Alpha, beta-ketoalkene reduction. A novel reduction pathway in mammalian tissues.

Reduction of an alpha, beta-unsaturated ketone to the corresponding saturated ketone in a 2-benzylidene-3-ketocoumaran derivative has been characterized with respect to subcellular and tissue distribution, species specificity, cofactor requirements, kinetic constants, and effects of inhibitors. In rats, the highest activity was found in the hepatic cytosolic (100,000g supernatant) fraction having an apparent substrate Km and Vmax of 5.6 microM and 1.3 nmol/min/mg protein, respectively. In rat hepatic cytosolic incubations, NADPH supported the reduction reaction having an apparent Km of 15 microM while NADH was 70% less effective in catalyzing the reaction than NADPH. The most effective inhibitors of the reaction were potassium cyanide and sulfhydryl reagents such as p-chloromercuribenzoic acid and iodoacetamide. Glutathione, dithiothreitol, and flavin mononucleotide were without consequential effect. Phenobarbital did not induce the activity in rats. Guinea pig hepatic cytosol was approximately 8 times more active than rat hepatic cytosol in catalyzing the reduction reaction. In rats, whole blood catalyzed the reduction of the substrate in the absence of added NADPH. Hemolysis resulted in a loss of activity which could be restored by addition of NADPH. Cinnamic acid was not reduced by the rat cytosolic fraction. The enzyme utilizes the A-side hydrogen atom at C-4 of NADPH.