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Serine carboxypeptidases: a new and versatile family of enzymes
Serine carboxypeptidases: a new and versatile family of enzymes S. James Remington University of Oregon, Eugene, USA Several structural and biochemical studies in the past year have revealed the potential application of a new family of serine proteinase, the serine carboxypeptidases, in peptide synthesis,carboxy-terminal peptide sequencing and the production of biologically active carboxy-terminal peptide amides. The recent determination of the high-resolution crystal structures for two members of the family, CPDW-II and CPDY, should permit protein engineering to further increase their utility. Current Opinion in Biotechnology 1993, 4:462-468 Introduction
tests of the utility of these enzymes in such procedures are also discussed.
Chemical peptide synthesis is expensive and on a large scale involves potentially very dangerous conditions; for example, the N-carboxyanhydride method, o n e of two general methods currently available, requires the extremely poisonous chemical p h o s g e n e [1]. Furthermore, m a n y bioactive peptides with drug-related or experimental applications cannot b e p r o d u c e d by genetic engineering in bacteria because they have amino- or carboxy-terminal blocking groups, for example, carb o x y a m i d o groups, that are essential for full activity (see [2-5]). These are the result of post-translational enzymatic processing, the machinery for which is lacking in bacteria. An attractive alternative to chemical peptide synthesis is enzymatic peptide synthesis. Such syntheses can proceed under mild, non-toxic conditions, and p r o b l e m s such as racemization are avoided. The starting materials for enzymatic peptide synthesis can be obtained by engineering genes for precursor peptides into bacteria (although these p o s e other special problems, such as instability of products), and the required bioactive material then generated by o n e or more enzymatically catalyzed steps i n vitro. Growth h o r m o n e releasing factor, a 40-44 amino acid peptide that contains a carboxy-terminal leucine amide which is essential for full activity [6,7], has recently b e e n synthesized b y this approach [8]. Using this method, a gene containing a carboxy-terminal Leu44-Gly45 w a s constructed, the inactive peptide p r o d u c e d in bacteria and subsequently converted to the biologically active carboxy-terminal leucine amide by use of an enzyme, peptidyl glycine0t-amidating monooxygenase [8,5]. This review focuses on recent results from structural studies of the serine carboxypeptidases, a class of enzymes that promise to be very useful for biosynthetic and analytic procedures. Several recent experimental
Serine carboxypeptidases Serine carboxypeptidases (CPDs) are ubiquitous in higher organisms and have several tissue-specific and cell c o m p a r t m e n t specific functions. They are serine exopeptidases/esterases that efficiently r e m o v e carboxy-terminal amino acids and amino acid amides f r o m peptides (for a comprehensive review, see [9]). T h e y also hydrolyze peptide esters and release amm o n i a from amino acid amides; metal ions are not required for activity. An unusual feature of these enz y m e s is that the p H o p t i m u m for activity toward p e p tide substrates is acidic, with activity at pH as low as 3 [9], in contrast to m e m b e r s of the trypsin or subtilisin families of endopeptidases, which are essentially inactive at pH < 7. O n the other hand, activity toward ester substrates is optimal at basic pH, which allows effective, and as will be seen, useful control o v e r the reaction catalyzed. Possibly one of the most useful enzymatic reactions of serine CPDs ;is the catalysis of transpeptidation. A reversible acyl e n z y m e intermediate is formed during the course of the reaction [10-12], and the product is released u p o n nucleophilic attack, usually b y water. Thus, the overall reaction is as follows: Peptide-CO-NHCHRCO2H + HO-Enz ---> Peptide-COOEnz + NH2-CHRCO2H Peptide-COOEnz + H20 --+Peptide CO2H + HO-Enz However, the e n z y m e can also catalyze the nucleophilic addition of an amino acid to the acyl e n z y m e intermediate (aminolysis) with high yield, permitting carboxy-terminal protein synthesis (see below).
Abbreviations CPD--carboxypeptidase. 462
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Serine carboxypeptidases Remington 463 Serine CPDs h a v e been characterized in detail from several sources, namely, w h e a t bran (CPDW-II) [13], malted barley [14], yeast (CPD-Y) [9,15,16] a n d Pencillium janthinellum (CPD-P) [17] as well as being isolated and characterized from many other sources [9]. Generally, they are glycoproteins with a subunit molecular mass o f - 60kDa. Thus far, they h a v e not b e e n found in bacteria. Substrate specificities have b e e n explored in some detail [9,13,14] and extensive kinetic analyses have been undertaken [10,11], primarily with CPD-Y. The enzymes a p p e a r to fall into three general classes of substrate specificity. The first class is highly specific and is involved in the regulation and processing of peptide hormones, for example h u m a n kidney prolyl CPD-inactivates angiotensin II and III, and m a y be involved in the regulation of blood pressure [18]. In yeast, the product of the KEX1 gene is a serine CPD that catalyzes the final step in maturation of the a-factor mating p h e r o m o n e by the stepwise removal of a pair of basic residues [19]. The less specific enzymes, which in general will be m o r e useful to biotechnology, segregate into two general classes with s o m e overlap: those with preference for basic groups o n either side of the scissile bond; and those with preference for hydrophobic groups on either side. CPDW-II is an example of the former, while CPD-Y is an example of the latter. To date, no serine CPD has been discovered that shows high activity toward carboxy-terminal proline or acidic residues. These enzymes are serine proteinases because they are inactivated by diisopropylfluorophosphate and chloromethyl ketone derivatives, two c o m p o u n d s which were u s e d in early studies to detect serine in the active site [20] and which also implicated histidine in the active site [21]. It w a s X-ray structural studies of the wheat enzyme, however, that first demonstrated the existence of the 'catalytic triad' aspartate, histidine and serine commoi~ to the other two families of serine proteinases2
Three-dimensional structure Liao and Remington [22] provided the first description of the three-dimensional structure of homodimeric CPDW-II at 3.5 A resolution and reported that the fold of the e n z y m e was completely different from that of the trypsin and subtilisin families of serine endopeptidases, although it contained the catalytic triad, Asp338, His397, Ser146 in the active site. As the folds of the three families are unrelated, they suggested that this is an example of convergent evolution to a c o m m o n catalytic mechanism. More recently, the structure of CPDW-II has b e e n fully refined at high-resolution (2.2A) [23"] a n d compared in detail with the structures of chymotrypsin and subtilisin. The structure of the yeast vacuolar enzyme, m o n o m e r i c CPD-Y (with - 2 5 % overall sequence h o m o l o g y to CPDW-II) has also b e e n determined and refined at 2.7 A resolution, and is very similar to CPDW-II (J Endrizzi, K Breddam, SJ Remington, unpublished data).
