Photoinactivation of porcine D-amino acid oxidase with flavin adenine dinucleotide.

FAD photosensitizes its own oxidation and that of D-amino acid oxidase. First order photoinactivation of the enzyme is paraIieIed by losses of cysteine, histidine, tyrosine, lysine, and, at saturating levels of FAD, tryptophan. Minimal photo-oxidation of methionine has also been found with illuminated enzyme samples. Native and partially photoinactivated enzyme have the same pH activity profile and K, values for D-alanine and for FAD from pH 8 to 10. The degree of photoinactivation correlates with the number of catalytically inactive enzyme molecules. Hence, an all-ornone inactivation is indicated. The binding ability of photoinactivated enzyme for benzoate (a substrate-competitive inhibitor) diminishes, while no changes in FAD-binding stoichiometry occur. This indicates that photochemical modification of the substrate-binding site is a cause of photoinactivation. Under conditions such that the native oxidase exists only as a monomer, the light-treated enzyme consists of monomer, dimer, tetramer, and polymer (or polymers). The remaining activity is associated only with the monomeric units, which are probably mixtures of fully active and completely inactivated monomer. Decrease in specific activity of the monomeric units isolated from partially photoinactivated enzyme is concomitant with partial photo-oxidation of cysteine and tyrosine only. Limited amounts of benzoate were found to slow down the photoinactivation by protecting tyrosyl residues. Hence, there are probable involvements of cysteinyl and tyrosyl residues in the catalytic activity of D-amino acid oxidase. Photoinactivation of oxidase is temperature dependent. From the biphasic Arrhenius plot with a transition point at 15”, values of energy of activation were calculated as 8.2