absolute configuration of l(3)-acyl-2-deoxy-2-phenylglycerols is determined by conversion of the diacylglycerol analogs to tropic acid and by O4 t

E•"0.100.05-

"

0.00i

i

12.5

15.0

i

17.5

I

20.0

I

it

22.5 Time (min)

FIG. 2. HPLC analysis of diastereomeric l(3)-oleoyl-2-O-hexadecyl-sn-glycerol carbamates. Column, Eurosphere 100, 5 tzm, 250 × 4 mm i.d. (Knauer); mobile phase A, a n-heptane-2-propanol (99.6 : 0.4, v/v); flow, 0.8 ml/min; detection, U V absorbance at 210 nm.

[21]

STEREOSELECTIVITY OF LIPASE FROM R h i z o p u s oryzae

367

comparison of the optical rotation to literature data. 13 It is worth noting that the elution order of l(3)-acyl-2-deoxy-2-phenylglycerol carbamates is o p p o s i t e ( R t sn-l,2 < R t sn-2,3) to that observed with all other analogs. Data obtained in hydrolysis and esterification reactions are summarized in Tables II and III, respectively. The stereoselectivity of the R. oryzae lipase is strongly influenced by the substituent in position sn-2 of the glycerol- or 1,3-propanediol backbone. The stereochemical preference in hydrolysis switches from position sn-1 to sn-3 by replacing the 2-acyl, 2-O-alkyl, or 2-deoxy-2-alkyl residues by 2deoxy-2-hexadecanoylamino or 2-deoxy-2-phenyl groups. The acyl chain length (oleic versus octanoic acid) in the sn-1 and sn-3 position has little effect on stereoselectivity,a3

T A B L E II STEREOSELECTIVITY OF NATIVE

Rhizopus oryzae

LIPASE

IN HYDROLYSIS OF TRIOLEOYLGLYCEROL AND ANALOGS a

Substrate O

II CH 2 - - O - - C - - R 1 7= o CH2--O--C--R R1~

R2=

C17H33

3

ee

(~)

sn

49.0 _+ 5.2

3

R2 O

II C15H31- C - - N -

I H O

II C17H33 - - C - - O - C16H33--O-

C16H33--

33.2 --_ 3.0

1

63.8 +- 4.1

1

18.8 --+ 2.4

1

63.8 2 2.2

3

"ee, Enantiomeric excess; sn, stereospecific numbering of the preferred sn position; values are given as mean _ standard deviation (n = 3).

368

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[211

TABLE [II STEREOSEEECTIVITY OF N A T I V E R h i z o p u s o r y z a e LIPASE IN ESTERIFICATION OF 2-MONOACYLGLYCEROL AND A N A L O G S IN t / - H E X A N E a

Substrates CHaOH ee

g

(%)

sn

C17H35 - - C - - O - -

21.9 --- 8.9

1

C16H33 - - O - -

52.6 - 1.9

1

C16H33--

89.7 - 8.3

1

CH2OH R2

o

I[

a

ee, Enantiomeric excess;sn, stereospecificnumbering of the preferred sn position;valuesare givenas mean _+ standard deviation (n = 3).

In esterification reactions the same stereopreference (for sn-1) was observed as in hydrolysis of 2-acyl-, 2-O-alkyl-, or 2-deoxy-2-alkyl substrates (see Tables II and III). In esterification reactions the sn-1 stereoselectivity increases concomitantly with a decrease in substrate polarity at the sn-2 position. Interestingly, on introducing the most nonpolar (2-deoxy-2-alkyl) residue the enantiomeric excess is much lower in hydrolysis as compared to the esterification reaction. Similar observations were made for the lipases from C h r o m o b a c t e r i u m viscosum, P s e u d o m o n a s cepacia, and Geotrichum candidum. Different organic solvents (dodecane, toluene, and light petroleum) as well as variation in the chain length of the acylating agent (oleic versus octanoic acid) do not affect the stereoselectivity of R O L (data not shown). As shown in comparative studies the same stereoselectivity is found for the native and the recombinant R O L in hydrolysis and in esterification reactions. Computer-Aided Modeling of L i p a s e - S u b s t r a t e I n t e r a c t i o n Table IV summarizes the modeling strategy that was applied to model the molecular basis of stereopreference of R O L toward triacylglycerols and analogs, z9 Four substrates are compared: trioctanoylglycerol ("ester") 29H.-C. Holzwarth, J. Pleiss, and R. D. Schmid, I. Mol. Catal. B. in press (1997).

[21]

STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

369

TABLE IV OVERVIEW ON MODELING STRATEGY

Modeling of the open form of ROL

1 1

Search for hydrophobic patches in the binding site

Docking of the tetrahedral intermediate of octanoyl ester (leaving fatty acid chain)

Docking of the sn-2 and the nonhydrolyzedsn-3/1 chain

Relaxation of the complex by energy minimization and molecular dynamicssimulation

Analysis: Comparison of sn-1 and sn-3 orientation

Internal energy of substrates

Stabilization of the oxyanion

Interaction with side chains

and the sn-2 analogs 2-X-1,3-dioctanoylglycerol, where 2-X = 2-O-octyl ("ether"), 2-deoxy-2-octanoylamino ("amide"), and 2-deoxy-2-phenyl ("phenyl"). Ester and ether substrates are preferentially hydrolyzed at sn-1, whereas amide and phenyl reverse R O L stereopreference to sn-3. Stereoselectivity is similar for the 1,3-dioctanoyl and the 1,3-dioleoyl substrates. 13

Modeling of Open Form of Rhizopus oryzae Lipase The structure of the open form of R O L was modeled by homology. 7 The structures of two reference proteins are available: lipases from R. delemar ( R D L ) 8,3° and Rhizomucor miehei (RML) 6 share 99 and 57% sequence identity with R O L , respectively. The R D L was crystallized in two "semiopen lid" conformations. The structure was determined at a resolution of 3oL. Swenson, R. Green, R. Joerger, M. Haas, K. Scott, Y. Wei, U. Derewenda, D. Lawson, and Z. S. Derewenda, Proteins 18, 301 (1994).

370

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[2 1]

2.6 A. The RML was crystallized in the closed form and complexed with two inhibitors: diethylpohosphate and hexylphosphonate ethyl ester (resolution, 1.9, 2.6, and 3.0 A). C, root-mean-square (RMS) deviation between the two RDL structures (excluding the lid region) is 0.7 A, between the two RML complexes 0.3 .A, and between RML and the two R D L structures 0.9 and 1.1 A. The model structure of ROL deviates from RML and the two structures of R D L by 0.8, 1.1, and 1.2 ,~. For 19 amino acids in contact with substrate the deviation from RML and R D L is only 0.3 and 0.5 A, respectively.

Search for Hydrophobic Acyl Chain-Binding Sites There are two major hydrophobic binding sites in ROL. Searching for hydrophobic regions using the METHYL probe of GRID9631 revealed a long and deep hydrophobic crevice of about 2 x 16 ~.29 This crevice was also described in other lipases that are homologous to ROL: lipase from Humicola lanuginosa 32 and RML. 6 In the latter, this site binds the hexyl chain of the inhibitor hexylphosphonate ethyl ester; therefore, it may represent the binding site for the leaving fatty acid chain. Using the D R Y 33 probe of GRID, a shallow hydrophobic dent of about 2 x 5 A w a s f o u n d 29 that is parallel to the hydrophobic crevice at a distance of 4-5 A. This site exists also in H. lanuginosa lipase, and may represent a binding site for a nonhydrolyzed fatty acid chain. 34

Docking of Tetrahedral Intermediate of 1 (3)-Octanoylglycerol The tetrahedral intermediate of sn-1 and sn-3 octanoylglycerol is docked to the binding site (Fig. 3). Docking is guided by the position of hexylphosphonate ethyl ester in RML. A covalent bond between Ser-145 and the tetrahedral carbon of the substrate is created. The oxyanion fits into the oxyanion hole formed by the backbone nitrogens of Thr-83 and Leu-146 and the side-chain hydroxyl group of Thr-83. The ester oxygen linked to glycerol and the Ser-145 side-chain oxygen are in hydrogen-bonding distance from the catalytic His-257. The acyl chain is positioned in the hydrophobic crevice.

31 p. j. Goodford, J. Med. Chem. 28, 849 (1985). 32 M. Norin, F. Haeffner, A. Achour, T. Norin, and K. Hult, Protein Sci. 3, 1493 (1994). 33Molecular Discovery, Ltd., "GRID96 Manual." Molecular Discovery, Ltd., Oxford, England, 1996. 34 D. M. Lawson, A. M. Brzozowski, S. Rety, C. Vemag, and G. G. Dodson, Protein Eng. 7, 543 (1994).

[211

STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

/

371

Leu-146

H/N Thr-83.----~:

HH'.::~.~.;io CHz'4t-'-Ser'145

L_v/

HzC-Io.180 ~-0.420 Substrate /Q6?i~i~.R~26 R2 CH2 7~.p11J'lo.226 Ho.~-- C~1" j~"-0.294 R1

4

His-257 FIG. 3. Partial charges of the catalytic Ser-145 and His-257 and the l(3)-octanoylglycerol substrate as tetrahedral intermediate(R1, alkylchainof leavingfatty acid;R2, glycerolmoiety). The oxyanionis stabilizedby three hydrogenbonds from the backboneof Leu-146and Thr-83 and the side chain of Thr-83.

Docking of sn-2 and Nonhydrolyzed sn-3H Chain It can be assumed that either of the two chains (sn-2 and the nonhydrolyzed sn-3/1 chain) binds to the hydrophobic dent. So there are four possibilities as to how a triacylglycerol can bind (Fig. 4): depending on whether the sn-1 or the sn-3 chain binds to the hydrophobic crevice the orientation has been called " s n - l " or "sn-3 orientation," respectively. If the sn-2 chain binds to the hydrophobic dent, the substrate binds in the "sn-2 down binding mode," otherwise in the "sn-2 up binding mode. ''29 For substrates with an aliphatic chain in sn-2 position (ester, ether, amide), the most probable binding mode is sn-2 down (Fig. 4a and b). The hydrophobic alkyl chains of the two nonhydrolyzed chains pack favorably to each other as do the polar ester groups of the triacylglycerol substrate. This cooperative effect stabilizes the complex. However, in the sn-2 up binding mode the polar ester group of the nonhydrolyzed sn-3(1) chain contacts the other two hydrophobic chains (Fig. 4c and d). Molecular dynamics simulations (see below) of ether, ester, and amide substrates in sn-2 up binding mode support this intuitive prediction, because the nonhydrolyzed sn-3(1) chain tends to move out of the hydrophobic dent, while the complex is stable in the sn-2 down binding mode. 29 For the phenyl

372

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

sn-3

[9.1]

sn-1 x

~.¥

hydrophobic dent

~

sn-2

~

sn-2

/ Ser

Ser

hydrophobic crevice

d sn-2 Y

H

X

/,

~

~.

.........

sn-2

~.~.

~,~ . . . .

~;~

Ser

," . . . .

Y

•. . . . . . .

";

Ser ~ x

Fic. 4. Four possible ways in which triacylglycerol or analog can bind to ROL. The long and the short gray-shaded regions indicate the hydrophobic crevice and the hydrophobic dent, respectively. The catalytic serine marks the cleavage site. The sn-2 chain is marked by a boldface line. X, Ester bond in the sn-1 and sn-3 chain; Y, sn-2 group of ester, ether, or amide. In the phenyl substrate, the Y and the aliphatic chain are replaced by a phenyl ring. The s u b s t r a t e can bind in sn-1 (a and c) or sn-3 (b and d) orientation. In the "sn-2 down" binding mode (a and b) the sn-2 chain binds to the hydrophobic dent, whereas in "sn-2 up" (c and d) it does not bind to the hydrophobic dent.

substrate, however, the sn-2 up binding mode is preferred. The nonhydrolyzed sn-3(1) aliphatic chain binds to the hydrophobic dent, and the phenyl group points out of the binding site.

Relaxation of Complex Energy minimization and molecular dynamics (MD) simulations of the complex of all four substrates each in sn-1 and sn-3 orientation are carried out using Sybyl 6.1 (Tripos, St. Louis, MO) with the Tripos force fieldY For the catalytic histidine and the oxyanion the partial charges are modified (Fig. 3) as calculated by the semiempirical method MNDO94/PM336 using the Unichem 3.0 interface (Cray Research, Mendota Heights, MN). Prior to molecular dynamics simulations the initial complexes are energy minimized. They are then gradually heated in four intervals of 2 psec each 35M. Clark, R. D. Cramer, III, and N. van Opdenbosch, J. Comput. Chem. 111,982 (1989). 36 j. j. p. Stewart, J. Comput. Chem. 10, 209 (1989).

[21]

STEREOSELECTIVITY OF LIPASEFROMRhizopus oryzae

373

TABLE V SUMMARY OF MODELING RESULTS a AND COMPARISON TO EXPERIMENTAL DATA

Glycerol conformation Thr-83 oxyanion hole Leu-258 interaction Experimental ee (rounded values)

Ether

Ester

Amide

Phenyl

sn-1 sn-1 sn-1

sn-1 sn-1 sn-3

sn-3 sn-1 sn-3

sn-3 sn-3 sn-3

60% (sn-1)

30% (sn-1)

50% (sn-3)

60% (sn-3)

a Three factors favor the sn-1 or sn-3 orientation, depending on the substrate: conformation of the glycerol backbone, participation of Thr-83 side chain in the oxyanion hole, and repulsion of substrate by Leu-258.

to 5, 30, 100, and 200 K and during 6 psec to 300 K. The step size is 1 fsec up to 100 K and 0.5 fsec at 200 and 300 K. The temperature coupling constant is set to 10 fsec, 37 the nonbonded interaction cutoff to 8 ,~. After this equilibration phase the system is further simulated for 4 psec. Averaging of structure and torsion angle values is performed only during this production phase.

Comparison of sn-1 and sn-3 Orientation: Internal Energy of Substrates As a consequence of changing the orientation of a substrate, the configuration at glycerol C-2 inverts (Fig. 4a and b). The local conformation of the glycerol backbone is expected to be different in the two orientations, because the tetrahedral intermediate is fixed at the active site and the position of the sn-2 chain in the hydrophobic dent is maintained. To estimate the difference in internal energy of the glycerol backbone in both orientations, a model of the glycerol backbone and the first three atoms of the sn-2 moiety is built. Torsion angles are assigned to the respective average values of the substrates during the MD simulation in sn-1 and sn-3 orientation, and the energy is evaluated by geometry optimization using MNDO94/ PM3, constraining the torsion angles. For ether and ester the sn-1 orientation is energetically preferred by 5 and 4 kcal/mol, respectively, whereas for amide and phenyl the sn-3 orientation is more favorable (10 and 4 kcal/ mol, respectively). This result corresponds to the measured stereopreference of the substrates (Table V).

Stabilization of Oxyanion A second interaction that is different in both orientations can be identified. For ester, ether, and amide substrates in sn-1 orientation and phenyl 37 H. J. C. Berendsen, J. P. M. Postma, and W. F. van Gunsteren, J. Chem. Phys. 81, 3684 (1984).

374

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

121]

in sn-3 orientation the side-chain hydroxyl of Thr-83 forms a hydrogen bond to the oxyanion (Fig. 3). This residue has previously been suggested to act as additional hydrogen donor to the tetrahedral intermediate of the substrate in RML. 6 The role of this hydroxyl group was further investigated by protein engineeringT'9: replacing Thr-83 by serine, the activity of the ROL mutant enzyme is still 22% that of wild type. Removing the hydroxyl group by replacing Thr-83 by valine or alanine inactivates the enzyme. In the unfavored orientation, the Thr-83 side chain is rotated. This may be due to repulsive interaction of the hydroxyl group with glycerol C-3. Instead of stabilizing the oxyanion, the hydroxyl of Thr-83 then points in the direction of Asp-92 (data not shown). Thus, the transition state is less stabilized, which leads to a slower reaction of the substrate in the unfavored orientation. Interaction with Rhizopus oryzae Lipase Side Chains Experimentally there is clear evidence that ROL is less sn-1 and more sn-3 selective as the chemical structure at sn-2 becomes more rigid and bulky. So for bulky substrates there should be either an additional stabilizing interaction in the sn-3 orientation or a destabilizing interaction in the sn-1 orientation. This interaction should be localized near the C-2 glycerol atom and the start of the sn-2 chain. In sn-1 orientation steric repulsion between the side chain of Leu-258 and the ester (Fig. 5) and amide group at sn-2 has been observed. 29 For the ether substrate, which is smaller and more flexible, no difference in local interactions can be found in the two orientations. For the phenyl substrate, the situation is similar: the sn-3 orientation is preferred, whereas in sn-1 orientation the phenyl ring interferes with Leu-258. The factors that favor the sn-1 or sn-3 orientation are summarized in Table V and compared to the experimental data on stereoselectivity. The model is in accordance with the experiments: the more factors favor an orientation, the higher the respective stereoselectivity.

Conclusion It is evident from the experimental data obtained in hydrolysis and esterification that the substituent in position sn-2 of the glycerol- or 1,3propanediol backbone plays a crucial role in the stereoselectivity observed for ROL. It is noteworthy to mention that modulating polarity and flexibility of the sn-2 group not only affects the stereoselectivity expressed as enantiomeric excess, but also results in a reversal of stereopreference (sn-1 versus sn-3) in hydrolysis. In esterification reactions the stereoselectivity increases

[211

STEREOSELECTIVITY OF LIPASE FROM Rhizopus oryzae

375

Leu-258

C (sn-1 or

sn-311

Fic. 5. The sn-2 carbonyl group of the ester substrate in sn-1 (light gray) and sn-3 (dark gray) orientations. The side chain of Leu-258 is pushed away by the substrate in the sn-1 orientation.

c o n c o m i t a n t l y with a decrease of substrate polarity at the sn-2 position. H o w e v e r , no explanation can be p r o v i d e d for the different stereoselectivity in hydrolysis and esterification of substrates with 2-deoxy-2-alkyl g r o u p in sn-2 position. By now, crystallographic data on binding of acyl chain analogs are available for lipases f r o m Rhizornucor rniehei, 5 Candida antarctica, 3s Candida rugosa, 39 and h u m a n pancreatic lipase. 4° A l t h o u g h the m e c h a n i s m of hydrolysis is similar in all lipases, the structure of their binding site and the s t e r e o p r e f e r e n c e t o w a r d triacylglycerols and analogs differ. I n contrast to esters of s e c o n d a r y alcohols, for which a c o m m o n rule has b e e n p r o p o s e d to predict stereoselectivity, 41 there is no such c o m m o n rule to predict stereoselectivity t o w a r d triacylglycerol substrates. In R. oryzae lipase, at least three factors g o v e r n stereoselectivity t o w a r d these substrates: the internal 38j. Uppenberg, N. Oehrner, M. Norin, K. Hult, G. J. Kleywegt, S. Patkar, V. Waagen, T. Anthonsen, and T. A. Jones, Biochemistry 34, 16838 (1995). 39p. Grochulski, F. Bouthillier, R. J. Kazlauskas, A. N. Serregi, J. D. Schrag, E. Ziomek, and M. Cygler, Biochemistry 33, 3494 (1994). 40M. P. Egloff, F. Marguet, G. Buono, R. Verger, C. Cambillau, and H. van Tilbeurgh, Biochemistry 34, 2751 (1995). 41R. J. Kazlauskas, Trends Biotechnol. 12, 464 (1994).

