PURIFICATION AND KINETIC CHARACTERIZATION OF PICKEREL LIVER ALCOHOL-DEHYDROGENASE WITH DUAL COENZYME SPECIFICITY

A major alcohol dehydrogenase isozyme that displays dual coenzyme spec ificity has been purified from pickerel liver by ion-exchange, gel fil tration, and affinity chromatographic procedures. The purified enzyme is chromatographically and electrophoretically homogeneous. It is dime ric and possesses common physical properties shared by other liver alc ohol dehydrogenases. Phosphorus-31 nuclear magnetic resonance spectros copy demonstrates that NADP(+) binds to two coenzyme sites of the pick erel 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(+) redu ction at low NAD(+) concentrations follows an ordered Bi-Bi mechanism with NAD(+) being the leading reactant.