Rat kidney L-2-hydroxyacid oxidase. Structural and mechanistic comparison with flavocytochrome b2 from baker's yeast.
Hydroxyacid oxidase from rat kidney is an FMN-dependent enzyme that catalyzes the oxidation of L-alpha-hydroxy acids as well as, more slowly, that of L-alpha-amino acids. We report here a modified purification method for the enzyme, which is found to possess one cofactor per subunit of Mr 39,000. Determination of its N-terminal sequence suggests the protein is homologous to spinach glycolate oxidase and baker's yeast lactate dehydrogenase. In the presence of a hydroxy acid and of bromopyruvate, under anaerobic conditions, the enzyme is found to catalyze both transhydrogenation and reductive bromide ion elimination. It had previously been observed that hydroxyacid oxidase could not catalyze chloride elimination from chlorolactate in the presence of oxygen [Cromartie, T.H., & Walsh, C.T. (1975) Biochemistry 14, 3482-3490]. The behavior of this enzyme toward halogeno substrates is therefore similar to that of baker's yeast L-lactate dehydrogenase and in part different from that of Mycobacterium smegmatis lactate oxidase and porcine kidney D-amino-acid oxidase. These findings can be rationalized on the basis of a common mechanism for all these enzymes, implying formation of a carbanion as a first step, with different rate-limiting steps in the overall reaction.
Urban
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Unité Associée au Centre National de la Recherche Scientifique UA 122, Hôpital Necker, Paris, France.
Chirat
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Lederer
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D000429:Alcohol Oxidoreductases / Q000032:analysis / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001965:Bromides / Q000378:metabolism; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002712:Chlorides / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D006880:Hydroxy Acids / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D010100:Oxygen / Q000494:pharmacology; D011773:Pyruvates / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D001965:Bromides; D002712:Chlorides; D006880:Hydroxy Acids; D011773:Pyruvates; C017092:bromopyruvate; D005486:Flavin Mononucleotide; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); C021380:L-2-hydroxyacid oxidase; D010100:Oxygen