Mechanism of Methylmalonyl-CoA Mutase: A Radical Enzyme

描述（由申请人提供）：哺乳动物中腺苷钴胺素（AdoCbl）依赖性异构酶的唯一例子是线粒体酶，甲基丙二酰辅酶a变化酶，它催化甲基丙二酰辅酶a重排为琥珀酰辅酶a。甲基丙二酸-辅酶a变异酶功能障碍导致甲基丙二酸尿症，导致有机酸代谢异常，严重时可致命。本研究的重点是阐明甲基丙二烯辅酶a突变的反应机制，并描述与致病性特定错义突变相关的生化惩罚。辅助因子AdoCbl在该反应中的作用是作为一个潜在的自由基库，在催化过程中部署，以完成化学上具有挑战性的碳骨架重排反应。在底物存在的情况下，这种酶能使钴碳（Co-C）键的均裂速率提高约万亿倍，其机制一直是争论不休的话题。我们建议(i)通过测试Y89（活性位点残基）作为分子楔子在底物结合后撬开Co-C键的假设来阐明Co-C键稳定的机制。我们将研究Y89上的取代对动力学耦合和隧道效应的影响，并使用光谱方法在底物类似物存在的情况下探测野生型和Y89变体中科林斯的电子环境。（ii）我们将阐明活性位点的“芳香走廊”残基在重排的立体化学控制中的作用，并通过脉冲EPR方法和光谱模拟估计自由基几何形状和自由基间距离。将设计用于稳定自由基中间体的底物类似物。（iii）我们将描述与甲基丙二酸尿症患者和每个亚基携带n端R93H突变或c端突变（在adocbll结合域）的功能性异二聚体中筛选出的一组错义突变相关的生化缺陷，以确定观察到的互补的基础。这些关于甲基丙二烯辅酶a突变酶的野生型和致病性变异的研究将使我们对一种新的和临床上重要的酶的理解取得进展。

DESCRIPTION (provided by applicant): The only example of an adenosylcobalamin (AdoCbl)-dependent isomerase in mammals is the mitochondrial enzyme, methylmalonyI-CoA mutase, which catalyzes the rearrangement of methylmalonyI-CoA to succinyI-CoA. Dysfunction of methylmalonyI-CoA mutase leads to methylmalonic aciduria, which causes aberrations in organic acid metabolism, and in severe cases, can be fatal. This proposal focuses on elucidating the reaction mechanism of methylmalonyI-CoA mutase, and characterizing the biochemical penalties associated with specific missense mutations that are pathogenic. The role of the cofactor, AdoCbl, in this reaction is to serve as a latent radical reservoir which is deployed during catalysis to accomplish a chemically challenging carbon skeleton rearrangement reaction. The enzyme orchestrates an ca. trillion fold rate enhancement of the homolytic cleavage of the cobalt-carbon (Co-C) bond in the presence of substrate, and its mechanism has been the subject of enduring debate. We propose to (i) elucidate the mechanism by which the Co-C bond is labilized by testing the hypothesis that Y89, an active site residue, is used as a molecular wedge to pry apart the Co-C bond following binding of substrate. We will examine the effect of substitutions at Y89 on kinetic coupling and tunneling and use spectroscopic methods to probe the electronic environment of the corrin in wild type and Y89 variants in the presence of substrate analogs. (ii) We will elucidate the roles of the "aromatic corridor" residues in the active site on stereochemical control of the rearrangement, and estimate radical geometries and interradical distances by pulsed EPR methods, and spectral simulations. Substrate analogs that are designed to stabilize the radical intermediates will be designed. (iii) We will characterize the biochemical deficits associated with a select group of missense mutations identified in methylmalonic aciduria patients and functional heterodimers carrying either an N-terminal R93H mutation or a C-terminal mutation (in the AdoCbl-binding domain) in each subunit to determine the basis of the observed complementation. These studies on the wild type and pathogenic variants of methylmalonyI-CoA mutase will make inroads into our understanding of a novel and clinically important enzyme.