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Proteases
Proteases Margaret A. Phillips and Robert J. Fletterick University of Texas Southwestern Medical Center, Dallas, Texas and University of California, San Francisco, California, USA Proteases control many physiological responses, thus the identification and characterization of specific proteases offers the prospect of new medical advances. Study of the structure-function relationships in protease-catalyzed hydrolysis of the peptide bond has advanced our understanding of enzyme catalysis and specificity. Three-dimensional structures have been solved for each mechanistic class of protease, and these studies have been extended using site-directed mutagenesis to characterize the amino acid residues required for enzyme function. Current Opinion in Structural Biology 1992, 2:713-720
Introduction The pancreatic proteases, exemplified by trypsin and carboxypeptidase, were among the earliest enzymes to be characterized. These enzymes have been extensively studied, both structurally and kinetically, as model sTsterns for understanding the molecular basis of catalysis and specificity in enzymes [ 1]. In addition to the digestive enzymes, proteases are essential components of physiological regulation [2]. These discoveries have led to an emerging interest in proteases as directors of biological processes and as potential chemotherapeutic targets. Proteases regulate catalytic activity through the limited proteolysis of inactive precursors, or zymogens. Peptide bond cleavage releases active protein through conformational rearrangements [3,4] or through removal of an inhibitory peptide [5"]. Active proteases are further regulated by specific protein inhibitors, the controlled balance between proteases and inhibitors being essential to maintenance of the normal physiological state [6]. Physiological responses to injury and invasion, such as blood coagulation [7], complement activation and fibrinolysis are all controlled by cascades of zymogen activation [2]. Study of the proteolytic events during fibrinolysis led to the development of tissue plasminogen activa tor as a therapeutic agent to dissolve blood clots after coronary thrombosis {8]. Programmed embryonic development is also dependent on specific proteases; for example, the easter gene, which encodes a trypsin-like serine protease, is essential for normal cell development in Drosophila [9]. Physiological responses that are regulated by hormones are often controlled by pro-hormone processing events, often involving both amino- and carboxyterminal cleavages [10,11]. For example, the reninangiotensin system controls blood pressure and vol-
ume homeostasis. Inhibitors of angiotensin-converting enzyme, used for the treatment of high blood pressure, were designed using the carboxypeptidase structure as a model for Zn 2+ protease action [12]. At the cellular level, proteases are involved in a multitude of pathways. These include processing of secretory proteins [13], regulated intracellular protein degradation [14], lysosomal protein degradation, and mast cell response to inflammatory or "allergic reaction [2]. Proteases have been implicated in a number of disease states. Increased levels of specific proteases appear to play a role in tumor metastasis [15] and arthritis [16], whereas an imbalance in the ratio of neutrophil elastase to its inhibitor, ¢{ 1 antitrypsin, is the underlying cause of emphysema [17]. In addition, proteases are essential to the life cycle of many viral and parasitic pathogens, functioning to process viral precursor proteins [18,19], to allow parasites to invade host cells [20,21] or to provide food [22]. Specific targeting of protease function is achieved through tight regulation, narrow distribution and limited substrate recognition. This specificity, as well as their physiological significance, makes proteases good chemotherapeutic targets. Rational drug design depends on a thorough understanding of the target protein. This review will focus on recent advances in understanding the structural basis of protease catalysis, substrate specificity and regulation.
Catalysis Proteases catalyze the hydrolysis of peptide bonds via nucleophilic attack of the targeted carbonyl bond (Fig. 1). The nature of the nucleophile participating in
Abbreviations HIV--human immunodeficiencyvirus; TS~transition state. ~) Current Biology Ltd ISSN 0959-440X
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714
Catalysis and regulation the hydrolytic mechanism is used to classify the proteases into four categories: serine proteases, cysteine proteases, metallo proteases and acid proteases. The members of each class are further categorized according to their evolutionary relationships. Unrelated proteins that have converged to similar mechanisms are: the serine proteases of the trypsin and subtilisin families; the metallo proteases of the carboxypeptidase and thermolysin families; and the thiol proteases of the papain and cysteine-activecenter viral protease families [23].
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that develops on histidine in the TS. Water-catalyzed hydrolysis of the acyl enzyme leads to the formation of products [3]. In trypsin, the catalytic triad is composed of Ser195, His57 and Asp102 (Fig. 2a). A charged tetrahedral intermediate is formed during both acylation and deacylation (Fig. la), and the oxyanion is stabilized through backbone amide hydrogens donated by residues Gly193 and Ser195. A wealth of data has been accumulated with the aim of understanding how the catalytic triad and the oxyanion hole function in hydrolysis. The importance of Ser195, His57 and Aspl02 in trypsin-catalyzed hydrolysis is underscored by the substantial reduction in catalytic rate observed upon the systematic mutation of each residue [24°°]. The free energy of TS binding decreased by 4 kcal m o l - 1 upon mutation of Aspl02 and by ~7kcalmo1-1 upon mutation of Ser195 or His57. Removal of the entire catalytic triad did not further destabilize the TS. A similar result was obtained for subtilisin in a study which also included mutations of the oxyanion hole. The oxyanion hole in subtilisin is formed by the sidechain amide of Asn155, the main-chain amide of Ser221 and the side-chain hydroxyl of Thr220, which (according to molecular dynamic studies) participates in a 4 ~ hydrogen bond. The reduction in TS binding energy was 4 kcal m o l - 1 for Asn155Ala, 2 kcal m o l - 1 for Thr220Ala and 6 kcal m o l - 1 for the double mutant [25"]. Mutation of Asn155 in combination with the catalytic histidine [26-] or serine [27] destabilized the TS to the same extent as was observed for the single-site mutation of histidine or serine. The extent of TS destabilization caused by replacing Thr220 with alanine was successfully predicted by free energy calculations [28°].
Fig. 1. General reaction mechanisms for (a) enzymes that form acyl enzyme intermediates (serine proteases are represented by OH; cysteine proteases are represented by S-), and (b) enzymes that use water as a nucleophile (metallo proteases are represented by Zn 2+, acid proteases are represented by COOH. The semicircle depicts the oxyanion binding site on the enzyme (E) and the arrows show electron flow during bond making and breaking. R, R' and R" represent side groups not involved in the reaction mechanism.
These studies demonstrate that the mutational effects of replacing catalytic residues may not be additive. The oxyanion hole and the catalytic triad of serine proteases function cooperatively to enhance the catalytic rate. Mutational effects on TS stabili W are only additive when the residues function independently and when the rate-limiting step and mechanism remain unchanged (e.g. the Thr220Ma/Asn155Ma double mutant of subtilisin).
Enzymes catalyze reactions by binding the transition state (TS) more tightly than the ground state. In catalyzing peptide-bond hydrolysis, proteases stabilize the TS by activating the nucleophile, stabilizing the oxyanion formed in the tetrahedral intermediate, and providing a proton to the amine leaving group. Each of the four classes of proteases achieve these functions in different but related ways.
In the absence of the key functional groups, trypsin and subtilisin still achieve increases in catalytic rate 104-105. fold greater than the uncatalyzed rate. Thus, in addition to the functional groups involved in the chemical reaction, interactions that contribute to binding and conformational positioning of the extended substrate in the TS contribute to rate enhancement during enzymatic catalysis.
Serine proteases Serine proteases are the best characterized of the four mechanistic classes. The peptide bond is cleaved by nucleophilic attack of the serine hydroxyl group on the scissile carbonyl bond, forming an acyl enzyme intermediate (Fig. la). An imidazole group in the active site abstracts the alcoholic proton and conveys it to the amine leaving group, while an aspartate stabilizes the positive charge
The activity of the His641Ma mutant of subtilisin was partially restored for substrates with histidine at the P2 position (see Fig. 3 for an explanation of the nomenclature). This rate enhancement was attributed to recruitment of the substrate histidine into the catalytic triad. The TS structure formed normal interactions with the oxyanion hole, but interaction with the remaining components of the triad was not optimal [26o°]. The reaction mechanism for trypsin has been described using classical and quantum mechanical theory [29°]. For
Proteases Phillips and Fletterick
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(d)
Fig. 2. Protease active-site structures. All complexes shown, except for that of carboxypeptidase A, represent ground-state structures. The molecule labeled as substrate shows the hypothetical position of the substrate and is based on the position of the inhibitor bound to the active site during the crystallographic experiment. Residues that are referred to in the text are labeled. (a) Trypsin complexed to bovine trypsin inhibitor, coordinates for which were obtained from the Brookhaven protein data bank (PDB), accession number ZPTC. The arrow points to the scissile carbonyl of the pseudo-substrate. (b) Papain, complexed to acetyl-Ala-Phe-methylenyl-Ala, the product of the reaction of a chloromethyl ketone with the enzyme, PDB accession number IPAD. The arrow indicates the methylene carbon, which in this structure is bound to the active site Cys. (c) Carboxypeptidase A complexed to the transition-state analog, Z-Cly-P(0)-Phe (N-Ubenzyloxycarbonyl-aminomethyll-hydroxy phosphinyll-t-phenylalanine); the coordinates were kindly provided by D Christianson. The tetrahedral phosphonate, which in the substrate would represent the scissile carbon, is labeled with an arrow. fd) Human immunodeficiency virus protease complexed to N-acetyl-Thr-lie-Nle-KH2-NH)-Nle-Glu-Arg, PDB accession number 4HVP (Nle, norleucine). The CH, group, which in the substrate would be the scissile carbonyl, is indicated with an arrow.
peptide hydrolysis, the rate-limiting step was formation of the first tetrahedral intermediate. Proton transfer from Ser195 to His57 occurred simultaneously with the attack of the serine hydroxyl on the scissile carbonyl bond; likewise, in deacylation, nucleophilic attack by water was concomitant with proton transfer. Asp102 stabilized the TS through an electrostatic contact with His57 and did not accept the proton. The buried charge on Asp102 was stabilized by W-214 and by backbone amides; k,_,t for the Ser214Lys mutant decreased to 1% of that for the wild type [30-l. This loss in activity was attributed to an alteration in the electric field of the catalytic triad.
