Abstract
The mechanism of action of bovine pancreatic carboxypeptidase. Aalpha (peptidyl-L-amino acid hydrolase; EC 3.4.12.2) has been investigated by application of cryoenzymologic methods. Kinetic studies of the hydrolysis of the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate have been carried out with both the native and the Co2+-substituted enzyme in the 25 to --45 degrees C temperature range. In the --25 to --45 degrees C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (ks) processes. In Arrhenius plots, ks extrapolates to kcat at 25 degrees C for both enzymes in aqueous solution, indicating that the same catalytic rate-limiting step is observed. The slow process is analyzed for both metal enzymes, as previously reported (Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc Natl. Acad. Sci. U. S. A. 73, 3882-3886), to involve the deacylation of a mixed anhydride acyl-enzyme intermediate. Near --60 degrees C the acyl-enzyme intermediate of both metal enzymes can be stabilized for spectral characterization. The pH and temperature dependence of ks reveals a catalytic ionizing group with a metal ion-dependent shift in pKa and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Co2+ enzyme. These parameters identify the ionizing catalytic group as the metal-bound water molecule. Extrapolation of the pKa data to 25 degrees C indicates that this ionization coincides with that observed in the acidic limb of the pH profile of log(kcat/Km(app)) for substrate hydrolysis under steady state conditions. The results indicate that in the esterolytic reaction of carboxypeptidase. A deacylation of the mixed anhydride intermediate is catalyzed by a metal-bound hydroxide group.
Highlights
Biology, Cummings Life Science Center, The University of Chicago, The mechanism of action of bovine pancreatic carboxypeptidase A
Temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast and the slow (k8) processes
In contrast to the interpretations of structural and chemical studies, our results indicate that the active species in ester hydrolysis catalyzed by carboxypeptidase A is a metalhydroxide complex
Summary
Cummings Life Science Center, The University of Chicago, The mechanism of action of bovine pancreatic carboxypeptidase A, Kinetic studies of the hydrolysis of substrate 0-( trans-p-chlorocinnahave been carried out with both the native and the Co’+-substituted enzyme in the 25 to -45°C temperature range. In the -25 to -45°C temperature range with enzyme in excess, a biphasic reaction is observed for substrate hydrolysis characterized by rate constants for the fast (kf) and the slow (k8) processes. The pH and temperature dependence of k, reveals a catalytic ionizing group with a metal ion-dependent shift in pK, and an enthalpy of ionization of 7.2 kcal/mol for the native enzyme and 6.2 kcal/mol for the Coz+ enzyme. The mechanism of action of carboxypeptidase A (peptidylL-amino acid hydrolase; EC 3.4.12.2) has been conjectural despite a wide variety of thorough kinetic, structural, and chemical studies (l-4). Structural requirements for substrate specificity and rapid hydrolysis are a free carboxyl group and an aromatic or branched aliphatic side chain of the COOH-terminal residue
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