Interactions of five- and six-membered cyclic esters with α-chymotrypsin: Kinetic evidence for covalent enzyme intermediates

Abstract

Interactions of α-chymotrypsin with 2-coumaranone ( I), 3,4-dihydrocoumarin ( II), o-hydroxy-α-toluenesulfonic acid sultone ( III), and β- o-hydroxyphenylethanesulfonic acid sultone ( IV) were studied in the presence of 14% acetonitrile at pH 7.0 by means of the proflavin displacement technique and by inhibition of N-acetyl- l-tryptophan ethyl ester (ATrEE) hydrolysis. Under saturating conditions of either I, II, or III, an enzyme intermediate was shown to accumulate using either the proflavin displacement technique or the ATrEE activity assay. The intermediates have characteristics of covalent enzyme-substrate compounds and are believed to decompose simultaneously by two pathways, one to give free enzyme and hydrolyzed cyclic ester, and the other to give the original cyclic ester and free enzyme. With α-chymotrypsin and III the observed first-order rate constant for decomposition of the intermediate by the two pathways was 0.19 ± 0.04 min −1, while the rate constant for the hydrolytic pathway alone was 0.013 ± 0.0009 min −1. These results indicate that the covalent-like intermediate with this sultone is not only capable of reverting to starting cyclic ester but prefers this pathway over hydrolysis. Sultone IV was found to bind to enzyme; but in contrast to the behavior of esters I–III, the binding did not result in accumulation of a covalent-like intermediate.