Mechanism of chymotrypsin: concurrent chemical and enzymic aminolysis of an acyl-enzyme of methylchymotrypsin. Efficiency of acid–base catalysis

Abstract

An acyl-enzyme of a modified chymotrypsin in which the nitrogen N^ε2 of histidine-57 is methylated has been prepared. The rates of deacylation of this acyl-enzyme in the presence of various amines of pK_a between 3.8 and 11 have been measured at several pH values. For hydrazine, two concurrent pathways of aminolysis are observed, one whose rate is proportional to the hydroxide ion concentration and one whose rate is proportional to the fraction of the active site in the non-protonated form with a pK_a close to 7. For the other amines, the rates of aminolysis have been measured above pH 8 and the occurrence of two pathways is detected in most cases. From the slope of the Brønsted plot and the absence of terms second order in amine, the rate-limiting step of the hydroxide ion catalysed pathway is described as serine anion expulsion from the anionic tetrahedral intermediate (T^–). The rate-limiting step of the enzymic pathway is the serine expulsion from T^– catalysed by the conjugate acid of the imidazolyl group of the active site. The efficiency of the acid catalysis of the decomposition of the anionic tetrahedral intermediate is estimated for the modified and the native enzyme. With the native enzyme, this contribution to the overall catalysis is very high. Attempts to observe large rate enhancements due to intrmolecular catalysis in the aminolysis by diamines were unsuccessful. When the active site is modified, the leaving group specificity observed with the native enzyme and amino-acid amides is lost.