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Home , Aspartic protease, Caspase, Caspase 1, Caspase 2, Caspase 6, Caspase 7

Death and Differentiation (1999) 6, 1054 ± 1059 ã 1999 Stockton Press All rights reserved 13509047/99 $15.00 http://www.stockton-press.co.uk/cdd Review Catalytic properties of the

HR Stennicke1 and GS Salvesen*,1 bond , and includes and exopepti- dases. Caspases are strict endopeptidases. From a mechan- 1 The Program for and Research, The Burnham Institute, istic point of view most utilize their ability to force the 10901 North Torrey Pines Road, La Jolla, California, CA92037, USA trigonal planar into a tetrahedal geometry as a * Corresponding author: GS Salvesen, The Program for Apoptosis and Cell prerequisite for . Thus, the majority of the available Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La binding energy is used for stabilizing this tetrahedral Jolla, California, CA 92037, USA. Tel: +1 (619) 646 3114; Fax: +1 (619) 646 3189; E-mail: [email protected] intermediate rather than merely forming an - complex. The ability to catalyze the hydrolysis of the peptide Received 20.5.99; accepted 21.9.99 bond at neutral pH and ambient temperatures therefore Edited by D Nicholson characterizes proteases, where various catalytic mechanisms are located in a variety of otherwise unrelated scaffolds. At least five distinct catalytic mechanisms have been Abstract discovered in proteases, but irrespective of the type of stands for -dependent aspartate specific residues involved in the particular mechanisms, the key , and is a term coined to define proteases related to features are identical. About the point in the reaction coordinate where the enzyme pulls the peptide bond into interleukin 1b converting enzyme and CED-3.1 Thus their tetrahedral geometry, a adds to the carbonyl enzymatic properties are governed by a dominant specificity of the scissile bond. In some families of proteases (the for substrates containing Asp, and by the use of a Cys side- metallo- and classes) this is simply done by chain for catalyzing peptide bond cleavage. The use of a Cys making a water molecule nucleophilic through specific side chain as a nucleophile during peptide bond hydrolysis is interactions to groups in the protease. In others (the common to several protease families. However, the primary cysteine, , and protease classes) a side- specificity for Asp turns out to be very rare among protease chain within the protease acts as the nucleophile, forming a families throughout biotic kingdoms. Of all known mammalian covalent adduct (an ester) with the substrate during . proteases only the caspase activator B, a serine Hydrolysis of substrate by these latter classes occurs by protease, has the same primary specificity. In addition to this displacement of the ester. The second step in peptide bond unusual primary specificity, caspases are remarkable in that hydrolysis is the protonation of the a-amino moiety of the leaving group, which is particularly important in cysteine certain of their have intrinsic proteolytic activity. proteases such as the caspases due to the lower stability of This latter property is essential to trigger the proteolytic the ester intermediate compared to regular esters. pathways that lead to apoptosis. Here we review the known In the case of the caspases it is the formation of a enzymatic properties of the caspases and their zymogens tetrahedral intermediate by promotion of a cysteine residue within the broad context of structure:mechanism:activity to act as a nucleophile that is the pivotal point in catalysis relationships of proteases in general. and thus, we will focus on this particular mechanism for promoting hydrolysis. The organization of the catalytic site, Keywords: protease; ; catalytic mechanism; caspase the geometry of the residues, and the distance between them, all have a variety of implications for protease Abbreviations: AMC, 7-amino-ymethyl-coumarin; +PA, tissue functions in vivo. This particular point becomes very clear when comparing two proteases that uses the same catalytic machinery to promote the breakage of the peptide bond, but on quite different scaffolds as it is the case for the caspase family and the family. Though they use Breaking the peptide bond essentially the same catalytic mechanism, these two The emergence of amino as the building blocks of families of cysteine proteases have some very significant allows for the combination of stability, strength, and differences in the promotion of catalysis as well as the structural features unique to the peptide bond. However, this organization of the substrate . strength and stability of the peptide bond also makes specific cleavage or degradation of proteins by hydrolysis much more Catalytic mechanism than a simple `just add water' problem. This is evident from the fact that non-catalyzed hydrolysis of proteins requires Like other cysteine proteases, the caspases contain a prolonged heating even in the presence of strong acids. catalytic Cys-His pair with Cys285 acting as the nucleophile Thus, that cleave peptide bonds (peptidases or and His237 acting as the general to abstract the proton proteases) have high energy barriers to overcome. The term from the catalytic Cys and promote the nucleophile (Figure 1). protease is synonymous with peptidase, meaning peptide The numbering of caspase residues follows the Catalytic properties of the caspases HR Stennicke and GS Salvesen 1055