376

ISOLATION, CLONING, EXPRESSION, AND ENGINEERING

[21]

energy of the glycerol backbone of the substrate, the participation of the Thr-83 in the oxyanion hole, and repulsive interaction between Leu-258 and the substrate. Combining experimental structure information, computer-aided modeling of substrate-lipase interaction, and experimental analysis, this model explains the observed reversal in stereopreference and qualitatively ranks substrates by stereoselectivity. Acknowledgments Financial support by the German Research Foundation, grants SP 135/11-1 and SCHM 1240/1-1, and by the Fund for the Promotion of Scientific Research in Austria, grant F102, are gratefully acknowledged.

C o n t r i b u t o r s to V o l u m e 2 8 4 Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

STEVE DANKO (18), Genencor International, Palo Alto, California 94304

PATRICK ADLERCREUTZ (12), Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lurid University, S-221 O0 Lund, Sweden L1LIA ALBERGHINA (14), Dipartimento Fisiologia e Biochimica Generali, Sezione Biochimica Comparata, Universit6 degli Studi di Milano, 20133 Milano, Italy

RICHARD C. DAVIS (9, 13), Lipid Research Laboratory, West Los Angeles VA Medical Center, Los Angeles, California 90073, and Department of Medicine, Universityof California, Los Angeles, Los Angeles, California 90024

EUNICE ASHIZAWA (18), Genencor International, Palo Alto, California 94304

FINN DRABLtbS (2, 3, 7), SINTEF Unimed, N-7034 Trondheim, Norway

LARS BL~.CKBERG(10), Department of Physiological Chemistry, University of Umegt, Ume& Sweden

RuI-DONG DUAN (10), Department of Cell Biology, University Hospital, S-221 85 Lund, Sweden

KIM BORCH (19), Enzyme Design, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark

LILIANE DUPUIS (15), Laboratoire de Lipolyse Enzymatique, UPR-9025 de I'IFR-1 du CNRS, 13402 Marseille codex 20, France

MATI'HEW BOSTON (18), Genencor International, Palo Alto, California 94304

MARIE PIERRE EGLOFF ( 5 ) , AFMB-CNRS, 13402 Marseille codex 20, France

RICHARD BOTT (18), Genencor International, Palo Alto, California 94304

MAARTEN R. EGMOND (6), UnileverResearch Laboratory, Vlaardingen, The Netherlands

YVES BOURNE (5), AFMB-CNRS, 13402Marseille codex 20, France

Assou EL BAqTARI (20), Inserm U260, 13385 Marseille codex 5, France

CHRISTIAN CAMBILLAU (5), AFMB-CNRS, 13402 Marseille codex 20, France

THOMAS GITLESEN (12), Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, S-221 O0 Lund, Sweden

STEPHANE CANAAN (15), Laboratoire de Lipolyse Enzymatique, UPR 9025 de I'IFR-1 du CNRS, 13402Marseille codex 20, France

LuTz HAALCK (21), Institute of Chemical and Biochemical Sensor Research, D-48149 Mfinster, Germany

IB GROTH CLAUSEN (19), Enzyme Design, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark JUAN A~rroNlo CONTRERAS (16), Department of Cell and Molecular Biology, Section for Molecular Signalling, Lund University, S-221 O0 Lund, Sweden

JOHN S. HILL (9, 13), Lipid Research Laboratory, West Los Angeles VA Medical Center, Los Angeles, California 90073, and Department of Medicine, University of California, Los Angeles, Los Angeles, California 90024

MIROSLAW CYGLER (1, 4), Biotechnology Research Institute, National Research Council of Canada, MontrOal, QuObec H4P 2R2, Canada

CECILIA HOLM (16), Department of Cell and Molecular Biology, Section for Molecular Signalling, Lurid University, S-221 O0 Lund, Sweden ix

X

CONTRIBUTORS TO VOLUME 284

ALISHA JARNAGIN (18), Genencor International Palo Alto, California 94304 DOMINIQUE LOMBARDO (20), lnserm U 260, 13385 Marseille c~dex 5, France MARINA LOTFI (14), Dipartimento Fisiologia e Biochimica Generali, Sezione Biochimica Comparata, Universit6 degli Studi di Milano, 20133 Milano, Italy MARK E. LOWE (8, 17), Departments of Pediatrics and of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 PAULO MARTEL (7), Instituto de Tecnologia Quimica e Tecnologica, P-2781 Oeiras, Portugal CHRISLAINE MARTINEZ (5), AFMB-CNRS, 13402 MarseiUe c~dex 20, France ERIC MAS (20), Inserm U260,13385 Marseille c~dex 5, France IGNATIOUS NCUBE (12), Department of Biochemistry, University of Zimbabwe, Harare, Zimbabwe FRITZ PALTAUF (21), Department of Biochemistry and Food Chemistry, Technical University Graz, A-8010 Graz, Austria SHAMKANTANANT PATKAR (19), Enzyme Design, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark EVAMARIA I. PETERSEN (3, 7), SINTEF Unimed, N-7034 Trondheim, Norway MARIA TERESA NEVES PETERSEN (3, 7), SINTEF Unimed, N-7034 Trondheim, Norway STEFFEN B. PETERSEN (2, 3, 7), SINTEF Unimed, N-7034 Trondheim, Norway J~3RGEY PLEISS (21), Institute of Technical Biochemistry, University of Stuttgart, D-70569 Stuttgart, Germany A. J. POULOSE (18), Genencor International, Palo Alto, California 94304 JOHN S. READ (12), Department of Biochemistry, University of Zimbabwe, Harare, Zimbabwe CAROL REQUADT (18), Genencor International, Palo Alto, California 94304 MIREILLE RIVI~RE (15), Laboratoire de Lipolyse Enzymatique, UPR 9025 de I'IFR-1 du CNRS, 13402 Marseille c~dex 20, France

M. LUISA Rr~A (11), Departemento de Bioquimica, Universidad de Vigo, 32003 Ourense, Spain MARIE-ODILE SADOULET (20), Inserm U 260, 13385 Marseille c~dex 5, France ROLF D. SCHMIO (11, 21), Institute of Technical Biochemistry, University of Stuttgart, D-70569 Stuttgart, Germany CLAUDIA SCI-IM~DT-DANNERT(11), Institute of Technical Biochemistry, University of Stuttgart, D-70569 Stuttgart, Germany MICHAEL C. SCHOTZ (9, 13, 16), Lipid Research Laboratory, West Los Angeles VA Medical Center, Los Angeles, California 90073, and Department of Medicine, University of California, Los Angeles, Los Angeles, California 90024 JOSEPH D. SCHRAG (1, 4), Biotechnology Research Institute, National Research Council of Canada, MontrOal, Quebec H4P 2R2, Canada FRITZ SPENER (21), Institute of Chemical and Biochemical Sensor Research, D-48149 Miinster, Germany PETER STADLER (21), Department of Biochemistry and Food Chemistry, Technical University Graz, A-8010 Graz, Austria BERIT STERNBY (10), Department of Medicine, University Hospital, S-221 85 Lund, Sweden ALLAN SVENDSEN (19), Enzyme Design, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark MARIANNE TnELLERSEN (19), Enzyme Design, Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark CARLA J. VAN BEMMEL (6), Unilever Research Laboratory, Vlaardingen, The Netherlands HERMAN VAN TILBEURGH (5), CBM-CNRS, MontpeUier, France ROBERT VERGER (16), Laboratoire de Lipolyse Enzymatique, UPR 9025 de I'IFRC1 du CNRS, 13402 Marseille e~dex 20, France CAXHERINE WICrmR-PLANQUARr (15), Laboratoire de Lipolyse Enzymatique, UPR

CONTRIBUTORS TO VOLUME 284 9025 de I'IFR-1 du CNRS, 13402 Marseille cddex 20, France HOWARDWONG (9, 13), Lipid Research Laboratory, West Los Angeles VA Medical Center, Los Angeles, California 90073, and Department of Medicine, Universityof California, Los Angeles, Los Angeles, California 90024

xi

SHAN WU (18), Genencor International, Palo Alto, California 94304 DAWN YANt~ (9, 13), Lipid Research Laboratory, West Los Angeles VA Medical Center, Los Angeles, California 90073, and Department of Medicine, Universityof California, Los Angeles, Los Angeles, California 90024

METHODS IN ENZYMOLOGY VOLUME I. Preparation and Assay of Enzymes Edited by SIDNEYP. COLOWICK AND NATHAN O. KAPLAN VOLUME II. Preparation and Assay of Enzymes

Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME III. Preparation and Assay of Substrates

Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME IV. Special Techniques for the Enzymologist Edited by SIDNEYP. COLOWICKAND NATHANO. KAPLAN VOLUME V. Preparation and Assay of Enzymes

Edited by SIDNEY P. COLOWlCK AND NATHAN O. KAPLAN VOLUME VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques

Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VII. Cumulative Subject Index

Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUMEVIII. Complex Carbohydrates

Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate M e t a b o l i s m Edited by WILLIS A. WOOD

VOLUMEX. Oxidation and Phosphorylation

Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUMEXI. Enzyme Structure Edited by C. H. W. HIRS VOLUMEXII. Nucleic Acids (Parts A and B)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUMEXIII. Citric Acid Cycle Edited by J. M. LOWENSTEIN VOLUMEXIV. Lipids Edited by J. M. LOWENSTEIN VOLUMEXV. Steroids and Terpenoids Edited by RAYMONDB. CLAYTON VOLUMEXVI. Fast Reactions Edited by KENNETHKUSTIN XV

xvi

METHODS IN ENZYMOLOGY

VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C) Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes

Edited by GERTRUDE E. PERLMANNAND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D) Edited by LAWRENCE GROSSMANAND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A) Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFE VOLUME XXVI. Enzyme Structure (Part C) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFE VOLUME XXVII. Enzyme Structure (Part D) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFE VOLUME XXVIII. Complex Carbohydrates (Part B) Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by LAWRENCE GROSSMANAND KIVlE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A)

Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME XXXII. Biomembranes (Part B) Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I - X X X

Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN

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xvii

VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B) Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C) Edited by W. A. WOOD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B) Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling Edited by WILLIAMB. JAKOBYAND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E) Edited by C. H. W. Hms AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLIX. Enzyme Structure (Part G) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C) Edited by VICTOR GINSBUR6 VOLUME LI. Purine and Pyrimidinc Nucleotide Metabolism Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIII. Biomembranes (Part D: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

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METHODS IN ENZYMOLOGY

VOLUMELV. Biomembranes (Part F: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUMELVI. Biomembranes (Part G: Bioenergetics) Edited by SIDNEYFLEISCHERAND LESTER PACKER VOLUMELVII. Bioluminescence and Chemiluminescence Edited by MARLENEA. DELuCA VOLUMELVIII. Cell Culture Edited by WILLIAMB. JAKOBYAND IRA PASTAN VOLUMELIX. Nucleic Acids and Protein Synthesis (Part G)

Edited by Kiviz MOLDAVEAND LAWRENCEGROSSMAN VOLUMELX. Nucleic Acids and Protein Synthesis (Part H)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME61. Enzyme Structure (Part H) Edited by C. H. W. HIRS AND SERGE N. TIMASHEEE VOLUME62. Vitamins and Coenzymes (Part D)

Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods)

Edited by DANIEL L. PURICH VOLUME64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems)

Edited by DANIEL L. PURICH VOLUME65. Nucleic Acids (Part I)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME66. Vitamins and Coenzymes (Part E)

Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME67. Vitamins and Coenzymes (Part F)

Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME68. Recombinant DNA Edited by RAY Wu VOLUME69. Photosynthesis and Nitrogen Fixation (Part C) Edited by ANTHONYSAN PIETRO VOLUME70. Immunochemical Techniques (Part A)

Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE VOLUME71. Lipids (Part C) Edited by JOHN M. LOWENSTEIN VOLUME72. Lipids (Part D) Edited by JOHN M. LOWENSTEIN

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xix

VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME75. Cumulative Subject Index Volumes XXXI, XXXII, X X X I V - L X Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 76. Hemoglobins Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI,AND EMILIA CHIANCONE VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A) Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part Edited by SIDNEY PESTKA

B)

VOLUME 80. Proteolytic Enzymes (Part Edited by LASZLO LORAND

C)

VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAMAND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSENAND LEON W. CUNNINGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites Edited by WILLIAM E. M. LANDSAND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereochemistry, and Rate Studies) Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER VOLUME 89. Carbohydrate Metabolism (Part Edited by WILLIS A. WOOD

D)

VOLUME 90. Carbohydrate Metabolism (Part E) Edited by WILLIS A. WOOD

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METHODS IN ENZYMOLOGY

VOLUME 91. Enzyme Structure (Part I) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)

Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity)

Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 94. Polyamines Edited by HERBERT TABORAND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61-74, 76-80

Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases)

Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B)

Edited by RAY W u , LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C)

Edited by RAY W u , LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by ANTHONYR. MEANS AND BERT W. O'MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides) Edited by P. MICHAELCONN VOLUME 104. Enzyme Purification and Related Techniques (Part C) Edited by WILLIAMB. JAKOBY VOLUME 105. Oxygen Radicals in Biological Systems Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A)

Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 107. Posttranslational Modifications (Part B)

Edited by FINN WOLD AND KIVIE MOLDAVE

METHODS IN ENZYMOLOGY

xxi

VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAK1S

VOLUME 109. Hormone Action (Part I: Peptide Hormones) Edited by LUTZ BIRNBAUMERAND BERT W. O'MALLEY VOLUME 110. Steroids and Isoprenoids (Part A)

Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 111. Steroids and Isoprenoids (Part B) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A)

Edited by KENNETH J. WIDDER AND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Compounds Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A)

Edited by HAROLD W. WYCKOEF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B) Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNIDI SABATO, JOHN J. LANGONE,AND HELEN VAN VUNAKIS

VOLUME 117. Enzyme Structure (Part J) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology Edited by ARTHUR WEISSBACHAND HERBERT WEISSBACH VOLUME 119. Interferons (Part C) Edited by SIDNEYPESTKA VOLUME 120. Cumulative Subject Index Volumes 81-94, 96-101 VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G) Edited by FRANK CHYTILAND DONALD B. McCoRMICK VOLUME 123. Vitamins and Coenzymes (Part H) Edited by FRANK CHYTILAND DONALD B. McCoRMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides) Edited by P. MICHAELCONN VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER

xxii

METHODS IN ENZYMOLOGY

VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology)

Edited by JURE P. SEGREST AND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERS AND JURE P. SEGREST VOLUME 130. Enzyme Structure (Part K) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and Cell-Mediated Cytotoxicity)

Edited by GIOVANNI DI SABATO AND JOHANNES EVERSE VOLUME 133. Bioluminescence and Chemiluminescence (Part B) Edited by MARLENE DELuCA AND WILLIAMD. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton)

Edited by RICHARD B. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B) Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C) Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D) Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E) Edited by VICTOR GINSBURO VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins) Edited by ANTHONYR. MEANS AND P. MICHAEL CONN VOLUME 140. Cumulative Subject Index Volumes 102-119, 121-134 VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids) Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines Edited by SEYMOUR KAUFMAN

METHODS IN ENZYMOLOGY

xxiii

VOLUME143. Sulfur and Sulfur Amino Acids Edited by WILLIAMB. JAKOBYAND OWEN GRIFFITH VOLUME144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME146. Peptide Growth Factors (Part A)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME147. Peptide Growth Factors (Part B)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME148. Plant Cell Membranes

Edited by LESTER PACKER AND ROLAND DOUCE VOLUME149. Drug and Enzyme Targeting (Part B)

Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME150. Immunochemical Techniques (Part K" In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by GIOVANNIDI SABATO VOLUME151. Molecular Genetics of Mammalian Cells

Edited by MICHAEL M. GOTI'ESMAN VOLUME152. Guide to Molecular Cloning Techniques

Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME153. Recombinant DNA (Part D) Edited by RAY Wu AND LAWRENCEGROSSMAN VOLUME 154. Recombinant DNA (Part E)

Edited by RAY W u AND LAWRENCE GROSSMAN VOLUME155. Recombinant DNA (Part F) Edited by RAY Wu VOLUME156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na,K-Pump)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME157. Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME158. Metalloproteins (Part A)

Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME159. Initiation and Termination of Cyclic Nucleotide Action Edited by JACKIED. CORBINAND ROGER A. JOHNSON VOLUME160. Biomass (Part A: Cellulose and Hemicellulose)

Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG

xxiv

METHODS IN ENZYMOLOGY

VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin) Edited by WILLIS A. WOOD AND SCOTTT. KELLO60 VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by GIOVANNIDI SABATO VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by GIOVANNIDI SABATO VOLUME 164. Ribosomes Edited by HARRY F. NOLLER, JR., AND KIVIE MOLDAVE VOLUME 165. Microbial Toxins: Tools for Enzymology Edited by SIDNEYHARSHMAN VOLUME 166. Branched-Chain Amino Acids

Edited by ROBERT HARRIS AND JOHN R. SOKATCH VOLUME 167. Cyanobacteria

Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides) Edited by P. MICHAELCONN VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A) Edited by JACEK HAWIGER VOLUME 170. Nucleosomes

Edited by PAUL M. WASSARMAN AND ROGER O. KORNBERG VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 175. Cumulative Subject Index Volumes 135-139, 141-167 VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics)

Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism)

Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES

M E T H O D S IN E N Z Y M O L O G Y

XXV

VOLUME178. Antibodies, Antigens, and Molecular Mimicry Edited by JOHN J. LANGONE VOLUME 179. Complex Carbohydrates (Part F) Edited by VICTORGINSBURG VOLUME180. RNA Processing (Part A: General Methods) Edited by JAMES E. DAHLBERGAND JOHN N. ABELSON VOLUME181. RNA Processing (Part B: Specific Methods) Edited by JAMES E. DAHLBERGAND JOHN N. ABELSON VOLUME182. Guide to Protein Purification Edited by MURRAYP. DEUTSCHER VOLUME183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences Edited by RUSSELLF. DOOLITTLE VOLUME184. Avidin-Biotin Technology Edited by MEIR WILCHEKAND EDWARDA. BAYER VOLUME185. Gene Expression Technology Edited by DAVID V. GOEDDEL VOLUME186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants) Edited by LESTER PACKERAND ALEXANDERN. GLAZER VOLUME187. Arachidonate Related Lipid Mediators Edited by ROBERT C. MURPHYAND FRANKA. FITZPATRICK VOLUME188. Hydrocarbons and Methylotrophy Edited by MARY E. LIDSTROM VOLUME189. Retinoids (Part A" Molecular and Metabolic Aspects) Edited by LESTER PACKER VOLUME190. Retinoids (Part B: Cell Differentiation and Clinical Applications) Edited by LESTERPACKER VOLUME191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEYFLEISCHERAND BECCA FLEISCHER VOLUME192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEYFLEISCHERAND BECCA FLEISCHER VOLUME193. Mass Spectrometry Edited by JAMES A. McCLOSKEY VOLUME194. Guide to Yeast Genetics and Molecular Biology Edited by CHRISTINEGUTHRIEAND GERALD R. FINK VOLUME195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase Edited by ROGER A. JOHNSONAND JACKIED. CORBIN

xxvi

METHODS IN ENZYMOLOGY

VOLUME 196. Molecular Motors and the Cytoskeleton Edited by RICHARDB. VALLEE VOLUME 197. Phospholipases Edited by EDWARD A. DENNlS VOLUME 198. Peptide Growth Factors (Part C) Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO VOLUME 199. Cumulative Subject Index Volumes 168-174, 176-194 VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression)

Edited by TONY HUNTER AND BARTHOLOMEW M. SEVrON VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases)

Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON VOLUME 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes) Edited by JOHN J. LANGONE VOLUME 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by JOHN J. LANGONE VOLUME 204. Bacterial Genetic Systems

Edited by JEFFREY H. MILLER VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules)

Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 206. Cytochrome P450

Edited by MICHAEL R. WATERMAN AND ERIC F. JOHNSON VOLUME 207. Ion Channels Edited by BERNARDO RUbY AND LINDA E. IVERSON VOLUME 208. P r o t e i n - D N A I n t e r a c t i o n s

Edited by ROBERT T. SAUER VOLUME 209. Phospholipid Biosynthesis

Edited by EDWARD A. DENNIS AND DENNIS E. VANCE VOLUME 210. Numerical Computer Methods Edited by LUDWIGBRANDAND MICHAELL. JOHNSON VOLUME 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA) Edited by DAVID M. J. LILLEYAND JAMES E. DAHLBERG

xxvii

METHODS IN ENZYMOLOGY

VOLUME213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by LESTERPACKER VOLUME214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis) Edited by LESTERPACKER VOLUME215. Platelets: Receptors, Adhesion, Secretion (Part Edited by JACEKJ. HAWIGER VOLUME 216. Recombinant DNA (Part Edited by RAY Wu

G)

VOLUME217. Recombinant DNA (Part Edited by RAY Wu

H)

VOLUME218. Recombinant DNA (Part Edited by RAY Wu

I)

B)

VOLUME219. Reconstitution of Intracellular Transport Edited by JAMES E. ROTHMAN VOLUME220. Membrane Fusion Techniques (Part Edited by NEJAT DOZGONE~

A)

VOLUME221. Membrane Fusion Techniques (Part Edited by NEJAT DOZGCrNE~

B)

VOLUME222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors) Edited by LASZLOLORANDAND KENNETHG. MANN VOLUME223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors) Edited by LASZLOLORANDAND KENNETHG. MANN VOLUME224. Molecular Evolution: Producing the Biochemical Data

Edited by ELIZABETH ANNE ZIMMER, THOMAS J. WHITE, REBECCA L. CANN, AND ALLAN C. WILSON VOLUME225. Guide to Techniques in Mouse Development Edited by PAUL M. WASSARMANAND MELVINL. DEPAMPHILIS VOLUME226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and MetaUoproteins) Edited by JAMES F. RIORDANAND BERT L. VALLEE VOLUME227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by JAMES F. RIORDANAND BERT L. VALLEE VOLUME228. Aqueous Two-Phase Systems Edited by HARRYWALTERAND GOTE JOHANSSON

xxviii

METHODS IN ENZYMOLOGY

VOLUME229. Cumulative Subject Index Volumes 195-198, 200-227 VOLUME230. Guide to Techniques in Glycobiology Edited by WILLIAMJ. LENNARZ AND GERALD W. HART VOLUME231. Hemoglobins (Part B: Biochemical and Analytical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME232. Hemoglobins (Part C: Biophysical Methods) Edited by JOHANNESEVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME233. Oxygen Radicals in Biological Systems (Part C) Edited by LESTERPACKER VOLUME234. Oxygen Radicals in Biological Systems (Part D) Edited by LESTERPACKER VOLUME235. Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors) Edited by VIRGINIAL. CLARKAND PATRIKM. BAVOIL VOLUME236. Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells)

Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME237. Heterotrimeric G Proteins Edited by RAVI IYENGAR VOLUME238. Heterotrimeric G-Protein Effectors Edited by RAVI IYENGAR VOLUME239. Nuclear Magnetic Resonance (Part C)

Edited by THOMAS L. JAMES AND NORMAN J. OPPENHEIMER VOLUME240. Numerical Computer Methods (Part B)

Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME241. Retroviral Proteases

Edited by LAWRENCE C. K u o AND JULES A. SHAFER VOLUME242. Neoglycoconjugates (Part A) Edited by Y. C. LEE AND REIKO T. LEE VOLUME243. Inorganic Microbial Sulfur Metabolism

Edited by HARRY D. PECK, JR., AND JEAN LEGALL VOLUME244. Proteolytic Enzymes: Serine and Cysteine Peptidases

Edited by ALAN J. BARRETT VOLUME245. Extracellular Matrix Components Edited by E. RUOSLAHTI AND E. ENGVALL VOLUME246. Biochemical Spectroscopy Edited by KENNETHSAUER VOLUME247. Neoglycoconjugates (Part B: Biomedical Applications) Edited by Y. C. LEE AND REIKO T. LEE

xxix

M E T H O D S IN E N Z Y M O L O G Y

VOLUME 248. Proteolytic Enzymes: Aspartic and Metallo Peptidases Edited by ALAN J. BARRETT VOLUME 249. Enzyme Kinetics and Mechanism (Part D: Developments in Enzyme Dynamics) Edited by DANIEL L. PURICH VOLUME 250. Lipid Modifications of Proteins Edited by PATRICKJ. CASEYAND JANICE E. BUSS VOLUME 251. Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals) Edited by LESTER PACKER VOLUME 252. Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation) Edited by LESTER PACKER VOLUME 253. Adhesion of Microbial Pathogens Edited by RON J. DOYLE AND ITZHAK OFEK VOLUME 254. Oncogene Techniques Edited by PETER K. VOGT AND INDER M. VERMA VOLUME 255. Small GTPases and Their Regulators (Part A: Ras Family) Edited by W. E. BALCH, CHANNINGJ. DER, AND ALAN HALL VOLUME 256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. BALCH, CHANNINGJ. DER, AND ALAN HALL VOLUME 257. Small GTPases and Their Regulators (Part C: Proteins Involved in Transport) Edited by W. E. BALCH, CHANNINGJ. DER, AND ALAN HALL VOLUME 258. Redox-Active Amino Acids in Biology Edited by JUDITH P. KLINMAN VOLUME 259. Energetics of Biological Macromolecules Edited by MICHAEL L. JOHNSONAND GARY K. ACKERS VOLUME 260. Mitochondrial Biogenesis and Genetics (Part Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN

A)

VOLUME 261. Nuclear Magnetic Resonance and Nucleic Acids Edited by THOMAS L. JAMES VOLUME 262. D N A Replication Edited by JUDITH L. CAMPBELL VOLUME 263. Plasma Lipoproteins (Part C: Quantitation) Edited by WILLIAM A. BRADLEY, SANDRA H. GIANTURCO,AND JERE P. SEGREST VOLUME 264. Mitochondrial Biogenesis and Genetics (Part B) Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN VOLUME 265. Cumulative Subject Index Volumes 228, 230-262

XXX

METHODS IN ENZYMOLOGY

VOLUME 266. Computer Methods for Macromolecular Sequence Analysis Edited by RUSSELL F. DOOLITTLE VOLUME 267. Combinatorial Chemistry Edited by JOHN N. ABELSON VOLUME 268. Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase) Edited by LESTER PACKER VOLUME 269. Nitric Oxide (Part B: Physiological and Pathological Processes) Edited by LESTER PACKER VOLUME 270. High Resolution Separation and Analysis of Biological Macromoleeules (Part A: Fundamentals) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 271. High Resolution Separation and Analysis of Biological Macromolecules (Part B: Applications) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 272. Cytochrome P450 (Part B) Edited by ERIC F. JOHNSON AND MICHAEL R. WATERMAN VOLUME 273. R N A Polymerase and Associated Factors (Part A) Edited by SANKAR ADHYA VOLUME 274. R N A Polymerase and Associated Factors (Part B) Edited by SANKAR ADHYA VOLUME 275. Viral Polymerases and Related Proteins Edited by LAWRENCE C. Kuo, DAVID B. OLSEN, AND STEVEN S. CARROLL VOLUME 276. Macromolecular Crystallography (Part A) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 277. Macromolecular Crystallography (Part B) Edited by CHARLES W. CARTER, JR., AND ROBERT U. SWEET VOLUME 278. Fluorescence Spectroscopy Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 279. Vitamins and Coenzymes, Part I Edited by DONALD B. McCoRMICK, JOHN W. SUTrlE, AND CONRAD WAGNER VOLUME 280. Vitamins and Coenzymes, Part J Edited by DONALD B. McCoRMICK, JOHN W. SuTrIE, AND CONRAD WAGNER VOLUME 281. Vitamins and Coenzymes, Part K Edited by DONALD B. McCoRMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 282. Vitamins and Coenzymes, Part L (in preparation) Edited by DONALD B. McCoRMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 283. Cell Cycle Control Edited by WILLIAM G. DUNPHY

METHODS IN ENZYMOLOGY

xxxi

VOLUME 284. Lipases (Part A: Biotechnology) Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 285. Cumulative Subject Index Volumes 263, 264, 266-289 (in preparation) VOLUME 286. Lipases (Part B: Enzyme Characterization and Utilization) (in preparation) Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 287. Chemokines (in preparation) Edited by RICHARD HORUK VOLUME 288. Chemokine Receptors (in preparation) Edited by RICHARD HORUK VOLUME 289. Solid Phase Peptide Synthesis (in preparation) Edited by GREGG B. FIELDS VOLUME 290. Molecular Chaperones (in preparation) Edited by GEORGE H. LORIMERAND THOMASO. BALDWIN

AUTHOR INDEX

377

Author Index

Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A Abkevich, V., 60 Abola, E., 30 Abouakil, N., 188, 190,191(45), 342,344-347, 347(44), 348(47), 349, 349(44), 352(20) Abrams, C. K., 185 Achour, A., 27, 370 Adams, A. P., 185 Adams, D. A., 288 Adlercreutz, P., 220, 223, 225, 227-229 Adrian, F.-J., 198, 217(25), 328 Ahle, S., 233 Ailhard, G., 3 Aires-Barros, M. R., 217, 327 Alberghina, L., 98, 102(45), 246-247, 251(6), 253, 254(6), 255, 256(22) Alden, R. A., 9 Alder, B. J., 134 Aleman-Gomez, J. A., 343, 344(25) Aletta, J. M., 352 Alford, A. A., 220 Allen, J., 353 Allen, M. P., 134 Alon, R. N., 87, 106, 106(14) Altschul, S. F., 30, 69 Ameis, D., 171, 187, 188(25), 189(25), 234, 244, 261, 262(12), 269(12), 343,

344(21), 350(21) Amic, J., 112 Ampe, G., 227, 228(19) Andalibi, A., 171,234 Anderson, R. A., 103, 261, 262(13), 269(13), 270(13), 272(13) Andersson, L., 186, 297 Andrews, P. C., 285, 292, 293(19), 297(4) Anelli, P. L., 361

Anfinsen, C. B., 85 Angal, S., 261,270(14) Anthonsen, H. W., 42, 51(49), 53(49), 74 Anthonsen, T., 14, 27(34), 31, 33(18), 375 Antosiewics, J., 137, 139(31) Aoubala, M., 261,265 Arakawa, T., 8, 237 Arand, M., 102, 106(61) Archambault, L., 263 Argos, P., 44 Argraves, W. S., 172, 233, 246(7) Arnoldsson, K. C., 186 Ashizawa, E., 298 Aslanidis, C., 262 Atkins, P. W., 135, 139(21) Atomi, H., 195,199(13), 201(13), 203(13), 206, 209(45), 354 Augustin, J., 233, 234(18) Awasthi, A. K., 262 Axelsen, P. H., 31, 32(11) Azim, C. A., 262

B Baba, T., 344 Baillargeon, M. W., 220 Bairoch, A., 31 Baker, C. J., 149 Balasubramanian, K. A., 261 Baldari, C., 257 BaUesteros, A., 247, 251(7), 253(7) Baptista, A., 42, 51(49), 53(49), 74, 134, 135(20) Baratti, J., 221 Barbaras, R., 233 Barfoed, M., 327, 333(31)

378

AUTHOR INDEX

Barnes, P. J., 354 Barnhart, R. L., 233 Barr, P. J., 211 Barrans, A., 233 Barrett, A. J., 105 Barrowman, J. A., 185 Barrowman, J. S., 261 Barter, P. J., 233 Barton, G. J., 35, 51, 52(66) Bashaum, C., 348 Bashford, D., 138, 141,144(34) Bass, H. B., 189 Bateman, D. F., 149 Batenburg, A. M., 96, 318 Battari, A. E., 340 Batteneli, 329 Battey, J. F., 288, 289(13) Baulard, A., 197, 197(23), 198(23), 205(23), 206(23), 214(23), 220(23) Baumann, W. J., 359 Beer, D., 353, 355(5), 375(5) Beer, H. D., 206, 354, 374(7) Beetham, J. K., 102, 106, 106(61) Beisiegel, U., 172, 233 Belev, T. N., 206, 355 Belfrage, P., 272-274, 274(7, 8, 11), 275(8), 277(9), 279-280, 280(7), 281, 281(5, 27), 283, 283(5) Benajiba, A., 349 Ben-Avram, C. M., 234 Benedik, M. J., 200 Bengtsson-Olivecrona, G., 171-172, 172(6), 232, 244 B6nicourt, C., 265 Benjamin, S. B., 185 Bennett, F. A., 349 Bensadoun, A., 233, 236, 237(29) Ben-Zeev, O., 234 Berendsen, H.J.C,, 373 Berger, J. E., 272 Berger, M., 221 Berglund, P., 17 Bergstr6m, B., 185-186, 343 Berka, T. R., 353 Bernard, A.-M., 106 Bernard, C., 328 Bernbach, S., 341 Bernback, S., 189, 261 Bernstein, F. C., 30, 86 Bersani, M. T., 329 Bersot, T., 236, 237(29)

Bertolini, M. C., 98, 254 Bianchetta, J., 239 Bilofsky, H., 44 Bilyk, A., 231 Biosym/MS1, 146-147 Birchbauer, A., 273 Birktoft, J. J., 9 Biswas, J., 342 Bjfrgell, P., 273, 280, 281(27) Bjorkling, F., 5, 15(14), 34(34), 35, 157, 301, 309(16), 321,328-329, 354, 370(6), 374(6) Bjursell, G., 187, 188(24), 343, 344(23), 344(27) Bl~ickberg, L., 185, 187-188, 188(1, 24), 189, 189(20), 190(20), 191-192, 261,341-343, 344(27), 346, 349-350, 350(53) Blacklow, S. C., 303 Blakenship, D. T., 233, 239(20) Blankenship, R. E., 206 Bleasby, A. J., 44 Blevins, G. T., 285, 297(4) Blevins, P. M., 292, 293(19) Blow, D. M., 4, 9, 307 Bluestone, J. A., 285 Blumberg, D. D., 286, 289(11) Blum-Kaelin, D., 285, 289(2), 296(2), 297(2) Blundell, T. L., 86 Bockris, J. O., 135, 137(23) Bodmer, M. W., 261,270(14) Boeckmann, B., 31 Boel, E., 3, 20(7), 109, 249, 318, 322, 355 Bohne, L., 196 Bois, A. G., 333 Bonicel, J., 190, 191(45), 265, 345 Bonnefis, M. J., 109 Bonten, E., 100 Borch, K., 317-318, 327, 333(31) Borchert, T. V., 325 Borensztajn, J., 233 BorgstrSm, B., 3, 108, 187, 187(8), 188(25), 189, 189(25), 192(15), 344(21), 350(21), 353 Borhan, B., 106 Born, M., 133 Bornscheuer, U., 354 Bos, J. L., 267 Bos, J. W., 96 Bosner, M. S., 191,343 Boston, M., 298 Bott, R., 298-299, 307, 312, 314(26), 318, 321(2)

AUTHOR INDEX Boudouard, M., 239 Bouet, F., 31, 32(11) Bourgeois, D., 109 Bourne, Y., 31, 33(17), 34(23), 97, 107-108, 110, 112(4, 15) BouthiUier, F., 5, 21(17), 23(19), 26, 31, 32(13), 33(21, 22), 98, 246-247, 247(2), 253(8), 300, 375 Bowie, J. U., 87, 106(13) Bradford, M. D., 235 Bradford, M. M., 275, 275(23a) Bradley, S., 217 Brady, L., 4, 15(8), 31, 34(26), 96, 157, 171, 300, 318 Brady, S. E., 233 Brahms, J. G., 188, 344 Branden, C., 49 Brannon, P. M., 294 Bratt, C., 186 Braunsteiner, H., 262 Brautaset, T., 134, 135(20) Breddam, K., 100, 102 Brenner, S., 51, 86, 87(10), 91, 100(10),

105(25) Breviario, D., 249, 250(12) Brewer, H. B., 181 Brewer, H. B., Jr., 20, 237, 244, 245(31) Briee, M. D., 30, 86 Bridger, W. A., 80 Bristline, R. G., 220 Brocca, S., 98, 102(45), 247, 249, 250(12), 251(6), 253, 254(6), 257, 259(25) Brock, T., 195 Brockersdorf, H., 326 Brockman, H. L., 3, 335, 342 Broda, P., 355 Brower, D. P., 231 Brown, M., 40 Brown, M. A., 233, 239(19) Brown, M. S., 262 Bruneau, N., 349, 351 Brunzell, J. D., 181, 237, 245(31) Bruzzone, R., 294 Bryant, S. H., 30, 35 Brzozowski, A. M., 4-5, 9(15), 15(8, 14, 15), 31, 34(26, 33, 34), 35, 96, 157, 171, 300301,309(16), 314, 315(28), 318, 321,354, 370, 370(6), 374(6) Buchwald, P., 107, 285,289(2), 296(2), 297(2) Buckley, J. T., 104-105 Bukatin, M. A., 134

