The imidazole-promoted inactivation of horse-liver alcohol dehydrogenase

The effect of imidazole on the inactivation of liver alcohol dehydrogenase by alkylation of Cys-46 with iodoacetate, bromoacetate, 2-bromopropionate, 3-bromopropionate, 2-bromobutyrate and iodoacetamide has been studied at pH 7.0. Imidazole promoted inactivation with all the compounds but 2-bromobutyrate. Enzyme inactivation with haloacids was faster in a ternary enzyme-imidazole-haloacid complex compared to a binary enzyme-haloacid complex. Inactivation with iodoacetamide, which is a direct biomolecular reaction, was faster with the binary enzyme-imidazole complex as compared to the free enzyme. Results of haloacid and iodoacetamide inactivation in the presence of imidazole were fitted, by nonlinear regression analysis, to the rate expressions for the proposed mechanisms and the kinetic parameters resulted. Imidazole was also found to promote inactivation of the cobalt-substituted and cadmium-substituted liver alcohol dehydrogenases. CYs-46 is alkylated as a metal-thiol complex. Imidazole, when binding to the active-site metal, donates sigma-electrons to the metal atom, which distributes the increased electron density further to the other ligands. The increased nucleophilicity of the sulphur of Cys-46 results in promoted alkylation. Proof that the imidazole promotion effect is caused by a displaced electron distribution in the active-site coordination unit is provided by imidazole also promoting the alkylation of the model thiol, zinc-mercaptoethanol.