A Birch-like mechanism in enzymatic benzoyl-CoA reduction: a kinetic study of substrate analogues combined with an ab initio model.
A Birch-like mechanism in enzymatic benzoyl-CoA reduction: a kinetic study of substrate analogues combined with an ab initio model.
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DOI：
10.1021/bi0113770
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发表时间：
2002-02
期刊：
影响因子：
2.9
通讯作者：
Henrik Möbitz;M. Boll
中科院分区：
文献类型：
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作者：
Henrik Möbitz;M. Boll
来自厌氧细菌 Thaueraaromatica 的苯甲酰辅酶 A 还原酶催化 ATP 驱动的苯甲酰辅酶 A 芳香部分的双电子还原。先前针对苯甲酰辅酶A还原酶提出了一种Birch机制，涉及向芳环交替进行单电子和单质子转移步骤。由于高氧化还原势垒，产生自由基阴离子的第一个电子转移步骤被认为是该反应中的限速步骤。这项工作着眼于底物还原机制，将许多底物类似物的动力学分析与基于苯甲酰辅酶A自由基阴离子的从头计算的电子密度的模型结合起来，该模型是所提出的Birch机制的过渡态模型。邻位取代的苯甲酰基-CoA 的 K(m) 和 k(cat) 均随着取代基受体强度的增加而增加 (F > Cl = H > OH > NH(2))。单氟苯甲酰辅酶A的异构体中，还原率按以下顺序降低：邻位>间位>对位； K(m) 值按以下顺序增加：meta > ortho > para。五环杂芳香酸硫羟酸酯的还原顺序为：噻吩>呋喃>吡咯； 2-异构体的还原速度比 3-异构体快得多。大多数这些结果都可以通过模型合理化。哈米特图表明反应机制仅具有轻微极性，表明涉及苯甲酰辅酶A的羰基氧的部分质子化和/或第一个电子和质子的同时转移。令人惊讶的是，苯甲酰-CoA 还原酶对吡啶-2-羰基-CoA (2.1) 表现出对 k(cat) 的氢动力同位素效应，但对苯甲酰-CoA (1.2) 的 k(cat) 表现出可忽略不计的氢动力同位素效应，表明吡啶-2-羰基-CoA 还原按照不同的机制进行。
Benzoyl-CoA reductase from the anaerobic bacterium Thauera aromatica catalyzes the ATP-driven two-electron reduction of the aromatic moiety of benzoyl-CoA. A Birch mechanism involving alternate one-electron and one-proton transfer steps to the aromatic ring was previously proposed for benzoyl-CoA reductase. Due to the high redox barrier, the first electron transfer step yielding a radical anion is considered the rate-limiting step in this reaction. Focusing on the mechanism of substrate reduction, this work combines the kinetic analysis of a number of substrate analogues with a model based on the ab initio calculated electron density of the radical anion of benzoyl-CoA, a transition state model of the proposed Birch mechanism. Both K(m) and k(cat) of ortho-substituted benzoyl-CoA increased in parallel with the substituent's acceptor strength (F > Cl = H > OH > NH(2)). Among the isomers of monofluorobenzoyl-CoA, reduction rates decreased in the following order: ortho > meta > para; the K(m) values increased in the following order: meta > ortho > para. Five-ring heteroaromatic acid thiol esters were reduced in the following order: thiophene > furan > pyrrole; the 2-isomers are reduced much faster than the 3-isomers. Most of these results could be rationalized by the model. A Hammett plot indicated that the reaction mechanism is only slightly polar, suggesting the involvement of a partial protonation of the carbonyl oxygen of benzoyl-CoA and/or a simultaneous transfer of the first electron and proton. Surprisingly, benzoyl-CoA reductase exhibited a hydrogen kinetic isotope effect on k(cat) for pyridine-2-carbonyl-CoA (2.1) but only a negligible one for benzoyl-CoA (1.2), indicating that pyridine-2-carbonyl-CoA reduction proceeds according to a varied mechanism.