SUBSTRATE AND INHIBITOR SPECIFICITY OF INTERLEUKIN-1-BETA-CONVERTING ENZYME AND RELATED CASPASES

Interleukin-1 beta-converting enzyme (ICE) is a novel cysteine proteas e responsible for the cleavage of pre-interleukin-1 beta (pre-IL-1 bet a) to the mature cytokine and a member of a family of related protease s (the caspases) that includes the Caenorhabditis elegans cell death g ene product, CED-3. In addition to their sequence homology, these cyst eine proteases display an unusual substrate specificity for peptidyl s equences with a P-1 aspartate residue. We have examined the kinetics o f processing pre-IL-1 beta to the mature form by ICE and three of its homologs, TX, CPP-32, and CMH-1. Of the ICE homologs, only TX processe s pre-IL-1 beta, albeit with a catalytic efficiency 250-fold less than ICE itself. We also investigated the ability of these four proteases to process poly(ADP-ribose) polymerase, a DNA repair enzyme that is cl eaved within minutes of the onset of apoptosis. Every caspase examined cleaves PARP, with catalytic efficiencies ranging from 2.3 x 10(6) M( -1) S-1 for CPP32 to 1.0 x 10(3) M(-1) S-1 for TX. In addition, we rep ort kinetic constants for several reversible inhibitors and irreversib le inactivators, which have been used to implicate one or more caspase s in the apoptotic proteolysis cascade. Ac-Asp-Glu-Val-Asp aldehyde (D EVD-CHO) is a potent inhibitor of CPP-32 with a K-i value of 0.5 nM, b ut is also potent as inhibitor of CMH-1 (K-i = 35 nM) and ICE (K-i = 1 5 nM). The x-ray crystal structure of DEVD-CHO complexed to ICE presen ted here reveals electrostatic interactions not present in the Ac-YVAD -CHO co-complex structure (Wilson, K. P., Black, J.-A. F., Thomson, J. A., Kim, E. E., Griffith, J. P., Navia, M. A., Murcko, M. A, Chambers , S. P., Aldape, R. A., Raybuck, S. A., and Livingston, D. J. (1994) N ature 370, 270-275), accounting for the surprising potency of this inh ibitor against ICE.