Chemical modification of tryptophan residues of d-amino acid oxidase from Rhodotorula gracilis

d-Amino acid oxidase was inactivated by N-bromosuccinimide (NBS) at 30°C and pH 8. The reaction followed pseudo-first order kinetics with second-order rate constants of 69.8 mM−1 min−1 for the apoenzyme and 0.63 mM−1 min−1 for the holoenzyme. The presence of substrates or benzoate protected the enzyme against inactivation. Difference absorption spectra at 280 nm, low consumption of NBS per mole of enzyme, the decrease in the fluorescence emission at 335 nm, integrity of the protein backbone and the absence of cysteine oxidation pointed to the modification of tryptophan residues. The statistical analysis of the residual fractional activity vs. the number of modified tryptophan residues led to the conclusion that one tryptophan residue is essential for the enzyme activity. This tryptophan residue was not involved in binding of FAD or dimerization of the enzyme.