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Home , Aminopeptidase, Assemblin, Carboxypeptidase, Catalytic triad, Chymotrypsinogen, Enzyme catalysis

IUBMB , 61(5): 510–515, May 2009

Critical Review

Serine Enrico Di Cera Department of and Molecular Biophysics, Washington University School of Medicine, Box 8231, St. Louis, MO, USA

the fold especially evident in the trypsins. This Summary new dimension changes our understanding of Over one third of all known proteolytic are serine function, regulation, and specificity. proteases. Among these, the trypsins underwent the most pre- dominant genetic expansion yielding the enzymes responsible for , blood , fibrinolysis, development, - tilization, , and immunity. The success of this expan- GENERAL PROPERTIES OF SERINE PROTEASES sion resides in a highly efficient fold that couples and Barrett and coworkers have devised a classification scheme regulatory interactions. Added complexity comes from the recent observation of a significant conformational plasticity of based on statistically significant similarities in sequence and the fold. A new paradigm emerges where two forms of structure of all known proteolytic enzymes and term this data- the protease, E* and E, are in allosteric equilibrium and deter- MEROPS (8). The classification system divides proteases mine biological activity and specificity. Ó 2009 IUBMB into clans based on catalytic mechanism and families on the ba- IUBMB Life, 61(5): 510–515, 2009 sis of common ancestry. Over one third of all known proteolytic enzymes are serine proteases grouped into 13 clans and 40 fam- Keywords serine proteases; catalysis; ; allostery. ilies. The family name stems from the nucleophilic Ser in the enzyme , which attacks the carbonyl moiety of the bond to form an acyl-enzyme intermediate (4). Nucleophilicity of the catalytic Ser is typically dependent on a PREAMBLE of Asp, His, and Ser residues, commonly referred A typical genome contains 2–4% of genes encoding for pro- to as the charge relay system (9). At least four distinct teolytic enzymes (1). Among these, serine proteases emerged folds as illustrated by trypsin, , prolyl , during as the most abundant and functionally diverse and ClpP peptidase utilize the Asp-His-Ser catalytic triad in group (2, 3). Because abundance is a measure of success in identical configuration to catalyze of peptide bonds evolutionary terms, the molecular mechanisms that ensure catal- (2). This is testimony to the success of the triad as a catalytic ysis and regulation in these enzymes deserve attention. Much machinery in four distinct evolutionary pathways. Many serine has been learned on how serine proteases catalyze the hydroly- proteases employ a simpler dyad mechanism where Lys or His sis of a and excellent reviews have covered this is paired with the catalytic Ser. Other serine proteases mediate subject (4, 5). Surprisingly, however, the rules encoding speci- catalysis via novel triads of residues, such as a pair of His resi- ficity in a given protease fold remain incompletely understood dues combined with the nucleophilic Ser. A summary of cata- (6). New developments from the structural investigation of a lytic units in all serine protease families is given in Table 1. number of serine proteases have recently challenged old para- Serine proteases are widely distributed in and found digms. Allostery, originally conceived to explain the properties in all kingdoms of cellular life as well as many viral genomes. of multisubunit enzymes (7), emerges as a key embodiment of However, significant differences exist in the distribution of each clan across species. For example, clan PA proteases are highly represented in , but rare constituents of prokaryotic Received 5 December 2008; accepted 28 December 2008 and plant genomes. Vertebrates boast an array of clan PA pro- Address correspondence to: Enrico Di Cera, Department of Biochem- istry and Molecular Biophysics, Washington University School of Med- teases responsible for a variety of extracellular processes. SB icine, Box 8231, St. Louis, MO 63110, USA. Tel: 1314-362-4185. and SC clans are most represented in other organisms. Serine Fax: 1314-362-4133. E-mail: [email protected] proteases are usually endoproteases and catalyze bond hydroly-

ISSN 1521-6543 print/ISSN 1521-6551 online DOI: 10.1002/iub.186 SERINE PROTEASES 511