3 convention. Interestingly, serine proteases (such as , drop in kcat. In the caspases this third component of the tissue plasminogen activator, and ) also frequently is not a side-chain, but rather is proposed to be contain a His residue that accompanies the catalytic Ser. In the backbone of residue 177, which in the model papain, where the cysteine caspase 1 is a proline and in caspase 3 a threonine.4,5 protease catalytic mechanism has been studied most Unfortunately, the importance of this third member is difficult extensively, it is believed that the Cys-His dyad exists as an to establish in the caspases since the interactions are to a ion-pair where the thiol proton of the catalytic Cys has been backbone moiety, which cannot easily be experimentally transferred to the acceptor His before substrate binding.2 Pre- verified by mutagenesis. Furthermore, alignment of the polarization of the catalytic nucleophile represents one of the known mammalian caspases (Accession PF00656)6 does major differences between the serine and cysteine proteases, not reveal any significant conservation in the region since in the serine proteases the nucleophile is believed to surrounding the putative third member of the triad, allowing develop along the reaction coordinate. While the ability of the us to raise the hypothesis that the putative third member may catalytic His to abstract a proton from the catalytic Cys is not exist in the caspase structure and mechanism. extremely important, the ability to protonate the a-amino group Investigations of the pH dependence of caspase of the scissile bond is expected to be at least as important in catalysis have provided useful insights into the catalytic the caspases. The reason for the importance of the mechanism. caspases exhibit only minor differ- protonation of this group lies in the properties and reactivity ences in pH profiles, and all are maximally active within the of the thiol ester: its susceptibility to nucleophilic attack. Thus, pH range and ionic strength of cell cytosols.7,8 They all if the a-amino group is not protonated the reformation of the exhibit a rather narrow bell-shaped pH dependence with peptide bond will be favored over the release of the leaving optima in the range 6.8 ± 7.2 signifying the existence of one group, and consequently proteolysis will not occur. active form of the enzyme with the increase in activity most In many proteases the side-chain of a third residue plays a likely due to the de-protonation of the catalytic Cys residue. significant role in the promotion of catalysis. The majority of In this respect the caspases closely resemble other the members of the papain family contain an Asn side-chain unrelated cysteine proteases such as papain in their that is believed to orient the in the Cys-His ion-pair, activity pH profiles although the pH-profile is much more thereby influencing catalysis.3 Indeed, when Asn175 is narrow than that found for papain.9 Interestingly, the replaced by an alanine in papain, the catalytic activity is substitution of the third member of the catalytic triad in decreased by more than 100-fold mainly due to a significant papain also results in a considerable narrowing of the pH profile due to the increased distance between the catalytic residues.3 This observation supports the lack of a third member of the catalytic triad in caspases since examination of the structure of papain complexed with reveals a distance between the catalytic residues to be 3.75 AÊ ,10 which is significantly less than the distance of 5.2 AÊ found in the caspase:aldehyde structures.4,5 Interestingly, the large distance between catalytic Cys and His residues in caspase structures argues against an ion pair, with the implication that the Cys may not be pre-polarized.

The substrate binding site The substrate binding site in all proteases is composed of a fairly large number of amino residues that secure proper alignment of the substrates prior to hydrolysis and help promote catalysis through stabilization of the transition state. The binding site is divided into a number of sub-sites (see Figure 2), each securing a single residue of the substrate by multiple interactions. In addition to interactions Figure 1 The minimal caspase catalytic site. The role of the catalytic site of with specific side-chains, binding of the peptide backbone proteases may be summarized as the ability to force formation of a tetrahedral intermediate, and the following explanation derives from the 3-D structures of also plays an important role in catalysis. caspases 1 and 3.4,5 The substrate chain (white text) lies in the binding site The substrate specificity of the caspases will be with the scissile bond positioned close to the catalytic residues. The backbone described elsewhere in this issue and we will cover the of Gly238 and Cys285 (which constitute the `' in elliptical essential details briefly for comparative purposes. Although shading) donate H-bonds to the carbonyl oxygen, thus polarizing the carbonyl group of the scissile bond. The carbon is now electrophilic and susceptible to no detailed studies have been done to address the P1 11 attack by the juxtaposed nucleophilic thiol of the catalytic Cys285 (triangle specificity since the original description of caspase 1, all shading). Prior to or during the nucleophilic attack on the carbonyl carbon the the caspases are believed to possess a strict preference for thiol group of Cys285 donates its proton to His237 (square shading), which Asp in the enzyme S1 pocket as previously indicated in the then can act as the catalytic acid by protonating the a-amino group of the P1' origin of the family name. In the papain family the primary amino acid. During deacylation of the enzyme His237 will help to polarize the water molecule required for completing the hydrolysis by forming the second specificity pocket is S2, which tends to prefer hydrophobic tetrahedral intermediate substrate side-chains. Indeed, there is no detectable S1 Catalytic properties of the caspases HR Stennicke and GS Salvesen 1056