Iaue crystallography has been used in an attempt to observe the chemical intermediates formed during inhibition of chymotrypsin by a mechanism-based inhibitor [31.]. In the future, this method may provide the means of observing the time-dependent structural changes that occur during enzyme-catalyzed reactions.
Cysteine proteases
The hydrolytic reaction catalyzed by cysteine proteases shares a number of features in common with that cata lyzed by serine proteases. It proceeds through an acyl enqme intermediate (Fig. la); an imidazole group assists in
715
716
Catalysisand regulation Enzyme
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Fig. 3. The protease polypeptide-binding site. Substrate amino acids extending away from the scissile bond towards the amino terminus are denoted P1, P2... Pn, whereas those extending towards the carboxyl terminus are denoted P1 ', P2' ... Pn'. The corresponding binding sites on the enzyme are donated $1, $2... Sn,
and $1', $2'... Sn'[59].
the nucleophilic attack but, unlike serine proteases, the active-site thiol readily forms an ion pair with the imidazole group in the ground state. The thiolate-imidazolium ion pair is stabilized by a hydrogen bond to a neutral asparagine and represents the nucleophile in the reaction [4]. In papain, the best characterized member of the cysteine protease family, this catalytic triad is made up of Cys25, His159 and Asn175 (Fig. 2b). The activity of papain towards thionoesters was thought to be inconsistent with the presence of an oxyanion hole. The amide proton of Cys25 and the side chain of Glnl9 are, however, in position to stabilize the oxyanion. Re placement of Gln19 with alanine or serine destabilized the TS by 2.4 and 3.8 kcal mo1-1, respectively, providing evidence that Glnl9 does stabilize the oxyanion [32"°]. Spectroscopy shows that papain is remarkably insensitive to the amino terminal to carboxy-terminal orientation of some peptides in the active site [33]. Resonance Raman spectroscopy was used to demon strate that the acyl group in N acTlglycine dithioacyl papain binds to the S1 and $2 subsites, whereas the acyl group of substrates with larger Pl side chains binds to the $1' and $2' subsites. The hydrolytic rate of the two sets of substrates only differed by a factor of three [32].
Metallo proteases Peptide-bond hydrolysis by metallo proteases proceeds via the nucleophilic attack of Zn2+-bound water on the scissile carbonyl bond (Fig. lb). The active site of carboxypeptidase A contains a general base (Glu270) which is positioned so as to remove the proton from the Zn 2+ -bound water, and a positively charged functional group (Arg127) positioned so as to stabilize the oxyanion (Fig. 2c) [34]. This mechanism is supported by the demonstration that phosphonate inhibitors are TS ana logs. The tetrahedral phosphonate moiety mimics the expected TS along the water-catalyzed pathway [35"]. The role of Arg127 in carboxypeptidase-catalyzed hydrolysis has been studied by site-directed-mutagenesis [36"]. Substitution with methionine or alanine destabilized the TS by 6~8 kcal m o l - 1 without significantly altering ground-state binding. The decreases in kcat/Km observed for the replacement of Arg127 with lysine, methio-
nine or alanine paralleled the decreases in binding affinity obtained for a matched set of phosphonate inhibitors; this correlation extended over a range of 7 kcal mol-1. These results demonstrate that phosphonate inhibitors mimic the electronic nature of the oxyanion, as well as the geometric components of the TS [36°']. The structure of leucine aminopeptidase bound to bestatin supports a general base mechanism for substrate hydrolysis, which is similar to the mechanism of carboxypeptidase-catalyzed hydrolysis [37"]. The active site contains two Zn 2 + molecules, but no evidence for a nucleophilic side chain has been found. Arg336 occupies a position in which it stabilizes the oxyanion, and Lys262 may provide a proton to the amine leaving group.
Acid proteases Acid proteases hydrolyze peptide bonds by a general base-catalyzed mechanism (Fig. lb). Hydrolysis proceeds by aspartate-assisted protonation of the scissile carbonyl oxygen, nucleophilic attack of water on the carbonyl bond (with transfer of a proton to a catalytic aspartate) and protonation of the amine leaving group by the acids to form products [38]. In contrast to the other protease classes, the tetrahedral intermediate, which is represented by an amide hydrate, is uncharged. Penicillopepsin binds difluorostatone-containing peptides as gem-diols [39"]. The structure resembles the tetrahedral intermediate expected along the watercatalyzed pathway. In addition, Asp213 is positioned to participate as the general base, Asp33 as the general acid, and a water molecule, bound between the two acids, acts as the nucleophile [39"']. Most acid proteases are monomeric, but the protease of human immunodeficiency virus type I (H1V-1) assembles the active-site acids, Asp25A and Asp25B, at the interface of two identical subunits, A and B (Fig. 2d) [40"°]. The catalytic mechanism of HW-1 protease appears to be identical to that of pepsin, and is characterized by the concerted transfer of two protons in the TS, with the collapse of the amide hydrate to form products being the rate-limiting step [40"].
Substrate specificity The primary specificity determinant usually includes the amino acids nearest the cleaved peptide bond; however, specificity can be manifest throughout the polypeptide-binding site (Fig. 3). At each site, amino acid preferences can be defined by collecting kinetic data on a series of peptide substrates. Structural data of enzyme-inhibitor complexes provides information about the enzyme residues involved in direct interactions with the substrate. It was generally believed that these interactions provided the basis for preferential substrate binding. It is becoming clear, however, that the structural determinants of specificity are not limited to these residues.
Proteases Phillips and Fletterick The P2 residue is the primary specificity determinant for the related cysteine proteases papain and cathepsin B; papain prefers phenytalanine to arginine by 900-fold, whereas cathepsin B accepts both with similar affinity [41.]. Serine in the $2 pocket of papain is replaced by glutamate in cathepsin B, accounting (in part) for the increased activity towards arginine substrates [41.]. Substitution of the $2 residues in papain with the corresponding residues from cathepsin B confers cathepsinB-like specificity on papain without compromising the catalytic rate [42-]. a-Lytic protease prefers small hydrophobic residues at the P1 site (Ala > P h e by 105-fold). The mutation of binding-site residues resulted in an enzyme with broad specificity and increased catalytic rate [43"]. The structural determinations of ten mutants complexed to boronic acid TS analogs revealed that increased flexibility within the binding pocket provides the basis for the broad specificity [43"]. This data set was used to develop an algorithm to aid in the design of proteases with desired specificity; an ¢vlytic protease with a preference for leucine at P1 was successfully engineered [44°].
polar interactions contribute significantly to the tight binding; the inhibitor phenylalanines are involved in a chain of four aromatic rings which form edge-to-face interactions [47-]. Neutral endopeptidase is a member of the thermolysin metallo protease family, but unlike thermolysin it also ha~s dipeptidyl carboxypeptidase activity. A novel arginine residue which may play a role in this activity has been identified by site-directed mutagenesis. Comparison with the thermolysin structure has suggested that the arginine is on the edge of the active site, in a position to interact with the terminal carboxylate [48.]. Leaving group specificity in chymotrypsin has been analyzed by characterizing acyl transfer to a series of glycine oligomers [49"]. Arginine was favored at Pl' and P3', consistent with the presence of two acidic residues in the S' sites.
Regulation Regulation by the propeptide
A different picture emerged for trypsin [45"]. The major specificity determinant for trypsin and chymotrypsin is the P1 residue; trypsin prefers Lys > Phe by 106-fold, whereas chymotrypsin prefers Phe > Lys by 103-fold. Re placement of the S1 residues in the trypsin pocket with the analogous residues from chymotrypsin caused a large reduction in catalytic rate. Chymotrypsin specificity was conferred on trypsin by the concurrent replacement of two surface loops. Interestingly, the loops do not form part of the substrate-binding site. Though catalytic efficiency was restored, the variant mutant trypsin does not bind substrate as well as wild-type trypsin.
Zymogens are activated by cleavage of an amino-terminal propeptide. Generally, the cleavage is followed by release of the propeptide, but in chymotrypsin the propeptide remains bound to the active enzyme through a disulfide bond. Activation occurs through the structural rearrangement of active-site residues [3]. Cleavage and release of the 44-amino-acid propeptide of pepsinogen also causes a major conformational change in the first 13 amino acids of the active enzyme (Ilel moves by 44A) [50]. These amino acids form part of the substrate-binding pocket, accounting for the inactivigTof the zymogen.
What factors contribute to the requirement for specificity determinants outside the substrate-binding pocket? Papain prefers large hydrophobic residues, yet its selectivity is not as stringent as that of trypsin (103-fold versus 106-fold substrate preference, respectively). To a large extent, specificity is manifest in the TS, not in the ground state. Flexible loops may function to increase selectivity through more productive TS binding.
An entirely different mode of zymogen activation is found in carboxypeptidase. Structural rearrangements of the enzymatic domain do not accompany cleavage. The propeptide makes extensive contacts throughout the entire substrate-binding region, preventing substrate access to the active site in a similar manner to protein inhibitors [5"]. Indeed, the cleaved propeptide inhibits carboxypeptidase A activity ( Ki = 2 nM).
The recruitment of surface loops in providing additional specificity is likely to be a general phenomenon. Structural comparison of enzymes in the carboxypeptidase family suggests that, in addition to binding determinants in the $1' pocket, a surface loop will also play a role in specificity [46..]. Experimental measurements of positional flexibilities suggest that the S' binding pocket and the surface loop are flexible, allowing for conformational changes upon amino acid replacement [46"].
