A radiometric kynurenine monooxygenase assay

Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of l-kynurenine to 3-hydroxyl- l-kynurenine in the kynurenine pathway of tryptophan metabolism. The enzyme requires NADH or NADPH as a cofactor. A discontinuous assay that utilizes l-[ 3H]kynurenine as substrate is described. The assay offers high precision and a wide range of accessible substrate and cofactor concentrations. The assay was used to measure kinetic isotope effects and the stereospecificity of oxidation of the cofactor. Hydride is transferred from the A-side (pro- R) of NADH and NADPH since primary deuterium isotope effects were observed for both cofactors when they were deuterated on the A-side but not on the B-side. The large isotope effect on V max K m for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.