The fold of CPDW-II and CPD-Y is shown schematically in Fig. 1, where the view is directly into the active site. The overall fold is quite typical for an enzyme and comprises an 0t +~ protein with a large central p-sheet consisting of 11 ~ strands flanked by 15 a-helices. Residues 180-310 form a largely 0t-helical insertion into the core structure, adopting an irregular structure which completely surrounds or forms part of the active-site cavity. Unlike the serine endopeptidases, the active-site cavity is a p r o n o u n c e d and rather hydrophobic depression in the surface of the molecule, suggesting that binding sites for several subsites amino-tei:minal to the scissile bond m a y exist. Although a n u m b e r of glycosytation sites are evident, they are generally located far from the active site and are unlikely to be involved in catalysis. Indeed, CPD-Y has b e e n s h o w n not to require carbohydrate for activity [24]. Although CPDW-II is a homodimer, there is no evidence that the dimeric structure is required for activity. One of the most surprising and fascinating results from the w o r k of Liao et al. [23"] is that the serine CPDs are m e m b e r s of a recently discovered and very large class of enzymes that have largely unrelated enzymatic activities, but topologically identical folds [25"']. This group includes: dienelactone hydrolase from Pseudomonas sp. B13 [26]; several triacylglycerol lipases, for example, from Geotrichum candidum [27] and Rhizomucor miehei [28]; acetylcholine esterase from Torpedo californica [29]; and haloalkane dehalogenase from Xanthobacter autotrophicus [30]. The structures of all of these enzymes have only recently been solved and there h a d b e e n no hint that they would have related folds (except that there is slight sequence h o m o l o g y b e t w e e n acetylcholine esterase and the G. candidum lipase). All contain a catalytic triad with the configuration nucleophile-acid-histidine in order of amino acid sequence, where the nucleophile is either serine, aspartate or cysteine and the acid is either aspartate or glutamic acid. The only similarity b e t w e e n the reactions catalyzed by these enzymes is that all are hydrolysis reactions. For this reason, the overall topoiogy of this e n z y m e family has b e e n named the 'a/J3 hydrolase fold' and is s h o w n schematically in Fig. 2. The configurations of the active-site residues in these enzymes are so similar [25"q that the exciting possibility is raised of 'swapping' parts of these enzymes by mutagenesis, thereby creating an activity for which no enzyme currently exists. There is little doubt that other m e m b e r s of this family still await discovery.
Active-site cavity The active site of CPDW-II is a d e e p hydrophobic pit containing a number of functional residues of interest. The catalytic triad Asp338-His397-Ser146 (Fig. 3) is similar, but not identical to that of the trypsin and subtilisin families of enzymes [23"], and is adjacent to an 'oxyanion hole' consisting of the backbone amides of Gly53 and Tyr147, which most likely stabilize a tetrahedral intermediate. Therefore, it is nearly certain that the enzymatic mechanism of the serine CPDs is
464
Protein engineering
Fig. 1. Stereo schematic diagram of the backbone topology of serine carboxypeptidase from wheat and yeast. The point of view is into the active site, with the active site serine located at the carboxyl terminus of the central strand of ~-sheet.
Fig. 2. Schematic diagram of the topology of the ~c/!8 hydrolase fold enzymes. 'A', representsthe acid; 'Nu', the nucleophile and 'H', the histidine of the catalytic triad. Lightly shaded loops are positions where insertions of up to 200 residues (largely (*-helical) can be inserted into the basis topology. identical to that of trypsin and subtilisin. Unlike those enzymes, however, a conserved tyrosine side chain contacts the active-site histidine ring in a perpendicular arrangement (Fig. 3) and m a y be responsible for the p r e s u m e d unusually low active-site pK a and consequent activity at pH < 7.
Analysis of the binding of the free amino acid arginine (a reaction product) suggested a mechanism for the interaction of the carboxylate of the substrate with the enzyme [23"] that is most unusual. The arginine carboxylate forms hydrogen b o n d s with the carboxylate of Glu145, the side-chain amide Asn51 and the amide of Gly52 (Fig. 4). Furthermore, the carboxylates of Glu145 and Glu65 form a hydrogen b o n d with each other, suggesting that one or both are protonated and have unusually high pK a values. It seems likely that low activity toward peptide substrates at p H 7 is a result of deprotonation of Glu145 and of consequent unfavorable electrostatic interactions with the substrate. This is consistent with the demonstration [9,14] that low activity toward peptides at high p H is because of an increased Kin, not a decrease in kcat. Presumably, as glutamine could in theory replace Glu145, thereby leading to a peptidase activity less d e p e n d e n t on pH, it is not at all clear w h y the serine CPDs have glutamic acid at this position. Perhaps it is important that the activity is under the control of pH, consistent with the e n z y m e ' s usual location in acidic cellular compartments, such as the vacuole in yeast [31]. Figure 4 illustrates part of the basis for substrate specificity for either hydrophobic or basic residues. CPDWII shows unusual dual specifici W and is most active toward basic groups at the carboxy-terminal position, but also efficiently hydrolyzes hydrophobic residues such as phenylalanine. As shown in Fig. 4, the side chain of the arginine residue lies in a narrow channel formed b y Tyr60 and Tyr239, which is terminated by Glu398
Serine carboxypeptidases Remington 465
GLY ~%~ 53 w
1.- . v i 18397
ASP8
4ISgg7
Fig. 3. Stereo drawing of the active-site configuration of serine carboxypeptidase from wheat (CPDW-II). The hydrogen bonds between the residues of the catalytictriad are shown as thin lines. W187 is a presumed water molecule that occupies the 'oxyanion hole' formed by the amides of Gly53 and Tyr147. Oxygen and nitrogen atoms are shown as solid circles, and carbon as open circles.
,5
TYR239
ERI46 ~L~2
i
TYR60
Fig. 4. Stereo drawing of arginine bound to the carboxy-terminal leaving group pocket of CPDW-II. The carboxylate of arginine interacts through hydrogen bonds with the carboxylate of Glu145, the amide nitrogen of Gly52 and the side-chain nitrogen of Asn51 (thin bonds). Tyr239, Tyr60, Glu398 and Glu272 form the side-chain binding pocket, providing dual specificity for basic and hydrophobic side chains.
and Glu272. The acidic groups appear to be the specificity determinants and m a k e generalized electrostatic interactions with the guanidinium group, whereas the tyrosine residues interact with the hydrophobic portion of either sort of side chain. Presumably it is this last feature that accounts for the dual specificity. In CPDY, Glu398 and -272 are replaced b y methionine and leucine, respectively, which accounts for the lower activity of CPD-Y toward basic side chains; however, all other catalytic residues are retained. It may therefore be possible to replace one or more of these specificity determinants in order to create, for example, a serine CPD specific for acidic peptides. An extensive cleft provides opportunity for several amino acids amino-terminal to the scissile b o n d to bind to the enzyme, but no studies have yet been reported describing these potential binding-site inter-
actions. It is clear, however, that the m o d e of substrate recognition b y serine CPDs is perhaps more general and differs from that of subtilisin and trypsin, which m a k e extended ~-sheet hydrogen b o n d interactions with peptide substrates ([23"] and references therein).
Applications of serine carboxypeptidases Peptide synthesis A n u m b e r of investigators have demonstrated protein synthesis using serine carboxypeptidases. Although in principle the hydrolysis reaction could be forced to proceed in the reverse direction by the use of nonaqueous s o l v e n t s - - v a l i d for any proteolytic enzyme, although some are inhibitory toward serine CPDs
466
Protein engineering
~ 1 0 Jl
R~ oII
P--NH--CH-C--OMe
+
CPD-Y
H2N--CH--C--X
~
~
~, OII
~2
O
II
P--NH--CH--C--NH--CH--C--X
+ MeOH
Fig. 5. Schemeshowing enzymatic peptide synthesis using serine carboxypeptidaseto add an amino acid to the acyl enzyme intermediate derived from a peptide ester. X can be either OH or NH2.