The propeptide of ~-lytic protease inhibits enzyme activity (K i = 0.1 nM), implying a similar mechanism of zymogen activation [51"]. In addition, the propeptide is required for bacterial secretion and for in vitro refolding of the enzyme. Refolding in the absence of the propeptide results in the formation of a stable folding intem~ediate. Folding of this intermediate to form active enzyme is catalyzed, with a rate increase of 107, by the addition of propeptide to the reaction [51"].
Substrate-enzyme interactions outside the primary specificity determinant also contribute to substrate preferences. By combining the specificity preferences of carboxypeptidase A at P2, P1 and PI' (the major determinant), an extremely tight-binding ( K I = 11 fM) phosphonate inhibitor, Z-Phe-Val-e(O)Phe has been designed [35"]. This inhibitor binds the enzyme 106-fold more tightly than Z-Gly-Gly-e(O)Phe [36"]. Structural characterization has suggested that hydrophobic and weakly
Proteins as regulators of protease activity Structural characterization of enzyme-inhibitor complexes provides information about the mechanism of inhibition and about specificity determinants within the substrate-binding region. The mechanism of hirudin inhibition of thrombin differs from that of most serine protease inhibitors. Hirudin does not contact the S1
717
718 Catalysisand regulation subsite; instead, binding specificity is achieved through extensive interactions with regions distal to the cleaved bond. These interactions include electrostatic contacts with the anion-binding recognition exosite, a main specificity determinant in thrombin [52"]. The serine protease inhibitor ecotin may inhibit through a similar mechanism. Ecotin is unrelated to other serine protease inhibitors. It inhibits a broad range of enzymes, implying that interaction with the protease extends beyond the $1 site [53]. Thrombin is both a procoagulant, through the activation of fibrinogen, and an anticoagulant through the activation of protein C. The balance between these functions is critical to the regulation of hemostasis and is controlled by structural changes in thrombin induced by the binding of thrombomodulin. Mutation of Glu192 to glutamine mimics the specificity changes observed upon binding of thrombin to thrombomodulin [54"]. Fibrinogen-clotting and thrombomodulin-binding activities can be dissociated by single amino acid replacements. Lys52 is re quired for fibrinogen cleavage, but not protein C a c t i vation, whereas the opposite was found to be tree for Arg70 [55"].
Regulation by cleavage In addition to zymogen activation, proteolytic events promote some novel mechanisms of regulation. For example, thrombin function is not limited to its role in coagulation: it is also the most potent stimulator of platelet aggregation. The cellular functions are regulated by a receptor, which has been cloned and characterized. Thrombin cleaves the receptor adjacent to a hirudin like sequence, liberating a new receptor amino-terminal peptide (residues 42-55), which binds and activates the receptor [56"']. The pentapeptide Ser-Phe-Leu-Leu Arg, which corresponds to residues 42-46, was the minimal sequence required for agonist activity. Deletion of the serine and substitution of the phenylalanine abolished receptor activity [57]. The core protein from Sindbis virus shares structural features with chymotrypsin, including a catalytic triad. This fnding supports the hypothesis that autocatalysis liberates the core protein from the precursor viral polyprotein. The carboxy-terminal tryptophan remains in the S1 subsite, inactivating the proteolytic function and relegating the core to a structural role [58"].
Conclusion Mthough a lot has been learned about the structural basis for catalysis and specificity, it is clear that there are additional factors awaiting elucidation. Understanding the molecular interactions that allow proteases to be specifically regulated will provide insight into drug development, as well as the d e n o v o design of enzymes with novel functions.
References and recommended reading Papers of particular interest, published within the annual period of re. view, have been highlighted as: • of special interest •. of outstanding interest 1.
NEURATH H: Proteolytic Enzymes, Past and Present. Fed Proc
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NEURATHH: Proteolytic Processing and Physiological Regulation. Trends Biochem Sci 1989, 14:26~271.
3.
POLGARL: Serine Proteases. In Mechanisms of Protease Action. Boca Raton: CRC Press; 1989:87-113.
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POI~3ARL: Cysteine Proteases. In Mechanisms of Protease Action. Boca Raton: CRC Press; 1989:124~147.
1985, 44:2907-2913.
5. ..
GUASCHA, COLt M, A\aLES FX, HUBER R: Three-dimensional Structure of Porcine Pancreatic Procarboxypeptidase A: a Comparison of the A and B Zymogens and Their D e terminants for Inhibition and Activation. J Mol Biol 1992, 224:141 157. The first structural characterization of a propeptide which inactivates its protease by a mechanism similar to that used by protein inhibitors. 6.
BODE W, HUBER R: Proteinase-Protein Inhibitor Interaction. Biomed Biochim Acta 1991, 50:437-446.
7
DAVIE EW, FUJIK~XWAK, KISIELW: The Coagulation Cascade: Initiation, Maintenance, and Regulation. 13iocheml~'t~3' 1991, 30:10363 10370.
8.
COLI£ND, GOLD HK: New Developments in Thrombolytic Therapy. Adv Exper Med Biol 1990, 281:333-354.
9.
JIN YS, ANDERSON KV: Dominant and Recessive Alleles of the Drosophila easter Gene Are Point Mutations at Conserved Sites in the Set Protease Catalytic Domain. Cell 1990, 60:873-881.
10.
SKIDGEI.RA: Basic Carboxypeptidases: Regulators of Peptide Hormone Activity. Trends Pharmacol Sci 1988, 9:299-304.
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DARBYNJ, SMYTH DG: Endopeptidases and Prohormone Processing. Biosci Rep 1990, 10:1 13.
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HOOPERNM: Angiotensin Converting Enzyme: Implications from Molecular Biology for its Physiological Functions. Im J Biochem 1991, 23:641q547.
13.
MISUMIY, ODA K, FUJI~AP,A T, TAI'CAMIN, TASHIROK, IKE|tAIZ.XY: Functional Expression of Furin Demonstrating its Intracellular Localization and Endoprotease Activity for Processing of Proalbumin and Complement Pro-C3. 1 Biol Chem 1991, 266:16954-16959.
14.
CROALLDE, DEMARTINO GN: Calcium-activated Neutral Protease (Calpain) System: Structure, Function, and Regulation. Physiol Re1' 1991, 71:813--g47
15.
MATmStANLM: Metalloproteinases and Their Inhibitors in Matrix Remodeling. Trends Genet 1990, 6:121-125.
16.
HASTYKa, REIFE ia, RANG a~l, ST[ART JM: The Role of Stromelysin in the Cartilage Destruction that Accompanies Inflammatory Arthritis. Arthritis Rheum 1990, 33:388397.
17.
CRYSTALRG: The Alpha l-Antitrypsin Gene and its Deftciency States. Trends Genet 1989, 5:411~417.
18.
KORANTBD: Viral Proteases: an Emerging Therapeutic Target. Crit Rev Biotechnol 1988, 8:14~157.
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HUFFJR: HIV Protease: a Novel Chemotherapeutic Target for AIDS. J Med Chem 1991, 34:2305-2314.
20.
DOENHOFFMJ, CURTISS RH, NGAIZAJ, MODHAJ: Proteases in the Schistosome Life Cycle: a Paradigm for Turnout Metastasis. Cancer Metastasis Rev 1990, 9:381-392.
21.
BOIrWERJ, SCHNEIDER P, ETGES R, BORDIER C: Peptide Substrate Specificity of the Membrane-bound Metalloprotease of Leishmania. Biochemisto~ 1990, 29:10113-10119.
Proteases 22.
23.
C-OLDBERG DE, SLATER AF, CERAMI A, HENDERSON GB: Hemoglobin Degradation in the Malaria Parasite Plasm o d i u m falciparurr~ an Ordered Process in a Unique Organelle. Proc Natl Acad Sci USA 1990, 87:2931-2935. BAZANJF, FLETTERlCK RJ: Detection of a Trypsin-fike Serine Protease Domain in Flaviviruses and Pestiviruses. Virology 1989, 171:637~539.
24. ..
COREY DR, CRAm CS: An Investigation into the Minimum Requirements for Peptide Hydrolysis by Mutation of the Catalytic Triad of Trypsin. J Am Chem Soc 1992, 114:1784-1790. This paper demonstrates the importance of binding energy to TS sta bilization in enzymatic reactions. 25. ,
BRAXTONS, WELkS JA: The Importance of a Distal Hydrogen Bonding Group in Stabilizing the Transition State in Subtilisin BPN'. J Biol Chem 1991, 266:11797-11800. This paper and [28*] describe a coordinated theoretical and experimental approach which tests the effect of a mutation on an enzymatic reaction. 26. ..
CARTERP, ABRAHMSEN L, WELTS JA: Probing the Mechanism and Improving the Rate of Substrate-assisted Catalysis in Subtilisin BPN'. Biochemistry 1991, 30:6142~5148. This study provides insight into designing a sequence-specific protease. 27.
WELLSJA: Additivity of Mutational Effects in Proteins. Bic~ chemistry 1990, 29:8509-8517.
28.
MIZUSHIMAN, SPELLMEYERD, HIRONO S, PEARLMAND, KOLLMAN • P: Free Energy Perturbation Calculations on Binding and Catalysis after Mutating Thr220 in Subtifisin. J Biol Chem 1991, 266:11801-11809. This paper describes the theoretical outcome of the mutations de ,scribed in [25"]. 29. •
DAGGETrV, SCHRODERS, KOLLMANP: Catalytic Pathway of Set Proteases - - Classical Quantum Mechanical Calculations. J Am Chem Soc 1991, 113:8926-8935. The mechanism of trypsin-catalyzed hydrolysis is described by molecular simulation and provides a testable hypothesis for the detailed func tion of serine proteases. 30. ••
MCGRATH ME, V&SQUEZ JR, CRAIK CS, YANG AS, HONIG B, FLETI-ERICK RJ: Perturbing the Polar Environment of Aspl02 in Trypsin - - Consequences of Replacing Conserved Ser214. Biochemistry 1992, 31:3059-3064. One of the few studies to address the importance of neighboring residues on active-site function, and a remarkable attempt to quanti Late the role of electrostatic potential in catalysis. 31.