than other cysteine proteases. Thus, the caspases are very distinct from the papain family proteases that display a

S2 predominant P2 specificity and no b-sheet formation with S4 S1’ the substrate. The caspases can be considered as proteases that have adopted a cysteine nucleophile to hydrolyze proteins bound in a more-or-less like substrate conformation. The differences in the specificity of the caspases are

largely due to interactions at the distant S4 sub-site, which results in the segregation of the caspases into three S different groups.19 Although this classification provides a S3 1 S2’ useful guide to specificity of the caspases it should be used with caution, mainly because of the different level of activity Figure 2 The protease substrate binding site. The peptide chain of a observed for the caspases. For example, the rate of substrate (or inhibitor) lies across the substrate-binding cleft of the protease. hydrolysis (kcat/KM) observed for caspase 1 on WEHD- The side-chains of each residue of the substrate are numbered sequentially, AMC is around 100-fold higher than that observed with 59 with the `P' designation, from the scissile bond (dashed). By convention, caspase-4 on the same substrate,8 and thus, may those towards the N-terminal of the substrate are called the `unprimed' side, and those toward the C-terminal of the substrate are called the `primed' side. approach the rate by which caspases belonging to other Complementary pockets on the surface of the protease are given the `S' groups cleave the same substrate. Additionally, some designation. Though the P sites constitute a single side-chain, the S sites are caspases, such as , requires subsite occupancy each usually composed of several different portions of the enzyme, since they beyond S4 for efficient hydrolysis and thus their activity may define a binding surface. Though this convention is extremely useful for be misrepresented by the P categorization.20 While the understanding protease specificity, it is important to remember that not all 4 subsites are occupied in different proteases, and subsites are not always truly large degree of importance of the P4 interactions are well independent60 described and clearly of biological importance, the caspases are far from unique in their requirements. Indeed preference throughout the papain family,12,13 and the many other proteases have a significant degree of

substrate backbone is even bound in a different way than substrate discrimination at the P4 position. A prime in the caspases.10 example of this is the subtilisin family which, though often

When compared to the papain family of proteases, this considered to have a rather broad primary (P1) specificity, way of accommodating the P1 residue provides an has a significant ability to discriminate between different P4 additional explanation for the drop in activity observed in substituents.21 the acidic pH range. This is because protonation of the side Proteases depend on transition state substrate binding

chain carboxylate of the P1 Asp residue would make it for catalysis, close to the tetrahedral intermediate, rather unable to bind to the basic S1 subsite of the caspases. That than to the ground state of the native substrate. Thus, the only the negatively charged form of the P1 Asp residue will binding energy contained in the system is used to promote result in productive binding of the substrate is supported by: catalysis rather than association with the substrate. This (i) the strict preference for Asp in this position as compared means that proteases generally bind quite weakly to their

to Asn, and (ii) the close similarity in the pH dependence in substrates in the ground-state (KM values in the mMrange) the low pH range between the caspases and other but very tightly to the transition state, which has important

