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

[Image: see text] 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...

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Detalles Bibliográficos
Autor principal: Malthouse, J. Paul G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2020
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081292/
https://www.ncbi.nlm.nih.gov/pubmed/32201777
http://dx.doi.org/10.1021/acsomega.9b03750
Descripción
Sumario:[Image: see text] 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.