A kinetic approach to evaluate nucleophilic reactivity of amino acid esters for aminolysis in papain-catalyzed peptide bond formation

Papain(EC 3.4.22.2) catalyzes not only hydrolytic reactions of protein substrates but also the peptide bond synthesis and, furthermore, the polymerization of N-unprotected several amino acid esters. Principally on the basis of reaction model for hydrolysis, a chemical mechanism has emerged for papain catalysis that involves acyl-transfer to and from the sulfhydryl moiety of a Cys residue in its active site. According to this mechanism, in papain-catalyzed polymerization of N-unprotected amino acid esters, the combination of papain with an acyl-donor substrate, an amino acid ester, to form a Michaelis-complex is followed by the formation of a covalent acyl-enzyme intermediate and an equivalent of alcohol. In the presence of a suitable nucleophile, another amino acid ester with a free amino group, the acyl-enzyme undergoes aminolysis to produce free enzyme and the peptide product. In the absence of a nucleophile, the acyl-enzyme will hydrolyze. As part of an investigation to evaluate aminolytic activity of amino acid esters in papaincatalyzed polymerization of amino acid esters, we have undertaken a study of the kinetics of reaction of this enzyme with prototype acyl-donor substrate Bz-Gly-OEt and several amino acid esters. In this paper, we report the results of our initial kinetic studies to lay a conceptual foundation and to present a method for analyzing aminolysis by kinetic experiments. The data for the reaction of papain with both Bz-Gly-OEt and amino acid esters are consistent with the mechanism involving an acyl-enzyme intermediate that can be deacylated by either reaction with water or an amino acid ester as a primary amine nucleophile, and also indicate that aminolysis proceeds through the binding of an amino acid ester to the acyl-enzyme in the deacylation step. The direct measurement of aminolytic behavior of amino acid esters allows us to estimate kinetic parameters of this aminolysis step.