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

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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.