Mechanism of action of carboxypeptidase A in ester hydrolysis.

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Abstract
The reaction of carboxypeptidase A (peptidyl-L-amino-acid hydrolase; EC 3.4.12.2) with the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-.beta.-phenyllactate was investigated in the temperature range 25.degree. to -40.degree. with use of organic-aqueous cosolvent mixtures. In the subzero temperature range the hydrolysis is characterized by a biphasic decrease in absorbance specific for the substrate. The kinetic data can be unambiguously analyzed as 2 consecutive 1st-order reactions with formation of a covalent acyl-enzyme intermediate. Deacylation of the covalent intermediate is rate-limiting in the subzero temperature range, and near -60.degree. it is sufficiently stable for spectral characterization. Consideration of the structure of the active site and of the catalytically functional residues of the enzyme leads to the conclusion that the intermediate is a mixed anhydride in which the .gamma.-carboxylate of glutamate-270 is acylated by the substrate. The temperature dependence of the rate constants of the acylation and deacylation steps explains why the intermediate of this enzyme-catalyzed reaction is observed only at low temperatures.