A Radical Enzyme and its Escorts

自由基酶及其护航者

• 批准号: 8000132
• 负责人: RUMA V BANERJEE
• 金额: $ 4.85万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000132
• 关键词: Active Sites; Address; Assimilations; Bacterial Proteins; Binding; Carbon; Cell Line; Chemicals; Chemistry; Chimeric Proteins; Circular Dichroism Spectroscopy; Cobalamin; Coenzymes; Complex; Cytoplasm; DNA Sequence Rearrangement; Data; Disease; Electronics; Enzymes; Fibroblasts; GTP-Binding Proteins; Genetic; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; In Vitro; Inborn Genetic Diseases; Individual; Kinetics; Laboratories; Magnetism; Mammals; Maps; Metabolic Pathway; Metabolism; Methionine; Methods; Methylmalonyl-CoA Mutase; Methylobacterium extorquens; Mitochondria; Mitochondrial Proteins; Modeling; Molecular Chaperones; Mutase; Mutation; Nucleotides; Orthologous Gene; Pathway interactions; Patients; Physiologic pulse; Principal Investigator; Property; Proteins; Reaction; Role; Skeleton; Solutions; Source; Spectrum Analysis; Structure; Supplementation; Surface; System; Techniques; Testing; Thermodynamics; Vitamin B 12; cobamamide; cofactor; crosslink; gene discovery; insight; methylmalonic aciduria; methylmalonyl-coenzyme A; polypeptide; programs; protein complex; protein protein interaction; succinyl-coenzyme A

DESCRIPTION (provided by applicant): Studies on patients with methylmalonic aciduria, an inborn error of B12 metabolism, had revealed that in addition to the mitochondrial adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, two other disease causing loci were involved. With the recent discovery of the genes encoding MMAA, a G- protein chaperone of unknown function, and adenosyltransferase, which synthesizes AdoCbl from cob(ll)alamin, the mitochondrial components dedicated to assimilation and utilization of B12 have been identified. In contrast, our quest for understanding the interactions and functional interplay between these proteins has just begun and is the subject of this proposal. Methylmalonyl-CoA mutase catalyzes the chemically daunting carbon skeleton rearrangement of methylmalonyl-CoA to succinyl CoA and deploys AdoCbl as a radical reservoir for this reaction. Our studies on this enzyme have furnished insights into how the mutase effects a trillion-fold rate enhancement of Co-C bond homolysis and the role of active site residues in controlling radical reactivity. In this proposal, we plan to address the following specific questions: (i) What is the reaction mechanism of the mutase and how does the bacterial ortholog of MMAA, MeaB, modulate it? We propose to use spectroscopic approaches (EPR, MCD and rapid reaction kinetics) to obtain further mechanistic insights into the mutase reaction and to characterize the influence of MeaB on the mutase reaction coordinate. We also plan to characterize a fusion protein, McmC, in which the mutase and its chaperone, MeaB, are encoded in a single polypeptide and to identify the surfaces of the two proteins that interact with each other, (ii) What are the kinetics of direct transfer of AdoCbl from adenosyltransferase to methylmalonyl-CoA mutase? We will determine and compare the kinetic and thermodynamic parameters associated with B12 binding to the mutase from solution versus its direct transfer from adenosyltransferase to test our model that delivery of B12 is chaperoned, (iii) How do MeaB and MMAA modulate AdoCbl synthesis by adenosyltransferase and its transfer to methylmalonyl-CoA mutase? We will use a combination of ex vivo (with patient cell lines) and in vitro (with a limited number of patient mutations in the mutase and in MMAA) studies to assess the function of the G-protein chaperone and will examine how MeaB modulates the kinetics of direct AdoCbl transfer between adenosyltransferase and the mutase.

描述（由申请人提供）：关于B12代谢的天生误差的甲基丙二酰酸尿症患者的研究表明，除线粒体腺苷 - 核心素（ADOCBL）依赖性酶，甲基甲基-COA突变酶，甲基甲基甲基-COA突变酶，两种其他疾病，还涉及其他疾病。随着最近发现编码MMAA的基因，MMAA是一个未知功能的G蛋白伴侣，以及腺基转移酶的基因，这些基因从COB（LL）Alamin合成了ADOCBL，该基因已确定了用于同化和利用B12的COB（LL）Alamin，已确定了B12的线粒体成分。相比之下，我们寻求理解这些蛋白质之间的相互作用和功能相互作用始于开始，并且是该提案的主题。甲基甲基甲酰辅酶A突变酶催化甲基甲硅烷-COA的化学艰巨的碳骨架重排向琥珀酰COA，并将Adocbl部署为这种反应的根治性储层。我们对该酶的研究提供了关于突变酶如何影响Co-C键同伊分析的率如何增强的率以及主动位点残基在控制根部反应性中的作用的见解。在此提案中，我们计划解决以下特定问题：（i）突变酶的反应机理是什么，MMAA的细菌直系同源物如何调节它？我们建议使用光谱方法（EPR，MCD和快速反应动力学）来获得对突变酶反应的进一步机械见解，并表征MEAB对突变酶反应坐标的影响。我们还计划表征融合蛋白MCMC，其中突变酶及其伴侣MEAB被编码在单个多肽中，并识别两个相互作用的两个蛋白质的表面，（ii）Adocbl直接转移到丙二酶转移到甲基甲基甲基甲基甲基甲基甲基酸酯mutasase的动力学是什么？我们将确定并比较与B12与突变酶结合的动力学和热力学参数与从溶液中与突变酶结合而不是其从腺苷基转移酶直接转移，以测试我们的模型，即B12的递送是伴随的，（iii）MEAB和MMAA如何通过腺苷基转移酶和甲基甲基甲基甲基甲基甲基甲基化酶调节ADOCBL？我们将使用EX VIVO（与患者细胞系）和体外（突变酶和MMAA中的患者突变数量有限）的组合来评估G蛋白伴侣蛋白的功能，并将检查MEAB如何调节丙糖基转移酶和突变酶之间直接adocbl转移的动力学。