Zhang K, Auld D S
Biostructure Institute, University City Science Center, University of Pennsylvania, Philadelphia 19104, USA.
Biochemistry. 1995 Dec 19;34(50):16306-12. doi: 10.1021/bi00050a010.
Carboxypeptidase A, ZnCPD, is typical of a wide range of exo- and endo-metalloproteases that have three protein ligands and a water molecule bound to a catalytic zinc atom and a glutamate residue in the active site that likely acts in conjunction with the Zn-bound water to bring about catalysis. Such enzymes generally have bell-shaped pH-activity profiles (EH2 in equilibrium with EH in equilibrium with E) where the concentration of the catalytic species EH is regulated by pKEH2 and pKEH. The present X-ray absorption fine structure, XAFS, study has determined the structure and pH behavior of the binary and ternary product complexes in order to examine the role of the Zn-bound water in catalysis. Increasing the pH from 7 to 10 of the ZnCPD.L-Phe complex results in the same type of progressive spectral changes in the near-edge XAFS spectrum as is seen for ZnCPD, but the changes are complete by more than 1 pH value below that observed for ZnCPD. The results are in agreement with kinetic studies that show E binds the protonated form of L-Phe more tightly than EH, thus in effect decreasing the value of pKEH. The XAFS results show the average interatomic distance. R. for the zinc ligands of the EH.L-Phe complex decreases by 0.02 A upon formation of the E.L-Phe complex, essentially identical to that obtained for the EH and E forms of the native enzyme. Addition of azide to ZnCPD.L-Phe at pH 7 markedly changes the zinc coordination sphere from 4 N/O atoms at 2.021 +/- O.06 A and 1.4 +/- 0.5 N/O atoms at 2.54 +/- 0.5 A to 3.9 N/O atoms at 1.995 +/- 0.006 A. The decrease in R of about 0.03 A in both the Zn- and CoCPD.L-Phe.N3-complexes is likely due to the ligand exchange from a neutral water to an anion. The XAFS spectra of the ternary complex is pH independent from 7 to 9, in agreement with the ionization of the water being the source of the spectral changes in the free enzyme and its binary L-Phe complex. The enzyme.azide.L-Phe complex is likely bound in a manner analogous to that expected for a post-transition state in a biproduct complex for peptide hydrolysis--that is, the carboxylate anion of the peptide bound to the Zn and the protonated form of L-Phe H-bonded to the catalytic Glu-270 carboxylate. The XAFS results on the "spectroscopically silent" ZnCPD are compared to nuclear magnetic resonance and electron absorption studies on CoCPD.
羧肽酶A（ZnCPD）是多种外切和内切金属蛋白酶的典型代表，这些酶有三个蛋白质配体和一个与催化锌原子结合的水分子，活性位点有一个谷氨酸残基，它可能与锌结合的水协同作用以实现催化。这类酶通常具有钟形的pH-活性曲线（EH2与EH与E处于平衡状态），其中催化物种EH的浓度由pKEH2和pKEH调节。目前的X射线吸收精细结构（XAFS）研究确定了二元和三元产物复合物的结构和pH行为，以研究锌结合水在催化中的作用。将ZnCPD.L-Phe复合物的pH从7提高到10，会导致近边XAFS光谱出现与ZnCPD相同类型的渐进光谱变化，但这些变化在比ZnCPD观察到的pH值低1个多单位时就完成了。结果与动力学研究一致，动力学研究表明E比EH更紧密地结合L-Phe 的质子化形式，从而实际上降低了pKEH的值。XAFS结果表明，EH.L-Phe复合物中锌配体的平均原子间距离R在形成E.L-Phe复合物时减少了0.02 Å，这与天然酶的EH和E形式所得到的结果基本相同。在pH 7时向ZnCPD.L-Phe中加入叠氮化物，会使锌配位球明显改变，从2.021±0.06 Å处的4个N/O原子和2.54±0.5 Å处的1.4±0.5个N/O原子变为在1.995±0.006 Å处的3.9个N/O原子。ZnCPD.L-Phe.N3复合物中Zn和Co的R均下降约0.03 Å，这可能是由于配体从一个中性水分子交换为一个阴离子。三元复合物的XAFS光谱在pH 7至9范围内与pH无关，这与水的电离是游离酶及其二元L-Phe复合物光谱变化的来源一致。酶-叠氮化物-L-Phe复合物可能以类似于肽水解双产物复合物中过渡后状态预期的方式结合——也就是说，肽的羧酸根阴离子与锌结合，L-Phe的质子化形式与催化性Glu-270羧酸根形成氢键。将关于“光谱学上沉默”的ZnCPD的XAFS结果与关于CoCPD的核磁共振和电子吸收研究进行了比较。
