Inactivation of cysteine proteases

The cysteine proteases papain and cathepsin B are inactivated by a Michael acceptor, a peptidyl-beta-chloro-alpha, beta-unsaturated ester (N-Ac-L-Phe-NHCH2-CCl=CH-COOMe). Inactivation occurred concomitant with chloride release which was stoichiometric with the amount of enzyme. This result is consistent with nucleophilic attack of the active site cysteine on the beta-carbon of the inhibitor, followed by expulsion of chloride ion. Inactivation by this class of compounds requires the carbon skeleton about the double bond to be in the trans configuration. The cis isomer was a competitive inhibitor. The difference in the mode of inhibition between the isomers is probably due to non-productive binding of the cis isomer due to bulky chlorine substituent in the beta-position.