Co(II) Radical Pair Dynamics in B12 Enzyme Catalysis

描述(由申请人提供)：拟议研究的长期目标是阐明在金属-自由基对产生和耦合自由基介导的反应的背景下，控制酶中催化的基本分子原理。这一目标将通过对鼠伤寒沙门氏菌中依赖辅酶B12(腺苷钴胺)的乙醇胺解氨酶进行全面的生化和物理研究来实现。我们开发了脉冲电子顺磁共振(EPR)波谱的新方法、新软件和新硬件。结合生化技术，我们开发了低温系统和时间分辨EPR方法，使开创性的实验工作能够直接解决乙醇胺解氨酶中的一步分解共价键断裂/成键反应。总体目标是定义指导反应的自由能图景，确定蛋白质和耦合溶剂波动的起源和作用，推动系统通过这一图景，并解析构成自由能图景的埃尺度分子结构。这些项目开发了一个被提议的酶催化的“构型催化”模型，并试图建立自由基重排作为一个范例，以确定在酶反应中对化学步骤的景观和蛋白质-溶剂的运动贡献。溶剂结构和动力学对酶反应的贡献也通过一套EPR光谱和松弛技术得到解决。研究结果将影响生物医学和人类健康的分子方法，包括抗微生物治疗、催化剂、抑制剂和生物纳米隔间的合理设计，以及自由基反应的控制。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to elucidate fundamental molecular principles that govern catalysis in enzymes, in the context of metal-radical pair generation and coupled radical-mediated reactions. This goal will be enacted through a comprehensive program of biochemical and physical studies of the coenzyme B12 (adenosylcobalamin) - dependent enzyme, ethanolamine ammonia-lyase, from Salmonella typhimurium. We have developed new methods, software and hardware for pulsed-electron paramagnetic resonance (EPR) spectroscopy. In conjunction with biochemical techniques, we have developed low-temperature systems and time- resolved EPR approaches that enable pioneering experimental efforts that address, directly, covalent bond- breaking/bond-making reactions in ethanolamine ammonia-lyase at single-step resolution. The general aims are to define the free energy landscape that guides the reactions, identify the origins and characterize the roles of the protein and coupled solvent fluctuations that propel the system over this landscape, and resolve the Angstrom-scale molecular structures that frame the free energy landscape. The projects develop a proposed "configurational catalysis" model for enzyme catalysis, and seek to establish the radical rearrangement as a paradigm for defining landscape and protein-solvent motional contributions to chemical steps in enzyme reactions. Contributions of solvent structure and dynamics to the enzyme reactions are also addressed by a suite of EPR spectroscopic and relaxation techniques. The outcomes of the research will impact molecular approaches in biomedicine and human health, including anti- microbial therapies, rational design of catalysts, inhibitors, and bio-nanocompartments, and control of free radical reactions.