Protein Dynamics in Dihydrofolate Reductase Catalysis

二氢叶酸还原酶催化中的蛋白质动力学

• 批准号: 7014242
• 负责人: PETER Edwin WRIGHT
• 金额: $ 32.63万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014242
• 关键词: NAD(H) phosphate; active sites; bacterial proteins; bioenergetics; chemical kinetics; conformation; dihydrofolate reductase; enzyme activity; enzyme substrate; gene mutation; intermolecular interaction; ligands; nuclear magnetic resonance spectroscopy; protein metabolism; thermodynamics; time resolved data

DESCRIPTION (provided by applicant): Proteins are dynamic molecular machines, undergoing motions on a wide range of time scales. Although there is considerable evidence, both from theory and experiment, that many enzymes are inherently flexible, the fundamental question of how, or even if, protein fluctuations couple to catalytic function remains unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? What is the time scale of active site conformational changes required for catalysis? How is the energy landscape of the enzyme modulated during the catalytic cycle? These and other issues will be addressed using state-of-the-art NMR methods to elucidate the dynamic properties of an exceptionally well- characterized enzyme, dihydrofolate reductase from E. coli, in all of the intermediate states formed along its reaction pathway. DHFR is the target for anti-folate drugs, such as the anticancer agent methotrexate and the antibacterial trimethoprim, and is of major biomedical significance. The proposed research will focus on characterization of microsecond-millisecond time scale fluctuations in the active site, on the same time scale as key events in DHFR catalysis. The NMR experiments will provide a detailed dynamic and thermodynamic description of slow motions of the active site loops in all intermediate states involved in the catalytic cycle and in carefully selected mutants that impair the catalytic process. These experiments build upon earlier research from this laboratory that mapped the active site conformational states of DHFR and its fast (ps-ns) time scale dynamics. The proposed research will provide novel insights into the coupling between protein motions and catalytic function in DHFR, as well as an understanding of how these motions are modulated by interaction with substrate, cofactor, and products at various stages in the catalytic cycle.

描述（由申请人提供）：蛋白质是动态分子机器，在广泛的时间尺度上进行动作。尽管从理论和实验中都有大量证据表明，许多酶本质上是灵活的，这是蛋白质波动如何催化功能的基本问题，或者即使是催化功能。蛋白质运动是否与化学转化相结合，还是主要控制底物，产物或辅因子的通量？催化所需的主动部位构象变化的时间尺度是什么？在催化循环中，酶的能量格局如何调节？这些问题和其他问题将使用最先进的NMR方法解决，以阐明沿其反应途径形成的所有中间状态中异常表征良好的酶，来自大肠杆菌的二氢叶酸还原酶。 DHFR是抗叶酸药物的靶标，例如抗癌剂甲氨蝶呤和抗菌甲甲氧酰甲甲氧备，并且具有主要的生物医学意义。拟议的研究将集中于活动位点中微秒毫秒时尺度波动的表征，与DHFR催化中的关键事件相同。 NMR实验将提供详细的动态和热力学描述，以描述参与催化循环的所有中间状态以及损害催化过程的精心选择的突变体中的活性位点循环缓慢运动。这些实验基于该实验室的早期研究，该研究绘制了DHFR及其快速（PS-NS）时间尺度动力学的活跃位点构象状态。拟议的研究将为DHFR中的蛋白质运动与催化功能之间的耦合提供新的见解，以及了解这些运动如何通过与底物，辅助因子和催化循环各个阶段的相互作用来调节这些运动的方式。