The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase
The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase
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DOI：
10.1021/ja208694g
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发表时间：
2012-02-15
影响因子：
15
通讯作者：
Raven, Emma Lloyd
中科院分区：
文献类型：
--
作者：
Efimov, Igor;Basran, Jaswir;Raven, Emma Lloyd
作为犬尿氨酸途径的一部分，吲哚胺2，3-双加氧酶催化依赖氧的L色氨酸(L-Trp)氧化成N-甲酰-酪氨酸(NFK)。30多年前，人们首次发现高浓度L-色氨酸对酶活性有抑制作用，但其抑制机制尚未建立。结合动力学和还原电位的测量，我们提出的证据表明，在与L-色氨酸(L-色氨酸)的周转过程中，人吲哚胺2，3-双加氧酶(HIDO)和一些定点突变体(L-色氨酸)的酶活性受到抑制可以归因于O-2和L-色氨酸的顺序有序结合。数据分析表明，在低浓度的L-色氨酸作用下，O-2首先结合，然后与L-色氨酸结合；在较高浓度的L-色氨酸作用下，结合的顺序正好相反。此外，我们还表明，血红素还原电位(E-m(0))在控制总体催化速度(从而抑制程度)方面具有调节作用，因为E-m(0)(在L-色氨酸存在下增加)与O-2结合的速率常数之间存在定量的相关性。这意味着随着底物的结合，Fe2+-O-2初始形成Fe3+-O-2(中心点-)的热力学变得不那么有利，我们认为这一氧化步骤在较高底物浓度下的减慢是抑制的根源。相反，我们表明，在该机制的最后一步，形式上需要还原血红素的亚铁酶的再生(和NFK的形成)是由底物结合酶中较高的还原电位促进的，并且两个常数(k(CAT)和E-m(0))也被证明是相关的。因此，总体催化活性在该机制的初始步骤和最后步骤对血红素还原潜力的相等和相反的依赖之间保持平衡。这种还原电位的调节为反应活性的调节提供了一种简单的机制，这可能会在这一酶家族中得到更广泛的应用。
Indoleamine 2,3-dioxygenase catalyzes the O-2-dependent oxidation of L-tryptophan (L-Trp) to N-formylky-nurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O-2 and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O-2 binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E-m(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E-m(0) (that increases in the presence of L-Trp) and the rate constant for O-2 binding. This means that the initial formation of ferric superoxide (Fe3+-O-2(center dot-)) from Fe2+-O-2 becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E-m(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.