Substrate-Protein Interaction in Human Tryptophan Dioxygenase: The Critical Role of H76
Substrate-Protein Interaction in Human Tryptophan Dioxygenase: The Critical Role of H76
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DOI：
10.1021/ja807969a
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发表时间：
2009-03-11
影响因子：
15
通讯作者：
Yeh, Syun-Ru
中科院分区：
文献类型：
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作者：
Batabyal, Dipanwita;Yeh, Syun-Ru
人类犬尿氨酸途径的初始和限速步骤涉及由两种血红素蛋白（色氨酸2，3-双加氧酶（hTDO）和吲哚胺2，3-双加氧酶（hIDO））催化的色氨酸氧化为N-甲酰犬尿氨酸。在hTDO中，保守的H76残基被认为充当活性位点碱基以使Trp的吲哚NH基团去质子化，这是Trp氧化反应的初始步骤。在hIDO中，该组氨酸被丝氨酸取代。为了研究H76的作用，我们研究了hTDO的H76 S和H76 A突变体。活性测定表明，突变引起的K（猫）的减少和K-M增加的两个突变体。k（cat）的降低是由于活性位点碱催化剂H76被较弱的碱（可能是水）取代，而K-M的增加是由于H76与底物以及蛋白质基质之间的特异性相互作用丧失。与各种色氨酸类似物的共振拉曼研究表明，该基板被定位在活性位点的铵，羧酸盐，和吲哚基团，通过复杂的H-键合和疏水相互作用。这种情况与L-Trp结合显著干扰hTDO的O-2-加合物的电子性质的观察结果一致。H76在hTDO中的重要结构和功能作用通过观察到活性三元复合物L-Trp-O-2-hTDO的电子构型对H76突变敏感而得到强调。
The initial and rate-limiting step of the kynurenine pathway in humans involves the oxidation of tryptophan to N-formyl kynurenine catalyzed by two hemeproteins, tryptophan 2,3-dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). In hTDO, the conserved H76 residue is believed to act as an active site base to deprotonate the indole NH group of Trp, the initial step of the Trp oxidation reaction. In hIDO, this histidine is replaced by a serine. To investigate the role of the H76, we have studied the H76S and H76A mutants of hTDO. Activity assays show that the mutations cause a decrease in k(cat) and an increase in K-M for both mutants. The decrease in the k(cat) is accounted for by the replacement of the active site base catalyst, H76, with a weaker base, possibly a water, whereas the increase in K-M is attributed to the loss of the specific interactions between the H76 and the substrate as well as the protein matrix. Resonance Raman studies with various Trp analogs indicate that the substrate is positioned in the active site by the ammonium, carboxylate, and indole groups, via intricate H-bonding and hydrophobic interactions. This scenario is consistent with the observation that L-Trp binding significantly perturbs the electronic properties of the O-2-adduct of hTDO. The important structural and functional roles of H76 in hTDO is underscored by the observation that the electronic configuration of the active ternary complex, L-Trp-O-2-hTDO), is sensitive to the H76 mutations.