Kinetic Studies of the Effect of pH on the Trypsin-Catalyzed Hydrolysis of N-α-benzyloxycarbonyl-l-lysine-p-nitroanilide: Mechanism of Trypsin Catalysis.

Abstract

The pH dependence of the trypsin-catalyzed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide has been studied at 25 °C. kcat/KM was maximal at alkaline pH values but decreased with decreasing pH. kcat/KM was dependent on free enzyme pKa values of 6.75 ± 0.09 and 4.10 ± 0.13, which were assigned to the ionization of the active site histidine-57 and aspartate-189, respectively. Protonation of either group abolished catalytic activity. kcat is shown to equal the acylation rate constant k2 over the pH range studied. k2 decreased on the protonation of two groups with pKa values of 4.81 ± 0.15 and 4.23 ± 0.19. We assign the pKa of 4.23 to the ionization of the aspartate-189 residue and the pKa of 4.81 to the oxyanion of the tetrahedral intermediate formed during acylation. We conclude that during acylation, breakdown of the catalytic tetrahedral intermediate is rate-limiting and that there is a strong interaction between the imidazolium ion of histidine-57 and the oxyanion of the catalytic tetrahedral intermediate, which perturbs their pKa values. From the pH dependence of k3, we conclude that deacylation depends on a pKa of 6.41 ± 0.22 and that the ionization of the carboxylate group of aspartate-189 does not have a significant effect on the rate of deacylation (k3). A catalytic mechanism is proposed to explain the pH dependence of catalysis.