A radiometric kynurenine monooxygenase assay.
Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of L-kynurenine to 3-hydroxy-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 Vmax/Km for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.
Wiseman
J S
JS
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Nichols
J S
JS
D000818:Animals; D002845:Chromatography; D007700:Kinetics; D007737:Kynurenine / Q000378:metabolism; D050601:Kynurenine 3-Monooxygenase; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011874:Radiometry; D051381:Rats; D011919:Rats, Inbred Strains; D012044:Regression Analysis; D014316:Tritium
D009243:NAD; D014316:Tritium; D007737:Kynurenine; D009249:NADP; D006899:Mixed Function Oxygenases; D050601:Kynurenine 3-Monooxygenase