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: 6692623
• 负责人: RUMA V BANERJEE
• 金额: $ 3.69万
• 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 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6692623
• 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） 甲基丙二酸单酰辅酶 A 变位酶，该家族中唯一在两者中均发现的成员 在细菌和人体中，催化甲基丙二酸单酰辅酶 A 的 1,2 重排 琥珀酰辅酶A。它的功能障碍会导致甲基醛酸尿症，这是一种先天性错误 新陈代谢的影响，在严重的情况下，可能是致命的。几年前，我们不好 报道了一种新颖且异常大的动力学同位素对裂解的影响 当同位素取代时辅因子的钴碳键 在底物中将氕转化为氘。这被解释为 钴-碳键均裂与氢原子偶联的证据 从底物中抽提产生底物自由基。大的 氘同位素效应（20oC 时为 35.6）表明隧道效应的贡献 尽管其他解释也是可能的，但这种氢原子转移是可能的。 最近通过监测温度依赖性对此进行了检查 同位素效应产生阿伦尼乌斯比率值 指前因子 (AH/AD) 和活化能差异 (EaD-EaH) 远远超出半古典范围。为了画出 从同位素效应的测量值得出的机械结论是 有必要从理论上估计它们对于替代途径的价值。这 提案侧重于结合实验方法进行评估 隧道是父资助的一部分，理论上 甲基丙二酸单酰辅酶A中的动力学同位素效应的计算 预稳态条件下变位酶催化的反应。我们将使用 可用的晶体学信息来构建活性位点模型 优化反应物、过渡态和假定的结构 使用半经验、DTF 和/或从头算方法的中间体 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。然后将计算隧道效应的贡献。这些 研究将使我们更好地理解和控制这一机制 临床重要酶的新反应。