A Radical Enzyme and its Escorts

描述（由申请人提供）：对甲基丙二酸尿症（B12代谢的先天性缺陷）患者的研究表明，除了线粒体腺苷钴胺素（NACBl）依赖性酶甲基丙二酰辅酶A β外，还涉及其他两个致病基因座。随着最近发现编码MMAA（一种功能未知的G-蛋白伴侣）和腺苷转移酶（其从cob（II）丙氨酸合成β-Cbl）的基因，已经鉴定了致力于B12的同化和利用的线粒体组分.相比之下，我们对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 β催化甲基丙二酰辅酶A到琥珀酰辅酶A的化学上令人生畏的碳骨架重排，并将β Cbl作为该反应的自由基库。我们对这种酶的研究已经提供了深入了解如何影响的Co-C键的均裂和活性位点残基在控制自由基反应性的作用的万亿倍的速率增强。在这项提案中，我们计划解决以下具体问题：（一）什么是反应机制的mMAA，MeaB，如何调节它的细菌直系同源物？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。我们还计划表征的融合蛋白，McmC，其中的mosaic和它的伴侣，MeaB，编码在一个单一的多肽，并确定两个蛋白质的表面相互作用，（ii）什么是直接转移的mosaic Cbl从腺苷转移酶的动力学甲基丙二酰辅酶A mosaic？我们将确定和比较动力学和热力学参数与B12结合到从溶液中与其直接转移从腺苷转移酶，以测试我们的模型，B12的交付是陪伴，（iii）如何做MeaB和MMAA调节β Cbl合成腺苷转移酶和其转移到甲基丙二酰辅酶A β？我们将使用离体（与患者细胞系）和体外（与有限数量的患者突变在MMAA和MMAA）研究的组合，以评估G蛋白伴侣的功能，并将检查MeaB如何调节腺苷转移酶和MMAA之间的直接转移的动力学。

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.