Diazomethyl ketone substrate derivatives as active-site-directed inhibitors of thiol proteases. Papain

The diazomethyl ketones of z-Phe-Phe inactivate papain by a stoichiometric reaction at the active-center thiol. Since the reagents are stable in mercaptoethanol, their reaction with papain is judged to be the result of complex formation characteristic of affinity-labeling reagents. The diazomethyl ketones react by a mechanism different from that of chloromethyl ketones, since the pH dependence of their inactivation of papain is different, the rate increasing with decreasing pH. This relationship has been observed in other cases, such as in the reaction of azaserine with glutamine amidotransferases [Buchanan, J. M. (1973), Adv. Enzmol. Relat. Areas Mol. Biol. 39, 91], and is interpreted as an indication of reaction with a thiol group in its protonated form.