Hydrolysis of alkyl ester and amide substrates by papain

The ratio of the rate constants of acylation of papain with some amino acid ester and amide substrates is unexpectedly low. The contribution to this low ratio by the N-acyl group and the amino acid side chain was studied by measuring the rate constants of substrates containing various acyl groups (benzoyl and benzyloxycarbonyl) and various side chains (glycine, alanine, norleucine, citrulline and arginine). The benzoyl esters were found to be less reactive than the corresponding benzyloxycarbonyl esters, whereas the benzoyl and corresponding benzyloxycarbonyl amides reacted with papain at similar rates. These findings can be explained by the dominance of hydrogen bond formation between the enzyme and amide substrates, which comprensates for the less favourable binding of the benzoyl group. It is also apparent from the similar acylation rate constants for norleucine, citrulline and arginine derivatives that the guanidyl group only slightly affects the reaction of arginine derivatives with papain.