Purification and kinetic characterization of pickerel liver alcohol dehydrogenase with dual coenzyme specificity.

A major alcohol dehydrogenase isozyme that displays dual coenzyme specificity has been purified from pickerel liver by ion-exchange, gel filtration, and affinity chromatographic procedures. The purified enzyme is chromatographically and electrophoretically homogeneous. It is dimeric and possesses common physical properties shared by other liver alcohol dehydrogenases. Phosphorus-31 nuclear magnetic resonance spectroscopy demonstrates that NADP+ binds to two coenzyme sites of the pickerel enzyme. Steady-state kinetic studies suggest that pickerel liver alcohol dehydrogenase catalyzes NAD(P)(+)-linked ethanol oxidation via a random pathway. While the NADP+ reduction involves the formation of an abortive complex at high NADP+ concentrations, the NAD+ reduction at low NAD+ concentrations follows an ordered Bi-Bi mechanism with NAD+ being the leading reactant.