Synergism Between Coenzyme and Carboxylate Binding to Liver Alcohol Dehydrogenase

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Abstract
The interaction of decanoate and benzoate with the substrate‐binding site in liver alcohol dehydrogenase has been characterized by fluonmetric equilibrium binding studies, using auraimne O as a reporter ligand. The affinity of the enzyme for the carboxylates examined decreases about 50‐fold on complex formation with NADH. The coenzyme‐competitive inhibitors ADP‐ribose and Pt(CN)2−4 have a similar destabilizing effect on decanoate binding, indicating that the effect of NADH derives from its negatively charged pyrophosphate group. Carboxylate binding to free enzyme requires the protonated form of an ionizing enzymic group with pKa 9.2, while there is no corresponding effect of pH on binary complex formation with auramine O. Carboxylate binding to the enzyme · NADH+ complex exhibits no significant dependence on pH over the pH range 7–10. These results, combined with previously reported data for decanoate binding to the enzyme · NAD+ complex, provide clear evidence that carboxylates (in contrast to auramine O) are bound at the active‐site zinc ion of the enzyme in a process regulated by the ionization state of zinc‐bound water. The synergistic effect of NADH and NAD+ on carboxylate binding can be qualitatively and quantitatively explained in terms of electrostatic interactions analogous to those proposed to account for the effect of coenzymes on the pKa of zinc‐bound water.