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: 7591836
• 负责人: PETER Edwin WRIGHT
• 金额: $ 32.29万
• 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/7591836
• 关键词: Active Sites; Address; Amino Acids; Anabolism; Anti-Bacterial Agents; Antineoplastic Agents; Arts; Binding; Catalysis; Chemicals; Chemistry; Complex; Coupled; Coupling; Data; Dihydrofolate Reductase; Enzymes; Escherichia coli; Event; Folate; Goals; Holoenzymes; Kinetics; Laboratory Research; Ligands; Maps; Measurement; Measures; Methods; Methotrexate; Modeling; Molecular Conformation; Molecular Machines; Motion; Movement; NADP; Pathway interactions; Pharmaceutical Preparations; Play; Population; Process; Property; Protein Dynamics; Proteins; Purines; Reaction; Relaxation; Research; Residual state; Role; Side; Solutions; Staging; Structure; Testing; Tetrahydrofolates; Thermodynamics; Time; Trimethoprim; Vertebral column; Work; base; cofactor; flexibility; inhibitor/antagonist; insight; millisecond; molecular dynamics; mutant; novel; polypeptide; protein function; purine; research study; sound; theories; three dimensional structure; thymidylate

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.

蛋白质是动态的分子机器，在广泛的时间尺度上进行运动。 虽然有大量的理论和实验证据表明，许多酶是 这是一个本质上灵活的基本问题，即蛋白质波动如何或是否与 催化剂的功能仍然没有答案。蛋白质运动是否与化学转化相关联， 还是它们主要参与控制底物、产物或辅因子的通量？是什么 催化所需的活性部位构象变化的时间尺度？能源前景如何 在催化循环中调节的酶的活性这些问题和其他问题将通过 国家的最先进的核磁共振方法，以阐明一个非常好的动态特性- 特征酶，二氢叶酸还原酶来自E.大肠杆菌，在所有的中间状态形成 沿着其反应途径。DHFR是抗叶酸药物如抗癌剂的靶点 甲氨蝶呤和抗菌剂甲氧苄氨嘧啶，具有重要的生物医学意义。的 拟议的研究将集中在微秒-毫秒时间尺度波动的特征上 在活性位点，在相同的时间尺度上的关键事件在DHFR催化。核磁共振实验 将提供一个详细的动态和热力学描述的缓慢运动的活动网站 在所有参与催化循环的中间状态和精心挑选的突变体中， 损害催化过程。这些实验建立在这个实验室早期的研究基础上， 绘制了DHFR的活性位点构象状态及其快速（ps-ns）时间尺度动力学。的 拟议的研究将提供新的见解蛋白质运动和催化之间的耦合， 在DHFR中的作用，以及对这些运动如何通过相互作用调节的理解 与底物、辅因子和产物在催化循环的各个阶段。