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Abstract

We have isolated alcohol dehydrogenase (ADH, E.C. 1.1.1.1) from beans germinating 3 days by ammonium sulfate precipitation of the sodium phosphate extract of the homogenate of seeds, followed by chromatography on DEAE-cellulose and gel chromatography on Sephadex G-200 and G-100; both chromatographic operations were repeated twice. The activity of ADH increased 960 times after these procedures. The enzyme whose M_r is 60 000 consists of two identical subunits of M_r = 30 000. Allyl alcohol is oxidized and acetaldehyde reduced at the highest rate of all the alcohols and aldehydes tested. The reaction rate decreases with the increasing length of the carbon chain of the substrate. In contrast, the rate of oxidation of alcohols with a double bond in their molecules increased. The pH-optimum of substrate oxidation (pH 8.5) is different from the pH-optimum of substrate reduction (pH 7.5). From kinetic studies of the effect of pH on V_max and K_m the pK-value of amino acids participating on substrate oxidation is 9.2 and 8.5 whereas amino acids with pK 8.3 and 6.8 participate on substrate reduction. We have verified the participation of the SH-groups in experiments with the inactivation of ADH by iodoacetate: the inactivation was weaker after the enzyme had been preincubated with NAD or AMP, adenosine or nicotinamide. Likewise pyridoxal phosphate inactivates bean ADH by modifying its ε-amino group of lysine. The degree of inactivation depends also on the pH and the ionic strength of the medium. The protective effect of NAD or its analogs shows that lysine is present in the active center of the enzyme in the coenzyme-binding site.