H2NOCH2 N/~NO2 R3 --Ala-OH
~CPD-Y
R3__NH2NOC/~ ~ N ~ h ~ aO
+
R3 - NH2
H I2N2O3HN-C-
/:C \p
N~
1
H
+
H2Noc
I
II
1
>
R3 - NH2
+
Alanine +
Photoproducts
Fig. 6. Schemesummarizingthe reactionsinvolved in the production of peptide amide employinga combination of enzymatic aminolysis by serine carboxypeptidase(CPD-Y)and photochemical deblocking. Serinecarboxpeptidasecatalyzesthe addition of a photolabile nucleophile (nitrobenzyl glycine amide)to a peptide (R3-Ala-OH)by transpeptidation, followed by photochemical deblocking (h~a)with long wavelength ultra-violet radiationto produce the peptide amide (R3-NH2) [38°]. The photochemical productsand excited-stateintermediatesshown were not characterizeddefinitively in this instance and should be consideredas tentative. [ 3 2 ] - an energetically more favorable approach is the aminolysis of acyl e n z y m e intermediates formed from peptide esters, for example see Fig. 5. This reaction can o n l y be accomplished with enzymes that f o r m acyl e n z y m e intermediates, i.e. serine or cysteine proteinases. The serine CPDs are perfectly suited for such reaction steps as their largest acceptable nucleophilic substrate is at most an amino acid amide. The kinetics of this reaction have b e e n investigated in minute detail [11,12] permitting thoughtful design of industrial processes. Hydrolysis of the product can be avoided in several ways. Breddam [33] has used a chemically modified CPD-Y in the semisynthesis of h u m a n insulin from porcine insulin to reduce u n w a n t e d side reactions; however, at pH > 9, the enzyme has almost no peptidase activity. At high p H the amino acid amide must be u s e d as the nucleophile, as amino acids do not bind to the enzyme. Hydrolysis reactions compete with aminolysis reactions to make the process less than 100 % efficient and the specificity of the enzyme further limits yield for certain side-chain combinations; however, the process directly yields a peptide amide if this is desired. For the process to continue, the peptide must first b e deamidated. In another study, Breddam [34] has described methyl mercury halide modifications to CPD-Y that increase amidase activity, w h i c h is inefficient in the wild-type enzyme [9]. Ideally, it would b e better to use unmodified or engineered enzymes to accomplish these tasks, especially in the case of mercury c o m p o u n d s , which migrate b e t w e e n sulfhydryls. Indeed, Schwarz et al. [35]
recently described an efficient two-step procedure for dipeptide synthesis in which the product is deamidated b y a newly discovered peptide amidase isolated from orange peel. Variations of this procedure could b e used to m a k e larger peptides as well. Finally, Aasmul-Olsen et al. [36] discuss several techniques for efficient singlestep synthesis of dipeptides by enzymes other than serine CPDs and, in addition, show that all combinations of L--L, D--L, L--D and D--D stereoisomers can be efficiently produced. Another possible use for these reactions is the carboxyterminal labeling of proteins. Berne et al. [37] have d e v e l o p e d a carboxy-terminal labeling procedure in which tritiated valine-NH2 is used as a nucleophile, and the resulting transpeptidation reaction with CPD-Y yields tritiated carboxyl termini for several proteins.
Peptide amidation Although peptide amidation has b e e n achieved for a range of amides, this is not yet possible with proline amide, glutamic amide or aspartic amide as the nucleophile, and some bioactive peptides, such as calcitonin, terminate with these amino acid amides. Genetic engineering of the active site of serine CPDs to permit the use of these nucleophiles may eventually overcome this limitation, but other approaches are also possible. Henricksen et al. [38"] have recently explored a very interesting and extremely efficient combination of enzymatic aminolysis of peptides and peptide esters with photolabile nucleophiles such as nitrophenyl derivatives (see Fig. 6). Photolysis of the blocking g r o u p
Serine c a r b o x y p e p t i d a s e s Remington 467 yields peptide amide, and in test cases 95 % yield of the product was reported.
zymes that do not at present exist, such as peptide dehalogenases.
This is an ingenious use of an enzyme with a non-natural substrate, and the general technique has many other potential uses, such as generation of bioactive peptides at a particular instant or in a specific tissue.
References and recommended reading
Protein s e q u e n c i n g
Edman degradation of amino-terminal amino acid sequences is convenient and reliable, but no comparable and simple procedure exists for carboxy-terminal protein sequencing. In many cases, even where the gene sequence is known, one must still determine whether the active protein conforms with the gene sequence or whether it has b e e n subject to post-translational modification. CPDs have long been used to degrade carboxy-terminal amino acids, but the metallocarboxypeptidases will not release carboxy-terminal proline residues. CPD-Y and CPD-P are n o w both commercially available and are useful in this regard because they are capable of releasing carboxy-terminal proline residues [9,17]. Indeed, the adaptability of these proteins is s h o w n by the fact that CPD-Y is active in 0.5 % sodium dodecyl sulphate and 6 M urea [9], which will solubilize most peptides. The rates of hydrolysis depend strongly on the nature of the side chains adjacent to the scissile b o n d (for a summary, see [9]), and it is difficult to unambiguously identify sequential pairs of residues that are rapidly released. However, variations in the reaction conditions, or the type of enzyme chosen can be manipulated to minimize this problem. For exampie, aspartate is more efficiently released by CPD-Y at pH 5.0 than at pH 7.0, whereas the opposite is true for histidine [9]. As a final note, these enzymes will release carboxy-terminal amino acid amides that must be detected using a different procedure than with amino acids. .Perhaps there are other naturally occurring serine CPDs specific for residues such as proline or acidic groups, the discovery of which would make possible the production of a convenient and inexpensive carboxy-terminal sequencing 'kit' suitable for most c o m m o n applications.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest 1.
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296:659-660. 5.
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BECH L, BREDDAM K: C h e m i c a l Modifications o f a Cyst e i n y l Residue Introduced i n the Binding Site o f Carb o x y p e p t i d a s e Y b y Site-Directed Mutagenesis. Carlsberg Res C o m m u n 1988, 53:381-393.
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Conclusions I have described the three-dimensional structures of two serine CPDs, but because of constraints of space, I have summarized only a part of the wealth of recent data concerning these enzymes and their potential applications in biotechnology. Although more effort needs to be directed toward an understanding of substrate specificity in CPDs, it is clear that they will be very useful in a wide variety of biosynthetic and analytic procedures. Genetic engineering promises to make these enzymes even more useful for industrial and laboratory procedures. The fact that the serine CPDs belong to such a large family of hydrolytic enzymes suggests that it may be possible to create en-
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BREDDAM K: C h e m i c a l l y Modifaed C a r b o x y p e p t i d a s e Y w i t h I n c r e a s e d A m i d a s e Activity. Carlsberg Res C o m m u n 1984, 49:535-554.
35.
SCHWARZA, WANDREY C, STEINKE D, KULA MR: A TWo-Step E n z y m a t i c S y n t h e s i s o f Dipeptides. Biotech Bioeng 1992, 39:132-140.
36.
AASMUL--OLSENS, THORBEK P, HANSEN S, WIDMER F: Enzymatic SIngle Step P r o c e s s e s for the Production o f D i p e p t i d e s from S i m p l e S t a r t i n g Materials. B i o m e d Biochim Acta 1991, 50:S106-S109.
37.
