Effect of Zinc(II) on the Refolding and Reactivation of Liver Alcohol Dehydrogenase

Abstract

Horse‐liver alcohol dehydrogenase requres Zn²⁺ for enzymatic activity. Reactivation experiments after dissociation and denaturation of the enxyme in 6 M guanidinium hydrochloride and subsequent separation of zinc prove thaty the effect of the metal on the rate and yield of reconstitution is complex. In the absence of Zn²⁺ no reactivation is detecatble, while excess of Zn²⁺ leads to inactive aggregates. Optimum reactivation yields are obtained at 10 μM Zn²⁺ after short incubation in the denaturant; increasing zinc concentration causes a decreasae of the rate of reactivation. The refolding of the zinc‐free enxyme is characterized by consecutive first‐order processes which may be separated from second‐order dimer formation. Addition of 10 μM Zn²⁺ during refolding may be used to block side reaction scompeting with the reconstitution. The transition from sigmoidal kinetics to secodn‐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.