2-KETO-4-HYDROXYBUTYRATE . SYNTHESIS, CHEMICAL PROPERTIES, AND AS A SUBSTRATE FOR LACTATE DEHYDROGENASE OF RABBIT MUSCLE

2-Keto-4-hydroxybutyric acid, obtained by reaction of DL-homoserine with pyridoxal in the presence of Cu2+ ions and isolated by ion-exchange column chromatography, has been crystallized as the γ-lactone and also as its 2,4-dinitrophenylhydrazone derivative. The physicochemical properties of these crystalline compounds have been examined. Crystalline lactate dehydrogenase from rabbit muscle catalyzes the reduction of 2-keto-4-hydroxybutyrate in the presence of reduced diphosphopyridine nucleotide. Maximal rates of reduction relative to pyruvate are 61, 43, and 25% for glyoxylate, 2-ketobutyrate, and 2-keto-4-hydroxybutyrate as substrates, respectively. The apparent Km for 2-keto-4-hydroxybutyrate is 4.6 X 10-3 M in 33 mM potassium phosphate buffer at pH 7.4 and 25° compared with values of 3.3 X 10-4 M for pyruvate, 1.8 X 10-2 M for glyoxylate, and 4.8 X 10-3 M for 2-ketobutyrate. Oxamate competitively inhibits 2-keto-4-hydroxybutyrate reduction; in contrast, oxalate is a noncompetitive inhibitor. The dissociation constants for the enzyme-inhibitor complexes (Ki values), measured in 33 mM Tris-Cl buffer at pH 7.4 and 25°, are 8.0 X 10-5 and 8.1 X 10-4 M for oxamate and oxalate, respectively. Competitive inhibition by oxamate also occurs with pyruvate, glyoxylate, and 2-ketobutyrate (Ki values: 8.3 X 10-5, 7.6 X 10-6, and 7.4 X 10-5 M, respectively) whereas oxalate is noncompetitive (Ki values: 8.0 X 10-4, 8.4 X 10-4, and 8.0 X 10-4 M, respectively). The equilibrium for the reaction of 2-keto-4-hydroxybutyrate with reduced diphosphopyridine nucleotide in the presence of lactate dehydrogenase favors reduction of the ketohydroxy acid; the Kequil is 1.2 X 10-10 M determined in 0.30 m Tris-Cl buffer at 25°. Reduced diphosphopyridine nucleotide oxidation is also favored with pyruvate, glyoxylate, and 2-ketobutyrate as substrates (Kequil values: 4.8 X 10-12, 1.0 X 10-9, and 3.0 X 10-11 M, respectively). One mole of reduced diphosphopyridine nucleotide is oxidized for every mole of 2-keto-4-hydroxybutyrate reduced; 2,4-dihydroxybutyric acid has been identified as the product of the reaction. The metabolic significance of this enzymic reduction of 2-keto-4-hydroxybutyrate is discussed. © 1969, American Chemical Society. All rights reserved.