Electrostatic field effects of coenzymes on ligand binding to catalytic zinc in liver alcohol dehydrogenase

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Abstract
The synergism between coenzyme and anion binding to horse liver alcohol dehydrogenase was examined by equilibrium measurements and transient-state kinetic methods to characterize electrostatic interactions of coenzymes with ligands which are bound to the catalytic Zn ion of the enzyme subunit. Inorganic anions typically exhibit an at least 200-fold higher affinity for the general anion-binding site than for catalytic Zn on complex formation with free enzyme. Acetate and SCN- interact more strongly with catalytic Zn in the enzyme .cntdot. NAD+ complex than with the general anion-binding site in free enzyme. CN- shows no significant affinity for the general anion-binding site, but combines to catalytic Zn in the absence as well as the presence of coenzymes. Coordination of CN- to catalytic Zn weakens the binding of NADH by a factor of 50, and tightens the binding of NAD+ to approximately the same extent through interactions which do not include any contributions from covalent adduct formation between CN- and NAD+. These observations provide unambiguous information about the magnitude of electrostatic field effects of coenzymes on anion (e.g. OH-) binding to catalytic Zn. They lead to the important inference that coenzyme binding must be strongly affected by ionization of Zn-bound water irrespective of the actual acidity of the latter group. Much debated pH dependence of coenzyme binding to liver alcohol dehydrogenase must derive from ionization of Zn-bound water. The assumption that such is not the case leads to the inference that there is no detectable effect of ionization of Zn-bound water on coenzyme binding over the pH range 6-12, a possibility which is definitely excluded by the present results.