Lee K J, Kim D H
Department of Chemistry, Pohang University of Science and Technology, Korea.
Bioorg Med Chem. 1998 Sep;6(9):1613-22. doi: 10.1016/s0968-0896(98)00082-0.
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-beta-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.
羧肽酶A（CPA）与甘氨酰 - 酪氨酸复合物的X射线晶体结构已有文献记载。晶体结构表明，甘氨酰 - 酪氨酸的酰胺羰基氧和末端氨基氮均以双齿方式与CPA的活性位点锌离子配位，从而使与锌结合的水分子被氨基取代。关于CPA的催化机制，一般认为，在酯底物的情况下，Glu - 270的羧酸盐作为亲核试剂攻击可裂解的羰基碳（酸酐途径），而在肽底物的情况下，与锌结合的水分子攻击可裂解的肽键（通用碱途径）。根据X射线晶体结构和提出的该酶催化机制，可以设想O -（羟基乙酰基）-L-β-苯乳酸（L - 1）的酯键会被Glu - 270的羧酸盐攻击而水解，生成酸酐中间体。然后，后者的中间体将通过羟基的攻击引发分子内重排，从而与Glu - 270羧酸盐形成酯键。这种酯的形成会损害CPA的催化活性。我们通过动力学分析证明，L - 1确实是该酶的一种失活剂，Kinact / KI值为0.057 M-1 s-1。我们还证明，N -（羟基乙酰基）-L - 苯丙氨酸（L - 2）使该酶失活，kinact / KI值为0.071 M-1 s-1，这表明羧酸盐开始攻击肽羰基碳，生成与L - 1使CPA失活时形成的相同酸酐中间体。预计肽类底物会形成四面体过渡态，而酸酐中间体的形成是基于L - 2与酶结合时，与锌结合的水分子被L - 2的羟基取代这一情况。