Table 1 General properties of serine proteases Clan Families Representative member Catalytic residues Primary specificity PA 12 Trypsin His, Asp, Ser A, E, F, G, K, Q, R, W, Y SB 2 Subtilisin Asp, His, Ser F, W, Y SC 2 Prolyl oligopeptidase Ser, Asp, His G, P SE 6 D-A, D-A Ser, Lys D-A SF 3 LexA peptidase Ser, Lys/His A SH 2 His, Ser, His A SJ 1 Lon peptidase Ser, Lys K, L, M, R, S SK 2 Clp peptidase Ser, His, Asp A SP 3 His, Ser F SQ 1 DmpA Ser A, G, K, R SR 1 Lys, Ser K, R SS 1 L,D-carboxypeptidase Ser, Glu, His K ST 5 Rhomboid His, Ser D, E

Multiple members of each clan may give rise to a wide variety of primary specificities, as documented by the PA, SJ, and SQ clans.

sis in the middle of a polypeptide chain. However, several fami- A number of key biological processes rely on clan PA pro- lies of exoproteases have been described that remove one or teases. Chief among them are blood coagulation and the more amino from the termini of target polypeptide chains. immune response, which involve cascades of sequential zymo- Within the metazoan lineage, a select subset of protease fami- gen activation. In both systems, the protease domain is com- lies underwent significant gene duplication and divergence (3). bined with one or more apple, CUB, EGF, fibronectin, kringle, In fact, four protease families by themselves account for over sushi, and domains. These domains are 40% of all proteolytic enzymes in . These are the ubiq- present on the N-terminus as an extension of the propeptide uitin-specific proteases responsible for regulated intracellular segment of the protease and typically remain attached to the protein turnover (10), the controlling growth fac- protease domain through a covalent disulphide bond. tors and function (11), prolyl (12), and Family S1 of clan PA is partitioned into two subfamilies, the trypsin-like serine proteases (2), which are also the largest S1A and S1B, that are phylogenetically distinct but share a group of homologous proteases in the genome. Of 699 common two b-barrel architecture. The S1B proteases are found proteases in man, 178 are serine proteases and 138 of them in all cellular life and are responsible for intracellular protein belong to the S1 protease family. Abundance of S1 proteases turnover. In contrast, the S1A proteases are the trypsins that suggests the protein fold presents a selective advantage relative mediate a variety of extracellular processes. S1A proteases have to other proteases. The trypsin fold of the S1 protease family a limited distribution in plants, , and the archaea. In (Fig. 1) nestles catalytic efficiency, substrate selectivity, and humans, they are phylogenetically grouped into six functional multiple levels of regulation in a scaffold that is readily associ- categories: digestion, coagulation and immunity, , ated with other auxiliary protein modules. Because of the , , and . A variety of enzymes are scopes of this review, we will limit our discussion to PA pro- involved in the breakdown of in the digestive system teases and the trypsins in particular. Properties of the members (14). Trypsins, , and are endoproteases of other clans listed in Table 1 are discussed in detail elsewhere that breakdown polypeptides into shorter chains. Further diges- (2). tion is mediated by a number of exoproteases (15, 16). Tryp- tases are major components in the secretory granules of mast cells that are unique among clan PA proteases because of their CLAN PA PROTEASES homotetrameric structure (17). Matriptases are membrane bound Clan PA proteases bearing the trypsin fold are the largest S1 proteases bearing primary substrate selectivity similar to family of serine proteases and perhaps the best studied group of trypsin (18). Physiological substrates of this subfamily of pro- enzymes. Digestive enzymes such as trypsin and teases are largely unknown, but high gene expression levels for cleave polypeptide chains at positively charged (Arg/Lys) or matriptases are associated with a variety of cancers (19, 20). large hydrophobic (Phe/Trp/Tyr) residues, respectively. Most Similar association with cancer has led to great interest in kal- clan PA proteases have trypsin-like substrate specificity and pre- likreins (21), a large family better known for its role in regula- fer Arg or Lys side chains at the P1 position of substrate (13). tion of blood pressure through the kinin system (22). Gran- 512 DI CERA zymes are mediators of directed apoptosis by natural killer cells shown that conversion of trypsin into chymotrypsin requires the and cytotoxic T cells that play key roles in the defense against expected D189S replacement, but also extensive additional viral infection (23). These enzymes are unique in their primary replacements at loops not directly in contact with substrate (30). specificity for acidic residues. Despite these replacements, however, the conversion is never complete and enzyme activity in the mutant constructs remains poor. Furthermore, the same strategy fails in the reverse conver- THE CATALYTIC MECHANISM sion of chymotrypsin into trypsin (31) or the conversion of tryp- Activation of many trypsin-like serine proteases requires sin into (32). Recent mutagenesis studies have shown proteolytic processing of an inactive precursor (24). that the simple S189D replacement in chymotrypsin is sufficient Cleavage of the proprotein precursor occurs at the identical to confer trypsin-like specificity when access to the primary position in all known members of the family, that is, between specificity pocket is enhanced with the A226G substitution (33), residues 15 and 16 ( numbering). The nascent a result that echoes the impressive recent successes in the rede- N-terminus induces in the enzyme sign of primary specificity in the different protein scaffold of through formation of an ion-pair with the highly conserved OmpT (34). Yet, the S189D/A226G mutant of chymotrypsin D194 that organizes both the and substrate-bind- remains a poor protease, 5,000-fold slower than trypsin at cleav- ing site (25, 26). Alternatively, proteases like tissue-type plas- ing substrates with Lys at the P1 position. Apparently, the struc- minogen activator carry Lys at position 156 that engages D194 ture-function link required to understand the molecular basis of with an ion-pair that confers a catalytically competent fold serine protease specificity is missing a key element. The consid- without proteolytic cleavage at residue 15 (27). erable conformational plasticity of the trypsin fold may hold Nearly all clan PA proteases utilize the canonical catalytic important clues. triad and hydrolyze the peptide bond via two tetrahedral inter- mediates (28). Initially, the hydroxyl O atom of the catalytic S195 attacks the carbonyl of the substrate peptide bond utilizing THE E* FORM H57 as a general base. The oxyanion tetrahedral intermediate is Recent kinetic studies on thrombin (35), the key serine pro- stabilized by the backbone N atoms of G193 and S195, which tease of the blood coagulation cascade, vouch for an unexpected together generate a positively charged pocket within the active plasticity of the trypsin fold. Thrombin exists in three forms at site known as the oxyanion hole. H-bonding interactions in the equilibrium under physiological conditions. The Na1-free form oxyanion hole contribute 1.5–3.0 kcal/mol to ground and transi- E and the Na1-bound form E:Na1 are the low and high activity tion state stabilization (29). Collapse of the tetrahedral intermedi- conformations of the enzyme, with E:Na1 being responsible for ate generates the acyl-enzyme intermediate and stabilization of the procoagulant, prothrombotic, and signaling functions. A third the newly created N-terminus is mediated by H57. A mole- form, E*, is in equilibrium with E and is basically inactive to- cule displaces the free polypeptide fragment and attacks the acyl- ward substrate and unable to bind Na1 (36). The structural dif- enzyme intermediate. The oxyanion hole stabilizes the second tet- ferences between E and E:Na1 are subtle (37), almost disap- rahedral intermediate of the pathway and collapse of this inter- pointingly small considered the significant functional differences mediate generates a new C-terminus in the substrate (4). observed between the two forms in solution. However, enzymolo- gists have long recognized that subtle short-scale rearrangements within the active site of an enzyme may lead to profound ener- PRIMARY SPECIFICITY getic changes in the ground and transition states (38, 39). On the The trypsin fold is remarkable for the even distribution of other hand, E* differs dramatically from E and E:Na1 in its col- catalytic residues across the entire polypeptide sequence (Fig. lapse of the 215–217 b-strand into the active site, a disruption of 1). Two six-stranded b-barrels come together asymmetrically to the oxyanion hole due to a flip of the peptide bond between host at their interface the residues of the catalytic triad. H57 E192 and G193 from a type II to a type I b-turn, and other and D102 belong to the N-terminal b-barrel with S195 and the changes around D189 and the Na1 site (40) (Fig. 2). Importantly, oxyanion hole (S195 and G193) hosted by the C-terminal b-bar- the ion-pair between I16 and D194 remains intact, suggesting rel. Eight surface exposed loops in the protein decorate the en- that E* is not equivalent to the zymogen form of thrombin. trance to the active site. Within the same fold, trypsins prefer Existing structures of the zymogen forms of trypsin (26), chymo- Arg/Lys side chains at the P1 position of substrate (13) but chy- trypsin (41), and (42) all feature a broken I16-D194 motrypsins prefer Phe/Tyr/Trp residues (2). The molecular ori- ion-pair and do not document a collapse of the 215–217 b-strand. gin of this difference resides in part in the architecture of the Stopped-flow experiments demonstrate that the E*–E conversion primary specificity pocket, shaped as a cavity about 10 A˚ deep takes place on a time scale \10 msec (36), as opposed to the and connected to the active site, at the bottom of which trypsins zymogen-protease conversion that evolves over a much longer carry D189 and chymotrypsins carry S189. The simplicity of (100–1,000 msec) time scale (43, 44). this arrangement belies the difficulty of swapping specificity E* is not a peculiarity of thrombin. Some of the most strik- between trypsin and chymotrypsin. Pioneering studies have ing features of the E* conformation, like the collapse of the SERINE PROTEASES 513

