Chemical modifications of D-amino acid oxidase. Evidence for active site histidine, tyrosine, and arginine residues.

1. D-amino acid oxidase is inactivated by reaction with a low molar excess of dansyl chloride at pH 6.6, with complete inactivation accompanied by incorporation of 1.7 dansyl residues per mol of enzyme-bound flavin. The presence of benzoate, a potent competitive inhibitor, protects substantially against inactivation. Evidence is presented that the inactivation is due to dansylation of an active site histidine residue. Reactivation may be obtained by incubation with hydroxylamine. Diethylpyrocarbonate also inactivates the enzyme and modifies the labeling pattern with dansyl chloride. 2. Butanedione in the presence of borate reacts rapidly to inactivate D-amino acid oxidase. Reactivation is obtained spontaneously on removal of borate, implicating reaction of butanedione with an active site arginine residue. 3. Fluorodinitrobenzene appears to behave as an active site-directed reagent when mixed with D-amino acid oxidase at pH 7.4. Complete inactivation is obtained with incorporation of 2.0 dinitrophenyl residues per mol of enzyme-bound flavin. Again benzoate protects against inactivation; only one dinitrophenyl residue is incorporated in the presence of benzoate. The active site residue attacked by fluorodinitrobenzene has been identified as tyrosine.