STODDARDBL, KOENIGSP, PORTER N, PETRATOS K, PETSKO GA, RINGE D: Observation of the Light-triggered Binding of Pyrone to Chymotrypsin by Laue X-ray Crystallography. Proc Natl Acad Sci USA 1991, 88:5503-5507. This paper describes a potentially powerful new method, which may allow for structural determination of enzyme complexes during cataly sis. •
32. ••
MENARDR, CARRmREJ, LmLAMME P, PLOUFFE C, KHOUm HE, VERNETT, TESSIER De, THOMAS DY, STORER AC: Contribution of the Glul9 Side Chain to Transition-state Stabilization in the Oxyanion Hole of Papain. Biochemistry 1991, 30:8924-8928. The authors provide experimental evidence that thiol proteases, like serine proteases, require oxyanion stabilization. This result supports the concept that ei'~,cnes work by complementing the charges that develop in the "iS through electrostatic interactions. 33.
TONGEPJ, MENARD R, STORER AC, RUZSlCSKA BP, CAREy PR: Markedly Different Acyl Papain Structures Deacylate at Similar Rates - - Resonance Raman Spectroscopic and Kinetic Evidence. J Am Cbem Soc 1991, 113:4297~t303.
34.
CHRISTIANSONDW, LIPSCOMBE XXFN:Carboxypeptidase A. Acc Chem Res 1989, 22:624;9.
35. ••
KAPLANAP, BARTu~Tr PA: Synthesis and Evaluation of an Inhibitor of Carboxypeptidase A with a Ki Value in the Femtomolar Range. Biochemistry 1991, 30:81658170.
Phillips and Fletterick
Potent enzyme inhibitors can be rationally designed where the stmc tural basis of catalysis and specificity has been characterized. 36. .
PHILLIPSMA, KAP1ANAP, RUTTERWJ, BARTLETT PA: Transitionstate Characterization: a New Approach Combining Inhibitor Analogues and Variation in Enzyme Structure. Biochemistry 1992, 31:959-963. This report demonstrates the importance of oxyanion stabilization in carboxypeptidase-catalyzed hydrolysis, and provides a broadly applicable approach for demonstrating TS analogy" for enzyme inhibitors. 37. .
BURLEYSK, DAVID PR, LIPSCOMB WN: Leu Aminopeptidase: Bestatin Inhibition and a Model for Enzyme-catalyzed Peptide Hydrolysis. Proc Natl Acad Sci USA 1991, 88:691645920. Structural analysis of the bestatin-leucine aminopeptidase complex provides evidence that metallo an',inopeptidases function by a mechanism similar to those of the well studied proteases carboxypeptidase and thermolysin. 38.
DAVIESDR: The Structure and Function of the Asp Proteinases. A n n u Key Biophys B i o p ~ s Chem 1990, 19:189--215.
39. ..
JAMESMNG, SIELECKIAR, HAYAKAWAK, GEm MH: Crystallographic Analysis of Transition State Mimics Bound to PeniciUopepsin: Difluorostatine-containing and Difluorostatonecontaining Peptides. Biochemistry 1992, 31:3872~886. A detailed structural analysis of the catalytic mechanism of an aspartyl protease. 40. ..
HYLANDLJ, TOMASZEK TA JR, MEEK TD: Human Immunodeficiency Virus-1 Protease. 2. Use of pH Rate Studies and Solvent Kinetic Isotope Effects to Elucidate Details of Chemical Mechanisms. Biochemistry 1991, 30:8454-8463. A detailed mechanistic study of the catalytic function of HIV 1 protease. 41. •
MUSILD, ZUClC D, TURK D, ENGH RA, MAYR I, HUBER R, POPOVICT, TURK V, TOWATAR1T, KATUNUMAN: The Refined 2.15h X-ray Crystal Structure of Human Liver Cathepsin B: the Structural Basis for its Specificity. F~14BO J 1991, 10:2321 2330. This paper details the structural features that distinguish cathepsin B substmte specificity from that of papain, and includes an explanation for the dipeptidyl carbox}qpeptidase activity,. 42. .•
KHOURIHE, VERNETT, MENARDR, PARLATIF, LAFLAMEP, TESSIER De, GOUR-SALIN B, THOMAS DY, STOKER AC: Engineering of Papain: Selective Alteration of Substrate Specificity by Sitedirected Mutagenesis. Biochemistry 1991, 30:8929-8936. Substrate specificity in papa.in can evolve through a very small number of point mutations in amino acids that directly contact the substrate. 43. •.
BONER, FUJISHIGEA, KETTNER CA, AGARD DA: Structural Basis for Broad Specificity in Mpha-lytic Protease Mutants. Biochemistry 1991, 30:10388-10398. Flexibility in the substrate binding site allows the enzyme to accommodate substrates of va135ng size. 44. •
WILSONC, MACEJE, AGARDDA: Computation Method for the Design of Enzymes with Altered Substrate Specificity. J Mol B i d 1991, 220:495 506. Specificity can be predicted if a large enough data set of the conformational effects of mutations has been compiled for a specific enzyme. 45. **
HEDSTROML, SZILAGYI L, RUTFER WJ: Converting Trypsin to Chymotrypsin: the Role of Surface Loops. Science 1992, 255:1249-1253. Specificity detemainants are not collfined to enz~ane residues that form direct contacts with the substrate. 46. ••
FAMINGZ, KOBE B, STEWART CB, RUTFER WJ, GOLDSMITH EJ: Structural Evolution of an Enzyme Specificity: the Structure of Rat Carboxypeptidase A2 at 1.9-A Resolution. J Biol Chem 1991, 266:24606-24612. This report provides insight into the structural basis of surface loops as specificity determinants. 47. .•
KIM H, LIPSCOMBX~rN:Comparison of the Structures of Three Carboxypeptidase A-Phosphonate Complexes Determined by X-ray Crystallography. Biochemistry 1991. 30:8171~q180.
719
720
Catalysis and regulation The authors provide evidence that very strong binding affinities can be produced from relatively weak interactions, if recruited in specific spatial arrangements. 48.
BEAUMONTA, BARBE B, LE MOUAL H, BOILEAU G, CRINE P, . FOURNIE-ZALUSKIMC, ROQUES BP: Charge Polarity Reversal Inverses the Specificity of Neutral Endopeptidase-24.11. J Biol Chem 1992, 267:2138-2141. This paper demonstrates the structural basis for dipeptidyt carboxypeptidase activity. 49. .
SCHELLENBERGERV, JAKUBKE HD, KASCHE V: Electrostatic Elfects in the Alpha-chymotrypsin-catalyzed Acyl Transfer. II. Efficiency of Nucleophiles Bearing Charged Groups in Various Locations. Biochim Biophys Acta 1991, 1078:8-11. One of the few studies to address leaving group specificity in serine proteases. 50.
BAKERD, SOHL JL, AGARD DA: A Protein-folding Reaction u n d e r Kinetic Control. Nature 1992, 356:263-265. role of propeptides is not limited to enzyme inhibition. For ~-lytic protease, the propeptide is required for catalysis of the protein folding reaction, demonstrating that protein folding is not always under thermodynamic control. 52. •.
RYDELTJ, TULINSKY A, BODE W, HUBER R: Refined Structure of the Hirudin-Thrombin Complex. J Mol Biol 1992, 221:583-601. Interactions that are important for the binding of himdin in the anionbinding recognition exosite of thrombin also play a role in the binding of biological substrates such as the thrombin receptor.
54. •
55. •
WU QY, SHEEHANJP, TSIANG M, LENTZ SR, BIRKTOFTJJ, SADLER JE: Single Amino Acid Substitutions Dissociate Fibrinogenclotting and Thrombomodulin-binding Activities of H u m a n Thrombin. Proc Natl Acad Sci USA 1991, 88:6775-6779. This study and [54*] provide insight into the mechanism by which thrombomodulin alters thrombin specificity. 56. ,.
COUGHLIN SR, VU TK, HUNG DT, WHEATON VI: Characterization of a Functional Thrombin Receptor. Issues and Opportunities. J Clin Invest 1992, 89:351-355. The authors present a novel mechanism for receptor activation, which explains the mechanism of thrombin-induced cellular events at the molecular level. 57.
SIELECKIAR, FUJ1NAGAM, READ RJ, JAMES MN: Refined Structure of Porcine Pepsinogen at 1.8A Resolution. J Mol Biol 1991, 219:671492.
51. ~e
53.
This study and [55*] provide insight into the mechanism by which thrombomodulin alters the substrate specificity of thrombin.
MCGRATHME, HINES WM, SAKANARIJA, FLETrERICK RJ, CRAIK CS: The Sequence and Reactive Site of Ecotin. A General Inhibitor of Pancreatic Ser Proteases from Escherichia coli. J Biol Chem 1991, 266:662045625. LE BONNIEC BF, ESMON CT: GIu-192--~GIn Substitution in Thrombin Mimics t h e Catalytic Switch I n d u c e d by Thrombomodulin. Proc Natl Acad Sci USA 1991, 88:7371-7375.
SCARBOROUGHRM, MAUGHTON M, TENG W, HUNG DT, ROSE J, Vu TKH, WHEATON Vl, TURCK CW, COUGHLIN SR: Tethered Ligand Agonist Peptides: Structural Requirements for Thrombin Receptor Activation. J Biol Chem 1992, 267:13146-13149.
58. ..
CHOI HK, TONG L, MINOR W, DUMAS P, BOEGE U, ROSSMANN MG, WENGLER G: Structure of Sindbis Virus Core Protein Reveals a Chymotrypsin-like Ser Proteinase and t h e Organization of t h e Virion. Nature 1991, 354:37-43. This paper reports the structure of a unique serine protease which, after a single catalytic event, becomes inactive as a protease and assumes a structural role in the viral particle. 59.
