Rudolph R, Gerschitz J, Jaenicke R
Eur J Biochem. 1978 Jul 3;87(3):601-6. doi: 10.1111/j.1432-1033.1978.tb12412.x.
Horse-liver alcohol dehydrogenase requires Zn2+ for enzymatic activity. Reactivation experiments after dissociation and denaturation of the enzyme in 6 M guanidinium hydrochloride and subsequent separation of zinc prove that the effect of the metal on the rate and yield of reconstitution is complex. In the absence of Zn2+ no reactivation is detectable, while excess of Zn2+ leads to inactive aggregates. Optimum reactivation yields are obtained at 10 muM Zn2+ after short incubation in the denaturant; increasing zinc concentration causes a decrease of the rate of reactivation. The refolding of the zinc-free enzyme is characterized by consecutive first-order processes which may be separated from second-order dimer formation. Addition of 10 muM Zn2+ during refolding may be used to block side reactions competing with the reconstitution. The transition from sigmoidal kinetics to second-order profiles by adding Zn2+ after completion of the aforementioned first-order process corroborates the proposed uni-bimolecular reactivation mechanism which implies the involvement of inactive monomers. These gain their enzymatic function as a consequence of dimerization. The effect of Zn2+ may be explained by a side reaction in the overall reaction scheme of reactivation and renaturation which allows the kinetic measurements to be quantitatively described.
马肝醇脱氢酶的酶活性需要Zn2+。在6 M盐酸胍中使该酶解离和变性，随后分离锌，再进行重新激活实验，结果表明金属对重组的速率和产率的影响很复杂。在没有Zn2+的情况下，检测不到重新激活，而过量的Zn2+会导致无活性的聚集体。在变性剂中短暂孵育后，在10 μM Zn2+时可获得最佳的重新激活产率；锌浓度增加会导致重新激活速率降低。无锌酶的重折叠以连续的一级过程为特征，这些过程可能与二级二聚体形成过程分开。在重折叠过程中添加10 μM Zn2+可用于阻止与重组竞争的副反应。在上述一级过程完成后添加Zn2+，使动力学从S形转变为二级曲线，这证实了所提出的单分子-双分子重新激活机制，该机制意味着无活性单体的参与。这些单体由于二聚化而获得酶功能。Zn2+的作用可以通过重新激活和复性的整体反应方案中的副反应来解释，这使得动力学测量能够得到定量描述。