tase, D216 collapses into the oxyanion hole (46). A collapsed 215–217 segment is observed in the structures of the high-tem- perature-requirement-like protease (48), complement (49), K (50), hepatocyte growth factor activator (51), kallikrein (52), and prostasin (47). A disrupted oxyan- ion hole is observed in (53) and the arteri- protease nsp4 (54). In all these cases the I16-D194 ion- pair remains intact, and therefore the conversion of E* into the active form E of the protease cannot involve the canonical zym- ogen-protease conversion or a -induced zymogen activa- tion as observed for the prothrombin-staphylocoagulase inter- action (55). The conformational transition of E* into E must follow other routes, as demonstrated recently for thrombin in unprecedented detail (45). Binding of ligands to E* away from the active site region organizes a network of H-bonds that indu- ces a long-range rearrangement of the 215–217 b-strand and a flip of the oxyanion hole back into the active incarnations of the E form (45).

ALLOSTERY AND SERINE PROTEASES Figure 1. Ribbon representation of trypsin (2PTN) (25), the All these recent observations support E* as an inactive form prototypic example of serine protease from family S1 in clan of the protease in allosteric equilibrium with the active form E, PA (Table 1), colored according to the spectrum from the regardless of the ability of the protease to bind Na1 and further N-terminus (violet) to the C-terminus (red). The catalytic triad convert E into E:Na1. In other words, serine proteases are allo- (sticks) is hosted at the interface of two similar -barrels. D189 b steric enzymes whose activity can be modulated by affecting (sticks) occupies the bottom of the primary specificity pocket the E*–E equilibrium. Allostery is often associated with large- and confers specificity toward Arg or Lys side chain at the P1 scale conformational transitions in multimeric proteins like he- position of substrate. moglobin (56), aspartate transcarbamylase (57), the nicotinic re- ceptor (58), or GroEL (59). However, the ability of monomeric 215–217 b-strand into the active site and disruption of the oxy- enzymes to express complex kinetic behavior like hysteresis anion hole, have been observed in other structures of serine pro- (60), (61), and (62) has long teases in the free form (Fig. 2). In the inactive form of aI-tryp- been recognized. The molecular plasticity documented in mono-

