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 degr ees C and pH 8. The reaction followed pseudo-first order kinetics with seco nd-order rate constants of 69.8 mM(-1) min(-1) for the apoenzyme and 0.63 m M(-1) min(-1) for the holoenzyme. The presence of substrates or benzoate pr otected the enzyme against inactivation. Difference absorption spectra at 2 80 nm, low consumption of NBS per mole of enzyme, the decrease in the fluor escence emission at 335 nm, integrity of the protein backbone and the absen ce of cysteine oxidation pointed to the modification of tryptophan residues . The statistical analysis of the residual fractional activity vs. the numb er of modified tryptophan residues led to the conclusion that one tryptopha n residue is essential for the enzyme activity. This tryptophan residue was not involved in binding of FAD or dimerization of the enzyme. (C) 2000 Els evier Science B.V. All rights reserved.