Mechanism of Methylmalonyl-CoA Mutase: A Radical Enzyme

描述(申请人提供)：哺乳动物中依赖腺苷钴胺(ADOCbl)的异构酶的唯一例子是线粒体酶，甲基丙二酸辅酶A变位酶，它催化甲基丙二酸辅酶A重排为琥珀酸辅酶A。甲基丙二酸辅酶A变位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。这项建议的重点是阐明甲基丙二酸辅酶A突变的反应机制，并表征与致病的特定错义突变相关的生化惩罚。辅因子ADOCbl在该反应中的作用是作为潜在的自由基库，在催化过程中部署，以完成具有化学挑战性的碳骨架重排反应。在底物存在的情况下，该酶能使钴-碳(Co-C)键的同质裂解速率提高约万亿倍，其机制一直是争论的主题。我们建议(I)通过检验以下假设来阐明Co-C键被标记的机制：Y89是一个活性中心残基，在与底物结合后作为分子楔子撬开Co-C键。我们将考察Y89位取代对动力学耦合和隧穿的影响，并使用光谱方法探索在底物类似物存在的情况下野生型和Y89变异体中Corin的电子环境。(Ii)我们将阐明活性中心的“芳香走廊”残基在重排的立体化学控制中的作用，并用脉冲EPR方法和光谱模拟估计自由基的几何构型和自由基间的距离。将设计用于稳定自由基中间体的底物类似物。(Iii)我们将对甲基丙二酸尿症患者中发现的一组错义突变和在每个亚基中携带N端R93H突变或C端突变(在ADOCbl结合域中)的功能性异源二聚体相关的生化缺陷进行表征，以确定观察到的互补的基础。这些对甲基丙二醛-辅酶A突变酶野生型和致病变异体的研究将有助于我们对一种新的临床重要酶的理解。

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.