title: The role of zinc-bound water in liver alcohol dehydrogenase catalysis.
abstract: To elucidate the role of zinc-bound water in liver alcohol dehydrogenase catalysis, chelation by 1,10-phenanthroline and 2,2-bipyridine was studied. The rate constants for association of both chelating agents to the active center zinc were pH-dependent with a pKa of 9.2 and preferential binding to a protonated form. The binary complex dissociation rate constants were pH-independent for both chelating agents. In the presence of saturating NAD+, the pKa for the equilibrium binding of 2,2-bipyridine was perturbed to 7.6, similar to the functional group previously shown to be involved in NAD+ binding. The presence of saturating imidazole resulted in pH-independent 2,2-bipyridine binding. These studies provide compelling evidence that the ionizing enzyme functional group involved in coenzyme binding, proton liberation, and conformational states is zinc-bound water. The limiting rate of chelation by 2,2-bipyridine was pH-independent, and no limiting rate was observed in the presence of saturating imidazole. These results indicate that the limiting rate of chelation is due to the rate of dissociation of zinc-bound water. The implications of this regarding the role of zinc in catalytic turnover of liver alcohol dehydrogenase are discussed.
mesh_terms: D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D006736:Horses; D006860:Hydrogen Bonding; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010618:Phenanthrolines / Q000494:pharmacology; D011485:Protein Binding; D014867:Water; D015032:Zinc / Q000494:pharmacology
chemical_list: D010618:Phenanthrolines; D014867:Water; D015082:2,2'-Dipyridyl; D000429:Alcohol Oxidoreductases; D015032:Zinc