Spectroscopic studies of ligand and substrate binding to human indoleamine 2,3-dioxygenase.
Spectroscopic studies of ligand and substrate binding to human indoleamine 2,3-dioxygenase.
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DOI：
10.1021/bi1005078
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发表时间：
2010-06-22
期刊：
影响因子：
2.9
通讯作者：
Yeh SR
中科院分区：
文献类型：
--
作者：
Lu C;Lin Y;Yeh SR
人吲哚胺2，3-双加氧酶（human indoleamine 2，3-dioxygenase，hIDO）是一种含血红素的细胞内酶，在非肝组织中通过犬尿氨酸途径催化L-色氨酸（L-Trp）代谢的起始和限速步骤。稳态动力学数据显示hIDO具有底物抑制行为，暗示酶中存在第二底物结合位点，尽管迄今为止还没有直接证据支持这一点。动力学数据还显示L-Trp（15 μM）的Km比L-Trp的Kd低约27倍（0.4 mM）的配体自由亚铁酶，表明O2结合进行L-Trp结合在催化循环。用氰化物作为结构探针，我们研究了与配体和底物结合hIDO的热力学和动力学参数。平衡滴定研究表明，氰化物加合物能与两个L-色氨酸分子结合，其Kd值分别为18 μM和26 mM，首次直接证明了hIDO中存在第二底物结合位点。动力学研究表明，预结合的L-Trp的酶延缓氰化物结合约13倍，而预结合的氰化物的酶促进L-Trp结合约22倍。这些数据支持这样的观点，即在酶的活性周转期间，在动力学上更有利于在L-Trp之前结合O2。
Human indoleamine 2,3-dioxygenase (hIDO) is an intracellular heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Steady-state kinetic data showed that hIDO exhibits substrate inhibition behavior, implying the existence of a second substrate binding site in the enzyme, although so far there is no direct evidence supporting it. The kinetic data also revealed that the Km of L-Trp (15 μM) is ~27-fold lower than the Kd of L-Trp (0.4 mM) for the ligand-free ferrous enzyme, suggesting that O2 binding proceeds L-Trp binding during the catalytic cycle. With cyanide as a structural probe, we have investigated the thermodynamic and kinetic parameters associated with ligand and substrate binding to hIDO. Equilibrium titration studies show that the cyanide adduct is capable of binding two L-Trp molecules, with Kd values of 18 μM and 26 mM. The data offer the first direct evidence of the second substrate binding site in hIDO. Kinetic studies demonstrate that prebinding of L-Trp to the enzyme retards cyanide binding by ~13-fold, while prebinding of cyanide to the enzyme facilitates L-Trp binding by ~22-fold. The data support the view that during the active turnover of the enzyme it is kinetically more favored to bind O2 prior to L-Trp.