The Effect of Side Chain Structure of Ester Substrates in Determining the Rate-Controlling Step

Presteady state and steady state analyses of the a-chymotrypsin [EC 3.4.21.l]-catalyzed hydrolysis of three specific ester substrates and three ring-substituted derivatives were carried out to elucidate the effect of hydrophobic interactions due to the different side chains of the substrates on the individual steps of the reaction. Hydrolysis of all the substrates except for iVa-acetyl-JVill-formyltryptophan methyl ester (Ac-Trp(CHO)-OMe) was controlled by the deacylation rate. In spite of their comparable Ka values, the substrates with small /rCat, such as./V'-acetyltryptophan methyl ester and iV'-acetyl-2-(2-nitro-4-carboxyphenyIsulfenyl)-tryptophan methyl ester, characteristically gave Km values one order of magnitude smaller than the others. For the reaction of Ac-Trp(CHO)-OMe, it was ascertained that the deacyla-tion step was not rate-controlling. It is suggested that the acylation step controls the rate in this case. The steps in the hydrolysis catalyzed by ar-chymo-trypsin [EC 3.4.21.1] are given in Eq. 1, • E + P, ( 1) where S represents substrate ester or amide, ES an enzyme-substrate complex, Kt its dissociation constant, EA an acyl enzyme, Px an alcohol or amine, and P, an acid. Kinetic investigations of a-chymotryptic hydrolysis of various ester sub-strates have indicated that the deacylation (k^) step controls the rate (7-7). In contrast, th