proteases with preferences for negatively charged P1 implications for the generation of inhibitors of these substituents (glutamyl endopeptidases from Streptomyces enzymes. Thus, in contrast to enzymes like , the griseus14 and V815). generation of mutant substrates should not result in an The other significant feature of enzyme substrate inhibitory form of the substrate, but merely in the particular interactions is the influence of peptide backbones on protein not showing any affinity for the protease any more. substrate/inhibitor binding. Although the importance of Thus, the use of catalytically incapacitated mutants of these backbone interactions is difficult to investigate in proteases as dominant negatives must be considered detail due to the problems associated with changing the limited due to the relative weak binding of substrates to structure of the backbone, this is a central part of the the ground state. Only caspases that are recruited, by virtue binding of the substrate during catalysis. In all the caspase of their N-peptides, to activation complexes should act as structures the backbone of the bound inhibitor forms an dominant negative inhibitors of the activation process. anti-parallel b-sheet like structure with the enzyme.4,5 Similar binding modes are found for the family and subtilisin family serine proteases,16,17 whereas Enzymatic characteristics of the caspases papain homologs do not show an elaborate The features described above determine the enzymatic network between the enzyme and the substrate back- properties of the caspases and thus, to a large extent their bone.10,18 Though placed on a very different scaffold, the biological function. As with other cysteine proteases the

dominant S1 specificity and b-sheet like backbone caspases require a reducing environment in order to retain full hydrogen-bond interactions with the substrate demon- activity, presumably because the catalytic thiol is susceptible strates that the overall mode of substrate binding observed to oxidation. This would tend to limit caspase activity to the with caspases has more in common with serine proteases reducing environment found inside cells, and argue against Catalytic properties of the caspases HR Stennicke and GS Salvesen 1057 an extracellular role. It is clear from work on the recombinant activate the proteases, the perhaps most well known caspases that they require quite high concentrations of example being that of trypsin.38 Recently, an analogous reducing agents such as DTT to achieve maximal activity mode of activation has been suggested for the caspases in (probably due to oxidation of the catalytic Cys during the activation of caspase-3 by RGD containing peptides.39 purification; HR Stennicke and GS Salvesen, unpublished The mechanism of this activation, which is postulated to be a results). The endogenous caspases in cell cytosols appear to direct effect, is unclear and indeed attempts in our laboratory be fully active in the presence of low concentration of reducing to repeat the original observation using cytosolic extracts as agents and recombinant material also retains full activity at well as immuno-precipitated or purified recombinant pro- these conditions once activated providing that there are metal caspase-3 have failed (HR Stennicke, QL Deveraux and GS chelators such as EDTA present (HR Stennicke and GS Salvesen, unpublished results). Salvesen, unpublished results). This makes apparent a In contrast to other mechanisms, caspase activation related aspect to the oxidation, which is the effect of transition primarily occurs through cleavage within a segment at an metals on the activity. The influence of such metal ions on the internal position in the . In caspase 1, removal of the activity of other cysteine proteases has been well established N-peptide has been implicated as a maturation step,40 but for a long time, predominantly due to the direct interaction with simply removing the N-peptide is not sufficient for activation. the catalytic thiol but also the ability of these metals to On the other hand, the biochemical properties of the short N- catalyze oxidation. It is therefore not surprising that the peptide caspases such as caspase-3, which are involved in caspases are sensitive to Zn2+, being completely inhibited in the execution of the apoptotic signal, are not affected by the mM range,7 although there are significant differences in their removal of the N-peptide.41 Additionally, caspase-9, which is affinity. is most readily inhibited by Zn2+, becoming a key initiator of the post-mitochondrial cell death pathway, completely inactivated by 0.1 mM, and caspase 3 is the least does not get its large N-peptide removed during activa- sensitive requiring more than 1 mM for complete inactivation tion.42 ± 45 Thus the function of the individual N-peptide in the presence of 20 mM 2-mercapto ethanol. The inhibition extensions of the caspases is not conserved. of caspases by Zn2+ may explain the inhibitory action of this It is generally agreed that initiator caspases transmit the metal on apoptosis,22,23 though the interaction is blocked by proteolytic signal by directly processing executioner thiol compounds,7 and therefore presumably highly depen- zymogens, and at least in vitro activation of the dent on the redox potential of the cell. Ca2+ has little effect on executioner caspase zymogens by the initiators is efficient the activity of the caspases at concentrations up to 100 mM, and fast (Table 1). However, the molecular mechanism although minor effect on the activity of caspase-7 have been leading to the active enzyme is currently not known since reported.8 Thus, the reported role of Ca2+ in apoptosis, see for there are little available biochemical data and no structural example,24 is unlikely to be due to any effect on the caspases. models available for the zymogens. One possibility is that the cleavage of the inter-domain linker results in formation of the catalytic dyad, which is distorted in the zymogen due Activation of the caspases to tension in the protein. The degree of distortion imposed In general the activation of most proteases takes place by on the catalytic dyad would then be a function of the length limited proteolysis leading to removal of an N-terminal of the inter-domain linker and indeed caspases containing activation peptide, also known as the propeptide. This relatively long inter-domain linkers (caspases 8 and 9) have mechanism of activation is used by the majority of all known been demonstrated to possess activity in their zymogen proteases with the notable exception of the caspases. In the forms. Possibly this is a key to the mechanism by which papain like enzymes such as B and L the activation occurs in the activator complexes. propeptide binds in the cleft much like a substrate but in reverse direction, physically preventing access to the substrate binding site.25,26 In this case the activation occurs The importance of zymogenicity by auto-catalytic removal of the propeptide in trans (an Interestingly, depending upon expression conditions of intermolecular reaction). In contrast the propeptides of the recombinant proteins, one can obtain either processed active serine proteases a-lytic protease and subtilisin bind more like caspase or unprocessed zymogen from the same construct, a natural substrate and the activation is unregulated, at least for caspases 3, 7 and 9.41,45,46 For example, a short occurring by cleavage of the bond between the residues induction time (530 min) yields unprocessed zymogens but 27 ± 30 occupying the S1 and S1' subsites of the enzymes. This longer ones (43 h) yield fully processed enzymes. Signifi- reaction occurs in cis (an intra molecular reaction). Interest- ingly the propeptides of all these proteases are potent inhibitors of the mature enzymes and usually characterized Table 1 Activation rates of executioner caspase zymogens by selected initiators 71 71 by low to sub-nano molar binding constants.31 ± 34 (M s ) In the activation mechanism utilized by members of the chymotrypsin family the propeptide does not physically block Pro-caspase 3 2.26106 0.76106 4.86106 the active site. Here activation occurs when propeptide Pro- 4.46106 1.26106 6.46106 cleavage reveals a new N-terminal a-amino group that inserts into the core of the protein, and secures formation of the Puri®ed recombinant zymogens of the executioner caspases 3 and 7 are activated most ef®ciently by the serine protease granzyme B, followed by the 35 ± 37 active site. In cases where such events are required it apical caspase 8, and then caspase 10. Data for caspase-3 are taken from41 has been demonstrated that addition of specific peptides can and for caspase-7 from HR Stennicke and GS Salvesen, unpublished results Catalytic properties of the caspases HR Stennicke and GS Salvesen 1058