BEe,~EP-F, BLANQUETS, SCHMITrERJ-M: C a r b o x y p e p t i d a s e Y - C a t a l y z e d T r a n s p e p t i d a t i o n o f Esterified Oligo- a n d P o l y p e p t i d e s and Its Use for Specific Carboxy-Ternainai Labell/ng of P r o t e i n s . J A m Chem Soc 1992, 114:2603-2610.
23.
LIAO D-I, BREDDAM K, SWEET RM, BULLOCKT, REMINGTON SJ: Refined Atomic Model o f Wheat S e r i n e Carb o x y p e p t i d a s e H a t 2.2 A Resolution. Biochemistry 1992, 31:9796-9812. A definitive structural study of the first serine CPD to be crystallized that reports a n u m b e r of u n u s u a l results. T h e high-resolution model of the enzyme is analyzed in considerable detail and compared with those of chymotrypsin a n d subtilisin. It is concluded that critical active-site features of the catalytic residues that contact substrate are essentially identical in the three families of serine proteinases; nevertheless, there are revealing differences. An explanation for the pH d e p e n d e n c e of peptidase activity is proposed based on a study of the binding of arginine (a product) to the enzyme. 24.
WINTHERJR, STEVENS TH, I#-~IELLANDoBRANDTMC: Yeast Carb o x y p e p t i d a s e Y R e q u i r e s G l y e o s y l a t i o n for Ef~cient I n t r a e e l l u l a r T r a n s p o r t , but Not for V a c u o l a r Sorting, in Vivo Stability, o r Activity. Eur J Biochem 1991, 197:681--689.
25. •o
OLLIS DL, CHEAH E, CYGLER M, DYKSTRA B, FROLOW F, FRAKENS, HAREL M, REMINGTON SJ, SILMAN I, SCHRAGJ, ET AL.: T h e 0~/~ H y d r o l a s e Fold. Protein Eng 1992, 5:197-211. A very clear a n d remarkable paper in w h i c h the structures of five e n z y m e s with urLrelated a m i n o acid s e q u e n c e s and activity are compared and s h o w n to have a c o m m o n fold and similar active-site features. The e n z y m e s have diverged from a c o m m o n ancestor in such a w a y that the arrangement a n d n u m b e r of the catalytic groups, but not their identity, are retained. Only the histidine of the catalytic triad (acid, histidine, nucleophile) is conserved a m o n g all m e m b e r s of the family. All of the e n z y m e s are hydrolases, hence the n a m e of the fold. Some very u n u s u a l features c o m m o n to all members, such as disallowed conformational angles of the active-site nucleophfles, are discussed in detail. 26.
PATHAKD, NGAI KL, OLLIS, D: X-Ray C r y s t a l l . o g r a p h i c S t r u c t u r e o f D i e n e l a c t o n e H y d r o l a s e at 2.8 A Resolution. J Mol Biol 1988, 204:435-445.
27.
SCHRAG JD, LI Y, WU S, CYGLER M: Ser-I-Iis-Glu T r i a d F o r m s t h e Catalytic Site of t h e Lipase f r o m G e o t r i c b u m c a n d i d u m . N a t u r e 1991, 351:761-764.
38.
HENRIKSENDB, BREDDAMK, MOLLERJ, BUCHARDTO: P e p t i d e Amidation by Chemical Protein Engineering. A Combination of Enzymatic and Photochemical Synthesis. J A m Chem Soc 1992, 114:1877-1878. This is a brief account of a very interesting and extremely efficient u s e of yeast serine CPD to add non-natural photolabile substrates (2-nitrobenzyl derivatives) to the carboxyl terminus of a peptide by a transacylation reaction and then photochemically deprotecting to yield the peptide amide. While the goal of this paper was to d e m o n strate efficient production of the peptide amide, photochemical procedures like this h a v e other potential uses, such as rapid generation of a bioactive peptide at a particular instant or in a particular tissue.
SJ Remington, Institute of Molecular Biology and Department of Physics, University of Oregon, Eugene, Oregon 97403, USA.
tests of the utility of these enzymes in such procedures are also discussed.
Chemical peptide synthesis is expensive and on a large scale involves potentially very dangerous conditions; for example, the N-carboxyanhydride method, o n e of two general methods currently available, requires the extremely poisonous chemical p h o s g e n e [1]. Furthermore, m a n y bioactive peptides with drug-related or experimental applications cannot b e p r o d u c e d by genetic engineering in bacteria because they have amino- or carboxy-terminal blocking groups, for example, carb o x y a m i d o groups, that are essential for full activity (see [2-5]). These are the result of post-translational enzymatic processing, the machinery for which is lacking in bacteria. An attractive alternative to chemical peptide synthesis is enzymatic peptide synthesis. Such syntheses can proceed under mild, non-toxic conditions, and p r o b l e m s such as racemization are avoided. The starting materials for enzymatic peptide synthesis can be obtained by engineering genes for precursor peptides into bacteria (although these p o s e other special problems, such as instability of products), and the required bioactive material then generated by o n e or more enzymatically catalyzed steps i n vitro. Growth h o r m o n e releasing factor, a 40-44 amino acid peptide that contains a carboxy-terminal leucine amide which is essential for full activity [6,7], has recently b e e n synthesized b y this approach [8]. Using this method, a gene containing a carboxy-terminal Leu44-Gly45 w a s constructed, the inactive peptide p r o d u c e d in bacteria and subsequently converted to the biologically active carboxy-terminal leucine amide by use of an enzyme, peptidyl glycine0t-amidating monooxygenase [8,5]. This review focuses on recent results from structural studies of the serine carboxypeptidases, a class of enzymes that promise to be very useful for biosynthetic and analytic procedures. Several recent experimental
Serine carboxypeptidases Serine carboxypeptidases (CPDs) are ubiquitous in higher organisms and have several tissue-specific and cell c o m p a r t m e n t specific functions. They are serine exopeptidases/esterases that efficiently r e m o v e carboxy-terminal amino acids and amino acid amides f r o m peptides (for a comprehensive review, see [9]). T h e y also hydrolyze peptide esters and release amm o n i a from amino acid amides; metal ions are not required for activity. An unusual feature of these enz y m e s is that the p H o p t i m u m for activity toward p e p tide substrates is acidic, with activity at pH as low as 3 [9], in contrast to m e m b e r s of the trypsin or subtilisin families of endopeptidases, which are essentially inactive at pH < 7. O n the other hand, activity toward ester substrates is optimal at basic pH, which allows effective, and as will be seen, useful control o v e r the reaction catalyzed. Possibly one of the most useful enzymatic reactions of serine CPDs ;is the catalysis of transpeptidation. A reversible acyl e n z y m e intermediate is formed during the course of the reaction [10-12], and the product is released u p o n nucleophilic attack, usually b y water. Thus, the overall reaction is as follows: Peptide-CO-NHCHRCO2H + HO-Enz ---> Peptide-COOEnz + NH2-CHRCO2H Peptide-COOEnz + H20 --+Peptide CO2H + HO-Enz However, the e n z y m e can also catalyze the nucleophilic addition of an amino acid to the acyl e n z y m e intermediate (aminolysis) with high yield, permitting carboxy-terminal protein synthesis (see below).
