A radiometric kynurenine monooxygenase assay

Journal Article · Mon Jan 01 00:00:00 EST 1990 · Analytical Biochemistry; (USA)

DOI:https://doi.org/10.1016/0003-2697(90)90010-7· OSTI ID:6897154

Wiseman, J S; Nichols, J S ^[1]

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1. Merrell Dow Research Institute, Cincinnati, OH (USA)

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.

• OSTI ID: 6897154
• Journal Information: Analytical Biochemistry; (USA), Vol. 184:1; ISSN 0003-2697
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
TRYPTOPHAN
METABOLISM
TRYPTOPHAN OXYGENASE
RADIOASSAY
BIOLOGICAL PATHWAYS
CHROMATOGRAPHY
DEUTERIUM
ISOTOPE EFFECTS
KYNURENINE
NAD
NADP
RATS
REGRESSION ANALYSIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
AMINO ACIDS
ANIMALS
AROMATICS
AZAARENES
AZOLES
CARBOXYLIC ACIDS
COENZYMES
ENZYMES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
INDOLES
ISOTOPE APPLICATIONS
ISOTOPES
KETO ACIDS
LIGHT NUCLEI
MAMMALS
MATHEMATICS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
OXYGENASES
PYRROLES
RODENTS
SEPARATION PROCESSES
STABLE ISOTOPES
STATISTICS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques