Utilization of Kynurenic Acid Produced from D-kynurenine in an in Vitro Assay of D-Amino Acid Oxidase Activity

D-2-Amino-4-(2-aminophenyI)-4-oxobutanoic acid (D-kynurenine) was used to assay D-amino acid oxidase (DAAO, EC 1.4.3.3) activity. D-Kynurenine is enzymatically converted by DAAO to 4-hydroxyquinoline-2-carboxylic acid (kynurenic acid), which emits fluorescence at 398 nm (excitation wavelength 251 m) in the presence of zinc ions. Using D-kynurenine as a substrate, fluorescence intensity originating from kynurenic acid can be used to assess DAAO activity. Under enzymatic reaction at pH 8.5 (37 degrees C), the K(m) and V(max) of D-kynurenine were 148 mu M and 5.53 mu mol/min per mg, respectively. Inhibition of DAAO by a commercial DAAO inhibitor was clearly observed in this assay.