Abbreviations CPD--carboxypeptidase. 462
© Current Biology Ltd ISSN 0958-1669
Serine carboxypeptidases Remington 463 Serine CPDs h a v e been characterized in detail from several sources, namely, w h e a t bran (CPDW-II) [13], malted barley [14], yeast (CPD-Y) [9,15,16] a n d Pencillium janthinellum (CPD-P) [17] as well as being isolated and characterized from many other sources [9]. Generally, they are glycoproteins with a subunit molecular mass o f - 60kDa. Thus far, they h a v e not b e e n found in bacteria. Substrate specificities have b e e n explored in some detail [9,13,14] and extensive kinetic analyses have been undertaken [10,11], primarily with CPD-Y. The enzymes a p p e a r to fall into three general classes of substrate specificity. The first class is highly specific and is involved in the regulation and processing of peptide hormones, for example h u m a n kidney prolyl CPD-inactivates angiotensin II and III, and m a y be involved in the regulation of blood pressure [18]. In yeast, the product of the KEX1 gene is a serine CPD that catalyzes the final step in maturation of the a-factor mating p h e r o m o n e by the stepwise removal of a pair of basic residues [19]. The less specific enzymes, which in general will be m o r e useful to biotechnology, segregate into two general classes with s o m e overlap: those with preference for basic groups o n either side of the scissile bond; and those with preference for hydrophobic groups on either side. CPDW-II is an example of the former, while CPD-Y is an example of the latter. To date, no serine CPD has been discovered that shows high activity toward carboxy-terminal proline or acidic residues. These enzymes are serine proteinases because they are inactivated by diisopropylfluorophosphate and chloromethyl ketone derivatives, two c o m p o u n d s which were u s e d in early studies to detect serine in the active site [20] and which also implicated histidine in the active site [21]. It w a s X-ray structural studies of the wheat enzyme, however, that first demonstrated the existence of the 'catalytic triad' aspartate, histidine and serine commoi~ to the other two families of serine proteinases2
Three-dimensional structure Liao and Remington [22] provided the first description of the three-dimensional structure of homodimeric CPDW-II at 3.5 A resolution and reported that the fold of the e n z y m e was completely different from that of the trypsin and subtilisin families of serine endopeptidases, although it contained the catalytic triad, Asp338, His397, Ser146 in the active site. As the folds of the three families are unrelated, they suggested that this is an example of convergent evolution to a c o m m o n catalytic mechanism. More recently, the structure of CPDW-II has b e e n fully refined at high-resolution (2.2A) [23"] a n d compared in detail with the structures of chymotrypsin and subtilisin. The structure of the yeast vacuolar enzyme, m o n o m e r i c CPD-Y (with - 2 5 % overall sequence h o m o l o g y to CPDW-II) has also b e e n determined and refined at 2.7 A resolution, and is very similar to CPDW-II (J Endrizzi, K Breddam, SJ Remington, unpublished data).
The fold of CPDW-II and CPD-Y is shown schematically in Fig. 1, where the view is directly into the active site. The overall fold is quite typical for an enzyme and comprises an 0t +~ protein with a large central p-sheet consisting of 11 ~ strands flanked by 15 a-helices. Residues 180-310 form a largely 0t-helical insertion into the core structure, adopting an irregular structure which completely surrounds or forms part of the active-site cavity. Unlike the serine endopeptidases, the active-site cavity is a p r o n o u n c e d and rather hydrophobic depression in the surface of the molecule, suggesting that binding sites for several subsites amino-tei:minal to the scissile bond m a y exist. Although a n u m b e r of glycosytation sites are evident, they are generally located far from the active site and are unlikely to be involved in catalysis. Indeed, CPD-Y has b e e n s h o w n not to require carbohydrate for activity [24]. Although CPDW-II is a homodimer, there is no evidence that the dimeric structure is required for activity. One of the most surprising and fascinating results from the w o r k of Liao et al. [23"] is that the serine CPDs are m e m b e r s of a recently discovered and very large class of enzymes that have largely unrelated enzymatic activities, but topologically identical folds [25"']. This group includes: dienelactone hydrolase from Pseudomonas sp. B13 [26]; several triacylglycerol lipases, for example, from Geotrichum candidum [27] and Rhizomucor miehei [28]; acetylcholine esterase from Torpedo californica [29]; and haloalkane dehalogenase from Xanthobacter autotrophicus [30]. The structures of all of these enzymes have only recently been solved and there h a d b e e n no hint that they would have related folds (except that there is slight sequence h o m o l o g y b e t w e e n acetylcholine esterase and the G. candidum lipase). All contain a catalytic triad with the configuration nucleophile-acid-histidine in order of amino acid sequence, where the nucleophile is either serine, aspartate or cysteine and the acid is either aspartate or glutamic acid. The only similarity b e t w e e n the reactions catalyzed by these enzymes is that all are hydrolysis reactions. For this reason, the overall topoiogy of this e n z y m e family has b e e n named the 'a/J3 hydrolase fold' and is s h o w n schematically in Fig. 2. The configurations of the active-site residues in these enzymes are so similar [25"q that the exciting possibility is raised of 'swapping' parts of these enzymes by mutagenesis, thereby creating an activity for which no enzyme currently exists. There is little doubt that other m e m b e r s of this family still await discovery.
Active-site cavity The active site of CPDW-II is a d e e p hydrophobic pit containing a number of functional residues of interest. The catalytic triad Asp338-His397-Ser146 (Fig. 3) is similar, but not identical to that of the trypsin and subtilisin families of enzymes [23"], and is adjacent to an 'oxyanion hole' consisting of the backbone amides of Gly53 and Tyr147, which most likely stabilize a tetrahedral intermediate. Therefore, it is nearly certain that the enzymatic mechanism of the serine CPDs is
464
Protein engineering
Fig. 1. Stereo schematic diagram of the backbone topology of serine carboxypeptidase from wheat and yeast. The point of view is into the active site, with the active site serine located at the carboxyl terminus of the central strand of ~-sheet.
Fig. 2. Schematic diagram of the topology of the ~c/!8 hydrolase fold enzymes. 'A', representsthe acid; 'Nu', the nucleophile and 'H', the histidine of the catalytic triad. Lightly shaded loops are positions where insertions of up to 200 residues (largely (*-helical) can be inserted into the basis topology. identical to that of trypsin and subtilisin. Unlike those enzymes, however, a conserved tyrosine side chain contacts the active-site histidine ring in a perpendicular arrangement (Fig. 3) and m a y be responsible for the p r e s u m e d unusually low active-site pK a and consequent activity at pH < 7.
Analysis of the binding of the free amino acid arginine (a reaction product) suggested a mechanism for the interaction of the carboxylate of the substrate with the enzyme [23"] that is most unusual. The arginine carboxylate forms hydrogen b o n d s with the carboxylate of Glu145, the side-chain amide Asn51 and the amide of Gly52 (Fig. 4). Furthermore, the carboxylates of Glu145 and Glu65 form a hydrogen b o n d with each other, suggesting that one or both are protonated and have unusually high pK a values. It seems likely that low activity toward peptide substrates at p H 7 is a result of deprotonation of Glu145 and of consequent unfavorable electrostatic interactions with the substrate. This is consistent with the demonstration [9,14] that low activity toward peptides at high p H is because of an increased Kin, not a decrease in kcat. Presumably, as glutamine could in theory replace Glu145, thereby leading to a peptidase activity less d e p e n d e n t on pH, it is not at all clear w h y the serine CPDs have glutamic acid at this position. Perhaps it is important that the activity is under the control of pH, consistent with the e n z y m e ' s usual location in acidic cellular compartments, such as the vacuole in yeast [31]. Figure 4 illustrates part of the basis for substrate specificity for either hydrophobic or basic residues. CPDWII shows unusual dual specifici W and is most active toward basic groups at the carboxy-terminal position, but also efficiently hydrolyzes hydrophobic residues such as phenylalanine. As shown in Fig. 4, the side chain of the arginine residue lies in a narrow channel formed b y Tyr60 and Tyr239, which is terminated by Glu398
Serine carboxypeptidases Remington 465
GLY ~%~ 53 w
1.- . v i 18397
ASP8
4ISgg7
Fig. 3. Stereo drawing of the active-site configuration of serine carboxypeptidase from wheat (CPDW-II). The hydrogen bonds between the residues of the catalytictriad are shown as thin lines. W187 is a presumed water molecule that occupies the 'oxyanion hole' formed by the amides of Gly53 and Tyr147. Oxygen and nitrogen atoms are shown as solid circles, and carbon as open circles.
