Reactivity of histidyl residues in D-amino acid oxidase from Rhodotorula gracilis.

Incubation of D-amino acid oxidase from the yeast Rhodotorula gracilis with excess dansyl chloride at pH 6.6 and 18 degrees C caused an irreversible inactivation of D-amino acid oxidase. Benzoate, a competitive inhibitor of the enzyme, completely protected the enzyme from inactivation. The dansylated-enzyme, isolated by gel-filtration, was in part still active while the substrate specificity was altered substantially. It was completely reduced by D-alanine in anaerobiosic conditions and did stabilize the red anion semiquinone upon photochemical reduction with EDTA. The results provide evidence for the presence of essential histidyl residue(s) in the active center of the yeast enzyme.