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.

The pH dependence of the steady state kinetic parameters K,(app) and $,t of the a-, acetylated 6-, and ar-chymotrypsincatalyzed hydrolysis of the specific amide substrate N-acetylr.-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 K,(app). In earlier experiments we have shown that in amide hydrolysis the steady state kinetic parameter K,(app) is a measure of an over-all enzyme-substrate dissociation constant (K’,). (a) K,(app) was observed to be pH-dependent in the hydrolysis of N-acetyl-r&yptophanamide 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 kat was found to be pa-independent in the pH region 8 to 10 for the &chymotrypsin-catalyzed reaction and in the pH region 8 to 9.2 for the a-chymotrypsin-catalyzed reaction. (c) The pH dependence of the steady state kinetic parameters kcat and K,(app) in the chymotrypsin-catalyzed hydrolysis of iV-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