A Radical Enzyme and its Escorts

自由基酶及其护航者

• 批准号: 7459980
• 负责人: RUMA V BANERJEE
• 金额: $ 20.76万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7459980
• 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; Depth; Disease; Electronics; Enzymes; Fibroblasts; GTP-Binding Proteins; Genetic; Guanosine Diphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; In Vitro; Inborn Genetic Diseases; Individual; Kinetics; Laboratories; Mammals; Maps; Metabolic Pathway; Metabolism; Methionine; Methods; Methylmalonyl-CoA Mutase; Methylobacterium extorquens; Mitochondria; Mitochondrial Proteins; Modeling; Molecular Chaperones; Mutase; Mutation; Nucleotides; Numbers; Object Attachment; Orthologous Gene; Pathway interactions; Patients; Physiologic pulse; Principal Investigator; Property; Proteins; Pulse taking; Rate; Reaction; Role; Skeleton; Solutions; Source; Spectrum Analysis; Structure; Supplementation; Surface; System; Techniques; Testing; Thermodynamics; Vitamin B 12; circular magnetic dichroism; cobamamide; cofactor; crosslink; gene discovery; insight; methylmalonic aciduria; methylmalonyl-coenzyme A; mouse Gdi2 protein; polypeptide; programs; 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（B12 代谢先天性缺陷）患者的研究表明，除了线粒体腺苷钴胺素 (AdoCbl) 依赖性酶甲基丙二酰辅酶 A 变位酶外，还涉及另外两个致病位点。随着最近发现编码 MMAA（一种功能未知的 G 蛋白伴侣）和腺苷基转移酶（从 cob(II)alamin 合成 AdoCbl）的基因，已鉴定出专门用于同化和利用 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，也是本提案的主题。甲基丙二酰辅酶 A 变位酶催化甲基丙二酰辅酶 A 到琥珀酰辅酶 A 的化学上令人畏惧的碳骨架重排，并使用 AdoCbl 作为该反应的自由基储存库。我们对这种酶的研究深入了解了变位酶如何使 Co-C 键均裂速率提高数万亿倍，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）变位酶的反应机制是什么以及MMAA的细菌直系同源物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）来进一步了解变位酶反应的机理，并表征 MeaB 对变位酶反应坐标的影响。我们还计划表征融合蛋白 McmC，其中变位酶及其分子伴侣 MeaB 在单个多肽中编码，并鉴定彼此相互作用的两种蛋白质的表面，(ii) AdoCbl 从腺苷基转移酶直接转移到甲基丙二酸单酰辅酶 A 变位酶的动力学是什么？我们将确定并比较 B12 从溶液中与变位酶结合与从腺苷基转移酶直接转移相关的动力学和热力学参数，以测试 B12 的递送是陪伴的模型，(iii) MeaB 和 MMAA 如何调节腺苷基转移酶的 AdoCbl 合成及其向甲基丙二酸单酰辅酶 A 变位酶的转移？我们将结合离体（使用患者细胞系）和体外（使用有限数量的患者变位酶和 MMAA 突变）研究来评估 G 蛋白伴侣的功能，并将研究 MeaB 如何调节腺苷转移酶和变位酶之间直接 AdoCbl 转移的动力学。