Co(II) Radical Pair Dynamics in B12 Enzyme Catalysis

项目摘要 这项拟议研究的目标是阐明基本的物理化学 控制酶催化的原理，重点是自由基催化 辅酶B12(腺苷钴胺)依赖的酶。这个目标将是 通过一项生化和物理研究计划制定的 所有三类B12酶的代表：甲基丙二酰辅酶A变位酶 (MCM，第I类；人和甲基勒索杆菌)，乙醇胺氨- 裂解酶(EAL，II类；鼠伤寒沙门氏菌)和赖氨酸-5，6-氨基变位酶 (III类；LAM，Sticklandii梭菌)。这些方法将扩展到 探讨钴胺(B12)胞内转运的分子机制 通过CBLC和CblD蛋白在人类中的途径，并组装和 细菌微室(BMC)的功能，这是EAL的体内背景 酵素。高分辨率脉冲激光成像的创新方法、软件和硬件 电子顺磁共振(EPR)谱，与时间平行- 拆分的单步反应动力学和稳态溶剂动力学 连续波EPR方法，应用于独特的低温系统，将 实现对结构和动态的全面表征 溶剂-蛋白质-反应耦合对自由基重排的贡献 B12酶的催化作用。重大的生物医学和人类健康结果 包括：(1)关于蛋白质和偶联溶剂作用的基本知识 酶功能的构型状态，在生理温度下潜伏， 为开发路线图贡献新的工具、模型和目标 在药物开发和酶工程中利用这些状态，(Ii) EAL是肠道微生物群在人类中发挥作用的贡献者，其特征 健康和疾病进展，包括与炎症性肠病的联系， 肥胖症和糖尿病，(Iii)细胞的组装和功能的特征 乙醇胺利用(EUT)BMC，是最近公认的、广泛使用的 一类原始的、被蛋白质包裹的细菌细胞器，(Iv)对 人类代谢性疾病的分子机制基础与CBL 基因簇，特别是人类CBLC(MMACHC， 甲基丙二酸尿C型和同型半胱氨酸尿症)和CblD蛋白。

Project Summary The goal of the proposed research is to elucidate fundamental physical-chemical principles that govern catalysis in enzymes, with a focus on radical catalysis in coenzyme B12 (adenosylcobalamin) –dependent enzymes. This goal will be enacted through a program of biochemical and physical studies of representatives of all three classes of B12 enzymes: methylmalonyl-CoA mutase (MCM, Class I; human and Methylobacter extorquens), ethanolamine ammonia- lyase (EAL, Class II; Salmonella typhimurium), and lysine-5,6-aminomutase (Class III; LAM, Clostridium stricklandii). The approaches will be extended to address molecular mechanism in the intracellular cobalamin (B12) trafficking pathway in humans by the CblC and CblD proteins, and to the assembly and function of the bacterial microcompartment (BMC), an in vivo context of the EAL enzyme. Innovative methods, sofware and hardware for high-resolution pulsed- electron paramagnetic resonance (EPR) spectroscopy, in parallel with time- resolved, single-step reaction kinetics and steady-state solvent dynamics from continuous-wave EPR methods, applied in unique, low-temperature systems, will enable comprehensive characterization of the structural and dynamical contributions of solvent-protein-reaction coupling to radical rearrangement catalysis in the B12 enzymes. Significant biomedical and human health outcomes include: (i) fundamental knowledge about the role of protein and coupled solvent configurational states in enzyme function, latent at physiological temperatures, that contribute new tools, models and targets to the developing roadmap for leveraging these states in drug development and enzyme engineering, (ii) characterization of EAL, a contributor to the role of the gut microbiome in human health and disease progression, including links with inflammatory bowel disease, obesity, and diabetes, (iii) characterization of the assembly and function of the ethanolamine utilization (eut) BMC, one of a recently-recognized, widespread class of primitive, protein-encased bacterial organelles, (iv) insights into the molecular mechanistic basis of human metabolic disorders identified with the cbl gene cluster, and in particular, the role of the human CblC (MMACHC, methylmalonic aciduria type C and homocystinuria) and CblD proteins.