379

Bulsink, Y.B.M., 103 Buono, G., 3, 20, 23(44), 25(4), 26(4), 31, 33(20), 108, 112(8), 157, 375 Buonu, G., 113 Burgdorf, T., 5, 6(20), 19(20), 95 Burke, D. M., 233, 239(20) Burke, J. A., 262 Burma, S., 262 Bursell, G., 187 Burton, B. K., 262 Busch, S. J., 233, 239(20) Butters, T. D., 266 Byrd, J. C., 348

C Cabral, J.M.S., 3, 25(4), 26(4), 217, 327 Caldwell, R., 298 Cambillau, C., 3, 5, 5(5), 7(5), 9(16), 14(5), 20, 20(7, 16), 23(16, 44), 25(4, 43), 26(4), 31, 32(14, 15), 33(17, 19, 20), 34(31), 77, 95, 97, 107-110, 112, 112(4, 6-8; 15), 120, 152, 153(62), 157-158, 158(4), 234, 244(24), 300-301,303, 314(19), 375 Camulli, E. D., 342 Canaan, S., 261 Canioni, P., 112 Cardin, A. D., 233, 239(20) Carey, M. C., 261 Carlsson, P., 187, 188(24), 343, 344(27) Caron, A. W., 263 Carrier, F., 329 Carriere, F., 162, 185, 297 Carri~re, F., 3, 20(7), 25(4), 26(4), 109, 261, 269 Carter, C. P., 343, 350 Castagnola, M., 329 Cernia, E., 329 Chan, L., 44, 171-172, 233-234, 239(19), 352 Chandra, A. K., 298 Chang, M.-K., 279 Chap, H., 109, 233 Chappell, D. A., 172, 233, 245, 246(7) Chapus, C., 31, 33(17), 97,108, 110,112(4,15) Chaut, J.-C., 171, 234 Cheah, E., 4, 6(12), 7(12), 42, 51(48), 88, 91(20), 93(20), 95(20), 97(20), 106(20), 299, 319 Chen, C., 234 Chen, Q., 189 Chen, S.-H., 171-172, 233, 239(19)

380

AUTHOR INDEX

Cheng, C. F., 236, 237(29) Chevenet, F., 36, 37(39) Chirgwin, J. M., 214, 286 Chomczynski, P., 286, 287(8) Chothia, C., 45, 86, 87(10), 100(10), 130, 132(1) Christen, M., 26 Christensen, M., 249, 355 Christiansen, L., 4, 15(8), 31, 34(26), 96, 157, 171, 300, 318 Christophe, J., 292 Chumley, F. G., 149, 150(53) Chung, G. H., 200 Ciccotti, G., 134 Clark, M., 372 Clark, M. A., 342, 352(18) Clark, P. I., 88 Clark, R., 271 Clausen, I. O., 17, 317-318, 325 Clay, M. A., 233 Cleasby, A., 5, 19(21), 31, 34(24), 95,120, 300 Cole, G. T., 149 Collen, M. J., 185 Collet, X., 233 Colson, C., 3, 196-197, 197(23), 198(15, 23), 199(15), 205(23), 206(23), 214(23), 217(15), 220(23) Colwell, N. S., 343, 344(25) Connelly, P. W., 233 Connolly, M. L., 137 Contreras, J. A., 272-274, 275(18), 280, 280(18), 281(18), 283(18) Coons, D., 337, 338(44, 45) Cornich, A., 195 Costanzi, C., 288 Coulson, A. R., 263 Cowan, D. A., 195 Cox, R. C., 14, 127, 149 Craik, C. S., 88 Cramer, R. D. III, 372 Creighton, T. E., 138-139 Crout, D.H.O., 26 Cuccia, L. A., 124 Cudrey, C., 3, 20(7), 26, 31, 34(31), 77, 109, 113, 123, 303, 314(19) Cullen, B., 161 Cummings, R. D., 344, 346(41), 352(41) Cygler, M., 3-5, 6(12, 13a), 7(12), 9(18), 14, 14(10), 15, 15(13a), 17, 19, 19(20), 21(13, 17, 18), 23(19), 25-27, 31, 32(13), 33(21,

22), 34(27, 30), 42, 50-51, 51(48, 63), 85, 88, 91(20), 93, 93(20), 95, 95(20), 96, 97(20), 98, 98(19), 99(28, 49), 101, 106(20), 165, 246-247, 247(2), 253, 253(8), 254, 254(17), 299-300, 318-319, 375

D Dagan, A., 267, 270(37) Dahlqvist, A., 185 Daley, R. E., 206, 211(39) Dambach, L. M., 262 Dang, Q., 233 Danielsson, B., 273-274, 275(18), 280, 280(18), 281(18), 283(18) Danko, S., 298 Dankowski, M., 337, 338(44, 45) D'Arcy, A., 4, 6(9), 20(9), 70, 71(7), 97, 108, 109(3), 112(3), 157, 244, 300 Dartois, V., 197, 197(23), 198(23), 205(23), 206(23), 214(23), 220(23) Dashti, A., 344, 346(41), 352(41) Datta, S., 233, 239(19) Dauberman, J., 298, 312 D'Auriol, L., 171,234 David, F., 106 Davis, L. G., 288, 289(13) Davis, R. C., 104, 171-172, 177, 181,232,235236,238, 240,242-245,273-274, 274(17), 275(18), 280, 280(18), 281(18), 283(18) Davranov, K., 15, 96 Daya-Mishne, N., 195 d'Azzo, A., 100 de Boer, G. F., 267 Debye, V. P., 133 de Caro, A., 261,265 DeCaro, H., 112 de Caro, J., 261 Deeth, H. C., 224(21), 228, 229(21), 230(21), 231(21) Deever, A.M.Th.J., 217 Degerman, E., 273-274, 275(18), 280, 280(18), 281(18), 283, 283(18) De Geus, P., 3, 5(5), 7(5), 14(5), 31, 32(14), 95, 120, 150, 152, 153(62), 300 de Haas, G. H., 14, 103, 121, 127, 149, 153, 217, 318, 361,366(25) Delhi, M., 337, 338(44, 45) Dekker, N., 361,366(25)

AUTHOR INDEX DeMeester, I., 106 de Meutter, J., 150 Demleitner, G., 103 de Montigny, C., 165 Deng, W. P., 325 dePersio, L. P., 165 Deprez, P., 186 Derewenda, U., 5, 6(24), 9(15), 15(14, 15), 17, 22(37), 31, 34(28, 29, 32-34), 35-36, 91-92, 96, 100(26), 103(27), 157, 300301,309(16), 314, 321, 354, 369, 370(6), 374(6) Derewenda, Z. S., 4-5, 6(24), 9(15), 15(14, 15), 17, 22(37), 31, 34(26, 28, 29, 32-34), 35-36, 91-92, 96, 100(26), 103(27), 157, 171,234, 244(24), 300-301,309(16), 314, 318, 321,354, 369, 370(6), 374(6) de Silva, H. V., 233 Desnuelle, P., 3, 108, 109(9), 299 de Vlieg, J., 14, 31, 32(15), 127, 149, 153, 318, 333 Devlin, J. J., 271 Devlin, P. E., 271 Dezan, C., 112 Diard, P., 172, 233 D~az, E., 101, 102(55), 103(55) Diaz-Maurino, T., 247, 251(7), 253(7) Dibner, M. D., 288, 289(13) Dichek, H. L., 20, 168, 181,237,244, 245(31) Dick, R. F., 285 Dickman, M. B., 149 Di Fiore, P. P., 82 Dijkman, R., 217, 361, 366(25) Dijkstra, B., 4, 6(12), 7(12), 42, 51(48), 87-88, 91(20), 93(20), 95(20), 97(20), 106, 106(20), 299, 319 Dill, K. A., 86 Ding, J., 88 Di Persio, L. P., 344, 350-351 Doctor, B. P., 50, 51(63), 101, 253, 254(17) Dodson, E., 4, 15(8), 31, 34(26), 96, 157, 171,300 Dodson, G. G., 3-5, 6(24), 15(8, 14), 20(7), 31, 34(26, 28, 32, 34), 35, 96, 109, 157, 171,300-301,309(16), 314, 315(28), 318, 321, 354, 370, 370(6), 374(6) Doerfler, W., 266 Doolittle, M. H., 171-172, 232, 235, 244 Douchet, I., 26, 354 Douste-Balzy, L., 109

381

Downs, D., 189, 344, 350 Drabl¢s, F., 28, 42, 49, 51, 51(49), 53(49), 6162, 69(1), 74, 130, 134, 135(20), 157, 261 Drtina, G. J., 26 Du, H., 263, 272(28) Duan, R.-D., 185-186, 192(15) Duax, W. L., 6, 21(25), 23(25), 31, 32(12), 98 Dugi, K. A., 20, 168, 171, 172(7), 244 Dunaway, M., 5, 6(20), 19(20), 95 D0nhaupt, A., 198, 217(26), 218(26) Dupuis, L., 261 Dupuy, P., 341 Durbin, R., 40

E Eberhardt, C., 104 Eckerskorn, C., 261, 262(12), 269(12) Eddy, S. R., 40 Edgren, G., 273-274, 275(18), 280, 280(18), 281(18), 283(18) Edholm, O., 321 Edlund, M., 192, 349-350, 350(53) Egan, J. J., 279 Egan, R. W., 342, 352(18) Egloff, M. P., 3, 5, 9(16), 20, 20(16), 23(16, 44), 25(4), 26(4), 31, 33(19, 20), 34(31), 77, 107-108, 112, 112(6, 8, 26), 114, 157, 158(4), 301,303, 314(19), 375 Egmond, M. R., 5, 14, 19(21), 31, 32(15), 34(24), 95-96, 119-120, 127, 149, 153, 300, 318, 333 Ehrat Sabatier, L., 31, 32(11) Eigtved, P., 246 Eisenberg, D., 40, 57, 87, 106(13) Ellis, L. A., 341 Elshourbagy, N. A., 80 Endo, T., 188, 345 Endrizzi, J. A., 100 Enerb/ich, S., 343 Enerb~ck, S., 187, 188(24), 343, 344(27) Erdmann, H., 206, 209(41), 214(41), 217(41), 218(41) Ergan, F., 4, 21(13) Erlanson, C., 108, 186, 187(8) Erman, M., 21(25), 23(25), 31, 32(12), 98 Escribano, M.-J., 188, 345-347, 348(47) Estell, D., 298 Etienne, J., 171, 234 Etzerodt, M., 172, 233

382

AUTHOR INDEX

Etzold, T., 44 Evnin, L. B., 88

F Faber, K., 246 Fan, J., 233 Farrell, A. M., 205 Fauvel, J., 109, 186, 34l Fazio, S., 236 Feairheller, S. H., 221 Feller, G., 273 Fernandez, V. M., 247, 251(7), 253(7) Ferrato, F., 3, 20(7), 26, 109, 123, 185, 261 Ferri, S., 91,100(26) Fersht, A., 57, 138-139, 153-154 Fett, W. F., 149 Feyten, M. P., 227, 228(19) Field, R., 261 Fietcher, A., 249 Figarella, C., 112, 187, 188(21), 189(21), 346347, 348(47) Fiil, N. P., 249, 250(12), 355 Fine, R., 132, 146 Fischer, W., 363 Fisher, E. A., 342, 349 Fitzgerald, M. C., 233, 239(20) Flanagan, M. A., 233, 239(20) Flores, T. P., 39, 86 Fltigge, U. I., 275, 275(23b) Foglia, T. A., 221 Fontaine, R. N., 343-344 Foster, T. J., 205 Foullois, B., 198, 199(24), 205(24), 206(24), 214(24), 220(24) Fouw6, B. L., 227, 228(19) Franc, J.-L., 188, 346-347, 347(44), 348(47), 349(44) Frane, J., 312, 314(26) Franken, S. M., 4, 6(12), 7(12), 42, 51(48), 87-88, 91(20), 93(20), 95(20), 97(20), 106(20), 299, 319 Franklin, M., 80 Franssen, M.C.R., 246 Fredrikson, G., 104, 272-274, 274(7, 11, 17), 279-280, 280(7) Fredrikzon, B., 185, 188(1) Freed, L. M., 341 Freidberg, T., 102, 106(61) Frenkel, D., 134

Frenken, L. G., 5, 19(21), 31, 34(24), 96 Frenken, L.G.J., 95, 120, 300, 318 Freudenberg, E., 187, 341 Frimurer, T., 321 Fritsch, E. F., 263, 288, 289(12), 303,325,355 Frolow, F., 4, 6(12), 7(12), 31, 32(10), 42, 51(48), 88, 91(20), 93(20), 95(20), 97(20), 106(20), 299, 319 Fry, G. L., 172, 245 Fuchs, R., 44 Fukase, F., 206, 214(40) Fukuda, M., 101, 102(54) Fulton, J. E., 217 Funk, W~, 363 Fusetti, F., 98, 102(45), 247, 251(6), 253, 254(6), 257, 259(25)

G Gaber, B. P., 30 Galeotti, V., 257 Galibert, F., 171, 234 Ganot, D., 261 Ganshaw, G., 312 Gargouri, Y., 103, 112, 185, 261,265 Garnon, J., 101, 102(56) Garqouri, Y., 333 Garsetti, D. E., 342, 352(18) Gastaldi, M., 343 Gatt, S., 267, 270(37) Gentry, M. K., 50, 51(63), 101,253, 254(17) Gerard, H. C., 149 Gerday, C., 273 Gerlt, J. A., 211 Gerstein, M., 45 Geselowitz, A. R., 145 Geyer, H., 266 Geyer, R., 266 Ghiara, P., 257 Ghosh, D., 21(25), 23(25), 31, 32(12), 98 Gibrat, J.-F., 35 Gibson, T. J., 69 Gilbert, E. J., 195 Gilbert, J., 17, 95, 104(30) Gill, S. C., 237 Giller, T., 107, 285, 289(2), 296(2), 297(2) Gillespie, D., 288 Gillin, F. D., 342 Gilson, M. K., 132, 137, 139(31), 141, 146 Gish, W., 30, 69

AUTHOR INDEX Gisi, C., 292 Gitlesen, T., 220, 223, 225, 228 Gjellesvik, D. R., 102, 192, 343, 344(26) Gjinovci, A., 294 Glassman, R. H., 82 Glick, J. M., 342 Gliemann, J., 172, 233 Glimcher, L. H., 285 Gloger, I. S., 80 Goderis, H. L., 227, 228(19) Goeldner, M., 31, 32(11) Goers, J. W., 181,245 Goldfine, I. D., 293 Goldman, A., 4, 6(12), 7(12), 31, 32(10), 88, 91(20), 93(20), 95(20), 97(20), 106(20), 299, 319 Goldstein, J. L., 262 Gomi, K., 354 Goodford, P. J., 370 Goosens, F., 106 Gormsen, E., 327, 329, 333(31) Gotto, A. M., 233 G6tz, F., 103, 205, 217 Grabowski, G. A., 263, 272(28) Graille, J., 226, 228(18), 283 Grandori, R., 98, 102(45), 247, 249, 250(12), 251(6), 254(6) Grant, D. F., 102, 106, 106(61) Gray, P. W., 104 Graycar, T., 298, 312 Green, R., 5, 6(24), 17, 22(37), 31, 34(28, 29), 96, 300, 321,354, 369 Greenberg, A. S., 279 Greten, H., 261,262(12), 269(12) Gribskov, M., 40 Griglio, S., 172, 233 Grochulski, P., 5, 9(18), 21(17, 18), 23(19), 26, 31, 32(13), 33(21, 22), 34(30), 246-247, 247(2), 253(8), 375 Groot, P. H., 80 Grouchulski, P., 98 Grusby, M. J., 285 Guffens, W. M., 227, 228(19) Guilhot, S., 171,234 Gulick, T., 191 Gulomova, K., 15, 96 Gunner, M. R., 141, 144(35) Gupta, A. K., 26, 31, 33(21), 247, 253(8) Gurd, F.R.N., 135 Gurd, R.F.N., 142

383

Gutnick, D. L., 87, 106, 106(14) Guy, O., 112, 186-188, 188(12, 21), 189(21), 341, 344, 346 Guy-Crotte, O., 346-347, 348(47)

H Haag, T., 100, 101(51) Haalck, L., 5, 19(22), 95, 353-354, 363(13), 366(13), 367(13) Haas, G. H., 335 Haas, M., 369 Haas, M. I., 321 Haas, M. J., 5, 6(24), 17, 22(37), 31, 34(28, 29), 96, 231, 300, 353-354, 355(4), 356(11), 357(4), 374(9) Habib, S., 262 Haeffner, F., 27, 370 Hagstrom, R., 146 Halban, P. A., 294 Hammock, B. D., 102, 106, 106(61) Hammon, A., 217 Hamosh, M., 185, 261,341 Hamosh, P., 185, 261,341 Han, J. H., 343, 344(22) Han, Q., 177, 245 Hanafusa, H., 82 Hanania, G.I.H., 135, 142 Hancock, R.E.W., 198, 217(25), 328 Hansen, M. W.,5, 9(23), 14(23), 15(23), 22(23), 23(23), 31, 34(25), 96, 254 Hansson, L., 192, 349-350, 350(53), 352 Harano, D., 263 Hardies, S. C., 214 Harel, M.,4, 6(12), 7(12), 31,32(10,11,16), 42, 50, 51(48, 63), 88, 91(20), 93(20), 95(20), 97(20), 101, 106(20), 253, 254(17), 299, 319 Harris, T.J.R., 261, 270(14) Harrison, D., 5, 21(17), 31, 32(13), 98, 246, 247(2), 300 Harrison, E. H., 157, 342 Harsuck, J. A., 189 Hartley, B. S., 9 Harvey, S. C., 134 Hashimoto, M., 355, 357(18) Hashimoto, T., 101,102(54) Hashimoto, Y., 197, 198(22), 199(22), 200, 206(22), 214(22), 220(22) Hasnain, S. E., 262

384

AUTHOR INDEX

Hatton, T. A., 327 Holtsberg, F. W., 342, 352(18) Haussler, D., 40 Holzwarth, H.-C., 368, 370(29), 371(20) Havel, R. J., 233 Homanics, G. E., 233 Hayakawa, K., 186 Homology, 69 Hayes, F.R.F., 154 Honda, H., 51,248, 249(10), 255(10) Heeht, H.-J., 5, 6(20), 19(20, 22), 95, 100, Honig, B., 132, 134, 141, 144(35), 146-147 101(51) H0nig, H., 27 Hegele, R. A., 233 Honikoff, J. G., 45 Hegyi, H., 45 Honikoff, S., 45 Heinzmann, C., 171, 234, 273, 274(11) Horowiz, P. M., 214 Heldt-Hansen, H. P., 329 Horton, R. M., 158 Hellquist, M., 343 Houdebine, L., 286, 287(9) Hemil~, H., 273 Howard, B. H., 325 Hendrickson, W. H., 309 Hsu, J.S.T., 236, 237(29) Hendriks, D., 106 Hua, Y., 233 Henning, W. D., 80 Huang, A.H.C., 221,231 Herbert, R. A., 195 Huang, W., 60 Hermes, J. D., 303 Huang, Y., 349 Hermetter, A., 359 Hubbard, R. E., 318 Hernell, O., 185, 187-188, 188(1, 24), 189, Hubbard, T., 86, 87(10), 100(10) 189(20), 190(20), 191-192, 261,341-343, Hube, B., 249 344(27), 346, 349-350, 350(53) Hueckel, E., 133 Hersl6f, B., 186 Huge-Jensen, B., 4-5, 15(8, 14), 31, 34(26, Hibler, D. W., 211 34), 35, 96, 157, 171, 249, 300-301, Hide, W. A., 44, 171,234 309(16), 318, 321-322, 354-355, 370(6), Higaki, J. N., 88 374(6) Higgins, D. G., 44, 69 Hughes, R. C., 266 Higuchi, R., 158 Hughes, S. A., 80 Hill, J. S., 171,232,236, 238,240, 242-243,245 Hui, D. Y., 165, 186, 342-344, 349-351 Hills, M. J., 221 Huldt, G., 342 Hilton, S., 105 Hult, K., 14-15, 17, 27, 27(34), 31, 33(18), 170, Hirsch, M., 60 318, 321,370, 375 Hirth, C., 31, 32(11) Hunt, H. D., 158 Hixenbaugh, E. A., 172, 232 Hunziker, W., 4, 6(9), 20(9), 70, 71(7), 97, Hjorth, A., 3, 20(7), 109 107-108, 109(3), 112(3), 157, 244, 285, Hjorth, S., 109, 162, 269, 329 289(2), 296(2), 297(2), 300 Ho, S. N., 158 Huser, H., 198 Hobbie, L., 351 Hoch, H. C., 149 I Hockney, R. C., 210 Hoffmann, B., 5, 19(22), 95 Iandolo, J. J., 205 Hol, W.G.J., 100 Iizumi, T., 98, 206, 214(40) Holland, K. T., 205 Ikeda, Y., 236 Holm, C., 104, 272-274, 274(11, 17), 275, Inlow, D., 263 275(18), 277(9), 279-280, 280(18), Inoue, I., 172, 245 281(18), 282(23), 283, 283(18), 284 Inouye, M., 211,325 Holm, L., 30, 35, 86, 100(6), 100(11) Inouye, S., 325 Holmberg, K., 219 Islam, A., 342 Holmquist, M., 15, 17, 27, 170, 318 Islam, S. A., 45

AUTHOR INDEX Isobe, M., 221 Ivanova, M., 283 Ivanova, T., 283 Iverius, P.-H., 172, 190, 245 Iwai, M., 221,353 Iwasaki, S., 51, 248, 249(10), 255(10)

385

Jonson, P. H., 130, 132(1) Jork, H., 363 JornvaU, H., 6, 21(25), 23(25), 31, 32(12), 98 Julien, R., 112, 333 Juneblad, K., 192, 350, 352 Junge, W., 186 Jtirgens, D., 198

d Jackson, J.D., 135 Jackson, K.W., 344, 346(41), 352(41) Jackson, R. L., 171, 172(4), 192, 233, 239(20), 281 Jaeger, K.-E., 196, 198, 200, 217(25), 328 Jaenicke, R.,194 Jfiger, S., 103 Jain, A., 262 Jakobi, G., 337, 338(44, 45) Janin, J., 130, 132(1) Jansen, D. B., 102 Jansen, H, 233 Jarnagin, A., 298 Jaspard, B., 233 Jenkins, D.J.A., 233 Jennens, M. L.,20,162,163(19),166,167(19), 285, 289(5), 290(5), 291(5), 292(5), 296, 296(5), 297(28) Jensen, R. G., 326 Jeohn, G. H., 200 Ji, Z.-S., 236 Jigami, Y., 354 Joerger, R., 17, 22(37), 31, 34(29), 96, 300, 353-354, 355(4), 356(11), 357(4), 369, 374(9) Johnson, K., 189,190(44) Johnson, L. N., 5, 19(21), 31, 34(24), 95, 120, 300 Johnson, M. S., 44, 45(55), 86 Johnson, W.J.,342 Jones, A.D., 106 Jones, A. T., 5 Jones, C.W., 195 Jones, D.H., 325 Jones, D. T., 86, 87(3) Jones, G. E.,349 Jones, L. E., 149 Jones, T.A., 9(23), 14,14(23),15(23),22(23), 23(23),27(34),31,33(18),34(25),96,254, 300, 375

K Kabat, E., 44 Kaelin, D. B., 107 Kaiser, R., 21(25), 23(25), 31, 32(12), 98 Kalk, K. H., 87, 106 Kameyama, K., 237 Kannius, M., 343, 344(23) Kant, A., 267 Karlsson, M., 274, 284 Karplus, M., 138, 141, 144(34) Karpp, A., 233 Katsuoka, N., 281 Kawaguchi, Y., 51,248, 249(10), 255(10) Kazemier, B., 102 Kazlauskas, R. J., 5, 23(19), 26, 31, 33(21, 22), 45, 48(59), 124, 247, 253(8), 375 Kempner, E. S., 157, 236, 238, 240-242, 242(36), 243-244, 244(36), 245 Kennard, O., 30, 86 Kerfelec, B., 31, 33(17), 97, 108, 112(4) Kern, H. F., 293 Kersting, S., 233 Khouri, H. E., 268, 270(39), 271(39) Kiewitt, I., 221 Kim, H.-K., 214, 217(51) Kim, H.-M., 214, 217(51) Kim, Y. S., 348 Kimbara, K., 101,102(54) Kimmel, A. R., 288 King, D. J., 261,270(14) King, M. M., 189, 341 Kingsley, D. M., 351 Kinnunen, P.K.J., 232 Kirchgessner, T. G., 171, 234, 273-274, 274(11), 285 Kirkwood, J. G., 133, 141 Kissel, J. A., 351 Kitamoto, K., 354 Klapper, I., 146 Klenk, H. D., 266

386

AUTHORINDEX

Kleywegt, G. J., 14, 27(34), 31, 32(16), 33(18), 375 Klibanov, A. M., 26 Klima, H., 262 Klisak, I., 187 Kmetz, P. J., 80 Knapp, J. S., 195 Kneser, K., 233 Knowles, J. R., 303 Knudsen, B. S., 82 Koana, T., 101,102(54) Kobata, A., 188, 345 Kobos, P. M., 17, 22(37), 96, 354 Koch, G., 267 Koepke, A., 105 Koetzle, T. F., 30, 86 Koivula, T. T., 273 Kolattukudy, P. E., 149-150, 298 Koller, W., 149 Komaromy, M., 20, 99, 172, 232 Konnert, J. H., 309 Koops, B. C., 14, 127, 149 Korn, E., 205 Koshlukova, S. E., 352 Koster, H., 303 K6tting, J., 198 Kouker, G., 196 Kounnas, M. Z., 172, 233, 246(7) Kovac, A., 354, 363(13), 366(13), 367(13) Kozarsky, K. F., 351 Krabisch, L., 280-281,281(27) Kraulis, P. J., 39 Kraut, J., 9, 301 Kretchmer, N., 294 Krieger, M., 351 Kriel, G., 268, 270(38) Krogh, A., 40 Krummel, B., 158 Kuan, S. F., 348 Kuenzi, R., 249 Kugimiya, W., 197, 198(22), 199(22), 200, 206(22), 214(22), 220(22), 355, 357(18) Kukral, J. C., 185 Kuksis, A., 189 Kullman, J., 292 Kumagai, C., 354 Kumar, B. V., 343, 344(25) Kunkel, T. A., 325 Kuroda, K., 266

Kurzban, G. P., 214 Kuusi, T., 232 Kuzel, P., 337, 338(44, 45) Kyger, E. M., 343, 344(24)

L Labb6, D., 101,102(56) Lackner, K. J., 262 Lad, P., 298 Lafont, H., 342, 352(20) Lafuente, F. J., 254 Lagrange, D., 172, 233 Lake, B. D., 262 Lalibert6, F., 268, 270(39), 271(39) Lalouel, J.-M., 172, 245 Landt, S., 106 Lang, D., 5, 6(20), 19(20, 22), 95 Lang, S., 198, 217(26), 218(26) Lange, L. G., 343, 344(24) Lange, L. G. III, 191 Langin, D., 104, 273 Langley, K. E., 237 Langrand, G., 221 Laposata, E. A., 172, 232 Larame6, L., 93, 98, 99(28), 254, 318 Larson, S., 5, 6(20), 19(20), 95 Laskowski, R. A., 53, 55(69) Lassen, S. F., 325 Lau, P.C.K., 101,102(56) Lauer, S. J., 233, 236 Laugier, R., 185, 261 Laurell, H., 273-274 Laurent, S., 283 Lauwereys, M., 3, 5(5), 7(5), 14(5), 31, 32(14), 95, 120, 150, 152, 153(62), 300 Lauwers, A., 357 Lawn, R. M., 343, 344(28) Lawson, D. M., 3, 5, 9(15), 15(14, 15), 20(7), 31, 34(33, 34), 35, 91, 100(26), 109, 157, 301,309(16), 314, 315(28), 318, 321,354, 369-370, 370(6), 374(6) Lech~ne de la Porte, P., 342, 351, 352(20) Leclaire, M., 341 Ledbetter, D. H., 233, 239(19) Lee, B., 137, 139(28) Lee, C. Y., 205 Lee, D. H., 189

AUTHOR INDEX Lee, G., 187, 188(25), 189(25), 343, 344(21), 350(21) Lee, G.-K., 273 Lee, H. S., 342 Lee, R. H., 145 Lee, T. C., 185 Lee, Y. P., 200 Leete, T. H., 187, 188(25), 189(25), 343, 344(21), 350(21) Lesk, A. M., 130, 132(1) Lesser, G. J., 145 Lesuisse, E., 3, 196, 198(15), 199(15), 217(15) Levenson, C., 271 Levitt, M., 86 Levy, R., 241 Lewis, J. H., 185 Leybold, K., 186 Li, N., 6, 21(25), 23(25), 31, 32(12), 98 Li, W.-H., 44, 171,233-234, 239(19) Li, X., 200 Li, Y., 4-5, 6(20), 9(18), 14(10), 19(20), 21(17, 18), 31, 32(13), 34(30), 88, 95, 98, 98(19), 246, 247(2) Li, Z., 273 Liao, D.-I., 88 Lidberg, U., 187, 343 Lidmer, A. S., 343, 344(23) Lie Ken Jie, M.S.F., 221 Lin, Y. H., 221, 231 Lindgren, K., 352 Lindstr6m, M., 189 Linke, M. J., 342 Lipman, D. J., 30, 66, 68(2, 4), 69, 69(2) Little, J. A., 233 Ljungstr6m, I., 342 Logsdon, C. D., 285, 293, 297(4) Lombardo, D., 31, 33(17), 97, 108, 110, 112(4, 15), 186-188, 188(12, 21), 189(21), 190, 191(45), 192, 340-347, 347(44), 348(47), 349, 349(44), 351, 352(20) Londos, C., 279 Long, G. C., 323 Longhi, S., 31, 32(15), 98,102(45), 247,251(6), 253, 254(6) Lookene, A., 172, 233 Loomes, K. M., 350 Lopez-Candales, A., 343 Lorens, J. B., 102, 343, 344(26)

387

Lotti, M., 98, 102(45), 246-247, 249, 251(6), 253, 254(6), 255, 256(22), 257, 259(25) Louie, C., 86 Louwrier, A., 26 Lowe, A. W., 292, 293(19) Lowe, G., 88 Lowe, M. E., 20, 114, 157, 160-162, 163(19), 164(18), 165(18), 166, 167(19), 170, 170(26), 269, 285,289(5), 290(5), 291(5), 292,292(5), 295-296, 296(5), 297,297(27, 28), 343 Lowe, P. A., 261,265, 270(14) Lowrey, A., 30 Luckow, V. A., 263 Lui, S.-W., 233, 239(19) Lundberg, L., 192, 343, 344(23), 349-350, 350(53) Lundh, G., 185 LUnsdorf, H., 206, 209(41), 214(41), 217(41), 218(41) Luo, C.-C., 171-172, 233, 239(19) Luo, J., 45 Lusis, A. J., 171, 187, 234, 273-274, 274(11) LUthy, R., 57, 87, 106(13) Lyons, A., 261,270(14)

M Mackay, K., 343, 344(28) Madej, T., 35 Maeda, N., 233 Maguire, G. F., 233 Mahley, R. W., 233, 236 Maiorella, B., 263 Maiti, I. B., 150 Majumdar, R., 80 Makhzoum, A., 195 Male, R., 102, 343, 344(26) Malewiak, M.-I., 172, 233 Malezet-Desmoulin, C., 343 Manent, J., 233 Manganaro, F., 189 Mangel, W. F., 88 Mangin, D., 283 Mangold, H. K., 359 Maniatis, T., 263, 288, 289(12), 303, 325, 355 Mannesse, M.L.M., 14, 127, 149 Mao, S.J.T., 233, 239(20) Marchot, P., 31, 34(23)

388

AUTHOR INDEX

Marfan, P., 187 Margotat, A., 342-343 Marguet, D., 106 Marguet, F., 3, 20, 23(44), 25(4), 26(4), 31, 33(20), 108, 112(8), 113, 157, 375 Mark, D. F., 271 Markvardsen, P., 325 Marsh, J. B., 342 Martel, P., 42, 51(49), 53(49), 74, 130, 134135, 135(20), 138(27), 139(27), 140(27), 141(27), 143(27), 144(27), 145(27), 150(27) Martin, G. A., 233, 239(20) Martindale, M. E., 189, 34l Martinelle, M., 15, 17, 170, 318 Martinez, C., 3, 5, 5(5), 7(5), 9(16), 14(5), 20(16), 23(16), 31, 32(14, 15, 17), 33(19), 34(31), 77, 95, 97,107-108,112(4, 7), 120, 152, 153(62), 157, 158(4), 300-301, 303, 314(19) Mas, E., 188, 340, 342, 345-347, 347(44), 348(47), 349, 349(44) Massie, B., 263 Masuda, Y., 255 Matthew, J. B., 135 Matthews, B. W., 110, 298 Matthyssens, G., 3, 5(5), 7(5), 14(5), 31, 32(14), 120, 150, 152, 153(62), 300 Mattiasson, B., 223, 225, 227-229 Mattson, F. H., 359 McCammon, J. A., 134, 137, 139(31) McCarthy, J.E.G., 206, 353-355, 355(5), 374(7), 375(5) McDonald, I. R., 134 McDonald, R. J., 286 McEven, P., 114 McFadden, G., 80 McGinnis, S., 312 McGrath, W. J., 88 McIntyre, T. M., 104 McLachlan, A. D., 40, 57 McNeill, G. P., 221 McPherson, A., 5, 6(20), 19(20), 95, 110-111 McQuarrie, D. A., 135, 137(24) Meighen, E. A., 91, 100(26) Meili, C., 249 Meinkoth, J. L., 288 Melford, K. H., 236, 237(29) Melo, E. P., 3, 25(4), 26(4) Mendoza, J. A., 214

Menge, U., 4, 15(8), 96, 157,171,195,196(12), 197(12), 199(12), 200(12), 206, 209(41), 214(41, 42), 217(12, 41), 218(41), 300,318 Merideth, G. D., 233, 239(20) Merkel, M., 261,262(12), 269(12) Metropolis, N., 134 Meyer, E. F., 30 Meyer, E. F., Jr., 86 Meyer, H. E., 198, 217(25), 328 Meyer, N., 172, 233 Mian, I. S., 40 Michel, R., 112 Miller, C. G., 273, 274(11) Miller, D. M., 214 Miller, J. H., 241 Miller, S., 130, 132(1) Miller, W., 30, 69 Miralles, F., 342, 346-347, 348(47) Mirny, L., 87, 106(14) Mitchinson, C., 298 Mitchison, G., 40 Moeller, B., 198, 199(24), 205(24), 206(24), 214(24), 220(24) Moessner, J., 293 Moestrup, S. K., 172 Mohandas, T., 187, 273, 274(11) Mollay, C., 268, 270(38) Monod, M., 249 Montanari, F., 361 Montesinos, J. L., 254 Montet, D., 226, 228(18) Montgomery, R. K., 261 Montreuil, J., 188, 346-347, 347(44), 348(47), 349(44) Mooss, M. C., Jr., 279 Moreau, H., 103, 185, 261,282 Moreau, R. A., 149 Morel, D. W., 342 Morlock-Fitzpatrick, K. R., 342, 349 Moss, D. S., 39 Moulin, A., 103 Moult, J., 133 Mozhaev, V. V., 194 Mueller, H. W., 262 Muhonen, L. E., 172 Mukerjee, B., 262 Mukherjee, K. D., 221 Mulders, S. M., 292, 293(19) Murakami, M., 262 Murray, J.A.C., 257

AUTHOR INDEX Murzin, A. G., 86, 87(10), 100(10) Musters, W., 129 Myers, E. W., 30, 69 Myers, J., 342, 352(18) Myher, J. J., 189

N Nabavi, N., 285 Nakamura, K., 206, 214(40) Nakanishi, M. K., 172 Nakase, T., 255 Namboodiri, K., 30 Nazrul, H., 342 Ncube, I., 220, 223, 225, 228 Near, J. C., 80 Negre, A. E., 267, 270(37) Nelson, R. M., 323 N6sa, M.-P., 112 Neves, M. T., 61, 130, 133, 150, 152(5), 157 Newnham, H. H., 233 Nicholls, A., 134, 147, 149 Nichols, R., 285, 297(4) Nicklen, S., 263 Nickloff, J. A., 325 Nicolas, A., 31, 32(15), 34(31), 77, 303, 314(19) Nielsen, M., 233 Nielsen, P. F., 109, 162, 269, 329 Nielsen, T. B., 327, 333(31) Nielson, M., 172, 233 Nikazy, J., 177, 181,234, 245 Nikkila, E. A., 232 Nikoleit, K., 205 Nilsson, ,~., 186, 189, 297 Nilsson, J., 187, 188(24), 343, 344(23, 27) Nilsson, N. O., 273, 274(7), 280, 280(7) Nilsson, S., 273, 274(8, 11), 275(8), 277(9) Nilsson-Ehle, P., 281 Nishikawa, K., 255, 256(22) Noble, M. E., 5,19(21), 31, 34(24), 95,120,300 No~l, J.-P., 103 Noma, A., 262, 353 Norin, M., 14, 27, 27(34), 31, 33(18), 321, 370, 375 Norin, T., 27, 370 Noronha, L. C., 221 Norskov, L., 4, 15(8), 31, 34(26), 96, 157, 171, 300, 318 Notebom, M.H.M., 210

