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）：对甲基丙二酸尿症（一种先天性B12代谢错误）患者的研究表明，除了线粒体腺苷钴胺素（AdoCbl）依赖酶甲基丙二酰辅酶a变化酶外，还涉及其他两个致病位点。随着最近发现的编码MMAA（一种功能未知的G蛋白伴侣）和腺苷转移酶（从cob(ll)alamin合成AdoCbl）的基因，已经确定了用于同化和利用B12的线粒体成分。相比之下，我们对了解这些蛋白质之间的相互作用和功能相互作用的探索才刚刚开始，这也是本提案的主题。甲基丙二酰辅酶a变化酶催化甲基丙二酰辅酶a的碳骨架重排为琥珀酰辅酶a，并部署AdoCbl作为该反应的自由基库。我们对该酶的研究揭示了该突变酶如何使Co-C键的均解速率提高数万亿倍，以及活性位点残基在控制自由基反应性中的作用。在本提案中，我们计划解决以下具体问题：(i)突变酶的反应机制是什么，MMAA的细菌同源物MeaB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）来进一步了解突变酶反应的机制，并表征MeaB对突变酶反应坐标的影响。我们还计划表征一种融合蛋白McmC，其中的突变酶及其伴侣MeaB被编码在一个单一的多肽中，并鉴定两种相互作用的蛋白的表面。（ii） AdoCbl从腺苷转移酶直接转移到甲基丙二酰辅酶a突变酶的动力学是什么？我们将确定并比较溶液中B12与突变酶结合的动力学和热力学参数，以及其从腺苷转移酶直接转移的动力学和热力学参数，以测试我们的模型，即B12的传递是有陪同的。（iii） MeaB和MMAA如何调节腺苷转移酶合成AdoCbl并将其转移到甲基丙二酰辅酶a突变酶？我们将结合离体（患者细胞系）和体外（在突变酶和MMAA中有有限数量的患者突变）研究来评估g蛋白伴侣的功能，并将研究MeaB如何调节AdoCbl在腺苷转移酶和突变酶之间的直接转移动力学。