title: D-amino acid oxidase from the yeast Trigonopsis variabilis.
abstract: D-Amino acid oxidase (EC 1.4.3.3) has been purified from the yeast Trigonopsis variabilis by the application of ion-exchange chromatography on DEAE-cellulose, salt precipitation, gel filtration, and hydroxyapatite adsorption. Alternatively the last two steps can be substituted by a single fast protein liquid chromatographic ion-exchange step (Mono Q). The enzyme appeared homogeneous in PAGE, but small amounts of impurities (not exceeding 5% of total protein) appeared in sodium dodecyl sulfate (SDS)-PAGE. Its Mr in SDS-PAGE is 39,000; it exhibits an isoelectric point of 4.8 and contains 7% (w/v) covalently bound carbohydrate. Its absorption spectrum is similar to hog kidney D-amino acid oxidase, indicating the presence of bound FAD, which, however, could not be separated from the enzyme under non-denaturing conditions. The enzyme is inhibited by SH-oxidizing agents, but not by metal-chelate formers and not by benzoate or toluene. It uses O2 exclusively as the only H acceptor. Km and Vmax values were determined for 15 D-amino acids, which, among 23 tested, were substrates of the enzyme. The enzyme has highest affinity for D-phenylalanine and D-leucine, but maximal activity is obtained with D-citrulline and D-isoleucine. The specific activity of the purified preparation is even higher than that of the commercially available hog kidney enzyme (21.7 vs 16 U/mg). The yeast enzyme may be a useful analytical and preparative tool in view of the difference between its substrate specificity and that of the hog enzyme.
authors.forename: E M
authors.initials: EM
authors.lastname: Kubicek-Pranz
authors.forename: M
authors.initials: M
authors.lastname: Röhr
mesh_terms: D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D003605:D-Amino-Acid Oxidase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D010101:Oxygen Consumption; D013379:Substrate Specificity; D013552:Swine; D015003:Yeasts / Q000201:enzymology*
chemical_list: D000963:Antimetabolites; D006023:Glycoproteins; D005182:Flavin-Adenine Dinucleotide; D003605:D-Amino-Acid Oxidase