The caspase family represents a new class of intracellular cysteine proteases with known or suspected roles in cytokine maturation and apoptosis. These enzymes display a preference for Asp in the P1 position of substrates. To clarify differences in the biological roles of the interleukin-1␤ converting enzyme (ICE) family proteases, we have examined in detail the specificities beyond the P1 position of caspase-1, -2, -3, -4, -6, and -7 toward minimal length peptide substrates in vitro. We find differences and similarities between the enzymes that suggest a functional subgrouping of the family different from that based on overall sequence alignment. The primary specificities of ICE homologs explain many observed enzyme preferences for macromolecular substrates and can be used to support predictions of their natural function(s). The results also suggest the design of optimal peptidic substrates and inhibitors.A growing body of evidence supports important roles for the interleukin-1␤ converting enzyme (ICE) 1 (1, 2) and its homologs (recently renamed caspases (3)) in cytokine maturation and apoptosis. The caspase gene family, defined by protein sequence homology but also characterized by conservation of key catalytic and substrate-recognition amino acids, includes caspase-2 (4), caspase-3 (5-7), caspase-4 (8 -10), caspase-5 (10), caspase-6 (11), caspase-7 (12-14), caspase-8 (15-17), caspase-9 (18, 19), and caspase-10 (17). Each is an intracellular cysteine protease that shares with the serine protease granzyme B specificity for Asp in the P1 position of substrates. The specific biological roles and interrelationships of these enzymes are for the most part unknown and are areas of active investigation in many laboratories.A role for caspase-1 in inflammation is supported by several lines of evidence. Caspase-1-deficient mice, and cells derived from those animals, are deficient in IL-1␤ maturation and are resistant to endotoxic shock (20,21). Peptidic inhibitors of caspase-1 can be effective in blocking maturation and release of IL-1␤ by cultured cells (1) and in whole animals (22, 23) and of inflammation in animal models (24,25). The selectivity of the inhibitors employed in these studies among the caspases has not been demonstrated, and so the precise role of each caspase in inflammation is uncertain. Nevertheless the results uphold the promise of caspase-1 and/or its homologs as targets for anti-inflammatory drug discovery.Caspases play important roles in apoptosis signaling and effector mechanisms. Sequence alignments reveal homology with Ced-3 (26), a nematode cysteine protease (27, 28) that is required for cell death. The viral proteins CrmA and p35 are antiapoptotic and act by inhibition of caspases (29,30). A bacterial invasin induces apoptosis by binding to and activating caspase-1 specifically (31). Caspase-3 is necessary and sufficient for apoptosis in one acellular model (6); however, in mice the essential function of this enzyme is limited to apoptosis in the brain (32). A hallmark of apoptosis is the pr...

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis.

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The peptidyl trifluoromethyl ketones Ac-Phe-CF3 (1) and Ac-Leu-Phe-CF3 (2) are inhibitors of chymotrypsin. They differ in Ki (20 and 2 microM, respectively) as well as in their kinetics of association with chymotrypsin in that 1 is rapidly equilibrating, with an association rate too fast to be observed by steady-state techniques, while 2 is "slow binding", as defined by Morrison and Walsh [Morrison, J. F., & Walsh, C. T. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 202], with a second-order association rate constant of 750 M-1 s-1 at pH 7.0 [Imperiali, B., & Abeles, R. (1986) Biochemistry 25, 3760]. The crystallographic structures of the complexes of gamma-chymotrypsin with inhibitors 1 and 2 have been determined in order to establish whether structural or conformational differences can be found which account for different kinetic and thermodynamic properties of the two inhibitors. In both complexes, the active-site Ser 195 hydroxyl forms a covalent hemiketal adduct with the trifluoromethyl ketone moiety of the inhibitor. In both complexes, the trifluoromethyl group is partially immobilized, but differences are observed in the degree of interaction of fluorine atoms with the active-site His 57 imidazole ring, with amide nitrogen NH 193, and with other portions of the inhibitor molecule. The enhanced potency of Ac-Leu-Phe-CF3 relative to Ac-Phe-CF3 is accounted for by van der Waals interactions of the leucine side chain of the inhibitor with His 57 and Ile 99 side chains and by a hydrogen bond of the acetyl terminus with amide NH 216 of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of seven peptidyl trifluoromethyl ketone (TFK) inhibitors of chymotrypsin have been prepared which differ at the P1 and P2 subsites. Inhibition equilibria and kinetics of association and dissociation with chymotrypsin have been measured. The association rate of Ac-Phe-CF3 was measured at enzyme concentrations between 8 nM and 117 microM in order to examine the relation between the ketone/hydrate equilibrium of trifluoromethyl ketones and the "slow binding" by these inhibitors. The association rate decreases at high enzyme concentrations, indicating that TFK ketone is the reactive species and that conversion of TFK hydrate to ketone becomes rate limiting under these conditions. Inhibitors with hydrophobic side chains at P2 bind more tightly but more slowly to chymotrypsin, indicating that formation of van der Waals contacts between the P2 side chain and the His 57 and Ile 99 side chains of chymotrypsin is a relatively slow process. Inhibitor properties were compared to the Michaelis-Menten kinetic constants of a homologous series of peptide methyl ester and peptide amide substrates. Plots of log Ki vs log (kcat/Km) are linear with slopes of 0.65 +/- 0.2, indicating that these inhibitors are able to utilize 65% of the total binding energy between chymotrypsin and its hydrolytic transition state.