389

Noteborn, M.H.M., 267 Nury, S., 26, 354 Nykjaer, A., 172, 233

O Obradors, N., 254 O'Brien, J. S., 262 Odgen, R. C., 288 Oefner, C., 31, 32(10), 88 Oehrner, N., 375 Oesch, F., 102, 106(61) Oh, T.-K., 214, 217(51) Ohama, T., 255 Ohkuma, M., 249, 255 Ohrner, N., 14, 27(34), 31, 33(18) Ohta, A., 255 Ohuchi, T., 82 Oishi, M., 101, 102(54) Oizumi, J., 186 Okabe, H., 262 Okayama, H., 234 Okkels, J. S., 318 Okoniewska, M., 93, 99(28), 318 Okumura, S., 221 Olesen, K., 102 Olive, C., 241 Olivecrona, G., 172, 233 Olivecrona, T., 171,172(6), 185, 187, 188(1), 232, 244, 341 Oliver, J., 5, 6(20), 19(20), 95 Ollis, D. L., 4, 6(12), 7(12), 42, 51(48), 87-88, 91(20), 93(20), 95(20), 97(20), 106(20), 299, 319 Olsen, O., 321 Olsson, H., 272 Onsager, L., 133 Orengo, C. A., 86, 87(3) Orimo, H., 262 O'Rourke, E. C., 271 Orrison, B. M., 294 Ortaggi, G., 329 Osada, J., 233 Osawa, S., 255 Osborne, J. C., Jr., 244 Osman, S. F., 149 Osterberg, E., 219 Osterland, T., 104 ~sterlund, T., 273-274, 274(17), 275(18), 280, 280(18), 281(18), 283(18)

390

AUTHOR INDEX

Otani, Y., 197, 198(22), 199(22), 200, 206(22), 214(22), 220(22), 355, 357(18) Otero, C., 247, 251(7), 253(7) Ouzounis, C., 30, 86, 100(6) Overington, J. P., 44, 45(55), 86 Owusu, R. K., 195 Ozgur, L. E., 342, 352(18)

P Paavola, L. G., 172, 232 Pablo, G., 217 Padfield, P. J., 294 Paltauf, F., 353-354, 359, 363(13), 366(13), 367(13) Palva, I., 273 Panaiotov, I., 283 Panesar, N., 294 Pangborn, W., 21(25), 23(25), 31, 32(12), 98 Parekh, S. M., 303 Park, S. M., 255 Parker, A. G., 106 Parrott, C., 181,237, 245(31) Pasqualini, E., 343 Pathak, D., 87 Patil, S. S., 149 Patkar, S., 5, 9(23), 14, 14(23), 15(23), 17, 22(23), 23(23), 27(34), 31, 33(18), 34(25), 36, 96, 254, 318, 375 Patkar, S. A., 5, 15(14), 34(34), 35, 157, 301, 309(16), 317-318, 321,327-329, 333(31), 354, 370(6), 374(6) Patrick, A. D., 262 Patsch, J. R., 233 Patsch, W., 233, 262 Pattabiraman, N., 30 Paul, C. H., 132 Payne, R. M., 285, 289(5), 290(5), 291(5), 292(5), 296(5), 297(2) Pearson, W. R., 30, 66, 68(2, 3), 69(2, 3) Pease, L., 158 Pereira, M. E., 342 Perret, B., 233 Perry, H., 44 Pesole, G., 255, 256(22) Peters, G. H., 321 Petersen, E. I., 61, 130, 133, 150, 152(5) Petersen, S. B., 28, 42, 49, 51, 51(49), 53(49), 61-62, 69(1), 74, 130, 132(1), 133-134, 135(20), 150, 152(5), 157, 261

Peterson, C. M., 172 Petrosino, J., 60 Pfeifer, O., 100, 101(51) Pharmacia, 325 Philipp, B., 186 Phillips, M. L., 236, 238, 240, 242-243, 245 Philo, J. S., 236, 238, 240, 242-243, 245 Piazza, G. J., 231 Pierce, D. A., 220 Pi6roni, G., 261, 265, 270(14) Pierre, M., 106 Pilon, C., 171,234 Pina, M., 226, 228(18) Pinot, F., 106 Pioch, D., 283 Pizzey, J. A., 349 Pizzi, E., 253 Pladet, M. W., 172, 245 Plapp, B. V., 158 Pleiss, J., 353, 368, 370(29), 371(29) Pletnev, V. Z., 21(25), 23(25), 31, 32(12), 98 Polg~ir, L., 105 Poon, P. H., 236, 238, 240, 242-243, 245 Popp, F., 205 Porro, D., 257, 259(25) Postma, J.P.M., 373 Poulose, A. J., 298, 318, 321(2) Power, S., 298 Prasad, S., 233 Prescott, S. M., 104 Preston, F. W., 185 Pries, F., 102 Przybylz, A. E., 286 Ptitsyn, O., 60 Puigserver, A., 285 Puissant, C., 286, 287(9) Pullen, J. K., 158

0 Quici, S., 361

R Rabino, R., 329 Racke, M. M., 233, 239(20) Raeder, U., 355 Rahaman, A., 134 Rangheard, M. S., 221 Ranjan, A., 262

AUTHOR INDEX Ransac, S., 3, 25(4), 26(4), 261, 282, 362 Rappaport, A. T., 124 Rashin, A. A., 132, 134 Rasmussen, P. B., 109, 162, 269, 329 Rathelot, J., 108, 112, 127(5) Ravelli, R. B., 31, 32(16) Rawlings, N. D., 105 Read, J. S., 220, 223, 225, 228 Rechsteiner, M., 350 Rechtin, A. E., 233, 239(20) Recktenwald, A., 93, 99(28), 165, 318 Reddy, A.K.N., 135,137(23) Reed, A. W., 224(21), 228, 229(21), 230(21), 231(21) Reid-Miller, M., 44 Reiner, D. S., 342 Remington, S. J., 4, 6(12), 7(12), 42, 51(48), 88, 91(20), 93(20), 95(20), 97(20), 100, 106(20), 299, 319 Requadt, C., 298 Rety, S., 314, 315(28), 370 Reue, K., 187, 188(25), 273, 343, 344(21), 350(21) Rhee, J. S., 200 Richards, F. M., 137, 139(28), 145(30) Richardson, J., 86, 87(5), 88(5) Riley, D.J.S., 191 Rindler-Ludwig, R., 262 Rivi~re, C., 261,265, 270(14), 282 Rivi6re, M., 261 Roberts, I. M., 261 Roberts, J. D., 325 Robertson, D. L., 105 Robertus, J. D., 9 Rochat, H., 112 Rodgers, J. R., 30, 86 Rogalska, E., 3, 25(4), 26, 26(4), 123, 190, 191(45), 344-345, 354, 362 Rogers, S., 350 Roigaard, H., 172, 233 Roman, V., 198, 199(24), 205(24), 206(24), 214(24), 220(24) Ronan, R., 181, 237, 245(31) Ronk, M., 187, 188(25), 189(25), 344(21), 350(21) Rose, G. D., 145 Rosemblum, J. L., 269 Rosenbaum, L. M., 262 Rosenblum, J. L., 114 Rosenbluth, A. W., 134

391

Rosenbluth, M. N., 134 Rosenstein, R., 205 Ross, A. C., 342 Rossmann, M. G., 307 Roudani, S., 342-343, 346-347, 347(44), 348(47), 349(44) Rovery, M., 110, 112(15), 239 Roxby, R., 141 Rozeboom, H. J., 87, 106 R~ia, M. L., 194-195, 199(13), 201(13), 203(13), 206, 209(45), 212, 213(49), 247, 251(7), 253(7) Rubin, B., 5, 21(17), 26, 31, 32(13), 33(21), 98, 246-247, 247(2), 253(8), 300 Rubin, R. P., 352 Rudenko, G., 100 Ruffiner, R., 249 Rugani, N., 5, 9(16), 20(16), 23(16), 31, 33(19), 108, 112, 112(7), 157, 158(4), 301 Russell, A. J., 154 Russell, R. B., 35, 45 Rutter, W. J., 286, 343, 344(22) Ruyssen, R., 357 Rydel, T., 5, 6(20), 19(20), 95

S Sacchi, N., 286, 287(8) Saccone, C., 255, 256(22) Sadoulet, M.-O., 340 Sagar, I. H., 298 Sahlin, P., 187, 343 Saiki, R. K., 158 Saimler, S., 262 Saito, S., 281 Sakurada, T., 262 Sali, A., 86 Salvayre, R. S., 267, 270(37) Sambrook, J., 263, 288, 289(12), 303,325, 355 Sampogna, R., 141, 144(35) Sander, C., 30, 35, 44, 86-87, 100(6, 11) Sando, G. N., 103, 261-262, 262(13), 269(13), 270(13), 272(13) Sanger, F., 263 Sanglard, D., 249 Santamarina-Fojo, S., 20, 168, 171, 172(7), 181, 237, 244, 245(31) Sarda, L., 3, 20, 25(43), 108-109, 109(9), 112, 112(6), 158, 265, 299, 333 Sarma, R., 307

392

AUTHOR INDEX

Sato, Y., 104 SauniOre, J.-F., 265, 341 Saxty, B. A., 80 Sbarra, V., 343 Schafer, M. P., 233, 239(20) Schalk, I., 31, 32(11) Schanck, K., 3, 196-197, 198(15), 199(15), 217(15) Schank, K., 197, 197(23), 205(23), 206(23), 214(23), 220(23) Scharp6, S., 106 Scheele, G. A., 293 Schein, C. H., 210 Schleifer, K. H., 205 Schmid, R. D., 5, 19(22), 95,194-195,196(12), 197(12), 199(12, 13), 200(12), 201(13), 203(13), 206, 209(41, 45), 212, 213(49), 214(41, 42), 217(12, 41), 218(41), 353354, 355(5), 368, 370(29), 371(29), 374(7), 375(5) Schmidt, B., 298 Schmidt-Dannert, C., 194-195, 196(12), 197(12), 199(12, 13), 200(12), 201(13), 203(13), 206, 209(45), 212, 213(49), 217(12) Schmitz, G., 262 Schneider, M. P., 221 Schneider, R., 44, 87 Schomburg, D., 5, 6(20), 19(20, 22), 95, 354, 374(7) Schottel, J. L., 36 Schotz, M., 187, 188(25), 189(25) Schotz, M. C., 20, 99, 104, 171-172, 177, 181, 187, 232, 234-235, 244-245, 272-274, 274(11, 17), 275(18), 280, 280(18), 281(18), 283(18) Schotz, M. G., 343, 344(21), 350(21) Schrag, J., 4, 6(12, 13a), 7(12), 31, 33(21, 22), 42, 51(48), 299, 319 Schrag, J. D., 3-5, 6(20), 9(18), 14, 14(10), 15, 15(13a), 17, 19(20), 21(13, 17, 18), 23(19), 26-27, 31, 32(13), 34(27, 30), 50-51, 51(63), 85, 88, 91(20), 93, 93(20), 95, 95(20), 96, 97(20), 98, 99(19, 28, 49), 101, 106(20), 165, 246-247, 247(2), 253, 253(8), 254, 254(17), 300, 318, 375 Schubert, W. K., 262 Schulte, G., 196 Sehumacher, C., 82 Scott, K., 369

Scow, R. O., 261 Sebastian, J., 298 Sebastiao, M., 42, 51(49), 53(49), 74 Seebart, K. E., 181,245 Seghezzi, W., 249 Selj6e, F., 106 Semenkovich, C. F., 171-172 Serrano, L., 57 Serre, L., 91, 100(26) Serreqi, A. N., 5, 23(19), 26, 31, 33(21, 22), 247, 253(8), 375 Shabati, Y., 195 Shaft, S., 233 Shakhnovich, E., 60 Shamir, R., 342 Sharp, K., 132, 141,144(35), 146-147 Sharp, P. M., 44 Shauger, A., 263 Shaykh, M., 149-150 Shenkin, P. S., 60 Sheriff, S., 263, 272(28) Shield, J. W., 318, 321(2) Shimada, Y., 98, 199, 206(28), 214(28) Shimanouchi, T., 30, 86 Shin, J. J., 262 Shirai, K., 192, 281 Shire, S. J., 135, 142 Shively, J. E., 187, 188(25), 189(25), 234, 273, 274(11), 343, 344(21), 350(21) Shotz, M. C., 285 Shukla, V.S.K., 221 Sidebottom, C. M., 88 Silman, I., 4, 6(12), 7(12), 31,32(10, 11,16), 42, 50, 51(48, 63), 88, 91(20), 93(20), 95(20), 97(20), 101, 106(20), 253, 254(17), 299, 319 Simpson, C., 298, 312 Sims, H. F., 285, 289(5), 290(5), 291(5), 292(5), 296(5) Singh, M., 206, 355 Sjolander, K., 40 Sj6vall, J., 185 Slabas, A. R., 88 Slotboom, A. J., 103 Smith, G. E., 162, 274 Smith, L. C., 172 Smith, M., 322 Smith, P., 5, 21(17), 31, 32(13), 98, 246, 247(2), 300 Smith, T., 40

AUTHOR INDEX Sobek, H., 100, 101(51) Solfi, C., 254 Sonnet, P. E., 220-221 Sonnhammer, E. L., 45 Sonnleitner, B., 195 Soro, S, 329 Soumanou, M. M., 354 Sowdhamini, R., 86 Sparkes, R. S., 187, 273, 274(11) Spener, F., 5, 19(22), 95, 353-354, 363(13), 366(13), 367(13) Spilburg, C. A., 191,343 Sprauer, A., 212, 213(49) Sprengel, R., 233, 234(18) Sridhar, P., 262 Stadler, P., 353-354, 357(13), 363(13), 366(13), 367(13) Stafforini, D. M., 104 Stahnke, G., 171,233, 234(18), 244 St~lfors, P., 273 Stanssens, P., 150 Steinberg, D,, 272 Steinhilber, W., 293 Stenman, G., 343 Stenmark, G., 187 Stenson Holst, L., 273 Sternberg, M. J., 45, 154 Sternby, B., 185-188, 188(25), 189, 189(25, 31), 192(31), 343, 344(21), 350(21) Stewart, J.J.P., 372 St6cklein, W., 195, 196(12), 197(12), 199(12), 200(12), 206, 214(42), 217(12) Stoll, B. J., 342 Strgdfors, P., 272, 274(7), 280, 280(7), 281(5), 283(5) Stratowa, C., 343, 344(22) Strauss, A. W., 114, 269 Strauss, J. F. III, 172, 232 Strickland, D. K., 172, 233, 245, 246(7) Strickland, L., 5, 6(20), 19(20), 95 Str6mberg, K., 187, 343 Str6mqvist, M., 192, 349-350, 350(53), 352 Stuckmann, M., 196, 356 Stura, E. A., 111 Sturman, J. A., 341 Sugiara, M., 221 Sugihara, A., 98, 195, 197, 199, 199(21), 206(21, 28), 214(21, 28), 217(21), 220(21) Sugiyama, H., 255 Sugo, T., 188, 345

393

Sullivan, T. R., 232 Sullivan, T. R., Jr., 172 Sultan, F., 233 Sumida, M., 273 Summers, M. D., 162, 263, 274 Sung, E., 337, 338(44, 45) Sung, M.-H., 214, 217(51) Sussman, J. L., 4, 6(12), 7(12), 31, 32(10, 11, 16), 50, 51(63), 87-88, 91(20), 93(20), 95(20), 97(20), 101, 106(14, 20), 253, 254(17), 299, 319 Suzuki, T., 255, 256(22) Svendsen, A., 17, 317-318, 319(5), 321,327, 329, 333(31) Svenson, K. L., 171,234, 273-274, 274(11) Svensson, A., 274 Svensson, I., 227, 229 Swarovsky, B., 293 Sweet, R. M., 88 Sweigard, J. A., 149, 150(53) Swenson, L., 5, 6(24), 17, 22(37), 31, 34(28, 29), 36, 96, 300, 321,354, 369 Syed Rahmatullah, M.S.K., 221 Szittner, R., 91,100(26) Sztajer, H., 195, 196(12), 197(12), 199(12), 200(12), 206, 209(41), 214(41, 42), 217(12, 41), 218(41)

T Tada, S., 354 Taipa, M. A., 217 Takagi, A., 236 Takagi, M., 101, 102(54), 249, 255 Takagi, T., 200, 237 Takahara, M., 211 Talley, G. D., 20, 244 Tamura, G., 354 Tanford, C., 141 Tang, J., 189, 341,344, 346(41), 350, 352(41) Tani, T., 195, 197, 199(21), 206(21), 214(21), 217(21), 220(21) Taniguchi-Morimura, J., 51, 248, 249(10), 255(10) Tanimoto, T., 249 Tasumi, M., 30, 86 Taylor, A, K., 187 Taylor, J. M., 233, 236 Taylor, P., 31, 34(23) Taylor, W. R., 86

394

AUTHOR INDEX

Teissere, M., 328 Teller, A. H., 134 Teller, E., 134 Terpstra, P., 102 Tessier, D. C., 165, 268, 270(39), 271(39) Tetling, S., 200 Thellersen, M., 317 Thim, L., 3-5, 15(8, 14), 20(7), 34(34), 35, 96, 109, 157, 162, 171,249, 250(12), 269, 300-301,309(16), 318, 321,329, 354-355, 370(6), 374(6) Thioulsouse, J., 36, 37(39) Thirstrup, K., 109, 162, 269, 329 Thiry, M., 273 Thomaas, D. Y., 318 Thomas, C. E., 233 Thomas, D. Y., 93, 98, 99(28), 165, 254, 268, 270(39), 271(39) Thomas, P. G., 154 Thompson, J. D., 69 Thomsen, W. E., 335 Thornton, J. M., 39, 53, 55(69), 86, 87(3) Thouvenot, J. P., 109 Thuren, T., 181, 245 Tilbeurgh, H., 20 Tildesley, D. J., 134 Timasheff, S., 8 Timmis, K. N., 101,102(55), 103(55) Tiruppathi, C., 261 Tjeenk, M. L., 103, 217 Tjoelker, L. W., 104 Tobback, P. P., 227, 228(19) Togni, G., 249 Toker, L., 31, 32(10), 88 Tolley, S., 4, 15(8), 31, 34(26), 96, 157, 171, 300, 318 Tominaga, Y., 98, 195, 197, 199, 199(21), 206(21, 28), 214(21, 28), 217(21), 220(21) Tonk, M., 343 Tooze, J., 49 Tornqvist, H., 273, 280-281,281(27) Toxvaerd, S., 321 Trabitzsch, U., 337, 338(44, 45) Tramontano, A., 98, 102(45), 247, 251(6), 253, 254(6) Tran, H., 172, 245 Triantaphylides, C., 221 Trimble, E. R., 294 Trong, H. L., 104