SCHECHTERI, BERGER A: O n the Size of t h e Active Site in Proteases. 1. Papain. Biochem Biophys Res Commun 1967, 27:157-162.
MA Phillips, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas 75235, USA_ RJ Fletterick, The Hormone Research Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA.
Introduction The pancreatic proteases, exemplified by trypsin and carboxypeptidase, were among the earliest enzymes to be characterized. These enzymes have been extensively studied, both structurally and kinetically, as model sTsterns for understanding the molecular basis of catalysis and specificity in enzymes [ 1]. In addition to the digestive enzymes, proteases are essential components of physiological regulation [2]. These discoveries have led to an emerging interest in proteases as directors of biological processes and as potential chemotherapeutic targets. Proteases regulate catalytic activity through the limited proteolysis of inactive precursors, or zymogens. Peptide bond cleavage releases active protein through conformational rearrangements [3,4] or through removal of an inhibitory peptide [5"]. Active proteases are further regulated by specific protein inhibitors, the controlled balance between proteases and inhibitors being essential to maintenance of the normal physiological state [6]. Physiological responses to injury and invasion, such as blood coagulation [7], complement activation and fibrinolysis are all controlled by cascades of zymogen activation [2]. Study of the proteolytic events during fibrinolysis led to the development of tissue plasminogen activa tor as a therapeutic agent to dissolve blood clots after coronary thrombosis {8]. Programmed embryonic development is also dependent on specific proteases; for example, the easter gene, which encodes a trypsin-like serine protease, is essential for normal cell development in Drosophila [9]. Physiological responses that are regulated by hormones are often controlled by pro-hormone processing events, often involving both amino- and carboxyterminal cleavages [10,11]. For example, the reninangiotensin system controls blood pressure and vol-
ume homeostasis. Inhibitors of angiotensin-converting enzyme, used for the treatment of high blood pressure, were designed using the carboxypeptidase structure as a model for Zn 2+ protease action [12]. At the cellular level, proteases are involved in a multitude of pathways. These include processing of secretory proteins [13], regulated intracellular protein degradation [14], lysosomal protein degradation, and mast cell response to inflammatory or "allergic reaction [2]. Proteases have been implicated in a number of disease states. Increased levels of specific proteases appear to play a role in tumor metastasis [15] and arthritis [16], whereas an imbalance in the ratio of neutrophil elastase to its inhibitor, ¢{ 1 antitrypsin, is the underlying cause of emphysema [17]. In addition, proteases are essential to the life cycle of many viral and parasitic pathogens, functioning to process viral precursor proteins [18,19], to allow parasites to invade host cells [20,21] or to provide food [22]. Specific targeting of protease function is achieved through tight regulation, narrow distribution and limited substrate recognition. This specificity, as well as their physiological significance, makes proteases good chemotherapeutic targets. Rational drug design depends on a thorough understanding of the target protein. This review will focus on recent advances in understanding the structural basis of protease catalysis, substrate specificity and regulation.
Catalysis Proteases catalyze the hydrolysis of peptide bonds via nucleophilic attack of the targeted carbonyl bond (Fig. 1). The nature of the nucleophile participating in
Abbreviations HIV--human immunodeficiencyvirus; TS~transition state. ~) Current Biology Ltd ISSN 0959-440X
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714
Catalysis and regulation the hydrolytic mechanism is used to classify the proteases into four categories: serine proteases, cysteine proteases, metallo proteases and acid proteases. The members of each class are further categorized according to their evolutionary relationships. Unrelated proteins that have converged to similar mechanisms are: the serine proteases of the trypsin and subtilisin families; the metallo proteases of the carboxypeptidase and thermolysin families; and the thiol proteases of the papain and cysteine-activecenter viral protease families [23].
(a) R'
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~
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that develops on histidine in the TS. Water-catalyzed hydrolysis of the acyl enzyme leads to the formation of products [3]. In trypsin, the catalytic triad is composed of Ser195, His57 and Asp102 (Fig. 2a). A charged tetrahedral intermediate is formed during both acylation and deacylation (Fig. la), and the oxyanion is stabilized through backbone amide hydrogens donated by residues Gly193 and Ser195. A wealth of data has been accumulated with the aim of understanding how the catalytic triad and the oxyanion hole function in hydrolysis. The importance of Ser195, His57 and Aspl02 in trypsin-catalyzed hydrolysis is underscored by the substantial reduction in catalytic rate observed upon the systematic mutation of each residue [24°°]. The free energy of TS binding decreased by 4 kcal m o l - 1 upon mutation of Aspl02 and by ~7kcalmo1-1 upon mutation of Ser195 or His57. Removal of the entire catalytic triad did not further destabilize the TS. A similar result was obtained for subtilisin in a study which also included mutations of the oxyanion hole. The oxyanion hole in subtilisin is formed by the sidechain amide of Asn155, the main-chain amide of Ser221 and the side-chain hydroxyl of Thr220, which (according to molecular dynamic studies) participates in a 4 ~ hydrogen bond. The reduction in TS binding energy was 4 kcal m o l - 1 for Asn155Ala, 2 kcal m o l - 1 for Thr220Ala and 6 kcal m o l - 1 for the double mutant [25"]. Mutation of Asn155 in combination with the catalytic histidine [26-] or serine [27] destabilized the TS to the same extent as was observed for the single-site mutation of histidine or serine. The extent of TS destabilization caused by replacing Thr220 with alanine was successfully predicted by free energy calculations [28°].
Fig. 1. General reaction mechanisms for (a) enzymes that form acyl enzyme intermediates (serine proteases are represented by OH; cysteine proteases are represented by S-), and (b) enzymes that use water as a nucleophile (metallo proteases are represented by Zn 2+, acid proteases are represented by COOH. The semicircle depicts the oxyanion binding site on the enzyme (E) and the arrows show electron flow during bond making and breaking. R, R' and R" represent side groups not involved in the reaction mechanism.
These studies demonstrate that the mutational effects of replacing catalytic residues may not be additive. The oxyanion hole and the catalytic triad of serine proteases function cooperatively to enhance the catalytic rate. Mutational effects on TS stabili W are only additive when the residues function independently and when the rate-limiting step and mechanism remain unchanged (e.g. the Thr220Ma/Asn155Ma double mutant of subtilisin).
Enzymes catalyze reactions by binding the transition state (TS) more tightly than the ground state. In catalyzing peptide-bond hydrolysis, proteases stabilize the TS by activating the nucleophile, stabilizing the oxyanion formed in the tetrahedral intermediate, and providing a proton to the amine leaving group. Each of the four classes of proteases achieve these functions in different but related ways.
In the absence of the key functional groups, trypsin and subtilisin still achieve increases in catalytic rate 104-105. fold greater than the uncatalyzed rate. Thus, in addition to the functional groups involved in the chemical reaction, interactions that contribute to binding and conformational positioning of the extended substrate in the TS contribute to rate enhancement during enzymatic catalysis.
Serine proteases Serine proteases are the best characterized of the four mechanistic classes. The peptide bond is cleaved by nucleophilic attack of the serine hydroxyl group on the scissile carbonyl bond, forming an acyl enzyme intermediate (Fig. la). An imidazole group in the active site abstracts the alcoholic proton and conveys it to the amine leaving group, while an aspartate stabilizes the positive charge
The activity of the His641Ma mutant of subtilisin was partially restored for substrates with histidine at the P2 position (see Fig. 3 for an explanation of the nomenclature). This rate enhancement was attributed to recruitment of the substrate histidine into the catalytic triad. The TS structure formed normal interactions with the oxyanion hole, but interaction with the remaining components of the triad was not optimal [26o°]. The reaction mechanism for trypsin has been described using classical and quantum mechanical theory [29°]. For
Proteases Phillips and Fletterick
(b)
\
‘, \ Oxyanm h o l e
Asp1 02
(d)
Fig. 2. Protease active-site structures. All complexes shown, except for that of carboxypeptidase A, represent ground-state structures. The molecule labeled as substrate shows the hypothetical position of the substrate and is based on the position of the inhibitor bound to the active site during the crystallographic experiment. Residues that are referred to in the text are labeled. (a) Trypsin complexed to bovine trypsin inhibitor, coordinates for which were obtained from the Brookhaven protein data bank (PDB), accession number ZPTC. The arrow points to the scissile carbonyl of the pseudo-substrate. (b) Papain, complexed to acetyl-Ala-Phe-methylenyl-Ala, the product of the reaction of a chloromethyl ketone with the enzyme, PDB accession number IPAD. The arrow indicates the methylene carbon, which in this structure is bound to the active site Cys. (c) Carboxypeptidase A complexed to the transition-state analog, Z-Cly-P(0)-Phe (N-Ubenzyloxycarbonyl-aminomethyll-hydroxy phosphinyll-t-phenylalanine); the coordinates were kindly provided by D Christianson. The tetrahedral phosphonate, which in the substrate would represent the scissile carbon, is labeled with an arrow. fd) Human immunodeficiency virus protease complexed to N-acetyl-Thr-lie-Nle-KH2-NH)-Nle-Glu-Arg, PDB accession number 4HVP (Nle, norleucine). The CH, group, which in the substrate would be the scissile carbonyl, is indicated with an arrow.
peptide hydrolysis, the rate-limiting step was formation of the first tetrahedral intermediate. Proton transfer from Ser195 to His57 occurred simultaneously with the attack of the serine hydroxyl on the scissile carbonyl bond; likewise, in deacylation, nucleophilic attack by water was concomitant with proton transfer. Asp102 stabilized the TS through an electrostatic contact with His57 and did not accept the proton. The buried charge on Asp102 was stabilized by W-214 and by backbone amides; k,_,t for the Ser214Lys mutant decreased to 1% of that for the wild type [30-l. This loss in activity was attributed to an alteration in the electric field of the catalytic triad.
