Cooperative Binding of l-Trp to Human Tryptophan 2,3-Dioxygenase: Resonance Raman Spectroscopic Analysis
Cooperative Binding of l-Trp to Human Tryptophan 2,3-Dioxygenase: Resonance Raman Spectroscopic Analysis
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DOI：
10.1093/jb/mvp002
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发表时间：
2009-04-01
影响因子：
2.7
通讯作者：
Shiro, Yoshitsugu
中科院分区：
文献类型：
--
作者：
Fukumura, Eiko;Sugimoto, Hiroshi;Shiro, Yoshitsugu
色氨酸2，3-双加氧酶（TDO）是一种四聚体酶，通过在吲哚环的2，3-键上添加O-2，催化l-色氨酸（l-Trp）氧化裂解为N-甲酰犬尿氨酸。该反应是哺乳动物犬尿氨酸途径中的第一步和限速步骤。在本研究中，我们测量了血红素袋重组人TDO（rhTDO）在铁的形式，诱导的L-Trp结合使用共振拉曼和光学吸收光谱的构象变化。血红素拉曼带在不同浓度的L-Trp的解卷积分析表明，野生型酶表现出同向性协同L-Trp结合，这是证实了在光学吸收光谱的变化。突变分析表明，Y 42 F突变体取消了合作的结合，而H76 A突变体大大降低了催化活性。这些数据和细菌TDO结构中报道的亚基间接触表明，rhTDO的Y 42通过参与相邻亚基的活性位点来负责l-Trp的合作结合。
Tryptophan 2,3-dioxygenase (TDO) is a tetrameric enzyme that catalyses the oxidative cleavage of l-tryptophan (l-Trp) to N-formylkynurenine by the addition of O-2 across the 2,3-bond of the indole ring. This reaction is the first and rate-limiting step in the kynurenine pathway in mammals. In the present study, we measured the conformational changes in the haem pocket of recombinant human TDO (rhTDO) in ferric form that are induced by l-Trp binding using both resonance Raman and optical absorption spectroscopies. The deconvolution analysis of the haem Raman bands at various concentrations of l-Trp revealed that the wild-type enzyme exhibits homotropic cooperativity in l-Trp binding, which was confirmed by a change in the optical absorption spectra. Mutation analysis showed that the Y42F mutant abolished the cooperative binding, and that the H76A mutant considerably reduced the catalytic activity. These data and the inter-subunit contacts reported in the bacterial TDO structure suggest that the Y42 of rhTDO is responsible for the cooperative binding of l-Trp by participating in the active site of the adjacent subunit.