Reaction of Liver Alcohol Dehydrogenase with Halogenoacids. Fate of the Iodide Anion Released by Carboxymethylation and Enzymic Catalysis of Iodide Solvolysis

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Abstract
The fate of the iodide liberated during carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase was determined with 125I-labeled iodoacetate. The [125I]iodoacetic acid was prepared from mesyloxyacetic acid and sodium [125I]iodide. When carboxymethylation of the enzyme is carried out in solution or in the crystalline state, no iodide is bound to the protein. The rate of iodide liberation during the reaction of iodoacetate, determined with an iodide-specific electrode, was biphasic: the fast phase corresponds to the carboxymethylation and the slow phase to iodide liberation due to the presence of protein. With 3-iodopropionate (2.5 mM), no inactivation was detected, but in the presence of the enzyme, 10 equivalents of iodide were liberated/subunit in 1 h. NADH does not inhibit this reaction. The electron density attributed to an iodide bound to the Zn atom of the crystalline enzyme is reinterpreted in view of these results as due to an imidazole bound to the active-site Zn. In the carboxymethylation, the reactivity of bromoacetate is higher than that of iodoacetate.