Effect of zinc(II) on the refolding and reactivation of liver alcohol dehydrogenase.

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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.