van Wart H E, Vallee B L
Biochemistry. 1978 Aug 8;17(16):3385-94. doi: 10.1021/bi00609a032.
Catalytically inactive, exchange-inert Co(III)-carboxypeptidase A has been prepared by reaction of Co(II)-carboxypeptidase A with the active-site-directed oxidizing agent m-chloroperbenzoic acid. Co(III)-carboxypeptidase A, isolated by affinity gel filtration chromatography, has the same amino acid composition and molecular weight as the starting material and contains 0.95 g-atom/mol of cobalt and 0.01 g-atom/mol of zinc. Its electron paramagnetic resonance, circular dichroic, magnetic circular dichroic, and visible absorption spectra are consistent with those of octahedral Co(III) model complexes. Co(III)-caboxypeptidase A is essentially devoid of catalytic activity toward both peptide and ester substrates of the native enzyme, and stopped-flow fluorescence studies with dansylated substrates show that it binds peptides, but not esters. Furthermore, the protein does not react with either type of substrate to yield a single turnover. The implications of these findings to the mechanism of action of carboxypeptidase A are discussed in the light of the "metal-carbonyl" and "metal-hydroxide" hypotheses. Since Co(III)-carboxypeptidase A does not bind esters, inner-sphere coordination to the metal appears to be necessary for ester binding. All attempts to prepare Co(III)-carboxypeptidase A by treatment of Co(II)-carboxypeptidase A with hydrogen peroxide according to previously published procedures (Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723) have been unsuccessful, and the present results do not confirm earlier reports that Co(III)-carboxypeptidase A exhibits esterase activity or that its activity is dependent on the method of preparation of the precursor Co(II)-carboxypeptidase A (Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253). These findings call for a reexamination of mechanistic conclusions based on the assumption that Co(III)-carboxypeptidase A is an active esterase.
通过将 Co(II)-羧肽酶 A 与活性位点导向的氧化剂间氯过苯甲酸反应，制备了催化无活性、交换惰性的 Co(III)-羧肽酶 A。通过亲和凝胶过滤色谱法分离得到的 Co(III)-羧肽酶 A 与起始原料具有相同的氨基酸组成和分子量，每摩尔含有 0.95 克原子的钴和 0.01 克原子的锌。其电子顺磁共振、圆二色性、磁圆二色性和可见吸收光谱与八面体 Co(III) 模型配合物的光谱一致。Co(III)-羧肽酶 A 对天然酶的肽和酯底物基本没有催化活性，对丹磺酰化底物的停流荧光研究表明它能结合肽，但不能结合酯。此外，该蛋白质与任何一种底物都不反应产生单周转。根据“金属羰基”和“金属氢氧化物”假说，讨论了这些发现对羧肽酶 A 作用机制的影响。由于 Co(III)-羧肽酶 A 不结合酯，因此金属的内球配位似乎是酯结合所必需的。按照先前发表的程序（Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723）用过氧化氢处理 Co(II)-羧肽酶 A 来制备 Co(III)-羧肽酶 A 的所有尝试均未成功，目前的结果也未证实早期的报道，即 Co(III)-羧肽酶 A 表现出酯酶活性或其活性取决于前体 Co(II)-羧肽酶 A 的制备方法（Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253）。这些发现要求重新审视基于 Co(III)-羧肽酶 A 是一种活性酯酶这一假设得出的机制结论。
