T. Schrader et Jr. Andreesen, PROPERTIES AND CHEMICAL MODIFICATION OF D-AMINO-ACID OXIDASE FROM TRIGONOPSIS-VARIABILIS, Archives of microbiology, 165(1), 1996, pp. 41-47
The basic properties of purified D-amino acid oxidase from the yeast T
rigonopsis variabilis were investigated. The pH optimum of activity wa
s 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 correspon
ded 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 his
tidine-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 substrat
e under anoxic conditions led to a substantial protection from inactiv
ation, which indicates a localization of the modified residues close t
o the active site. The pK(a) of the reacting group was determined to b
e 7.7, and the rate of inactivation reached a limiting value of 0.031
min(-1).