Mechanism of cysteine protease inactivation by peptidyl epoxides

Peptidyl epoxides are time- and concentration-dependent selective cysteine protease inhibitors. The lack of recovery of enzymic activity and the retention of 1 molar equivalent of radioactive inhibitor associated with the enzyme on dialysis, shown in this study, indicate that they form a covalent irreversible equimolar complex with the enzyme. It is also shown that the peptidyl epoxide inhibitors alkylate the active-site thiol. This alkylation only occurs when the enzyme is in its native conformation, as the denatured enzyme does not undergo alkylation by the inhibitor to any appreciable extent. Finally, the inactivation process is compared with a model reaction between a peptidyl epoxide and a protected cysteine in neutral and basic aqueous media. The inactivation of cathepsin B by Cbz-Phe-(O-benzyl)-Thr-epoxide is accelerated by 5.5 orders of magnitude relative to the rate of the model reaction at pH 10.0 and 25 degrees C, and estimated to be at least 10(8) times faster than the model reaction at pH 7.0. These results, in conjunction with the selectivity exhibited by peptidyl epoxides at all levels, point to a mechanism-based inhibition, and may have mechanistic implications regarding the catalysis carried out by cysteine proteases.