A Radical Enzyme and its Escorts

描述(申请人提供)：对甲基丙二酸尿症患者的研究表明，除了线粒体依赖的腺苷钴胺(ADO Cbl)依赖的酶，甲基丙二酰辅酶A突变酶外，还有另外两个致病基因。随着编码未知功能的G蛋白伴侣MMAA基因和从Cob(11)Alamin合成ADOCb1的腺苷转移酶基因的发现，专用于同化和利用B12的线粒体成分已经被确定。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变位酶催化化学上令人望而生畏的甲基丙二酰辅酶A为琥珀酰辅酶A的碳骨架重排，并将ADOCbl部署为该反应的自由基库。我们对该酶的研究为研究变位酶如何影响数万亿倍的Co-C键均裂速率以及活性中心残基在控制自由基反应中的作用提供了理论依据。在这项提案中，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的细菌同源基因MeaB是如何调节它的？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。我们还计划鉴定一种融合蛋白MCMC，其中变位酶及其伴侣MeaB编码在一个多肽中，并鉴定两种相互作用的蛋白质的表面，(Ii)ADOCb1从腺糖基转移酶到甲基丙二酰辅酶A变位酶的直接转移的动力学是什么？我们将确定和比较B12与溶液中的变位酶结合的动力学和热力学参数与其直接从腺苷转移酶转移的动力学和热力学参数，以测试我们的模型，即B12的传递是伴随的。(Iii)MeaB和MMAA如何通过腺苷转移酶调节ADOCb1的合成及其向甲基丙二酰辅酶A变位酶的转移？我们将使用体外(与患者细胞系)和体外(在变位酶和MMAA中有有限数量的患者突变)相结合的研究来评估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的直接Adobe Cbl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.