Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase

被引:56
作者
Breton, J [1 ]
Avanzi, N [1 ]
Magagnin, S [1 ]
Covini, N [1 ]
Magistrelli, G [1 ]
Cozzi, L [1 ]
Isacchi, A [1 ]
机构
[1] Pharmacia & Upjohn Inc, Dept Biol, I-20014 Nerviano, MI, Italy
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2000年 / 267卷 / 04期
关键词
3-hydroxykynurenine; kynurenine; mono-oxygenase; neurotoxicity; tryptophan pathway;
D O I
10.1046/j.1432-1327.2000.01104.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1092 / 1099
页数:8
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