,5
TYR239
ERI46 ~L~2
i
TYR60
Fig. 4. Stereo drawing of arginine bound to the carboxy-terminal leaving group pocket of CPDW-II. The carboxylate of arginine interacts through hydrogen bonds with the carboxylate of Glu145, the amide nitrogen of Gly52 and the side-chain nitrogen of Asn51 (thin bonds). Tyr239, Tyr60, Glu398 and Glu272 form the side-chain binding pocket, providing dual specificity for basic and hydrophobic side chains.
and Glu272. The acidic groups appear to be the specificity determinants and m a k e generalized electrostatic interactions with the guanidinium group, whereas the tyrosine residues interact with the hydrophobic portion of either sort of side chain. Presumably it is this last feature that accounts for the dual specificity. In CPDY, Glu398 and -272 are replaced b y methionine and leucine, respectively, which accounts for the lower activity of CPD-Y toward basic side chains; however, all other catalytic residues are retained. It may therefore be possible to replace one or more of these specificity determinants in order to create, for example, a serine CPD specific for acidic peptides. An extensive cleft provides opportunity for several amino acids amino-terminal to the scissile b o n d to bind to the enzyme, but no studies have yet been reported describing these potential binding-site inter-
actions. It is clear, however, that the m o d e of substrate recognition b y serine CPDs is perhaps more general and differs from that of subtilisin and trypsin, which m a k e extended ~-sheet hydrogen b o n d interactions with peptide substrates ([23"] and references therein).
Applications of serine carboxypeptidases Peptide synthesis A n u m b e r of investigators have demonstrated protein synthesis using serine carboxypeptidases. Although in principle the hydrolysis reaction could be forced to proceed in the reverse direction by the use of nonaqueous s o l v e n t s - - v a l i d for any proteolytic enzyme, although some are inhibitory toward serine CPDs
466
Protein engineering
~ 1 0 Jl
R~ oII
P--NH--CH-C--OMe
+
CPD-Y
H2N--CH--C--X
~
~
~, OII
~2
O
II
P--NH--CH--C--NH--CH--C--X
+ MeOH
Fig. 5. Schemeshowing enzymatic peptide synthesis using serine carboxypeptidaseto add an amino acid to the acyl enzyme intermediate derived from a peptide ester. X can be either OH or NH2.
H2NOCH2 N/~NO2 R3 --Ala-OH
~CPD-Y
R3__NH2NOC/~ ~ N ~ h ~ aO
+
R3 - NH2
H I2N2O3HN-C-
/:C \p
N~
1
H
+
H2Noc
I
II
1
>
R3 - NH2
+
Alanine +
Photoproducts
Fig. 6. Schemesummarizingthe reactionsinvolved in the production of peptide amide employinga combination of enzymatic aminolysis by serine carboxypeptidase(CPD-Y)and photochemical deblocking. Serinecarboxpeptidasecatalyzesthe addition of a photolabile nucleophile (nitrobenzyl glycine amide)to a peptide (R3-Ala-OH)by transpeptidation, followed by photochemical deblocking (h~a)with long wavelength ultra-violet radiationto produce the peptide amide (R3-NH2) [38°]. The photochemical productsand excited-stateintermediatesshown were not characterizeddefinitively in this instance and should be consideredas tentative. [ 3 2 ] - an energetically more favorable approach is the aminolysis of acyl e n z y m e intermediates formed from peptide esters, for example see Fig. 5. This reaction can o n l y be accomplished with enzymes that f o r m acyl e n z y m e intermediates, i.e. serine or cysteine proteinases. The serine CPDs are perfectly suited for such reaction steps as their largest acceptable nucleophilic substrate is at most an amino acid amide. The kinetics of this reaction have b e e n investigated in minute detail [11,12] permitting thoughtful design of industrial processes. Hydrolysis of the product can be avoided in several ways. Breddam [33] has used a chemically modified CPD-Y in the semisynthesis of h u m a n insulin from porcine insulin to reduce u n w a n t e d side reactions; however, at pH > 9, the enzyme has almost no peptidase activity. At high p H the amino acid amide must be u s e d as the nucleophile, as amino acids do not bind to the enzyme. Hydrolysis reactions compete with aminolysis reactions to make the process less than 100 % efficient and the specificity of the enzyme further limits yield for certain side-chain combinations; however, the process directly yields a peptide amide if this is desired. For the process to continue, the peptide must first b e deamidated. In another study, Breddam [34] has described methyl mercury halide modifications to CPD-Y that increase amidase activity, w h i c h is inefficient in the wild-type enzyme [9]. Ideally, it would b e better to use unmodified or engineered enzymes to accomplish these tasks, especially in the case of mercury c o m p o u n d s , which migrate b e t w e e n sulfhydryls. Indeed, Schwarz et al. [35]
recently described an efficient two-step procedure for dipeptide synthesis in which the product is deamidated b y a newly discovered peptide amidase isolated from orange peel. Variations of this procedure could b e used to m a k e larger peptides as well. Finally, Aasmul-Olsen et al. [36] discuss several techniques for efficient singlestep synthesis of dipeptides by enzymes other than serine CPDs and, in addition, show that all combinations of L--L, D--L, L--D and D--D stereoisomers can be efficiently produced. Another possible use for these reactions is the carboxyterminal labeling of proteins. Berne et al. [37] have d e v e l o p e d a carboxy-terminal labeling procedure in which tritiated valine-NH2 is used as a nucleophile, and the resulting transpeptidation reaction with CPD-Y yields tritiated carboxyl termini for several proteins.
Peptide amidation Although peptide amidation has b e e n achieved for a range of amides, this is not yet possible with proline amide, glutamic amide or aspartic amide as the nucleophile, and some bioactive peptides, such as calcitonin, terminate with these amino acid amides. Genetic engineering of the active site of serine CPDs to permit the use of these nucleophiles may eventually overcome this limitation, but other approaches are also possible. Henricksen et al. [38"] have recently explored a very interesting and extremely efficient combination of enzymatic aminolysis of peptides and peptide esters with photolabile nucleophiles such as nitrophenyl derivatives (see Fig. 6). Photolysis of the blocking g r o u p
Serine c a r b o x y p e p t i d a s e s Remington 467 yields peptide amide, and in test cases 95 % yield of the product was reported.
zymes that do not at present exist, such as peptide dehalogenases.
This is an ingenious use of an enzyme with a non-natural substrate, and the general technique has many other potential uses, such as generation of bioactive peptides at a particular instant or in a specific tissue.
