Evidence for the general base mechanism in carboxypeptidase A-catalyzed reactions: partitioning studies on nucleophiles and H2(18)O kinetic isotope effects.
Methanol does not detectably compete with water in carboxypeptidase-catalyzed cleavage of any substrate, although it is preferentially reactive in a model for the proposed nucleophilic mechanism for the enzyme that involves an anhydride intermediate. To test for such a common intermediate in the cleavage of related peptide and ester substrates, a method has been developed to examine H2(16)O-H2(18)O kinetic isotope-partitioning effects. The finding that benzoylglycylphenylalanine has an isotope effect of 1.019 +/- 0.002 while benzoylglycyl-beta-L-phenyl-lactate shows a small inverse isotope effect excludes most versions of a nucleophilic mechanism having a common anhydride intermediate. The bulk of the available evidence strongly favors the previously proposed general base mechanism.
D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D007553:Isotope Labeling; D007700:Kinetics; D010103:Oxygen Isotopes; D011485:Protein Binding; D013379:Substrate Specificity
D010103:Oxygen Isotopes; D002268:Carboxypeptidases; D043422:Carboxypeptidases A