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首页> 外文期刊>Biochemistry >Role of Active Site Residues in Promoting Cobalt?Carbon Bond Homolysis in Adenosylcobalamin-Dependent Mutases Revealed through Experiment and Computation
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Role of Active Site Residues in Promoting Cobalt?Carbon Bond Homolysis in Adenosylcobalamin-Dependent Mutases Revealed through Experiment and Computation

机译：在 腺苷钴胺 依赖性 变位酶 促进 钴 ？ 碳键 均裂 活性位点残 的 作用，通过 实验 和 计算后

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Adenosylcobalamin (AdoCbl) serves as a source of reactive free radicals that are generated by homolytic scission of the coenzyme’s cobalt?carbon bond. AdoCbl-dependent enzymes accelerate AdoCbl homolysis by ～10~(12)-fold, but the mechanism by which this is accomplished remains unclear. We have combined experimental and computational approaches to gain molecular-level insight into this process for glutamate mutase. Two residues, glutamate 330 and lysine 326, form hydrogen bonds with the adenosyl group of the coenzyme. A series of mutations that impair the enzyme’s ability to catalyze coenzyme homolysis and tritium exchange with the substrate by 2?4 orders of magnitude were introduced at these positions. These mutations, together with the wild-type enzyme, were also characterized in silico by molecular dynamics simulations of the enzyme?AdoCbl?substrate complex with AdoCbl modeled in the associated (Co?C bond formed) or dissociated [adenosyl radical with cob(II)alamin] state. The simulations reveal that the number of hydrogen bonds between the adenosyl group and the protein side chains increases in the homolytically dissociated state, with respect to the associated state, for both the wild-type and mutant enzymes. The mutations also cause a progressive increase in the mean distance between the 5′-carbon of the adenosyl radical and the abstractable hydrogen of the substrate. Interestingly, the distance between the 5′-carbon and substrate hydrogen, determined computationally, was found to inversely correlate with the log k for tritium exchange (r = 0.93) determined experimentally. Taken together, these results point to a dual role for these residues: they both stabilize the homolytic state through electrostatic interactions between the protein and the dissociated coenzyme and correctly position the adenosyl radical to facilitate the abstraction of hydrogen from the substrate.
机译：腺苷钴胺素（Adocbl）用作通过辅酶钴的均溶解碳键的均透明群产生的活性自由基的来源。 Adocbl依赖性酶加速Adocbl均匀〜10〜（12） - 折叠，但是完成的机制仍不清楚。我们结合了实验和计算方法，以获得对谷氨酸异构酸的这种方法进行分子层面的洞察。两个残基，谷氨酸330和赖氨酸326，与辅酶的腺苷基团形成氢键。在这些位置引入了一系列损害酶催化酶催化和氚交换的酶催化酶的能力。这些突变与野生型酶一起通过酶的分子动力学模拟在硅β·β··β络合物中的特征在于硅基β·粘合剂，其与形成的（CO-C键形成）或与玉米棒（II）解离[腺苷基）Alamin]状态。模拟表明，对于野生型和突变酶，腺苷基团与蛋白质侧链之间的氢键和蛋白质侧链之间的氢键数增加。突变也会导致腺苷自由基的5'碳与基材的抽象氢之间的平均距离增加。有趣的是，5'-碳和底物氢之间的距离被发现，发现与实验确定的氚交换（R = 0.93）的LOG K相反。总之，这些结果指向这些残留物的双重作用：它们通过蛋白质和解离辅酶之间的静电相互作用稳定均匀状态并正确地定位腺苷基团以促进来自基材的氢的抽象。

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