[pH-dependence of tryptophan ethyl ester hydrolysis by alpha-chymotrypsin]

The hydrolysis of L-tryptophane ethyl ester catalyzed by alpha-chymotrypsin and the effect of ethyl ster of D-tryptophane on the course of the reaction were studied. A kinetic pattern of a three-step enzymatic reaction based on the assumption that the enzyme complex with the protonated form of the substrate is the only reactive one, was proposed. It was shown that the limiting step of the enzymatic reaction consists in a formation of intermediate acyl enzyme. The pH-dependence of the bimolecular rate constant (kcat/Km) for the enzymatic hydrolysis of L-tryptophane ethyl ester is bell-shaped and is described by a pattern including ionization of two groups with pKa values of 7,0 +/- 0,1; and 7,5 +/- 0,1; the value of pKa equal to 7,5 +/- 0,1 corresponds to substrate ionization (pKas for the amino group of L-tryptophane ethyl ester is 7,6). The constants for the binding of protonated and non-protonated substrate forms by the enzyme were calculated from the step-wise dependence of the Km values of pH. An analysis of the bell-shaped dependence of the catalytic constant of enzymatic hydrolysis included determination of pK values of the ionogenic groups of the enzyme--substrate complex (pK'a = 6,8 +/- 0,1 and pK''a = 7,3 +/- 0,1).