Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives
Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives
复制标题
DOI：
10.1074/jbc.m200637200
复制
发表时间：
2002-06-14
影响因子：
4.8
通讯作者：
Scrutton, NS
中科院分区：
文献类型：
--
作者：
Khan, H;Harris, RJ;Scrutton, NS
通过停流分光光度法、氧化还原电位法和 X 射线晶体学研究了季戊四醇四硝酸酯还原酶与还原性和氧化性底物的反应。我们在季戊四醇四硝酸酯 (PETN) 还原酶的还原半反应中表明，NADPH 结合形成酶-NADPH 电荷转移中间体，然后氢化物从烟酰胺辅酶转移到 FMN。在氧化半反应中，双电子还原酶与多种底物发生反应，包括硝基酯炸药（三硝酸甘油和PETN）、硝基芳香族炸药（三硝基甲苯（TNT）和苦味酸）和α,β-不饱和羰基化合物（2-环己烯酮）。硝基芳族底物 TNT 对黄素的氧化在动力学上与其氢化物-Meisenheimer 络合物的形成无法区分，这与黄素 N5 原子在底物缺电子芳香核处的氢化物直接亲核攻击的机制一致。与苦味酸和 TNT 结合的氧化酶复合物的晶体结构与从还原黄素到硝基芳香族底物的直接氢化物转移一致。抑制剂 2,4-二硝基苯酚 (2,4-DNP) 的结合模式与苦味酸和 TNT 观察到的模式相似。然而，在这个位置，芳香核没有被激活以从黄素 N5 原子进行氢化物转移，因此导致与 2,4-DNP 缺乏反应性。我们对 PETN 还原酶的研究进一步建立了与老黄酶蛋白质家族的密切关系，但同时强调了与老黄酶的反应性相比的重要差异。我们的研究为 PETN 还原酶与硝基芳香族炸药化合物 TNT 和苦味酸反应的能力以及用 2,4-DNP 抑制酶活性的能力提供了结构和机制原理。
The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.