Figure 2. A: The E* form shows a collapse of the 215–217 b-strand into the active site. The surface (wheat) refers to trypsin ori- ented as in Fig. 1. Sticks refer to the superimposed structures of thrombin (3BEI, green) (45), aI-tryptase (1LTO, cyan) (46), and prostasin (3DFJ, yellow) (47). B: Details of the collapse that completely occludes access to the active site. Black labels refer to trypsin. White labels indicate the positions of W215 and E217 in the E* form of thrombin. 514 DI CERA meric proteins like serine proteases is further testimony to the 12. Rea, D. and Fulop, V. (2006). Structure-function properties of prolyl importance of allostery as an intrinsic property of all dynamic oligopeptidase family enzymes. Biochem. Biophys. 44, 349–365. 13. Schechter, I. and Berger, A. (1967). On the size of the active site in proteins closely intertwined with catalysis (63). This plasticity proteases. I. . Biochem. Biophys. Res. Commun. 27, 157–162. undoubtedly dictates many aspects of protease function, regula- 14. Rothman, S. S. (1977). The digestive enzymes of the : a mix- tion, and even substrate specificity (35, 64, 65). ture of inconstant proportions. Annu. Rev. Physiol. 39, 373–389. What is the physiological advantage of the E*–E equilib- 15. Whitcomb, D. C. and Lowe, M. E. (2007). Human pancreatic digestive rium? For each protease within its biological niche activity can enzymes. Dig. Dis. Sci. 52, 1–17. be fine tuned by properly setting the E*–E equilibrium. In the 16. Steinhoff, M., Vergnolle, N., Young, S. H., Tognetto, M., Amadesi, S., Ennes, H. S., Trevisani, M., Hollenberg, M. D., Wallace, J. L., case of complement factors, , tryptase, and some Caughey, G. H., Mitchell, S. E., Williams, L. M., Geppetti, P., Mayer, coagulation factors activity must be kept to a minimun until E. A., and Bunnett, N. W. (2000). Agonists of proteinase-activated re- binding of a trigger factor ensues. Stabilization of E* may ceptor 2 induce inflammation by a neurogenic mechanism. Nat. Med. 6, afford a ‘‘resting’’ state of the protease waiting for action, a 151–158. 17. Pereira, P. J., Bergner, A., Macedo-Ribeiro, S., Huber, R., Matschiner, strategy implemented by other enzymes (66–68). The role of E* G., Fritz, H., Sommerhoff, C. P., and Bode, W. (1998). Human b-tryp- can be exploited in engineering studies aimed at stabilizing a tase is a ring-like tetramer with active sites facing a central pore. Nature protease in the inactive form until a cofactor unravels its full 392, 306–311. catalytic power. In fact, thrombin has been redesigned to dis- 18. Lin, C. Y., Anders, J., Johnson, M., Sang, Q. A., and Dickson, R. B. play activity only in the presence of thrombomodulin and pro- (1999). Molecular of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity. . , 18231– tein C along the pathway (69, 70). Notably, the J. Biol. Chem 274 18236. structures of two anticoagulant variants have been solved (71, 19. Ramsay, A. J., Reid, J. C., Velasco, G., Quigley, J. P., and Hooper, J. 72) and show collapse of the 215–217 b-strand and disruption D. (2008). The type II transmembrane serine protease matriptase-2— of the oxyanion hole as seen in E* (40). These are exciting new identification, structural features, enzymology, expression pattern and developments that challenge old paradigms about the structural potential roles. Front. Biosci. 13, 569–579. rigidity of the trypsin fold and the molecular basis of specific- 20. Netzel-Arnett, S., Hooper, J. D., Szabo, R., Madison, E. L., Quigley, J. P., Bugge, T. H., and Antalis, T. M. (2003). Membrane anchored serine ity. Future work on the properties of E* in serine proteases proteases: a rapidly expanding group of cell surface proteolytic enzymes promises to be ground-breaking. with potential roles in cancer. Cancer Metastasis Rev. 22, 237–258. 21. Diamandis, E. 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