Iaue crystallography has been used in an attempt to observe the chemical intermediates formed during inhibition of chymotrypsin by a mechanism-based inhibitor [31.]. In the future, this method may provide the means of observing the time-dependent structural changes that occur during enzyme-catalyzed reactions.
Cysteine proteases
The hydrolytic reaction catalyzed by cysteine proteases shares a number of features in common with that cata lyzed by serine proteases. It proceeds through an acyl enqme intermediate (Fig. la); an imidazole group assists in
715
716
Catalysisand regulation Enzyme
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Fig. 3. The protease polypeptide-binding site. Substrate amino acids extending away from the scissile bond towards the amino terminus are denoted P1, P2... Pn, whereas those extending towards the carboxyl terminus are denoted P1 ', P2' ... Pn'. The corresponding binding sites on the enzyme are donated $1, $2... Sn,
and $1', $2'... Sn'[59].
the nucleophilic attack but, unlike serine proteases, the active-site thiol readily forms an ion pair with the imidazole group in the ground state. The thiolate-imidazolium ion pair is stabilized by a hydrogen bond to a neutral asparagine and represents the nucleophile in the reaction [4]. In papain, the best characterized member of the cysteine protease family, this catalytic triad is made up of Cys25, His159 and Asn175 (Fig. 2b). The activity of papain towards thionoesters was thought to be inconsistent with the presence of an oxyanion hole. The amide proton of Cys25 and the side chain of Glnl9 are, however, in position to stabilize the oxyanion. Re placement of Gln19 with alanine or serine destabilized the TS by 2.4 and 3.8 kcal mo1-1, respectively, providing evidence that Glnl9 does stabilize the oxyanion [32"°]. Spectroscopy shows that papain is remarkably insensitive to the amino terminal to carboxy-terminal orientation of some peptides in the active site [33]. Resonance Raman spectroscopy was used to demon strate that the acyl group in N acTlglycine dithioacyl papain binds to the S1 and $2 subsites, whereas the acyl group of substrates with larger Pl side chains binds to the $1' and $2' subsites. The hydrolytic rate of the two sets of substrates only differed by a factor of three [32].
Metallo proteases Peptide-bond hydrolysis by metallo proteases proceeds via the nucleophilic attack of Zn2+-bound water on the scissile carbonyl bond (Fig. lb). The active site of carboxypeptidase A contains a general base (Glu270) which is positioned so as to remove the proton from the Zn 2+ -bound water, and a positively charged functional group (Arg127) positioned so as to stabilize the oxyanion (Fig. 2c) [34]. This mechanism is supported by the demonstration that phosphonate inhibitors are TS ana logs. The tetrahedral phosphonate moiety mimics the expected TS along the water-catalyzed pathway [35"]. The role of Arg127 in carboxypeptidase-catalyzed hydrolysis has been studied by site-directed-mutagenesis [36"]. Substitution with methionine or alanine destabilized the TS by 6~8 kcal m o l - 1 without significantly altering ground-state binding. The decreases in kcat/Km observed for the replacement of Arg127 with lysine, methio-
nine or alanine paralleled the decreases in binding affinity obtained for a matched set of phosphonate inhibitors; this correlation extended over a range of 7 kcal mol-1. These results demonstrate that phosphonate inhibitors mimic the electronic nature of the oxyanion, as well as the geometric components of the TS [36°']. The structure of leucine aminopeptidase bound to bestatin supports a general base mechanism for substrate hydrolysis, which is similar to the mechanism of carboxypeptidase-catalyzed hydrolysis [37"]. The active site contains two Zn 2 + molecules, but no evidence for a nucleophilic side chain has been found. Arg336 occupies a position in which it stabilizes the oxyanion, and Lys262 may provide a proton to the amine leaving group.
Acid proteases Acid proteases hydrolyze peptide bonds by a general base-catalyzed mechanism (Fig. lb). Hydrolysis proceeds by aspartate-assisted protonation of the scissile carbonyl oxygen, nucleophilic attack of water on the carbonyl bond (with transfer of a proton to a catalytic aspartate) and protonation of the amine leaving group by the acids to form products [38]. In contrast to the other protease classes, the tetrahedral intermediate, which is represented by an amide hydrate, is uncharged. Penicillopepsin binds difluorostatone-containing peptides as gem-diols [39"]. The structure resembles the tetrahedral intermediate expected along the watercatalyzed pathway. In addition, Asp213 is positioned to participate as the general base, Asp33 as the general acid, and a water molecule, bound between the two acids, acts as the nucleophile [39"']. Most acid proteases are monomeric, but the protease of human immunodeficiency virus type I (H1V-1) assembles the active-site acids, Asp25A and Asp25B, at the interface of two identical subunits, A and B (Fig. 2d) [40"°]. The catalytic mechanism of HW-1 protease appears to be identical to that of pepsin, and is characterized by the concerted transfer of two protons in the TS, with the collapse of the amide hydrate to form products being the rate-limiting step [40"].
Substrate specificity The primary specificity determinant usually includes the amino acids nearest the cleaved peptide bond; however, specificity can be manifest throughout the polypeptide-binding site (Fig. 3). At each site, amino acid preferences can be defined by collecting kinetic data on a series of peptide substrates. Structural data of enzyme-inhibitor complexes provides information about the enzyme residues involved in direct interactions with the substrate. It was generally believed that these interactions provided the basis for preferential substrate binding. It is becoming clear, however, that the structural determinants of specificity are not limited to these residues.
Proteases Phillips and Fletterick The P2 residue is the primary specificity determinant for the related cysteine proteases papain and cathepsin B; papain prefers phenytalanine to arginine by 900-fold, whereas cathepsin B accepts both with similar affinity [41.]. Serine in the $2 pocket of papain is replaced by glutamate in cathepsin B, accounting (in part) for the increased activity towards arginine substrates [41.]. Substitution of the $2 residues in papain with the corresponding residues from cathepsin B confers cathepsinB-like specificity on papain without compromising the catalytic rate [42-]. a-Lytic protease prefers small hydrophobic residues at the P1 site (Ala > P h e by 105-fold). The mutation of binding-site residues resulted in an enzyme with broad specificity and increased catalytic rate [43"]. The structural determinations of ten mutants complexed to boronic acid TS analogs revealed that increased flexibility within the binding pocket provides the basis for the broad specificity [43"]. This data set was used to develop an algorithm to aid in the design of proteases with desired specificity; an ¢vlytic protease with a preference for leucine at P1 was successfully engineered [44°].
polar interactions contribute significantly to the tight binding; the inhibitor phenylalanines are involved in a chain of four aromatic rings which form edge-to-face interactions [47-]. Neutral endopeptidase is a member of the thermolysin metallo protease family, but unlike thermolysin it also ha~s dipeptidyl carboxypeptidase activity. A novel arginine residue which may play a role in this activity has been identified by site-directed mutagenesis. Comparison with the thermolysin structure has suggested that the arginine is on the edge of the active site, in a position to interact with the terminal carboxylate [48.]. Leaving group specificity in chymotrypsin has been analyzed by characterizing acyl transfer to a series of glycine oligomers [49"]. Arginine was favored at Pl' and P3', consistent with the presence of two acidic residues in the S' sites.
Regulation Regulation by the propeptide
A different picture emerged for trypsin [45"]. The major specificity determinant for trypsin and chymotrypsin is the P1 residue; trypsin prefers Lys > Phe by 106-fold, whereas chymotrypsin prefers Phe > Lys by 103-fold. Re placement of the S1 residues in the trypsin pocket with the analogous residues from chymotrypsin caused a large reduction in catalytic rate. Chymotrypsin specificity was conferred on trypsin by the concurrent replacement of two surface loops. Interestingly, the loops do not form part of the substrate-binding site. Though catalytic efficiency was restored, the variant mutant trypsin does not bind substrate as well as wild-type trypsin.
Zymogens are activated by cleavage of an amino-terminal propeptide. Generally, the cleavage is followed by release of the propeptide, but in chymotrypsin the propeptide remains bound to the active enzyme through a disulfide bond. Activation occurs through the structural rearrangement of active-site residues [3]. Cleavage and release of the 44-amino-acid propeptide of pepsinogen also causes a major conformational change in the first 13 amino acids of the active enzyme (Ilel moves by 44A) [50]. These amino acids form part of the substrate-binding pocket, accounting for the inactivigTof the zymogen.
What factors contribute to the requirement for specificity determinants outside the substrate-binding pocket? Papain prefers large hydrophobic residues, yet its selectivity is not as stringent as that of trypsin (103-fold versus 106-fold substrate preference, respectively). To a large extent, specificity is manifest in the TS, not in the ground state. Flexible loops may function to increase selectivity through more productive TS binding.
An entirely different mode of zymogen activation is found in carboxypeptidase. Structural rearrangements of the enzymatic domain do not accompany cleavage. The propeptide makes extensive contacts throughout the entire substrate-binding region, preventing substrate access to the active site in a similar manner to protein inhibitors [5"]. Indeed, the cleaved propeptide inhibits carboxypeptidase A activity ( Ki = 2 nM).
The recruitment of surface loops in providing additional specificity is likely to be a general phenomenon. Structural comparison of enzymes in the carboxypeptidase family suggests that, in addition to binding determinants in the $1' pocket, a surface loop will also play a role in specificity [46..]. Experimental measurements of positional flexibilities suggest that the S' binding pocket and the surface loop are flexible, allowing for conformational changes upon amino acid replacement [46"].
The propeptide of ~-lytic protease inhibits enzyme activity (K i = 0.1 nM), implying a similar mechanism of zymogen activation [51"]. In addition, the propeptide is required for bacterial secretion and for in vitro refolding of the enzyme. Refolding in the absence of the propeptide results in the formation of a stable folding intem~ediate. Folding of this intermediate to form active enzyme is catalyzed, with a rate increase of 107, by the addition of propeptide to the reaction [51"].
