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（腺苷钴胺素Cbl）依赖性异构酶的唯一实例是线粒体酶甲基丙二酰辅酶A，其催化甲基丙二酰辅酶A重排为琥珀酰辅酶A。甲基丙二酸单酰辅酶A的功能障碍导致甲基丙二酸尿症，其引起有机酸代谢的异常，并且在严重的情况下，可以是致命的。该提案的重点是阐明甲基丙二酰辅酶A的反应机制，并表征与致病的特定错义突变相关的生化惩罚。在该反应中，辅因子α Cbl的作用是作为潜在的自由基库，其在催化过程中被部署以完成具有化学挑战性的碳骨架重排反应。这种酶会协调一种。在底物存在下，钴-碳（Co-C）键的均裂速率增强万亿倍，其机制一直是持久争论的主题。我们建议（i）阐明的机制，通过测试的假设，Y 89，一个活性位点的残基，是作为一个分子楔撬开钴-C键结合底物后，钴-C键不稳定。我们将研究在Y89上的取代对动力学耦合和隧道效应的影响，并使用光谱方法来探测底物类似物存在下野生型和Y89变体中咕啉的电子环境。(ii)我们将阐明的作用的“芳香走廊”残基的活性位点上的立体化学控制的重排，并估计自由基的几何形状和interradical距离的脉冲EPR方法，和光谱模拟。将设计用于稳定自由基中间体的底物类似物。(iii)我们将表征与甲基丙二酸尿症患者和功能异源二聚体中确定的一组错义突变相关的生化缺陷，所述功能异源二聚体在每个亚基中携带N-末端R93 H突变或C-末端突变（在C57 Cbl结合结构域中），以确定所观察到的互补的基础。这些对甲基丙二酰辅酶A的野生型和致病性变体的研究将使我们对一种新的临床重要酶的理解有所进展。

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.