Tsuchiya, K., 354 Tsujisaka, Y., 221,353 Tsujita, T., 335 Tsunasawa, S., 98 Tsunesawa, S., 199, 206(28), 214(28) Tu, L., 233 Tuparev, G., 30, 86, 100(6) Turkenburg, J. P., 4-5, 15(8, 14), 31, 34(26, 34), 35, 96, 157, 171, 300-301, 309(16), 318, 321,354, 370(6), 374(6)

U Ueda, T., 255, 256(22) Ueshima, M., 199, 206(28), 214(28) Unger, J., 104 Uppenberg, J., 5, 9(23), 14, 14(23), 15(23), 22(23), 23(23), 27(34), 31, 33(18), 34(25), 96, 254, 300, 375 Upton, C., 80, 104 Usson, N. U., 186

V Valent, B., 149, 150(53) Valero, F., 254 Valla, S., 134, 135(20) van Bemmel, C. J., 119 Van Cauwenbergh, S. M., 227, 228(19) van der Eb, A. J., 267 van der Hijden, H. T., 14, 127, 149 van der Ploeg, J., 102 van Gemeren, I. A., 129 van Gunsteren, W. F., 134, 373 van Hondel, C.A.M.J.J., 129 Vanhoof, G., 106 Vanoni, M., 247 Van Oort, M. G., 217 van Opdenbosch, N., 372 van P6e, K.-H., 100, 101(51) van Tilbeurgh, H., 3, 5, 9(16), 20, 20(16), 23(16, 44), 25(4, 43), 26(4), 31, 33(19, 20), 34(31), 77, 107-108, 112, 112(6-8), 157158, 158(4), 301, 303, 314(19), 375 Vantuien, P., 233, 239(19) Vaughan, M., 272, 281 Vaz, L., 42, 51(49), 53(49), 74 Vemag, C., 370

AUTHOR INDEX Verger, R., 3, 5, 9(16), 20, 20(7, 16), 23(16, 44), 25(4, 43), 26, 26(4), 31, 33(19, 20), 34(31), 77, 103, 108-109, 112, 112(6-8), 113, 121, 123, 157-158, 185, 261, 265, 270(14), 272, 282-283,292,297, 301,303, 314(19), 333, 335, 354, 362, 375 Verheij, H. M., 14, 103, 127, 205, 217 Verhey, H. M., 153, 318, 333 Verma, C., 314, 315(28) Vernet, T., 93, 98, 99(28), 165, 254, 268, 270(39), 271(39), 318 Verrips, C. T., 31, 32(15), 96, 129, 318 Verschueren, K.H.G., 4, 6(12), 7(12), 88, 91(20), 93(20), 95(20), 97(20), 106, 106(20), 299, 319 Vezina, C., 233 Vieu, C., 233 Vilas, U., 268, 270(38) Villeneuve, P., 226, 228(18) Virtanen, I., 232 Vis, H. L., 341 Visher, R. C., 266 Visser, C., 96 Vogt, G., 44 Volpenhein, R. A., 359 von Heijne, G., 269 von Hippel, P. H., 237 Vriend, G., 30, 86, 100(6), 106

W Waagen, V., 14, 27(34), 31, 33(18), 375 Wagner, A. C., 292, 293(19) Wagner, F., 198, 217(26), 218(26) Wahl, G. M., 288 Wahl, S., 212, 213(49) Wainwright, T. E., 134 Waite, M., 181,245 Waknitz, M. A., 172 Wallace, A. C., 53, 55(69) Walther, B. T., 192 Wang, C.-S., 187, 189,189(19), 190(19,22, 44), 192(19), 341, 344, 346(41), 350, 352(41) Wang, H., 285 Wang, J., 233 Wang, K., 177, 245 Wang, S. C., 342 Wang, Y., 101, 102(56)

395

Ward, S. C., 342 Warner, T. G., 262 Watanabe, K., 255, 256(22) Waterman, M., 40 Wawrzak, Z., 21(25), 23(25), 31, 32(12), 98 Weber, W., 172 Wehtje, E., 227 Wei, Y., 5, 6(24), 17, 22(37), 31, 34(28, 29), 36, 91, 96, 100(26), 300, 321,354, 369 Weiner, P. K., 134 Weissfloch, A.N.E., 124 Wek, S. A., 279 Wells, J. A., 298 Wells, R., 350 Wells, T. N., 80 Wen, J., 236, 238, 240, 242-243, 245 Weng, J., 30 Wenzig, E., 217 Wessel, D., 275, 275(23b) Wicker-Planquart, C., 261, 285 Wickner, W., 206, 211(39) Wiegand, R. C., 343, 344(24) Wilke, D., 198, 199(24), 205(24), 206(24), 214(24), 220(24) Will, H., 171, 233, 234(18), 244 Williams, G.J.B., 30, 86 Williams, J. A., 285, 292-293,293(19), 297(4) Williams, S. E., 172, 245 Wilson, I. A., 111 Wilson, L., 298 Wimmer, H., 363 Wims, M., 171 Windust, J., 88 Winkler, F. K., 4, 6(9), 20(9), 51, 70, 71(7), 97-98, 99(49), 108, 109(3), 112(3), 157, 244, 300 Winkler, K. E., 342 Winkler, U. K., 196,198,200,217(25), 328,356 Wishart, M. J., 285, 292, 293(19), 297(4) Withers-Martinez, C., 109 Withiam-Leitch, M., 352 Witholt, B., 102 Wldike, H., 162 Wodak, S. J., 30 Wohlfahrt, G., 353-354, 355(5), 374(7), 375(5) WOldike, H., 3, 20(7), 109, 269 W¢ldike, H., 329 Wolever, T. S., 233

396

AUTHOR INDEX

Wong, H., 171-172, 177, 181, 187, 188(25), 189(25), 232-236, 238, 240, 242-245, 246(7), 343, 344(21), 350(21) Wong, K. R., 105 Woods, C. W., 233, 239(20) Wu, S., 4, 14(10), 88, 98(19), 298 Wu, T., 44

Yeh, J.-C., 344, 346(41), 352(41) Yokogawa, T., 255, 256(22) Yoo, O. J., 200 York, C. M., 341 Yosida, S., 281 Yu, C., 221,231

Z X Xu, Y. Y., 350

Y Yadwad, V. B., 221 Yamaguchi, S., 5, 6(24), 31, 34(28), 96, 300, 321 Yamamoto, A., 236 Yamashita, O., 104 Yang, A. S., 141, 144(35), 146 Yang, D., 171,232,236, 238, 240,242-243,245 Yano, K., 101, 102(54), 249 Yao, C., 149 Yarranton, G. T., 261,270(14) Yates, M. T., 233 Yeaman, S., 273 Yee, D. P., 86

Zakour, R. A., 325 Zambalik, J. L., 187 Zambaux, J., 187, 188(25), 189(25), 343, 344(21), 350(21) Zantema, A., 267 Z~ivsdosky, P., 194 Zechner, R., 328 Zehfus, M. H., 145 Zhang, S. H., 233 Zhu, Z. Y., 86 Zimmerman, G. A., 104 Ziomek, E., 5, 15, 23(19), 31, 33(22), 93, 96, 99(28), 165, 247, 318, 375 Zolfaghari, R., 342 Zoller, M. J., 322 Zuegg, H., 25 Zuegg, J., 27 Zundel, M., 329

SUBJECT INDEX

397

Subject Index

A ACC_RUN, computation of solvent accessibility, 145 Acetyleholinesterase analysis of conserved motifs with MULTIM, 70, 74 a/fl-hydrolase fold enzyme identification by sequence comparison, 101-102 Affinity chromatography, lipases heparin affinity chromatography carboxyl ester lipase from human milk, 190-191 hepatic lipase, 235-239 reversible inhibitor matrices, 328

B Bacillus thermocatenulatus lipases

biological functions, 220 BTL-1 aggregation, 198, 217 amino acid analysis, 198-199 amino terminal sequencing, 198 physical properties, 199-200 purification anion-exchange chromatography, 197 extraction, 197 gel electrophoresis, 197 large-scale fermentation, 196 screening of bacteria, 195-196 specific activity, 214 substrate specificity, 199-200 BTL-2 aggregation behavior, 217-219 amino terminal sequencing, 205, 213 catalytic triad, 203, 205 detergent effects on activity, 214-215

gel electrophoresis, 205-206 gene cloning, 200-201 homology with other lipases, 205, 219 overexpression in Escherichia coli expression level analysis, 207, 209 heat shock induction, 206-207, 209-211 processing, 209-211 vectors, 206-207 pH optimum, 214, 220 physical properties, 200, 205-206, 213-214 purification protein under native promoter, 205 recombinant protein in Escherichia coli, 211-213 sequence analysis, 202-205 specific activity, 214 substrate specificity, 215, 217 thermostability, 194-195, 214, 220 Baculovirus-insect cell expression system, see Gastric lipase; Hormone-sensitive lipase; Lysosomal acid lipase; Pancreatic lipase; Pancreatic lipase-related proteins 1,3-O-Benzylideneglycerol, synthesis, 358-359 Bile salt-activated lipase, see Carboxyl ester lipase Bromoperoxidase A2, a/fl-hydrolase fold enzyme identification by sequence comparison, 102 structure, 101 BTL-1, see Bacillus thermocatenulatus lipases BTL-2, see Bacillus thermocatenulatus lipases

398

SUBJECT INDEX

C Candida cylindracea cholesterol esterase

active site residues, 51, 53 geometric constraints and conserved residues, 53, 55 hydrogen bonding, 55-56 ligand binding, 53 nonpolar interaction analysis, 57, 59, 60 Candida rugosa lipase genes chromosomal localization, 250-251 cloning, 247-249 CUG serines mutagenesis for heterologous expression, 259-260 occurrence and frequency, 255-257 regulation of expression, 254 sequence analysis, 251-254 glycosylation, 246 heterologous expression in Saccharom y c e s cerevisiae, 257, 259-260 industrial applications, 246 isoenzyme separation, 247, 329 Michaelis-Menten kinetics of hydrolysis triolein, 230-232 trivernolin, 230-232 three-dimensional structure a/13-Hydrolase fold, 98 inhibitor complexes, 5, 23-25 lid domain, 21-22 oxyanion hole, 9, 14 Carboxyl ester lipase, see also Candicla cylindracea cholesterol esterase analysis of conserved motifs with MULTIM, 74, 76 bile salt recognition, 188 catalytic triad, 344 dietary supplementation with recombinant protein, 342 discovery, 187 gene cloning, 187, 343 locus, 187 structure, 343-344 glycosylation carbohydrate content, 187-188 drug blocking of glycosylation, 348-349 feto-acinar pancreatic protein, 346-348 linkage in disease, 188, 347

O-linked oligosaccharide structure, 346 pancreatic enzyme linkage types, 188, 344-345 N-linked oligosaccharide role in activity, 349, 351-352 N-linked oligosaccharide structure, 345-346 O-linked oligosaccharide role in activity, 349-352 site-specific mutagenesis of sites, 348350, 352 nomenclature, 186-187 physiological functions, 185, 341-342, 352 purification history of methods, 189 human milk enzyme abundance, 341 casein removal, 190 cream removal, 190 gel filtration, 191 heparin affinity chromatography, 190-191 human pancreatic enzyme bile salt removal, 194 cation-exchange chromatography, 194 cholate affinity chromatography, 192-193 gel electrophoresis, 194 gel filtration, 193-194 stability of purified enzyme, 188 structure, 188 substrate specificity, 186, 188-189, 340-341 CE, see Continuum electrostatics Cholesterol esterase, see Carboxyl ester lipase; C a n d i d a cylindracea cholesterol esterase Citrate lyase, analysis of conserved motifs with MULTIM, 80 lcle, see C a n d i d a cylindracea cholesterol esterase Colipase, see Pancreatic lipase Conformation, see Three-dimensional structure, lipases CONNECT, see MULTIM Conserved residue, see Sequence analysis Continuum electrostatics, see Electrostatic interactions CRL, see Candida rugosa lipase

SUBJECT INDEX Cutinase analysis of conserved motifs with MULTIM, 77, 80 electrostatic potential visualization, 152-153 Fusarium solani pisi enzyme backbone structure, 125 denaturation by detergents, 126-127 a/fl-hydrolase fold, 301 interracial activation, 125-127 oxyanion hole, 301 rotational correlation time with lithium dodecyl sulfate as measured by time-resolved fluorescence, 125-126 substrate specificity and mechanism, 127-129 a/B-hydrolase fold, 95, 100 pH optima, 150 physiological function, 299 simulated pH titration curves, 150-152 substrate specificity, 149

D DelPhi, calculation of electrostatic potential, 145-147 2-Deoxy-2-hexadecanoylamino-sn-glycerol, synthesis, 360-361 2-Deoxy-2-hexadecyl-sn-glycerol, synthesis, 361-362 2-Deoxy-2-phenyl-sn-glycerol, synthesis, 362

E Electrostatic interactions ACC_RUN, computation of solvent accessibility, 145 catalysis role, 132-133 continuum electrostatics charge sources, 136 Coulomb's law, 134 counterions, 136-137 dielectric constants, 135-136, 154 molecular surface determination, 137 Poisson-Boltzmann equation in modeling, 135, 137, 140, 145-146 contributions to total electrostatic energy, 132-133

399

DelPhi, calculation of electrostatic potential, 145-147 dipole types, 135 force fields of proteins, 132 Grasp, visualization of electrostatic potential, 147, 152 modeling approaches, 133-134 molecular recognition role, 130, 132, 154 pH effects on proteins, modeling applications in protein engineering, 153 cutinase, 150-152 importance of modeling, 137

pKa calculation with HendersonHasselbach equation, 139 effective value, 138, 141-144 intrinsic value, 138, 140, 142-143 perturbation by environment, 138-141 titratable residues, 138 types in modeling, 138-141 Poisson-Boltzmann equation in modeling, 135, 137, 140 TITRA program flow of program, 141-143 mean field titration, 144-145 site-site coupling factors, 144 Tanford-Kirkwood sphere model, 141-142 surface-positioned residues, 130 Esterase, see also specific esterases conserved residue identification classification of residues, 44-45 definition of conserved positions, 45, 49 explaining conservation patterns active site residues, 51, 53 geometric constraints and conserved residues, 53, 55 hydrogen bonding, 55-56 ligand binding, 53 nonpolar interaction analysis, 57, 59, 60 limitations, 60-61 odfl-hydrolase fold in serine esterases, 106 MULTIM analysis of conserved motifs CONNECT program, 67 database searching for homologous sequences, 69

400

SUBJECT INDEX

file types, 63-64 FILTER program, 66-67 flow chart, 63 MOTIF program, 64-66 program overview, 62 multiple sequence alignment construction, 43-44 sequence data set adding new sequences to initial alignment, 40-41 defining, 39-40 iteration of search process, 41-43 searching with hidden Markov approach, 40-41 three-dimensional data set alignment with Stamp program, 35-36, 39 identification of relevant structures, 30-31, 35 importance in sequence analysis, 29 structural subset identification, 36

F FAP, see Feto-acinar pancreatic protein Fatty acid selectivity, lipases determination hydrolysis of triglyceride mixtures in isooctane, 228-229 lipase preparation, 227 solvent selection, 226-227 substrate selection, 226 transesterification of tricaprylin with fatty acid mixtures, 229-230 water activity control, 227 industrial applications, 220-221 Feto-acinar pancreatic protein, see Carboxyl ester lipase FILTER, see MULTIM

G Gastric lipase, human baculovirus-insect cell expression system cell infection, 264 glycosylation of protein, 266-267 polyhedrin gene substitution, 262-263 signal peptides, effects on expression and secretion, 265-266, 268-269, 271-272 vector construction, 263-264

gene cloning, 263 a//3-hydrolase fold identification, 103-104 physiological function, 261 GCL, see G e o t r i c h u m c a n d i d u m lipase G e o t r i c h u m c a n d i d u m lipase a//3-hydrolase fold enzyme identification by sequence comparison, 101-102 structure, 98-99 lid-forming domain over active site, 21-22 substrate specificity, 220 Grasp, visualization of electrostatic potential, 147, 152

H Haloalkane dehalogenase, a//3-hydrolase fold enzyme identification by sequence comparison, 102-102, 106 Heparin affinity chromatography carboxyl ester lipase from human milk, 190-191 hepatic lipase, 235-239 Hepatic lipase engineering construction with polymerase chain reaction, 174-177 domain-exchanged chimeric lipase with lipoprotein lipase construction, 175-177 effects on activity, 181 expression, 177 structure, 173-174 structure-function elucidation, 172-173 human enzyme homology with other lipases, 244 kinetic properties, 236-237 purification of recombinant enzyme dextran sulfate chromatography, 236 heparin affinity chromatography, 235-239 hydrophobic affinity chromatography, 235 hydroxylapatite chromatography, 235 recombinant protein expression in Chinese hamster ovary cells, 234-235 structure, 233-234 subunit structure determination dimerization, 244-246

SUBJECT INDEX gel filtration and intensity light scattering, 237-239 radiation inactivation, 241-244 sedimentation equilibrium ultracentrifugation, 239, 241 physiological function, 171-172, 232-233 2-O-Hexadecyl-sn-glycerol, synthesis, 359-360 Hexadecyl methane sulfonate, synthesis, 359 High-performance liquid chromatography, lipase stereoselectivity assay enantiomer separation, 364-367 enzymatic hydrolysis of triacylglycerols and analogs, 363-364 esterification of monoacylglycerols and analogs with oleic acid, 364 preparative thin-layer chromatography, 364-365 principle, 362-363 HL, see Hepatic lipase Hormone-sensitive lipase assay lipid substrates, 280-281 monomolecular films with surface barostat detection buffer, 282-283 enzyme preparation, 283 incubation conditions, 283-284 limitations, 282 principle, 282 trough, 282 water-soluble substrates, 281-282 gene cloning, 273-274 a//3-hydrolase fold identification, 104, 274 phosphorylation, 280, 284 physiological role, 272-273 purification from baculovirus-insect cell expression system comparison of properties to native enzymes, 280 detergent selection, 278-279 human enzyme, 278 rat enzyme anion-exchange chromatography, 276-278 detergent solubilization, 276 homogenization of cells, 275 hydrophobic affinity chromatography, 277

401

large-scale suspension cultures, 275 recombinant baculovirus generation, 274 sequence homology with other lipases, 273-274 stability of purified enzymes, 279 substrate specificity, 280 HPLC, see High-performance liquid chromatography HSL, see Hormone-sensitive lipase Humicola lanuginosa lipase assays olive oil as substrate, 330-331 tributyrine as substrate, 330 interracial activation, 319-320 lipid contact zone, 319 site-specific mutagenesis effects on activity, 331-333 engineering goals activity, 321-322 binding improvement to negativelycharged surfaces, 320 hydrophobicity increase in lipid contact zone, 321 lid opening improvement, 321 stability, 321-322 gene cloning, 322 influence of alcohol ethoxylates studied in mixed monolayers, 334-336 mutant construction, 322-326 purification of mutants expressed in Aspergillus oryzae

D96L mutant, 331-332 hydrophobic interaction chromatography, 331 ion-exchange chromatography, 329, 331 W89 mutants, 332 wash effects, 339-340 surfactant effects assay, 334-336 mechanisms of activity alteration, 334 stability of enzyme, 333 wash effect evaluation, 336-340 a//~-Hydrolase fold catalytic triad catalytic acid, 93, 107 handedness and stereoselectivity, 9394, 99 histidine, 93

402

SUBJECT INDEX

nucleophile elbow, 91-94 sequence, 88 discovery in lipases, 4, 87-88, 94, 318-319 distribution in proteins, 107 enzyme identification by sequence comparison, 101-103 identification in lipases with limited sequence similarity hormone-sensitive lipase, 104 lingual, gastric, and lysosomal acid lipase, 103-104 platelet-activating factor acetylhydrolase, 104 Staphylococcus hyicus lipase, 103 uncharacterized lipases, 104-105 site-specific mutagenesis analysis, 99, 103-104, 106 structure, 6, 88, 91 bromoperoxidase A2, 101 Candida antarctica B lipase, 96 Candida rugosa lipase, 98 cutinase, 95, 100 features common to lipases, 99-100 Geotrichum candidum lipase, 98-99 pancreatic lipase, 97-99 Pseudomonas lipases, 95-96, 299-301 R h i z o m u c o r rniehei lipase, 96-97 sequence dependence, 87, 103 serine esterases, 106 serine peptidases, 101,105-106 thioesterase, 100-101 versatility, 98-99

Industrial applications, lipases, 119, 220221,246, 303, 317-318, 336-340 Interfacial activation, lipases cutinase, 125-127 Humicola lanuginosa lipase, 319-320 lid domain role, 158, 168-170 models, 121 overview, 3 Pseudomonas lipases, 121 Isoelectric focusing, lipases, 328

L Lid domain, see Three-dimensional structure, lipases

y-Linolenic acid, enrichment by lipases, 221 Lipase, see specific lipases Lipase fold, see cd/3-Hydrolase fold Lipoprotein lipase analysis of conserved motifs with MULTIM, 70 engineering construction with polymerase chain reaction, 174-180 domain-exchanged chimeric lipase with hepatic lipase construction, 175-177 effects on activity, 181 expression, 177 structure, 173-174 factor Xa cleavage site incorporation construction, 177-179 description of mutants, 174 effects on heparin binding, 182-183 monomer-repeat lipase activity, 183-184 construction, 179-180 description, 174 heparin binding, 184 structure-function elucidation, 172-173 physiological function, 171-172 subunit orientation, 183-184, 244-245 LPL, see Lipoprotein lipase Lysosomal acid lipase, human assay, 267 baculovirus-insect cell expression system cell infection, 264 polyhedrin gene substitution, 262-263 signal peptides, effects on expression and secretion, 267-269, 271-272 vector construction, 263-264 deficiency in disease, 262 gene cloning, 263 a//3-hydrolase fold identification, 103-104 physiological function, 262

M MOTIF, see MULTIM MULTIM, analysis of conserved motifs acetylcholinesterases, 70, 74 bile salt-activated lipase, 74, 76 citrate lyase, 80 combinatorial explosion, 84-85 CONNECT program, 67 cutinase, 77, 80

SUBJECT INDEX database searching for homologous sequences, 69 file types, 63-64 FILTER program, 66-67 flow chart, 63 limitations, 83-85 lipoprotein lipases, 70 MOTIF program, 64-66 nodule-25 protein, 76-77 phosphatidylinositol phosphatase, 82-83 program overview, 62 succinate-CoA ligase, 80 virus envelope proteins, 80, 82

N Nodule-25 protein, analysis of conserved motifs with MULTIM, 76-77

O 2-Octadecanoyl-sn-glycerol, synthesis, 360 Oxyanion hole, s e e Three-dimensional structure, lipases

P Pancreatic lipase classification, 107-108 conformations, 108 crystallization guinea pig related protein, 2 horse enzyme, 110-111 human enzyme, 109-110 human lipase-human colipase-Cll complexes, 114 human lipase-porcine colipase complex, 112-113 human lipase-porcine colipase-Cll complexes, 113 human lipase-porcine colipasephosphatidylcholine complex, 113 lipase-phospholipase chimeric enzyme, 111 porcine lipase-porcine colipase complex, 114 crystal packing classic lipases, 114-115 guinea pig related protein, 2 lipase-colipase complex, 115

403

lipase-colipase-inhibitor complexes, 115-119 lipase-phospholipase chimeric enzyme, 115 a//3-hydrolase fold, 97-99 lid-forming domain over active site deletion effects, 109 structure, 20, 108 physiological function, 171 signal peptide, application in baeulovirus-insect cell expression of heterologous proteins, 269, 271-272 site-specific mutagenesis of human pancreatic lipase carboxy terminal domain truncation, 166-167 catalytic triad residue mutations aspartate, 164-166 histidine, 164 serine, 163-164 expression systems COS cell transfectants, 160-161 baculovirus-insect cell system, 161-162 lid domain mutation effects colipase interactions, 168-169 interfacial activation, 168-170 substrate specificity, 168 mutagenesis by polymerase chain reaction, 158-160 purification of recombinant lipases, 162-163 tertiary structure evaluation, 163 Pancreatic lipase-related proteins expression and purification from baculovirus-insect cell system, 296-297 gene regulation, 285-286, 290-291 PLRP1, inactivity and physiological function, 297 PLRP2 activity, 297 RNA analysis blotting to detect specific message RNA dot blots, 288-291 gel electrophoresis and transfer to membranes, 288 hybridization conditions, 289 probes, 289 isolation guanidium thiocyanate-phenolchloroform extraction, 286-288

404

SUBJECT INDEX

pancreas handling, 286 temporal expression, 290-291 tissue distribution, 289 secretion analysis antibody preparation, 291-292 effects of animal age in rats, 295 immunoblotting, 293, 295 primary acinar cell preparation, 293-295 tissue culture of acinar cells, 292-293 sequence homology with pancreatic lipase, 285 PCR, see Polymerase chain reaction PDB, see Protein Data Base Phosphatidylinositol phosphatase, analysis of conserved motifs with MULTIM, 82-83 pK~, pH effects on proteins and modeling applications in protein engineering, 153 calculation with Henderson-Hasselbach equation, 139 cutinase, 150-152 effective pK., 138, 141-144 importance of modeling, 137 intrinsic pKa, 138, 140, 142-143 perturbation by environment, 138-141 Poisson-Boltzmann equation in modeling, 135, 137, 140 TITRA program flow of program, 141-143 mean field titration, 144-145 site-site coupling factors, 144 Tanford-Kirkwood sphere model, 141-142 titratable residues, 138 types of pKa in modeling, 138-141 PLRP1, see Pancreatic lipase-related proteins PLRP2, see Pancreatic lipase-related proteins Polymerase chain reaction construction of engineered lipases, 174-180 site-specific mutagenesis, 158-160, 323-324 Protein Data Base families of proteins, 86 keyword searching of entries, 30-31, 35 Protein fold, see also a/fl-Hydrolase fold estimation of number of unique folds, 86 sequence dependence, 86-87

Pseudomonas lipases

assay interracial activation, 121 reaction conditions using pH-stat equipment, 121 calcium binding, 327 c~//3-hydrolase fold, 95-96, 299-301 lid-forming domain over active site, 17, 19, 119-120 Pseudomonas glumae lipase backbone structure, 120-121 stereoselectivity, 123-124 Pseudomonas mendocina lipase assays immobilized substrate, 304 pH-stat titration, 305 crystallization, 306-307, 309 E600 inhibitor binding, 315 Fusarium solani pisi cutinase homology, 301,303, 315 industrial applications, 303 R202L/Y203L mutant crystal lattice interactions, 309, 312, 314, 316 crystallization, 306-307, 309, 316 refinement of crystal structure, 307, 309 three-dimensional structure compared to native enzyme, 312-314 triglyceride substrate specificity, 304-305, 314-315, 317 site-specific mutagenesis construction of mutants, 303 effects on activity, 304-305 selection, 303 substrate specificity, 298-299, 315 R

Radiation inactivation, subunit structure determination of hepatic lipase gel electrophoresis, 242, 244 inactivation curves, 242-244 principle, 241 sample preparation and irradiation, 241-242 Reverse micelle, lipase purification, 327 Rhizopus oryzae lipase assay activity, 357

SUBJECt INDEX

stereoselectivity enzymatic hydrolysis of triacylglycerols and analogs, 363-364 esterification of monoacylglycerols and analogs with oleic acid, 364 high-performance liquid chromatography of enantiomers, 364-367 preparative thin-layer chromatography, 364-365 principle, 362-363 factors governing stereoselectivity, 374-376 gene, 353 homology with other lipases, 353-354 processing, 353 purification native enzyme, 356-357 recombinant enzyme from Escherichia coli

chromatography, 357 gene cloning, 354-355 inclusion body solubilization, 355-356 transformation and expression, 355 substrate interactions, computer-aided modeling docking of sn-2 and sn-3/1 chain, 371-372 docking of tetrahedral intermediate of l(3)-octanoylglycerol, 370 hydrophobic acyl chain-binding sites, 370 open form of enzyme, 369-370 oxyanion stabilization, 373-374 relaxation of complex, 372-373 steric effects, 374 ' substrate orientation, 373 substrate preparation acylation of monoacylglycerol analogs, 362 1,3-O-benzytideneglycerol, 358-359 2-deoxy-2-hexadecanoylamino-snglycerol, 360-361 2-deoxy-2-hexadecyl-sn-glycerol, 361-362 2-deoxy-2-phenyl-sn-glycerol,362 hexadecyl methane sulfonate, 359 2-O-hexadecyl-sn-glycerol, 359-360 2-octadecanoyl-sn-glycerol, 360 substrate specificity, 353-354, 367-368, 374-376

405

S Sequence analysis BTL-2, 202-205 Candida rugosa lipases, 251-254 conserved residue identification classification of residues, 44-45 definition of conserved positions, 45, 49 explaining conservation patterns active site residues, 51, 53 geometric constraints and conserved residues, 53, 55 hydrogen bonding, 55-56 ligand binding, 53 nonpolar interaction analysis, 57, 59, 60 limitations, 60-61 hormone-sensitive lipase, 273-274 ct//3-hydrolase fold enzyme identification by sequence comparison, 101-103 MULTIM analysis of conserved motifs acetylcholinesterases, 70, 74 bile salt-activated lipase, 74, 76 citrate lyase, 80 combinatorial explosion, 84-85 CONNECT program, 67 cutinase, 77, 80 database searching for homologous sequences, 69 file types, 63-64 FILTER program, 66-67 flow chart, 63 limitations, 83-85 lipoprotein lipases, 70 MOTIF program, 64-66 nodule-25 protein, 76-77 phosphatidylinositol phosphatase, 82-83 program overview, 62 succinate-CoA ligase, 80 virus envelope proteins, 80, 82 multiple sequence alignment, 43-44, 69 overview, 28-29 sequence data set adding new sequences to initial alignment, 40-41 defining, 39-40 iteration of search process, 41-43 searching with hidden Markov approach, 40-41

406

SUBJECT INDEX

three-dimensional data set alignment with Stamp program, 35-36, 39 identification of relevant structures, 30-31, 35 importance in sequence analysis, 29 structural subset identification, 36 Serine peptidases catalytic triad, 105-106 a//3-hydrolase fold enzyme identification by sequence comparison, 102 structure, 101,105-106 Site-specific mutagenesis glycosylation sites, 348-350, 352 human pancreatic lipase carboxy terminal domain truncation, 166-167 catalytic triad residue mutations aspartate, 164-166 histidine, 164 serine, 163-164 expression systems baculovirus-insect cell system, 161-162 COS cell transfectants, 160-161 lid domain mutation effects colipase interactions, 168-169 interfacial activation, 168-170 substrate specificity, 168 mutagenesis by polymerase chain reaction, 158-160 purification of recombinant lipases, 162-163 tertiary structure evaluation, 163 Humicola lanuginosa lipase effects on activity, 331-333 engineering goals activity, 321-322 binding improvement to negativelycharged surfaces, 320 hydrophobicity increase in lipid contact zone, 321 lid opening improvement, 321 stability, 321-322 gene cloning, 322 influence of alcohol ethoxylates studied in mixed monolayers, 334-336 mutant construction, 322-326

purification of mutants expressed in Aspergillus oryzae D96L mutant, 331-332 hydrophobic interaction chromatography, 331 ion-exchange chromatography, 329, 331 W89 mutants, 332 wash effects, 339-340 a//3-hydrolase fold, 99, 103-104, 106 lid domain, 17, 109, 167-170 Pseudomonas mendocina lipase construction of mutants, 303 effects on activity, 304-305 R202L/Y203L mutant crystal lattice interactions, 309, 312, 314, 316 crystallization, 306-307, 309, 316 refinement of crystal structure, 307, 309 three-dimensional structure compared to native enzyme, 312-314 triglyceride substrate specificity, 304-305, 314-315, 317 selection, 303 Stamp program, alignment of three-dimensional structures, 35-36, 39 Stereoselectivity, lipases catalytic triad handedness and stereoselectivity, 93-94, 99 Pseudomonas glumae lipase stereoselectivity, 123-124 Rhizopus oryzae lipase assay enzymatic hydrolysis of triacylglycerols and analogs, 363-364 esterification of monoacylglycerols and analogs with oleic acid, 364 high-performance liquid chromatography of enantiomers, 364-367 preparative thin-layer chromatography, 364-365 principle, 362-363 factors governing stereoselectivity, 353-354, 367-368, 374-376 three-dimensional structure analysis, 26-27 Suecinate-CoA ligase, analysis of conserved motifs with MULTIM, 80

SUBJECT INDEX

T Thioesterase, a//3-hydrolase fold, 100-101 Three-dimensional structure, lipases Candida rugosa lipase inhibitor complexes, 5, 23-25 oxyanion hole, 9, 14 catalytic triad accessibility, 4, 7-8, 23 structure, see cd/3-Hydrolase fold conformations closed, 8-9 crystallization condition effects, 8, 10-11 open, 8-9 evaluation after site-specific mutagenesis, 163, 312-314 lid-forming domains over active site Candida antarctica lipase, 14-15, 22-23 Candida rugosa lipase, 21-22 Geotrichum candidum lipase, 21-22 mobility, 22-23 open versus closed conformation, 15, 17, 19, 157-158, 319 pancreatic lipase, 20, 108 Pseudomonas lipases, 17, 19, 119-120 Rhizomucor miehi lipase, 15, 17, 22 site-specific mutagenesis studies, 17, 109, 167-170 variation between lipases, 6-7, 14-15, 17, 19-22 lipase fold, see a/fl-Hydrolase fold orientation on membrane, 25-26 oxyanion hole, 9, 14, 77, 80 Pseudomonas mendocina lipase R202L/ Y203L mutant crystal lattice interactions, 309, 312, 314, 316 crystallization, 306-307, 309, 316 refinement of crystal structure, 307, 309 three-dimensional structure compared to native enzyme, 312-314 triglyceride substrate specificity, 304305, 314-315, 317 Rhizomucor mucor lipasc, inhibitor complexes, 4-5 Rhizopus oryzae lipase substrate interactions, computer-aided modeling

407

docking of sn-2 and sn-3/1 chain, 371-372 docking of tetrahedral intermediate of l(3)-octanoylglycerol, 370 hydrophobic acyl chain-binding sites, 370 open form of enzyme, 369-370 oxyanion stabilization, 373-374 relaxation of complex, 372-373 steric effects, 374 substrate orientation, 373 sequence analysis alignment with Stamp program, 35-36, 39 identification of relevant structures, 30-31, 35 structural subset identification, 36 three-dimensional data set, importance in sequence analysis, 29 species for solved structures, 4-6 stereospecificity analysis, 26-27 substrate-binding sites, 23-25 TITRA, modeling of pH effects on proteins flow of program, 141-143 mean field titration, 144-145 site-site coupling factors, 144 Tanford-Kirkwood sphere model, 141-142 Triolein, Michaelis-Menten kinetics of lipase hydrolysis, 230-232 Trivernolin extraction from Vernonia oil, 224 Michaelis-Menten kinetics of lipase hydrolysis, 230-232 properties, 226 purification, 224-225 vernolic acid preparation by enzymatic hydrolysis, 225-226

V Vernolic acid preparation by enzymatic hydrolysis of trivernolin, 225-226 properties, 226 Vernonia galamensis lipase assay, 222 fatty acid selectivity determination comparison to other plant seed lipases, 231-232

408

SUBJECT INDEX

hydrolysis of triglyceride mixtures in isooctane, 228-229 lipase preparation, 227 solvent selection, 226-227 substrate selection, 226 transesterification of tricaprylin with fatty acid mixtures, 229-230 water activity control, 227 immobilization on polypropene, 224, 227 Michaelis-Menten kinetics of hydrolysis triolein, 230-232 trivernolin, 230-232 pH optimum, 224 purification differential centrifugation, 222-223 extraction, 222 size-exclusion chromatography, 223 size, 223-224 solubility, 223 substrate specificity, 221-222

W Wash effect, evaluation of lipases commercial systems, 336 dose-response curves, 338-339 miniwash system condition for washing, 338 detergents, 338 equipment, 337 fabric, 337 soil, 338

X X-ray crystallography, see a//3-Hydrolase fold; Pancreatic lipase; P s e u d o m o n a s lipases; Three-dimensional structure, lipases

ISBN 0--12m182185--4

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