Substrate-enzyme interactions outside the primary specificity determinant also contribute to substrate preferences. By combining the specificity preferences of carboxypeptidase A at P2, P1 and PI' (the major determinant), an extremely tight-binding ( K I = 11 fM) phosphonate inhibitor, Z-Phe-Val-e(O)Phe has been designed [35"]. This inhibitor binds the enzyme 106-fold more tightly than Z-Gly-Gly-e(O)Phe [36"]. Structural characterization has suggested that hydrophobic and weakly
Proteins as regulators of protease activity Structural characterization of enzyme-inhibitor complexes provides information about the mechanism of inhibition and about specificity determinants within the substrate-binding region. The mechanism of hirudin inhibition of thrombin differs from that of most serine protease inhibitors. Hirudin does not contact the S1
717
718 Catalysisand regulation subsite; instead, binding specificity is achieved through extensive interactions with regions distal to the cleaved bond. These interactions include electrostatic contacts with the anion-binding recognition exosite, a main specificity determinant in thrombin [52"]. The serine protease inhibitor ecotin may inhibit through a similar mechanism. Ecotin is unrelated to other serine protease inhibitors. It inhibits a broad range of enzymes, implying that interaction with the protease extends beyond the $1 site [53]. Thrombin is both a procoagulant, through the activation of fibrinogen, and an anticoagulant through the activation of protein C. The balance between these functions is critical to the regulation of hemostasis and is controlled by structural changes in thrombin induced by the binding of thrombomodulin. Mutation of Glu192 to glutamine mimics the specificity changes observed upon binding of thrombin to thrombomodulin [54"]. Fibrinogen-clotting and thrombomodulin-binding activities can be dissociated by single amino acid replacements. Lys52 is re quired for fibrinogen cleavage, but not protein C a c t i vation, whereas the opposite was found to be tree for Arg70 [55"].
Regulation by cleavage In addition to zymogen activation, proteolytic events promote some novel mechanisms of regulation. For example, thrombin function is not limited to its role in coagulation: it is also the most potent stimulator of platelet aggregation. The cellular functions are regulated by a receptor, which has been cloned and characterized. Thrombin cleaves the receptor adjacent to a hirudin like sequence, liberating a new receptor amino-terminal peptide (residues 42-55), which binds and activates the receptor [56"']. The pentapeptide Ser-Phe-Leu-Leu Arg, which corresponds to residues 42-46, was the minimal sequence required for agonist activity. Deletion of the serine and substitution of the phenylalanine abolished receptor activity [57]. The core protein from Sindbis virus shares structural features with chymotrypsin, including a catalytic triad. This fnding supports the hypothesis that autocatalysis liberates the core protein from the precursor viral polyprotein. The carboxy-terminal tryptophan remains in the S1 subsite, inactivating the proteolytic function and relegating the core to a structural role [58"].
Conclusion Mthough a lot has been learned about the structural basis for catalysis and specificity, it is clear that there are additional factors awaiting elucidation. Understanding the molecular interactions that allow proteases to be specifically regulated will provide insight into drug development, as well as the d e n o v o design of enzymes with novel functions.
References and recommended reading Papers of particular interest, published within the annual period of re. view, have been highlighted as: • of special interest •. of outstanding interest 1.
NEURATH H: Proteolytic Enzymes, Past and Present. Fed Proc
2.
NEURATHH: Proteolytic Processing and Physiological Regulation. Trends Biochem Sci 1989, 14:26~271.
3.
POLGARL: Serine Proteases. In Mechanisms of Protease Action. Boca Raton: CRC Press; 1989:87-113.
4.
POI~3ARL: Cysteine Proteases. In Mechanisms of Protease Action. Boca Raton: CRC Press; 1989:124~147.
1985, 44:2907-2913.
5. ..
GUASCHA, COLt M, A\aLES FX, HUBER R: Three-dimensional Structure of Porcine Pancreatic Procarboxypeptidase A: a Comparison of the A and B Zymogens and Their D e terminants for Inhibition and Activation. J Mol Biol 1992, 224:141 157. The first structural characterization of a propeptide which inactivates its protease by a mechanism similar to that used by protein inhibitors. 6.
BODE W, HUBER R: Proteinase-Protein Inhibitor Interaction. Biomed Biochim Acta 1991, 50:437-446.
7
DAVIE EW, FUJIK~XWAK, KISIELW: The Coagulation Cascade: Initiation, Maintenance, and Regulation. 13iocheml~'t~3' 1991, 30:10363 10370.
8.
COLI£ND, GOLD HK: New Developments in Thrombolytic Therapy. Adv Exper Med Biol 1990, 281:333-354.
9.
JIN YS, ANDERSON KV: Dominant and Recessive Alleles of the Drosophila easter Gene Are Point Mutations at Conserved Sites in the Set Protease Catalytic Domain. Cell 1990, 60:873-881.
10.
SKIDGEI.RA: Basic Carboxypeptidases: Regulators of Peptide Hormone Activity. Trends Pharmacol Sci 1988, 9:299-304.
11.
DARBYNJ, SMYTH DG: Endopeptidases and Prohormone Processing. Biosci Rep 1990, 10:1 13.
12.
HOOPERNM: Angiotensin Converting Enzyme: Implications from Molecular Biology for its Physiological Functions. Im J Biochem 1991, 23:641q547.
13.
MISUMIY, ODA K, FUJI~AP,A T, TAI'CAMIN, TASHIROK, IKE|tAIZ.XY: Functional Expression of Furin Demonstrating its Intracellular Localization and Endoprotease Activity for Processing of Proalbumin and Complement Pro-C3. 1 Biol Chem 1991, 266:16954-16959.
14.
CROALLDE, DEMARTINO GN: Calcium-activated Neutral Protease (Calpain) System: Structure, Function, and Regulation. Physiol Re1' 1991, 71:813--g47
15.
MATmStANLM: Metalloproteinases and Their Inhibitors in Matrix Remodeling. Trends Genet 1990, 6:121-125.
16.
HASTYKa, REIFE ia, RANG a~l, ST[ART JM: The Role of Stromelysin in the Cartilage Destruction that Accompanies Inflammatory Arthritis. Arthritis Rheum 1990, 33:388397.
17.
CRYSTALRG: The Alpha l-Antitrypsin Gene and its Deftciency States. Trends Genet 1989, 5:411~417.
18.
KORANTBD: Viral Proteases: an Emerging Therapeutic Target. Crit Rev Biotechnol 1988, 8:14~157.
19.
HUFFJR: HIV Protease: a Novel Chemotherapeutic Target for AIDS. J Med Chem 1991, 34:2305-2314.
20.
DOENHOFFMJ, CURTISS RH, NGAIZAJ, MODHAJ: Proteases in the Schistosome Life Cycle: a Paradigm for Turnout Metastasis. Cancer Metastasis Rev 1990, 9:381-392.
21.
BOIrWERJ, SCHNEIDER P, ETGES R, BORDIER C: Peptide Substrate Specificity of the Membrane-bound Metalloprotease of Leishmania. Biochemisto~ 1990, 29:10113-10119.
Proteases 22.
23.
C-OLDBERG DE, SLATER AF, CERAMI A, HENDERSON GB: Hemoglobin Degradation in the Malaria Parasite Plasm o d i u m falciparurr~ an Ordered Process in a Unique Organelle. Proc Natl Acad Sci USA 1990, 87:2931-2935. BAZANJF, FLETTERlCK RJ: Detection of a Trypsin-fike Serine Protease Domain in Flaviviruses and Pestiviruses. Virology 1989, 171:637~539.
24. ..
COREY DR, CRAm CS: An Investigation into the Minimum Requirements for Peptide Hydrolysis by Mutation of the Catalytic Triad of Trypsin. J Am Chem Soc 1992, 114:1784-1790. This paper demonstrates the importance of binding energy to TS sta bilization in enzymatic reactions. 25. ,
BRAXTONS, WELkS JA: The Importance of a Distal Hydrogen Bonding Group in Stabilizing the Transition State in Subtilisin BPN'. J Biol Chem 1991, 266:11797-11800. This paper and [28*] describe a coordinated theoretical and experimental approach which tests the effect of a mutation on an enzymatic reaction. 26. ..
CARTERP, ABRAHMSEN L, WELTS JA: Probing the Mechanism and Improving the Rate of Substrate-assisted Catalysis in Subtilisin BPN'. Biochemistry 1991, 30:6142~5148. This study provides insight into designing a sequence-specific protease. 27.
WELLSJA: Additivity of Mutational Effects in Proteins. Bic~ chemistry 1990, 29:8509-8517.
28.
MIZUSHIMAN, SPELLMEYERD, HIRONO S, PEARLMAND, KOLLMAN • P: Free Energy Perturbation Calculations on Binding and Catalysis after Mutating Thr220 in Subtifisin. J Biol Chem 1991, 266:11801-11809. This paper describes the theoretical outcome of the mutations de ,scribed in [25"]. 29. •
DAGGETrV, SCHRODERS, KOLLMANP: Catalytic Pathway of Set Proteases - - Classical Quantum Mechanical Calculations. J Am Chem Soc 1991, 113:8926-8935. The mechanism of trypsin-catalyzed hydrolysis is described by molecular simulation and provides a testable hypothesis for the detailed func tion of serine proteases. 30. ••
MCGRATH ME, V&SQUEZ JR, CRAIK CS, YANG AS, HONIG B, FLETI-ERICK RJ: Perturbing the Polar Environment of Aspl02 in Trypsin - - Consequences of Replacing Conserved Ser214. Biochemistry 1992, 31:3059-3064. One of the few studies to address the importance of neighboring residues on active-site function, and a remarkable attempt to quanti Late the role of electrostatic potential in catalysis. 31.
