Transition State Charge Distribution in Reactions of an Acetyltyrosylchymotrypsin Intermediate

Abstract

Abstract The previously reported results implicating general base catalysis in the deacylation of furoylchymotrypsin have been confirmed with a specific acylenzyme, acetyltyrosylchymotrypsin. For nonaromatic amines extending over a range of basicity of 107·8 the rate varies by a factor of only four; the rate constants for the reaction of these amines are equal to approximately 200 m-1 s-1. Methoxyamine and ammonia are 16- and 10-fold, respectively, less reactive than the other amines. With four para-substituted anilines of pK 2.2 to 6.3 the rate is less than that for nonaromatic amines of equivalent basicity and the Brφnsted slope is equal to 0.52. Some unknown factor apparently prevents effective proton abstraction from attacking anilines. There is a deuterium isotope effect on the rates of reaction of ethylamine, formylhydrazine, and p-chloroaniline with the acetyltyrosylenzyme and this effect is equal to that for hydrolysis of the acylenzyme. The effects of basicity and deuterium oxide are consistent with a mechanism involving concerted (and effective) proton abstraction from the attacking amine. An alternate stepwise mechanism involving a rate-limiting diffusion of protonated His-57 away from an anionic tetrahedral intermediate, which predicts a zero Brφnsted β for basic amines and a β near unity for amines of pK less than 7, is not supported by the results.