cantly, even very short expression times and low inducer restricted to . There should be homologs consisting of concentrations have failed to yield caspase 8 zymogens in our the same fold that would help to shed on the origin of the hands (HR Stennicke and GS Salvesen, unpublished data). family. One of the most notable properties of the caspases is

Caspase 8 processes itself extremely rapidly upon hetero- the dominance of the S1 pocket, which caspases share in logous expression in E. coli, suggesting that the zymogen principle with serine proteases of the chymotrypsin family, and must possess significant intrinsic proteolytic activity, allowing in particular with granzyme B.56 Does this principle extend to

for autoprocessing. Indeed, a non-processing caspase 8 other cysteine protease families that also have a dominant P1 zymogen possesses 1% of the activity of the fully processed specificity? Are there other families that use a dominant S1 enzyme,47 a very substantial activity compared with most pocket, but with different specificity, positioned on the protease zymogens. These observations are the basis for the currently unique caspase fold? induced proximity hypothesis for the in vivo activation of Such a possibility has been proposed for the bacterial

caspase 8, whose assembly is forced by ligation of death proteases and R- (dominant P1 receptors in a process mediated by specific adaptor specificity for Arg), K-gingipain (dominant P1 specificity for 48 proteins. This clustering of zymogens possessing intrinsic Lys), and the plant and legumains (dominant P1 enzymatic activity forces processing in trans, and activation of specificity for Asn). Though all are cysteine proteases, they the first protease in the death pathway to may share the caspase fold, and therefore represent very apoptosis.47,49,50 An analogous activation of pro-caspase 9 distant homologs.57 Thus, we may so far only have found a by zymogen clustering may account for the origin of the post- few of the members of the family sharing the caspase fold mitochondrial death pathway,51,52 and the mechanism may and may still find a number of proteases that will eventually be highly conserved since the worm caspase CED-3 may be close the evolutionary gap between the bacterial and the similarly activated.53,54 These observations stress the mammalian members. importance of zymogenicity (defined in Table 2) in triggering the cell death signaling pathway. The interesting range of zymogenicity values displayed References by members of the caspase family is mirrored by members 1. 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