Inactivation of cysteine proteases by peptidyl epoxides: characterization of the alkylation sites on the enzyme and the inactivator

Erythro peptidyl epoxides are selective inactivators of cysteine proteases. The alkylation site, both on the enzyme papain and on the epoxide itself, was characterized. The inactivation of papain with the peptidyl epoxide ery thro benzyloxycarbonyl-Phe-Alaepoxide was followed by total hydrolysis by a cid. Mass spectral analysis of the hydrolysate revealed, in addition to the expected amino acids, a unique signal of m/z 209 (MH+). Its high-resolutio n mass spectrum and daughter peak analysis correspond to the product of alk ylation on cysteine and the expected fragmentation. A similar MS pattern wa s obtained for a synthetic model compound corresponding to the expected hyd rolysis product. A C-13 NMR analysis of papain inactivated by a specificall y C-13-labelled peptidyl epoxide indicated that the alkylation of the enzym e's cysteine residue occurs on the primary carbon of the epoxide moiety.