Aromatic α-Keto Acid Reductase from Rat Kidney

Abstract

Abstract An NADH-dependent aromatic α-keto acid reductase has been purified from the soluble fraction of rat kidney homogenate by ammonium sulfate precipitation, chromatography on calcium phosphate gel cellulose, and filtration on Sephadex G-100. The purified enzyme exhibited a single homogeneous peak (s20,w020,w = 4.8 S) in an ultracentrifuge and was nearly homogeneous on acrylamide gel disc electrophoresis. Keto acids corresponding to halogenated tyrosines or thyroid hormone are active substrates for this enzyme while aliphatic α-keto acids are not. The properties of this enzyme differ from those of a similar reductase isolated from dog heart. Thus, in reduction reactions with the rat enzyme, NADH, but no NADPH, serves as the hydrogen donor. Inhibition studies indicate that sulfhydryl groups are not necessary for the activity of this enzyme. 3,5-Diiodo-2-hydroxybenzoic acid (diiodosalicylic acid), but not 2,5-dihydroxybenzoic acid, is an inhibitor (competitive type) for the enzyme. The enzyme reaction strongly favors the formation of aromatic α-hydroxy acids. Assuming a molecular weight of 71,000, the enzyme has a molecular activity of 14,600 moles of 3,5-dibromo-4-hydroxyphenylpyruvic acid reduced per mole of enzyme per min at a pH close to the optimum, 6.5. The changes in the fluorescence of NADH or the enzyme protein that occur as a result of complex formation with the enzyme have been utilized in determining the stoichiometry and dissociation constant of the complex. The data indicate that the minimal weight of the enzyme protein, 38,700 g, binds to 1 mole of NADH with the dissociation constant of 0.2 x 10-6 m, and suggest that 1 mole of the enzyme has two binding sites for NADH.