Abstract
The pH dependence of the trypsin-catalyzed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide has been studied at 25 °C. k cat/K M was maximal at alkaline pH values but decreased with decreasing pH. k cat/K M was dependent on free enzyme pK a 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. k cat is shown to equal the acylation rate constant k 2 over the pH range studied. k 2 decreased on the protonation of two groups with pK a values of 4.81 ± 0.15 and 4.23 ± 0.19. We assign the pK a of 4.23 to the ionization of the aspartate-189 residue and the pK a 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 pK a values. From the pH dependence of k 3, we conclude that deacylation depends on a pK a 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 (k 3). A catalytic mechanism is proposed to explain the pH dependence of catalysis.