References and recommended reading
Protein s e q u e n c i n g
Edman degradation of amino-terminal amino acid sequences is convenient and reliable, but no comparable and simple procedure exists for carboxy-terminal protein sequencing. In many cases, even where the gene sequence is known, one must still determine whether the active protein conforms with the gene sequence or whether it has b e e n subject to post-translational modification. CPDs have long been used to degrade carboxy-terminal amino acids, but the metallocarboxypeptidases will not release carboxy-terminal proline residues. CPD-Y and CPD-P are n o w both commercially available and are useful in this regard because they are capable of releasing carboxy-terminal proline residues [9,17]. Indeed, the adaptability of these proteins is s h o w n by the fact that CPD-Y is active in 0.5 % sodium dodecyl sulphate and 6 M urea [9], which will solubilize most peptides. The rates of hydrolysis depend strongly on the nature of the side chains adjacent to the scissile b o n d (for a summary, see [9]), and it is difficult to unambiguously identify sequential pairs of residues that are rapidly released. However, variations in the reaction conditions, or the type of enzyme chosen can be manipulated to minimize this problem. For exampie, aspartate is more efficiently released by CPD-Y at pH 5.0 than at pH 7.0, whereas the opposite is true for histidine [9]. As a final note, these enzymes will release carboxy-terminal amino acid amides that must be detected using a different procedure than with amino acids. .Perhaps there are other naturally occurring serine CPDs specific for residues such as proline or acidic groups, the discovery of which would make possible the production of a convenient and inexpensive carboxy-terminal sequencing 'kit' suitable for most c o m m o n applications.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest •. of outstanding interest 1.
BACKLOCKTJ, HIRSCHMANN R, VEBER D: T h e P r e p a r a t i o n and Use o f N-Carboxyanhydrates and N-Thiocarb o x y a n h y d r a t e s for Peptide B o n d Formation. In The Peptides, vol 9. Edited by Udenfried S, Meien-hofer. New York: Academic Press; 1987:39-102.
2.
H1LSTEDL, REHFELJF: cz-Carboxyamidation o f A n t r a l Pro-
gastrin.
1987, 262:16953-16957.
BRADBURY AF, FINNIEMDA, SMYTHDG: Mechanism of Cterminal Amide Formation b y Pituitary Enzymes. Nature 1982, 298:686-688.
4.
TATEMOTOK, CARLQUISTM, MUTt V: Neuropeptide Y-a Novel Brain Peptide w i t h Structural Similarities to Peptide YY and Pancreatic Polypeptide. Nature 1982,
296:659-660. 5.
RAMERSE, CHENG H, PALCIC M1VI, VEDERAS JC: Formation o f Peptide Amides b y Peptidylglycine cz-Amidating M o n o o x y g e n a s e : a New Assay and Stereochemistry o f Hydrogen Loss. J Am Chem Soc 1988, 110:8526-8529.
6.
GUILLEMIN R, BRAZEAU R, BOHLEN P, ESCH F, LING N, WEHRENBERG ~ : Growth Hormone-Releasing Fact o r from a H u m a n Pancreatic Tumor that Caused Acromegaly. Science 1982, 218:575-587.
7.
RIVIER J, SPIESS J, VALE W: Characterization o f the Rat H y p o t h a l a m i c C o r t i c o t r o p i n - R e l e a s i n g Factor. Proc Natl A c a d Sci USA 1983, 80:4851-4855.
8.
ENGELS JW, GLAUDER J, MOLLNER H, TRIPIER D, UHLMANN E, WETEKAM W: E n z y m a t i c Amidation of Recombinant (Leu 27) Growth Hormone Releasing H o r m o n e - - G l y 45. Protein Eng 1987, 1:195-199.
9.
BREDDAMK: Serine Carboxypeptidases. A Review. Cadsberg Res C o m m u n 1986, 51:83-128.
10.
MARTIN BM, OLIVER RWA, JOHANSEN JT, WISWANATHA T: The Carboxypeptidase Y Catalyzed H y d r o l y s i s o f I n d o l e a c r y l o y l i m i d a z o l e . Carlsberg Res C o m m u n 1980, 45:69-78.
11.
CHRISTENSENU, DROHSE FIB, MOLGAARD L: M e c h a n i s m of Carboxypeptidase-Y-Catalyzed Peptide Semisynthesis. E u r J Biochem 1992, 210:467-473.
12.
DROHSE JB, BREDDAM K, CHRISTENSEN U: M e c h a n i s m of Carboxypeptidase Y Catalyzed H y d r o l y s i s and Amltxolysis Reactions. Biocatalysis 1991, 5:109-120.
13.
BREDDAMK, SORENSON, SB, SVENDSENI: Prinlary Structure and Enzymatic Properties o f Carboxypeptidase II from Wheat Bran. Carlsberg Res C o m m u n 1987, 52:297-311.
14,
BREDDAMK: E n z y m a t i c P r o p e r t i e s o f Malt C a r b o x y p e p tidase II i n H y d r o l y s i s and Amirtolysis Reactions. Carlsberg Res C o m m u n 1985, 50:309-323.
15.
BECH L, BREDDAM K: C h e m i c a l Modifications o f a Cyst e i n y l Residue Introduced i n the Binding Site o f Carb o x y p e p t i d a s e Y b y Site-Directed Mutagenesis. Carlsberg Res C o m m u n 1988, 53:381-393.
16.
WINTHER JR, KIELLANI>-BRANDTMC, BREDDAMK: Increased H y d r o p h o b i c i t y o f the S l ' B i n d i n g Site i n C a r b o x y p e p tidase Y O b t a i n e d b y Site-Directed Mutageuesis. Cadsberg Res C o m m u n 1985, 50:273-284.
Conclusions I have described the three-dimensional structures of two serine CPDs, but because of constraints of space, I have summarized only a part of the wealth of recent data concerning these enzymes and their potential applications in biotechnology. Although more effort needs to be directed toward an understanding of substrate specificity in CPDs, it is clear that they will be very useful in a wide variety of biosynthetic and analytic procedures. Genetic engineering promises to make these enzymes even more useful for industrial and laboratory procedures. The fact that the serine CPDs belong to such a large family of hydrolytic enzymes suggests that it may be possible to create en-
J Biol Cbem
3.
468
Protein e n g i n e e r i n g 17.
ZIESKE LR, HSI K-L, CHEN L, YUAN P-M: S t r u c t u r a l Det e r m i n a t i o n o f t h e Essential Serine and G l y c o s y l a t i o n Sites o f C a r b o x y p e p t i d a s e P. Arch Biochem Biophys 1992, 295:76-83.
18.
ODYACE, ERD~3S EG: H u m a n P r o l y l c a r b o x y p e p t i d a s e . In Methods Enzymol, vol 80. Edited by Lorand L. New York: Academic Press; 1981:460-466.
28.
BRZOZOWSI, a AM, DEREWENDA U, DEREWENDA ZS, DODSON GG, LAWSONDM, TURKENBURGJP, BJORKLINGF, HUGE--JENSEN B, PATI~R, SA, THIM L: A Model for Interfacial Activation i n Lipases f r o m t h e Structure o f a F u n g a l H p a s e - I n h i b i t o r C o m p l e x . Nature 1991, 351:491-494.
29.
