H-Tunneling in Methylmalonyl-CoA Mutase

甲基丙二酰辅酶A变位酶中的H-隧道

• 批准号: 6622173
• 负责人: RUMA V BANERJEE
• 金额: $ 3.72万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6622173
• 关键词: Poland; acid thiol ligase; active sites; carbon; catalyst; cobalt; computer simulation; cooperative study; enzyme mechanism; enzyme structure; hydrolysis; ionic bond; mathematical model; methylmalonic aciduria; methylmalonyl coA epimerase

DESCRIPTION (provided by applicant) Methylmalonyl-CoA mutase, the only member of the family that is found in both bacteria and in man, catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. Its dysfunction leads to methylinalonic aciduria, an inborn error of metabolism that, in severe cases, can be fatal. A few years ago, we bad reported a novel and anomalously large kinetic isotope effect on the cleavage of the cobalt carbon bond of the cofactor when an isotopic substitution from protium to deuterium was made in the substrate. This was interpreted as evidence that homolysis of the cobalt-carbon bond is coupled to hydrogen atom abstraction from the substrate leading to a substrate radical. The large deuterium isotope effect (35.6 at 20oC) suggests the contribution of tunneling to this hydrogen atom transfer although other explanations are also possible. This has recently been examined by monitoring the temperature dependence of the isotope effect which yields values for the ratio of the Arrhenius preexponential factors (AH/AD) and for the difference in activation energies (EaD-EaH) that lie well outside the semiclassical range. In order to draw mechanistic conclusions from the measured values of isotope effects it is necessary to estimate theoretically their values for alternative pathways. This proposal focuses on combining the experimental approach for evaluating tunneling that is a component of the parent grant, with theoretical calculations of kinetic isotope effects in the methylmalonyl-CoA mutase-catalyzed reaction under presteady-state conditions. We will use available crystallographic information to build a model of the active site and optimize structures of the reactants, transition states, and putative intermediates using semiempirical, DTF, and/or ab initio methods within recently developed QM/MM techniques. Vibrational analysis performed on these structures will allow us to calculate isotope effects within the semiclassical approximation. The tunneling contribution will be then calculated. These studies will allow us to better understand and control the mechanism of this novel reaction in the clinically important enzyme.

描述（由申请人提供） 甲基丙二酰辅酶A β是该家族中唯一一个在两种疾病中都存在的成员。 在细菌和人类中，催化甲基丙二酰辅酶A的1，2重排， 琥珀酰辅酶A。它的功能障碍导致甲基亚麻酸尿症，这是一种先天性缺陷 在严重的情况下，这可能是致命的。几年前，我们 报道了一种新的和巨大的动力学同位素效应的分裂 当来自钴的同位素取代时， 在衬底中进行了从氚到氘的转换。这被解读为 钴-碳键的均裂与氢原子偶联的证据 从基质中提取导致基质自由基。大 氘同位素效应（20 ℃时为35.6）表明隧道效应的贡献 尽管其它解释也是可能的。 这最近已经通过监测的温度依赖性进行了检查， 同位素效应产生的值的比例Arbidius 指前因子（AH/AD）和活化能差 （EaD-EaH），位于半经典范围之外。为了画 从同位素效应的测量值得出的机械结论， 有必要从理论上估计它们对替代途径的价值。这 建议的重点是结合实验方法进行评估 隧道，这是一个组成部分的父赠款，与理论 甲基丙二酰辅酶A中同位素动力学效应的计算 在预稳态条件下的突变酶催化反应。我们将使用 可用的晶体学信息来构建活性位点的模型， 优化反应物、过渡态和假定的 中间体使用半经验，DTF和/或从头计算方法， 最近开发的QM/MM技术。对这些进行振动分析 结构将使我们能够计算半经典内的同位素效应 近似然后将计算隧穿贡献。这些 研究将使我们更好地了解和控制这一机制， 临床重要酶的新反应。