Enzymatic Catalysis in the Affinity Labelling of Liver Alcohol Dehydrogenase with Haloacids

Abstract

The inactivation at pH 7.0 of horse liver alcohol dehydrogenase by iodoacetamide and a series of 6 haloacids was studied, and the kinetic constants determined. Enzyme inactivation was compared with the model alkylation of a metal-thiol and a thiolate anion free in solution. Inactivation of liver alcohol dehydrogenase by iodoacetamide is a direct thiol alkylation, while inactivation by selective alkylation of Cys-46 by the haloacids is facilitated by reversible complex formation. Inactivation half-time for the haloacids ranged over 4-190 min, a difference mainly caused by dissimilar chemical reactivities rather than diverse fitting in the active site. The thiol of Cys-46 is alkylated as a zinc-thiol complex. It is, as such, not especially reactive; it has a nucleophilic reactivity similar to that observed with the model compound free in solution. Affinity labeling of liver alcohol dehydrogenase by haloacids compared with alkylation of the similar group free in solution illustrates enzymatic catalysis by reversible complex formation. With the present series of substrates a rate enhancement of up to 58,000 is seen.