Affinity Labelling of Alcohol Dehydrogenases

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Abstract
DL-.alpha.-Bromo-.beta.(5-imidazolyl)-propionic acid is a potential affinity labeling reagent for metalloenzymes. It was used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis-Menten-type reaction, where 1 molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2''-dipyridyl, 1,10-phenanthroline and cyclohexanone, which all combine with the active-site Zn. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent decreased the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys-46, reversible binding occurred at the active-site Zn atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found. The potential affinity labeling and crosslinking reagent, symmetrical 1,3-dibromoacetone although unstable, was also used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis-Menten type at pH 7.0. Several ligands known to complex with the active-site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo-first-order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. Dibromoacetone reacted like a monofunctional reagent.