Evidence that Ionization of Zinc‐Bound Water Regulates the Anion‐Binding Capacity of the Coenzyme‐Binding Site in Liver Alcohol Dehydrogenase

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Abstract
1. The mechanistic and structural origin of the pKa 9.2 dependence of coenzyme association and anion binding to liver alcohol dehydrogenase has been investigated by titrimetric and spectrophotometric binding studies involving ligands (imidazole, 1,10‐phenanthroline and 2,2′‐bipyridine) which combine to the catalytic zinc ion of the enzyme subunit with displacement of zinc‐bound water.2. Imidazole abolishes the pKa 9.2 dependence of NADH binding to the enzyme. The pH dependence of ADP‐ribose and Pt(CN)2‐4 binding is similarly abolished by imidazole, as well as by 1,10‐phenanthroline and 2,2′‐bipyridine. It is concluded from these results that the pKa 9.2 dependence of coenzyme and anion binding most likely derives from ionization of zinc‐bound water.3. Evidence is presented showing that the protonation state of the pKa 9.2 group also regulates ligand binding to the catalytic zinc ion, which lends strong support to the conclusion that this ionizing group can be identified as zinc‐bound water. The pKa, 9.2 dependence of bipyridine binding is abolished by ADP‐ribose and Pt(CN)2‐4, indicating that complex formation at the anion‐binding subsite of the coenzyme‐binding site perturbs the pKa of zinc‐bound water to a value above 10.4. The cooperative interrelationship between ionization of zinc‐bound water and complex formation at the anion‐binding subsite is proposed to be attributable to a competition between the zinc‐bound hydroxyl ion and external anionic ligands for salt bridge formation with the guanidinium group of Arg‐47. This explains why ionization of zinc‐bound water affects the anion‐binding capacity of the enzyme and why complex formation at the anion‐binding subsite affects the pKa of zinc‐bound water.5. The kinetics of complex formation with bipyridine are consistent with a two‐step binding mechanism in which a pKa 9.2 dependent rapid pre‐equilibration between enzyme and ligand is followed by a pH independent rate‐limiting formation of the chromophoric binary complex. Desorption of zinc‐bound water, therefore, is likely to take place in the primary association step rather than in the subsequent rate‐limiting step. This renders the possibility less likely that productive ternary complexes formed during catalysis may contain a zinc‐bound penta‐coordinate water molecule.