Peptide synthesis catalyzed by the serine proteinases chymotrypsin and trypsin.

The ratio of the initial rates of aminolysis and hydrolysis in peptide semisynthesis catalyzed by chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) was found to depend non-linearly on the concentration of the added nucleophile. This is in agreement with a mechanism for the peptide semisynthesis where nucleophile binding to the acyl-enzyme precedes the aminolysis reaction. The acyl-enzyme-nucleophile complex can still be deacylated by water. A temperature optimum was observed for peptide synthesis for valinamide as nucleophile. This and the similarity of the P'1 specificity in peptide hydrolysis and nucleophile specificity in peptide semisynthesis also support the mechanism including the nucleophile binding. The influence of added nucleophiles on the acylation step during peptide synthesis was studied by determining kcat and Km for the appearance of the leaving group from the acyl donor. Acceptor (= nucleophile) specificity was shown to be more important for high ratios of aminolysis: hydrolysis than donor specificity. The maximum product concentration during kinetically controlled peptide semisynthesis was found to be independent of the enzyme content.