Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase.
The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.
Breton
J
J
Department of Biology, Pharmacia & Upjohn, Nerviano, Italy. jerome.breton@eu.pnu.com
Avanzi
N
N
Magagnin
S
S
Covini
N
N
Magistrelli
G
G
Cozzi
L
L
Isacchi
A
A
D000818:Animals; D019556:COS Cells; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D007737:Kynurenine / Q000031:analogs & derivatives / Q000378:metabolism; D050601:Kynurenine 3-Monooxygenase; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000378:metabolism / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014162:Transfection
D002712:Chlorides; D007477:Ions; D010545:Peroxides; D011993:Recombinant Fusion Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C005045:3-hydroxykynurenine; D007737:Kynurenine; D009249:NADP; D011732:Pyridoxal Phosphate; D006899:Mixed Function Oxygenases; D050601:Kynurenine 3-Monooxygenase; D019255:NADPH Oxidases