Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates I. THE pH DEPENDENCE OF THE CATALYTIC HYDROLYSIS OF N-ACETYL-l-TRYPTOPHANAMIDE BY THREE FORMS OF THE ENZYME AT ALKALINE pH

Abstract The pH dependence of the steady state kinetic parameters Km (app) and kcat of the δ-, acetylated δ-, and α-chymotrypsin-catalyzed hydrolysis of the specific amide substrate N-acetyl-l-tryptophanamide, has been investigated in the neutral and alkaline pH regions. A completely automatic technique for measuring the hydrolysis of amides was used. New results and important aspects of the chymotrypsin-catalyzed hydrolysis of a specific amide substrate which have emerged from this investigation are the following. The pH dependence of the catalytic reaction at alkaline pH is adequately accounted for by the pH dependence of Km(app). In earlier experiments we have shown that in amide hydrolysis the steady state kinetic parameter Km(app) is a measure of an over-all enzyme-substrate dissociation constant (K's). (a) Km(app) was observed to be pH-dependent in the hydrolysis of N-acetyl-l-tryptophanamide as catalyzed by all three forms of the enzyme. Analysis of this pH dependence shows that an ionizing group with pK(app) ∼ 8.5 is involved; an ionizing group of the enzyme with this pK(app) has been implicated in all chymotrypsin-catalyzed reactions that have been studied. (b) The catalytic rate constant kcat was found to be pH-independent in the pH region 8 to 10 for the δ-chymotrypsin-catalyzed reaction and in the pH region 8 to 9.2 for the α-chymotrypsin-catalyzed reaction. (c) The pH dependence of the steady state kinetic parameters kcat and Km(app) in the chymotrypsin-catalyzed hydrolysis ofN-acetyl-l-tryptophanamide is consistent with the findings of our earlier studies of the individual steps in the reaction between chymotrypsin and diisopropyl fluorophosphate and in the chymotrypsin-catalyzed hydrolysis of N-acetyl-l-phenylalaninamide: studies which indicated that the pH dependence of chymotrypsin-catalyzed hydrolysis above pH 8 is due to the effect of pH on the formation of chymotrypsin complexes and not on the bond-breaking step. (d) The experiments reported here are not in agreement with earlier studies of Bender and his co-workers, which indicated that the pH dependence of chymotrypsin-catalyzed reactions above pH 8 is due to the effect of pH on the bond-breaking step, the formation of chymotrypsin-substrate complexes being pH-independent. The δ form of chymotrypsin is a more efficient catalyst than the α form in the hydrolysis of N-acetyl-l-tryptophanamide, as indicated by both the observed maximum rate of reaction and the binding of substrate to enzyme. Both the values and the pH dependencies of the steady state kinetic parameters are different for these two forms of the enzyme, but are the same for δ- and acetylated δ-chymotrypsin. Only limited and qualified information regarding differences in the catalytic properties of α- and δ-chymotrypsin has been previously available. These and previous experiments are consistent with the hypothesis that the pH dependence of chymotrypsin-catalyzed reactions at alkaline pH is due to a pH-dependent equilibrium between two major conformations of the enzyme, with the same ionizing group of pK (app) ∼ 8.5 controlling both the equilibrium between enzyme conformations and the decrease in catalytic properties of the enzyme. This hypothesis leads to several predictions, discussed in this paper, which can be tested experimentally.