Revised Manuscript Received January 5, 1994® abstract: Interleukin-1| 8 converting enzyme (ICE) is a cysteine protease in monocytes that is essential for the proteolytic activation of interleukin-1 (3, an important mediator of inflammation. Peptide (acyloxy)-methyl ketones designed with the appropriate peptide recognition sequence (Ac-Tyr-Val-Ala-Asp-CH2-OC (O) Ar) are potent, competitive, irreversible inhibitors. Mass spectrometry and sequence analysis indicate that inactivation proceeds through expulsion of the carboxylate leaving group to form a thiomethyl ketone with the active site Cys285***.

The second-order inactivation rate is independent of leaving group pKa, with an approximate value of 1 X 106 M_1 s-1. This rateconstant is directly proportional to the reaction macroviscosity, indicating that the rate-limiting step in inactivation is association of enzyme and inhibitor, rather thanany bond-forming reactions. Affinity labeling of THP. 1 monocytic cell cytosol with a biotinylated tetrapeptide (acyloxy) methyl ketone for 28 half-lives resulted in labeling of only ICE, demonstrating the selectivity of these inhibitors. These inhibitors are relatively inert toward other bionucleophiles such as glutathione (< 5 X 10-4 M_1 s_1), making them excellent candidates for in vivostudies of enzyme inhibition.