SUSSMANJL, HAREL M, FROLOW F, OEFNER C, GOLDMAN A, TOKER L, SILMAN I: A t o m i c S t r u c t u r e o f Acetylc h o l i n e s t e r a s e f r o m Torpedo c a l i f o r n i c a : a Prot o t y p i c A c e t y l c h o l i n e - B i n d i n g P r o t e i n . Science 1991, 253:872-879.
30.
FRANKENSM, ROZEBOOM HJ, KALK KH, DIJKSTRA BW: C r y s tal S t r u c t u r e o f H a l o a l k a n e D e h a l o g e n a s e : a n EnZyme t o D e t o x i f y H a l o g e n a t e d A l k a n e s . EMBO J 1991, 10:1297-1302.
19.
COOPERA, BUSSEYH: Characterization o f the Yeast KEXl G e n e P r o d u c t : a C a r b o x y p e p t i d a s e I n v o l v e d i n Proe e s s i n g Secreted P r e c u r s o r P r o t e i n s . Mol Cell Biol 1989, 9:2706-2714.
20.
HAYASHIR, MOORE S, STEIN WH: S e r i n e at the Active C e n t e r o f Yeast C a r b o x - y p e p t i d a s e . J Biol Chem 1973, 248:8366-8369.
21.
HAYASH1R, Btd Y, HAT& T: Evidence for a n E s s e n t i a l H i s t i d i n e i n C a r b o x y p e t i d a s e Y. R e a c t i o n w i t h the Chlormethylketone Derivative of BenzyloxocarbonylL - P h e n y l a l a n i n e . J Biol Chem 1975, 250:5221-5226.
31.
WIEMKENA, SCHELLENBERGM, URECHK: Vacuoles: t h e Sole C o m p a r t m e n t of Digestive EnZymes i n Y e a s t ( S a c c h a r o m y c e s c e r e v i s i a e ) ? Arch Microbiol 1979, 123:23-35.
22.
LIAO D-I, REMINGTON SJ: S t r u c t u r e o f W h e a t S e r i n e Carb o x y p e p t i d a s e II at 3.5 fik Resolution. J Biol Chem 1990, 265:6528-6531.
32.
KUNIGIS: P h y s i c o c h e m i c a l Factors C o n t r o l l i n g t h e Peptide S y n t h e s i s b y S e r i n e C a r b o x y p e p t i d a s e s . B i o m e d Biochim Acta 1991, 50:$32-$37.
33.
BREDOAM K, JOHANSEN TJ: S e n a i s y n t h e s i s o f H u m a n Ins u l i n Utili~itxg Chemically Modified C a r b o x y p e p t i d a s e Y. Carlsberg Res C o m m u n 1984, 49:463-472.
34.
BREDDAM K: C h e m i c a l l y Modifaed C a r b o x y p e p t i d a s e Y w i t h I n c r e a s e d A m i d a s e Activity. Carlsberg Res C o m m u n 1984, 49:535-554.
35.
SCHWARZA, WANDREY C, STEINKE D, KULA MR: A TWo-Step E n z y m a t i c S y n t h e s i s o f Dipeptides. Biotech Bioeng 1992, 39:132-140.
36.
AASMUL--OLSENS, THORBEK P, HANSEN S, WIDMER F: Enzymatic SIngle Step P r o c e s s e s for the Production o f D i p e p t i d e s from S i m p l e S t a r t i n g Materials. B i o m e d Biochim Acta 1991, 50:S106-S109.
37.
BEe,~EP-F, BLANQUETS, SCHMITrERJ-M: C a r b o x y p e p t i d a s e Y - C a t a l y z e d T r a n s p e p t i d a t i o n o f Esterified Oligo- a n d P o l y p e p t i d e s and Its Use for Specific Carboxy-Ternainai Labell/ng of P r o t e i n s . J A m Chem Soc 1992, 114:2603-2610.
23.
LIAO D-I, BREDDAM K, SWEET RM, BULLOCKT, REMINGTON SJ: Refined Atomic Model o f Wheat S e r i n e Carb o x y p e p t i d a s e H a t 2.2 A Resolution. Biochemistry 1992, 31:9796-9812. A definitive structural study of the first serine CPD to be crystallized that reports a n u m b e r of u n u s u a l results. T h e high-resolution model of the enzyme is analyzed in considerable detail and compared with those of chymotrypsin a n d subtilisin. It is concluded that critical active-site features of the catalytic residues that contact substrate are essentially identical in the three families of serine proteinases; nevertheless, there are revealing differences. An explanation for the pH d e p e n d e n c e of peptidase activity is proposed based on a study of the binding of arginine (a product) to the enzyme. 24.
WINTHERJR, STEVENS TH, I#-~IELLANDoBRANDTMC: Yeast Carb o x y p e p t i d a s e Y R e q u i r e s G l y e o s y l a t i o n for Ef~cient I n t r a e e l l u l a r T r a n s p o r t , but Not for V a c u o l a r Sorting, in Vivo Stability, o r Activity. Eur J Biochem 1991, 197:681--689.
25. •o
OLLIS DL, CHEAH E, CYGLER M, DYKSTRA B, FROLOW F, FRAKENS, HAREL M, REMINGTON SJ, SILMAN I, SCHRAGJ, ET AL.: T h e 0~/~ H y d r o l a s e Fold. Protein Eng 1992, 5:197-211. A very clear a n d remarkable paper in w h i c h the structures of five e n z y m e s with urLrelated a m i n o acid s e q u e n c e s and activity are compared and s h o w n to have a c o m m o n fold and similar active-site features. The e n z y m e s have diverged from a c o m m o n ancestor in such a w a y that the arrangement a n d n u m b e r of the catalytic groups, but not their identity, are retained. Only the histidine of the catalytic triad (acid, histidine, nucleophile) is conserved a m o n g all m e m b e r s of the family. All of the e n z y m e s are hydrolases, hence the n a m e of the fold. Some very u n u s u a l features c o m m o n to all members, such as disallowed conformational angles of the active-site nucleophfles, are discussed in detail. 26.
PATHAKD, NGAI KL, OLLIS, D: X-Ray C r y s t a l l . o g r a p h i c S t r u c t u r e o f D i e n e l a c t o n e H y d r o l a s e at 2.8 A Resolution. J Mol Biol 1988, 204:435-445.
27.
SCHRAG JD, LI Y, WU S, CYGLER M: Ser-I-Iis-Glu T r i a d F o r m s t h e Catalytic Site of t h e Lipase f r o m G e o t r i c b u m c a n d i d u m . N a t u r e 1991, 351:761-764.
38.
HENRIKSENDB, BREDDAMK, MOLLERJ, BUCHARDTO: P e p t i d e Amidation by Chemical Protein Engineering. A Combination of Enzymatic and Photochemical Synthesis. J A m Chem Soc 1992, 114:1877-1878. This is a brief account of a very interesting and extremely efficient u s e of yeast serine CPD to add non-natural photolabile substrates (2-nitrobenzyl derivatives) to the carboxyl terminus of a peptide by a transacylation reaction and then photochemically deprotecting to yield the peptide amide. While the goal of this paper was to d e m o n strate efficient production of the peptide amide, photochemical procedures like this h a v e other potential uses, such as rapid generation of a bioactive peptide at a particular instant or in a particular tissue.
SJ Remington, Institute of Molecular Biology and Department of Physics, University of Oregon, Eugene, Oregon 97403, USA.