Studies on the inactivation of the flavoprotein D-amino acid oxidase from Trigonopsis variabilis.
Inactivation of D-amino acid oxidase occurred by different mechanisms. The enzyme showed a rapid loss of activity in the presence of micromolar amounts of Cu2+ and Hg2+. It was also sensitive to oxidative inactivation by Fe2+ and H2O2 when both reagents were added in millimolar amounts. When oxidatively inactivated D-amino acid oxidase and a corresponding non-treated control were modified with the sulfhydryl-modifying, fluorescent reagent monobromobimane and subsequently digested with endoproteinase Glu-C, Cys-298 was identified to be a target for oxidative modification according to differences in the known peptide profile of fluorescence intensity. Another reason for the observed loss of enzyme activity in crude extracts was the specific proteolytic digestion of D-amino acid oxidase, which was dependent on the growth phase of the cells used. This cleavage was catalyzed by a serine-type proteinase and was the introductory step for the further complete degradation of the enzyme. In addition, a coenriched 50-kDa protein, identified as NADPH-specific glutamate dehydrogenase, significantly decreased the stability of the D-amino acid oxidase activity. Treatment of apo-D-amino acid oxidase from T. variabilis with monobromobimane resulted in a significantly increased fluorescence of two peptides, neither of which contained any cysteine residue. Thus, an involvement of cysteine residues in binding the FAD coenzyme should be excluded.
Schräder
T
T
Institut für Mikrobiologie, Universität Halle, Germany.
Andreesen
J R
JR
D000595:Amino Acid Sequence; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology*; D003300:Copper / Q000494:pharmacology; D003605:D-Amino-Acid Oxidase / Q000037:antagonists & inhibitors*; D010450:Endopeptidases / Q000378:metabolism; D005656:Fungal Proteins / Q000037:antagonists & inhibitors*; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007501:Iron / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008628:Mercury / Q000494:pharmacology; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction
D001643:Bridged Bicyclo Compounds; D002413:Cations, Divalent; D005656:Fungal Proteins; D007527:Isoenzymes; D009249:NADP; D003300:Copper; D006861:Hydrogen Peroxide; D007501:Iron; D005969:Glutamate Dehydrogenase; D003605:D-Amino-Acid Oxidase; D010450:Endopeptidases; D008628:Mercury; C028381:monobromobimane