Phosphate Binding to Liver Alcohol Dehydrogenase Studied by the Rate of Alkylation with Affinity Labels

Abstract

Phosphate anions interact with the anion binding site of alcohol dehydrogenase from horse liver. In protection experiments against the 2 affinity labels, iodoacetic acid and bromo-imidazolylpropionic acid, the Kd for the enzyme-phosphate complex at pH 7.0 is, based on total phosphate, found to be 20 .+-. 5 mM. The 1,4-piperazinediethane-sulfonate anion has a lower affinity for the anion binding site, the Kd at pH 7.0 being 130 .+-. 20 mM. The anion-independent Kd for the reversible enzyme-affinity label complexes are at pH 7.0, 1.35 .+-. 0.2 mM for iodoacetic acid and 0.39 .+-. 0.05 mM for bromo-imidazolylpropionic acid. These findings have important implications with respect to past and future work on this well known enzyme.