Acylation of α- and δ-Chymotrypsins by p-Nitrophenyl Acetate: ENZYME-SUBSTRATE COMPLEX FORMATION AND pH DEPENDENCE

Abstract

Abstract The acylation step of the α-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate has been studied at pH 7.1 and 25° in 2-propanol-water mixtures. By the use of higher substrate concentrations than those used previously under the same conditions, it was shown that, contrary to a previous report, the reaction of acylation does follow saturation kinetics. The binding constant, Ks, and the acylation rate constant, K2, were determined. The acylation step of the δ-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate in acetonitrile-water was also shown to follow saturation kinetics. The pH dependences of k2 and Ks are similar to those reported for α-chymotrypsin, although the decrease in k2 at high pH is less for δ-chymotrypsin. Thus, the pH dependence of acylation by p-nitrophenyl acetate is qualitatively different from that of specific substrates where k2 is pH independent and Ks increases with pH. An explanation for this peculiar pH dependence of chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate is offered.