Sytkowski A J, Vallee B L
Biochemistry. 1978 Jul 11;17(14):2850-7. doi: 10.1021/bi00607a024.
The preparation of metal hybrid species of horse liver alcohol dehydrogenase is made possible by the development of carefully delineated systems of metal in equilibrium metal exchange employing equilibrium dialysis. The conditions which are optimal for the site-specific replacement of the catalytic and/or noncatalytic zinc atoms of the native enzyme by cobalt are not identical with those which are utilized for substitution with 65Zn. Thus, while certain 65Zn hybrids can be prepared by exploiting the differential effects of buffer anions, the cobalt hybrids are generated by critical adjustments in the pH of the dialysate. Factors which may determine the mechanism of metal replacement reactions include acid-assisted, ligand-assisted, and metal-assisted dechelation, steric restriction, and ligand denticity as well as physicochemical properties of the enzyme itself. The spectral characteristics of the catalytic and noncatalytic cobalt atoms reflect both the geometry of the coordination complexes and the nature of the ligands and serve as sensitive probes of these loci in the enzyme.
通过开发使用平衡透析的精心设计的金属平衡金属交换系统，制备马肝醇脱氢酶的金属杂化物种成为可能。用钴对天然酶的催化和/或非催化锌原子进行位点特异性置换的最佳条件，与用于用65Zn置换的条件并不相同。因此，虽然某些65Zn杂化物可以通过利用缓冲阴离子的差异效应来制备，但钴杂化物是通过对透析液pH值的关键调整而产生的。可能决定金属置换反应机制的因素包括酸辅助、配体辅助和金属辅助脱螯合、空间位阻、配体齿数以及酶本身的物理化学性质。催化和非催化钴原子的光谱特征既反映了配位络合物的几何形状，也反映了配体的性质，并作为酶中这些位点的敏感探针。
