The alkaline pH dependence of chymotrypsin reactions: postulation of a pH-dependent intramolecular competitive inhibition.

The two groups observed in k2/K, may both belong to k2 (acylation) or to K* (binding), or one may belong to k2 and the other to K,. Several studies2 indicate that the pH of 6.8 is part of the k2 step. This finding is consistent with the presence of pK 7.0 in the k3 step, since one would expect that the same catalytic group should be operative in both steps, if k2 and k3 are the microscopic reverse of one another. Both these pK's can be assigned to the imidazole group of histidine 57 which has been demonstrated by a stoichiometric inhibition reaction to be necessary for enzymatic activity.3 The ionization of pK 8.9 cannot as easily be assigned, however. Since it does not appear in the k3 step,' it is presumably not a catalytic group. In an earlier paper this ionization was assigned to the k2 step,' and was postulated to control a conformational change of the enzyme in that step.6 Recently, we have found that the ionization of pK 8.9 occurs in the pH dependence of the reaction of L-1-chloro-3-tosylamido-4-phenylbutanone with chymotrypsin,4 a reaction which results in the simple alkylation of histidine 57 of the enzyme.3 Since the nonenzymatic reaction of this reagent with imidazole cannot account for a pH dependence of 8.9, this finding implies that this pH dependence may be involved in the binding step of chymotrypsin and casts doubt on previous assignment of this pH dependence to the k2 step. Because of these doubts and because the function of the group of pK 8.9 has not been completely elucidated, we have carried out further experiments on the pH dependence of chymotrypsin-catalyzed reactions and experiments designed to probe the action of the group of pK 8.9 The kinetics of the acylation of a-chymotrypsin by p-nitrophenyl acetate were repeated and again show (Table 1) a binding constant (Ks) which is independent of pH and a rate constant, k2, which shows a bell-shaped pH-rate constant profile with pK's of 6.8 and -9, as stated previously. ' However, the kinetics of the a-chymotrypsin-catalyzed hydrolysis of N-acetylL-tryptophanamide show a Km dependent on an acidic group of pK -9 and a kcat dependent only on a basic group of the pK 6.8, and independent of pH up to pH 9.4.5 These data are shown in Table 2. Good agreement between the pH dependence of Km and the K1 of N-acetyl-L-tryptophanamide [as determined