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Mechanism of Action of Carboxypeptidase A in Ester Hydrolysis
Marvin W. Makinen, Kazuo Yamamura and E. T. Kaiser
Proceedings of the National Academy of Sciences of the United States of America
Vol. 73, No. 11 (Nov., 1976), pp. 3882-3886
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/66416
Page Count: 5
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The reaction of carboxypeptidase A (peptidyl-L-amino-acid hydrolase; EC 126.96.36.199) with the specific ester substrate O-(trans-p-chlorocinnamoyl)-L-β -phenyllactate has been investigated in the temperature range 25 degrees to -40 degrees with use of organic-aqueous cosolvent mixtures. In the subzero temperature range the hydrolysis reaction is characterized by a biphasic decrease in absorbance specific for the substrate. The kinetic data can be unambiguously analyzed as two consecutive first-order reactions with formation of a covalent acyl-enzyme intermediate. Deacylation of the covalent intermediate is shown to be rate-limiting in the subzero temperature range, and near -60 degrees 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 γ -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.
Proceedings of the National Academy of Sciences of the United States of America © 1976 National Academy of Sciences