Properties and chemical modification of D-amino acid oxidase from Trigonopsis variabilis

The basic properties of purified D-amino acid oxidase from the yeast Trigonopsis variabilis were investigated. The pH optimum of activity was between pH 8.5 and 9.0, and the native molecular masses of holo- and ape-enzyme were determined to be 170 kDa; higher aggregates corresponded to molecular masses of 320 and 570 kDa. The apparent V-max and K-m values for different substrates varied between 3.7 to 185 U/mg and 0.2 to 17.3 mM, respectively. The reaction of D-amino acid oxidase with sulfite was followed by the typical spectral modifications of the FAD resembling the reduced enzyme; a Kd Of 30 mu M was calculated for the N(5)-adduct. The red anionic flavin radical of the enzyme was stable; benzoate had no influence on the spectral properties. A complete loss of enzyme activity was observed after chemical modification by the histidine-specific reagent diethyl pyrocarbonate. The inactivation showed pseudo-first-order kinetics, with a second-order rate constant of 13.6 M(-1) min(-1) at pH 6.0 and 20 degrees C. The addition of a substrate under anoxic conditions led to a substantial protection from inactivation, which indicates a localization of the modified residues close to the active site. The pK(a) of the reacting group was determined to be 7.7, and the rate of inactivation reached a limiting value of 0.031 min(-1).