Mechanism of action of cysteine proteinases: oxyanion binding site is not essential in the hydrolysis of specific substrates

Abstract

To study the possible stabilization of the oxyanion of the tetrahedral intermediate formed in the course of the catalyses by cysteine proteinases, papain, chymopapain, papaya peptidase A and ficin, N-(benzyloxycarbonyl)phenylalanylthioglycine O-ethyl ester was synthesized and its hydrolysis compared with that of the corresponding oxygen ester, a highly specific substrate of the above enzymes. The substitution of sulfur for the carbonyl oxygen hardly affected the 2nd-order rate constant of acylation and diminished catalytic activity by about 1 order of magnitude in deacylation. These results contrast with those obtained with serine proteinases, where the hydrolysis of thiono esters could not be detected. From the results the following conclusions can be drawn. Stabilization of the tetrahedral intermediate at an oxyanion binding site is not essential with cysteine proteinases. Because of the lack of general base catalysis, cysteine proteinases have a less constrained transition-state structure than serine proteinases.