The Imidazole‐Promoted Inactivation of Horse‐Liver Alcohol Dehydrogenase

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Abstract
The effect of imidazole on the inactivation of liver alohol 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 inativation 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 bimolecular reaction, was faster with the binary enzyme‐imidazole complex as compared to the free enzyme. Results of haloacid and iodoaetamide 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 obalt‐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 σ‐electrons to the metal atom, which distributes the inccreased 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‐meraptoethanol.