The Catalytic Mechanism of Oxalate Decarboxylase Studied by Advanced EPR Experiments

无机化学、生物无机化学和金属有机化学项目的这项研究奖支持佛罗里达大学的Alexander Angerhofer教授研究细菌酶草酸脱羧酶(OxDC)催化草酸中碳-碳键断裂的分子机制。它存在于肠道细菌中，有助于防止肾结石的形成。本项目将使用先进的电子顺磁共振(EPR)技术来研究蛋白质亚基中两个锰离子的电子结构和局部配位环境。将使用高达25特斯拉的高磁场的多频率方法来识别活动地点。这些实验将产生有关底物和抑制剂与酶结合的重要动力学、热力学和结构信息，并测试当前的工作假说，即锰和相关的结合氧分子在草酸盐C-C键的断裂中充当氧化还原穿梭。冷冻-淬火实验将被用来研究酶-底物相互作用的预平衡动力学，并产生催化过程的随时间变化的快照。虽然OxDC的研究本身是有意义的，并可能导致未来治疗各种草酸相关的病理(高草酸尿)，但该酶也将成为蛋白质如何调节和影响催化反应机制的一个例子。已经产生了定点突变，它们降低了草酸脱羧酶，但提高了草酸氧化酶的活性。与这些突变体的合作将有助于阐明活性部位的结构如何指导化学。该项目与佛罗里达大学HHMI资助的生命科学倡议紧密结合在一起，该倡议通过尽早将本科生带入研究实验室来服务于本科生。预计至少有五名最有前途的本科理科专业学生将在学习初期参与到这个项目中来。他们将接受Angerhofer博士和他的合作者的密集指导，并有机会进行尖端研究，这不仅将丰富他们的教育，还将为他们提供未来在自然科学领域的职业前景。

This research award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports work by Professor Alexander Angerhofer at the University of Florida to study the molecular mechanism by which the bacterial enzyme oxalate decarboxylase (OxDC) catalyzes the cleavage of the carbon-carbon bond in oxalic acid. Its presence in gut bacteria helps prevent the formation of kidney stones. Advanced electron paramagnetic resonance (EPR) techniques will be used in this project to study the electronic structure and local coordination environment of its two Mn-ions in the protein subunit. A multi-frequency approach using high magnetic fields of up to 25 Tesla will be used to identify the active site. These experiments will yield important kinetic, thermodynamic, and structural information about substrate and inhibitor binding to the enzyme and test the current working hypothesis in which Mn and an associated bound oxygen molecule act as redox shuttles in the breaking of the oxalate C-C bond. Freeze-quench experiments will be employed to investigate the pre-equilibrium kinetics of enzyme-substrate interaction and yield time-dependent snapshots of the catalytic process. While the investigation of OxDC is of interest in its own right and may lead to future treatments of various oxalate-related pathologies (hyperoxaluria), the enzyme will also serve as an example of how the protein can modulate and affect catalytic reaction mechanisms. Site-directed mutants have been generated that have decreased oxalate decarboxylase but increased oxalate oxidase activity. Work with these mutants will help to elucidate how the structure of the active site can guide the chemistry. This project is tightly integrated into the HHMI-funded Science for Life initiative at the University of Florida which serves its undergraduate population by bringing them into the research lab early on. At least five of the most promising undergraduate science majors are expected to be involved in this project early in their years of study. They will receive intensive mentoring by Dr. Angerhofer and his collaborators and be given the opportunity to perform cutting-edge research which will not only enrich their education but also provide for them a perspective for a future career in the natural sciences.