Dahl K H, McKinley-McKee J S
J Inorg Biochem. 1981 Aug;15(1):79-87. doi: 10.1016/s0162-0134(00)80137-3.
The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition to bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn greater than Cd greater than Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co greater than Zn greater than Cd.
通过对天然锌（II）酶以及用钴（II）或镉（II）取代后的马肝醇脱氢酶进行金属导向亲和标记，研究了其活性位点金属。溴咪唑基丙酸与钴酶的可逆结合使源于催化钴原子的可见吸收带从655 nm蓝移至630 nm。咪唑与钴（II）酶的结合使655 nm的吸收带红移至667 nm。加入溴咪唑基丙酸后，该667 nm的吸收带又蓝移回630 nm。这证明了标记物与活性位点金属直接结合，并与咪唑竞争。标记物对三种酶中可逆结合位点的亲和力顺序为锌大于镉大于钴。在形成可逆复合物后，溴咪唑基丙酸使半胱氨酸-46烷基化，半胱氨酸-46是活性位点金属的蛋白质配体之一。每个可逆复合物中这种金属-巯基键的亲核反应性顺序为钴大于锌大于镉。
