ISSN:
1432-072X
Keywords:
Key wordsd-amino acid oxidase
;
Trigonopsis
;
variabilis
;
Chemical modification
;
Histidine residues
;
Km
;
Vmax
;
Sulfite
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract 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 apo-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 K d of 30 μM 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°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 pKa of the reacting group was determined to be 7.7, and the rate of inactivation reached a limiting value of 0.031 min–1.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002030050294
Permalink
