J. Biol. Chem., Vol. 255, Issue 4, 1509-1514, 02, 1980
The role of zinc-bound water in liver alcohol dehydrogenase catalysis
SA Evans and JD Shore
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.
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