Effect of zinc(II) on the refolding and reactivation of liver alcohol dehydrogenase.
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.
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D006146:Guanidines / Q000494:pharmacology; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D015032:Zinc / Q000494:pharmacology*
D006146:Guanidines; D000429:Alcohol Oxidoreductases; D015032:Zinc