Anion Binding to Liver Alcohol Dehydrogenase

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Abstract
Complex formation at the general anion-binding site of the [horse] liver alcohol dehydrogenase subunit was characterized by transient-state kinetic methods using NADH as a reporter ligand. Equilibrium dissociation constants for anion binding at the site are reported. They conform basically to the lyotropic series of affinity order, with exceptionally tight binding of sulfate. The particular specificity for sulfate might be a general characteristic of anion-binding enzymic arginyl sites. Anionic species of phosphate and pyrophosphate buffer solutions do not interact significantly with the general anion-binding site over the pH range 8-10. At lower pH, phosphate binding becomes significant due to complex formation with the monovalent H2PO4- species. The latter interaction corresponds to a dissociation constant of about 60 mM, indicating that phosphate binding is comparatively weak also at low pH. Previously reported pH dependence data for coenzyme bindng to liver alcohol dehydrogenase cannot be much affected by coenzyme-competitive effects of buffer anion binding. Kinetic parameter estimates now determined for NADH binding in weakly buffered solutions agree within experimental precision with those obtained previously from measurements made in buffer solutions of 0.1 M ionic strength.