STODDARDBL, KOENIGSP, PORTER N, PETRATOS K, PETSKO GA, RINGE D: Observation of the Light-triggered Binding of Pyrone to Chymotrypsin by Laue X-ray Crystallography. Proc Natl Acad Sci USA 1991, 88:5503-5507. This paper describes a potentially powerful new method, which may allow for structural determination of enzyme complexes during cataly sis. •
32. ••
MENARDR, CARRmREJ, LmLAMME P, PLOUFFE C, KHOUm HE, VERNETT, TESSIER De, THOMAS DY, STORER AC: Contribution of the Glul9 Side Chain to Transition-state Stabilization in the Oxyanion Hole of Papain. Biochemistry 1991, 30:8924-8928. The authors provide experimental evidence that thiol proteases, like serine proteases, require oxyanion stabilization. This result supports the concept that ei'~,cnes work by complementing the charges that develop in the "iS through electrostatic interactions. 33.
TONGEPJ, MENARD R, STORER AC, RUZSlCSKA BP, CAREy PR: Markedly Different Acyl Papain Structures Deacylate at Similar Rates - - Resonance Raman Spectroscopic and Kinetic Evidence. J Am Cbem Soc 1991, 113:4297~t303.
34.
CHRISTIANSONDW, LIPSCOMBE XXFN:Carboxypeptidase A. Acc Chem Res 1989, 22:624;9.
35. ••
KAPLANAP, BARTu~Tr PA: Synthesis and Evaluation of an Inhibitor of Carboxypeptidase A with a Ki Value in the Femtomolar Range. Biochemistry 1991, 30:81658170.
Phillips and Fletterick
Potent enzyme inhibitors can be rationally designed where the stmc tural basis of catalysis and specificity has been characterized. 36. .
PHILLIPSMA, KAP1ANAP, RUTTERWJ, BARTLETT PA: Transitionstate Characterization: a New Approach Combining Inhibitor Analogues and Variation in Enzyme Structure. Biochemistry 1992, 31:959-963. This report demonstrates the importance of oxyanion stabilization in carboxypeptidase-catalyzed hydrolysis, and provides a broadly applicable approach for demonstrating TS analogy" for enzyme inhibitors. 37. .
BURLEYSK, DAVID PR, LIPSCOMB WN: Leu Aminopeptidase: Bestatin Inhibition and a Model for Enzyme-catalyzed Peptide Hydrolysis. Proc Natl Acad Sci USA 1991, 88:691645920. Structural analysis of the bestatin-leucine aminopeptidase complex provides evidence that metallo an',inopeptidases function by a mechanism similar to those of the well studied proteases carboxypeptidase and thermolysin. 38.
DAVIESDR: The Structure and Function of the Asp Proteinases. A n n u Key Biophys B i o p ~ s Chem 1990, 19:189--215.
39. ..
JAMESMNG, SIELECKIAR, HAYAKAWAK, GEm MH: Crystallographic Analysis of Transition State Mimics Bound to PeniciUopepsin: Difluorostatine-containing and Difluorostatonecontaining Peptides. Biochemistry 1992, 31:3872~886. A detailed structural analysis of the catalytic mechanism of an aspartyl protease. 40. ..
HYLANDLJ, TOMASZEK TA JR, MEEK TD: Human Immunodeficiency Virus-1 Protease. 2. Use of pH Rate Studies and Solvent Kinetic Isotope Effects to Elucidate Details of Chemical Mechanisms. Biochemistry 1991, 30:8454-8463. A detailed mechanistic study of the catalytic function of HIV 1 protease. 41. •
MUSILD, ZUClC D, TURK D, ENGH RA, MAYR I, HUBER R, POPOVICT, TURK V, TOWATAR1T, KATUNUMAN: The Refined 2.15h X-ray Crystal Structure of Human Liver Cathepsin B: the Structural Basis for its Specificity. F~14BO J 1991, 10:2321 2330. This paper details the structural features that distinguish cathepsin B substmte specificity from that of papain, and includes an explanation for the dipeptidyl carbox}qpeptidase activity,. 42. .•
KHOURIHE, VERNETT, MENARDR, PARLATIF, LAFLAMEP, TESSIER De, GOUR-SALIN B, THOMAS DY, STOKER AC: Engineering of Papain: Selective Alteration of Substrate Specificity by Sitedirected Mutagenesis. Biochemistry 1991, 30:8929-8936. Substrate specificity in papa.in can evolve through a very small number of point mutations in amino acids that directly contact the substrate. 43. •.
BONER, FUJISHIGEA, KETTNER CA, AGARD DA: Structural Basis for Broad Specificity in Mpha-lytic Protease Mutants. Biochemistry 1991, 30:10388-10398. Flexibility in the substrate binding site allows the enzyme to accommodate substrates of va135ng size. 44. •
WILSONC, MACEJE, AGARDDA: Computation Method for the Design of Enzymes with Altered Substrate Specificity. J Mol B i d 1991, 220:495 506. Specificity can be predicted if a large enough data set of the conformational effects of mutations has been compiled for a specific enzyme. 45. **
HEDSTROML, SZILAGYI L, RUTFER WJ: Converting Trypsin to Chymotrypsin: the Role of Surface Loops. Science 1992, 255:1249-1253. Specificity detemainants are not collfined to enz~ane residues that form direct contacts with the substrate. 46. ••
FAMINGZ, KOBE B, STEWART CB, RUTFER WJ, GOLDSMITH EJ: Structural Evolution of an Enzyme Specificity: the Structure of Rat Carboxypeptidase A2 at 1.9-A Resolution. J Biol Chem 1991, 266:24606-24612. This report provides insight into the structural basis of surface loops as specificity determinants. 47. .•
KIM H, LIPSCOMBX~rN:Comparison of the Structures of Three Carboxypeptidase A-Phosphonate Complexes Determined by X-ray Crystallography. Biochemistry 1991. 30:8171~q180.
719
720
Catalysis and regulation The authors provide evidence that very strong binding affinities can be produced from relatively weak interactions, if recruited in specific spatial arrangements. 48.
BEAUMONTA, BARBE B, LE MOUAL H, BOILEAU G, CRINE P, . FOURNIE-ZALUSKIMC, ROQUES BP: Charge Polarity Reversal Inverses the Specificity of Neutral Endopeptidase-24.11. J Biol Chem 1992, 267:2138-2141. This paper demonstrates the structural basis for dipeptidyt carboxypeptidase activity. 49. .
SCHELLENBERGERV, JAKUBKE HD, KASCHE V: Electrostatic Elfects in the Alpha-chymotrypsin-catalyzed Acyl Transfer. II. Efficiency of Nucleophiles Bearing Charged Groups in Various Locations. Biochim Biophys Acta 1991, 1078:8-11. One of the few studies to address leaving group specificity in serine proteases. 50.
BAKERD, SOHL JL, AGARD DA: A Protein-folding Reaction u n d e r Kinetic Control. Nature 1992, 356:263-265. role of propeptides is not limited to enzyme inhibition. For ~-lytic protease, the propeptide is required for catalysis of the protein folding reaction, demonstrating that protein folding is not always under thermodynamic control. 52. •.
RYDELTJ, TULINSKY A, BODE W, HUBER R: Refined Structure of the Hirudin-Thrombin Complex. J Mol Biol 1992, 221:583-601. Interactions that are important for the binding of himdin in the anionbinding recognition exosite of thrombin also play a role in the binding of biological substrates such as the thrombin receptor.
54. •
55. •
WU QY, SHEEHANJP, TSIANG M, LENTZ SR, BIRKTOFTJJ, SADLER JE: Single Amino Acid Substitutions Dissociate Fibrinogenclotting and Thrombomodulin-binding Activities of H u m a n Thrombin. Proc Natl Acad Sci USA 1991, 88:6775-6779. This study and [54*] provide insight into the mechanism by which thrombomodulin alters thrombin specificity. 56. ,.
COUGHLIN SR, VU TK, HUNG DT, WHEATON VI: Characterization of a Functional Thrombin Receptor. Issues and Opportunities. J Clin Invest 1992, 89:351-355. The authors present a novel mechanism for receptor activation, which explains the mechanism of thrombin-induced cellular events at the molecular level. 57.
SIELECKIAR, FUJ1NAGAM, READ RJ, JAMES MN: Refined Structure of Porcine Pepsinogen at 1.8A Resolution. J Mol Biol 1991, 219:671492.
51. ~e
53.
This study and [55*] provide insight into the mechanism by which thrombomodulin alters the substrate specificity of thrombin.
MCGRATHME, HINES WM, SAKANARIJA, FLETrERICK RJ, CRAIK CS: The Sequence and Reactive Site of Ecotin. A General Inhibitor of Pancreatic Ser Proteases from Escherichia coli. J Biol Chem 1991, 266:662045625. LE BONNIEC BF, ESMON CT: GIu-192--~GIn Substitution in Thrombin Mimics t h e Catalytic Switch I n d u c e d by Thrombomodulin. Proc Natl Acad Sci USA 1991, 88:7371-7375.
SCARBOROUGHRM, MAUGHTON M, TENG W, HUNG DT, ROSE J, Vu TKH, WHEATON Vl, TURCK CW, COUGHLIN SR: Tethered Ligand Agonist Peptides: Structural Requirements for Thrombin Receptor Activation. J Biol Chem 1992, 267:13146-13149.
58. ..
CHOI HK, TONG L, MINOR W, DUMAS P, BOEGE U, ROSSMANN MG, WENGLER G: Structure of Sindbis Virus Core Protein Reveals a Chymotrypsin-like Ser Proteinase and t h e Organization of t h e Virion. Nature 1991, 354:37-43. This paper reports the structure of a unique serine protease which, after a single catalytic event, becomes inactive as a protease and assumes a structural role in the viral particle. 59.
SCHECHTERI, BERGER A: O n the Size of t h e Active Site in Proteases. 1. Papain. Biochem Biophys Res Commun 1967, 27:157-162.
MA Phillips, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas 75235, USA_ RJ Fletterick, The Hormone Research Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA.