| Abstract: | The fate of the iodide liberated during carboxymethylation of Cys-46 in horse liver alcohol dehydrogenase has been 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 during the reaction of iodoacetate, determined with an iodide-specific electrode, has been found to be 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 per subunit in 1 hr. NADH does not inhibit this reaction. The electron density attributed to an iodide bound to the zinc atom of the crystalline enzyme is reinterpreted in view of these results as due to an imidazole bound to the active-site zinc. In the carboxymethylation, the reactivity of bromoacetate is higher than that of iodoacetate. |
