Abstract
Kinetic and thermodynamic parameters were evaluated for the acylation and the deacylation steps in the hydrolysis of p-nitrophenyl acetate by α-chymotrypsin at pH 7.8 and at temperatures between 15 and 35°C by the use of stopped-flow and ordinary ultraviolet spectrophotometers. In contrast to the temperature dependencies of k2 and Ks reported in the literature (P.A. Adams and E.R.Swart, Biochem.J., 161, 83(1977)), no kinetic anomaly was observed in either of the steps, but reasonable straight lines were obtained in both Arrhenius and van't Hoff plots. On the other hand, in the chymotryptic hydrolysis of N-benzoyl-L-alanine methyl ester a sharp kinetic anomaly was found. The discrepancy in the case of p-nitrophenyl acetate is discussed in connection with a possible conformational change of the enzyme, an alteration of the rate-limiting step or differences in the experimental procedures. The cause of the anomaly observed in the case of N-benzoyl-L-alanine methyl ester is also discussed in detail.
