Design of mechanism-based carboxypeptidase A inactivators on the basis of the X-ray crystal structure and catalytic reaction pathway Original Research Article

The X-ray crystal structure of the complex of carboxypeptidase A (CPA) and Gly-Tyr, has been documented. The crystal structure reveals that both the amide carbonyl oxygen and the terminal amino nitrogen of Gly-Tyr coordinate to the active site zinc ion of CPA in a bidentate fashion, whereby the zinc-bound water molecule is displaced by the amino group. As to the catalytic mechanism of CPA, it is generally believed that while in the cases of ester substrates the carboxylate of Glu-270 functions as the nucleophile which attacks the scissile carbonyl carbon (anhydride pathway), in the case of peptide substrates the zinc-bound water molecule attacks the scissile peptide bond (general base pathway). In light of the X-ray crystal structure and the proposed catalytic mechanism for the enzyme, it is envisioned that the ester bond of O-(hydroxyacetyl)-l-β-phenyllactic acid (l-1) would be hydrolyzed by the attack of the carboxylate of Glu-270 to generate an anhydride intermediate. The latter intermediate would then undergo an intramolecular rearrangement initiated by the attack of the hydroxyl to result in to form an ester bond with the Glu-270 carboxylate. This ester formation impairs the catalytic activity of CPA. We have demonstrated using kinetic analysis that l-1 is indeed an inactivator for the enzyme having the kinact/KI value of 0.057 M−1 s−1. We have also demonstrated that N-(hydroxyacetyl)-l-phenylalanine (l-2) inactivates the enzyme with the kinact/KI value of 0.071 M−1 s−1, suggesting that the carboxylate becomes to attack the peptide carbonyl carbon to generate the same anhydride intermediate as that formed in the inactivation of CPA by l-1. The formation of the anhydride intermediate rather than a tetrahedral transition state that is expected for peptide type substrates was envisioned to occur on the ground that the zinc-bound water molecule is displaced by the hydroxyl of l-2 upon binding to the enzyme.