Structural Fluctuations in the Catalytic Cycle of DHFR

DHFR 催化循环中的结构波动

• 批准号: 6648999
• 负责人: Daniel McElheny
• 金额: $ 4.64万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6648999
• 关键词: catalyst; cofactor; conformation; dihydrofolate reductase; enzyme mechanism; enzyme structure; enzyme substrate; nuclear magnetic resonance spectroscopy; postdoctoral investigator; protein purification; radiotracer; structural biology; tetrahydrofolates

DESCRIPTION (provided by applicant): Dihyrdrofolate reductase (DHFR) plays a central role in maintaining intracellular levels of tetrahydrofolate through the NADPH-dependant reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF). THF is required for normal cellular metabolism since it serves as a precursor in synthesis of several metabolites. Consequently DHFR serves as an important drug target for rapidly proliferating disease such as cancer and bacterial infections. A detailed structural picture exists for intermediate steps throughout the complex catalytic cycle providing a unique basis to correlate structure with dynamics, hence providing a deeper understanding of the catalytic cycle. NMR studies will be implemented to study relatively slow molecular motions (us-ms) for the various DHFR complexes. Highly developed Carr-PurcelI-Meiboom-Gill (CPMG) NMR experiments are well suited for studying motions at the millisecond time scale and will be implemented as such. Molecular motions occurring within this regime are thought to be of utmost importance as this is the rate at which the critical hydride transfer step occurs (~ 950/s). After establishing the backbone dynamics of the enzyme, an alternative look at motions from the substrate and cofactor will also be attempted by isotopic labeling of each for study by NMR.

描述（由申请人提供）：二氢叶酸还原酶（DHFR）通过nadph依赖性地将7,8-二氢叶酸（DHF）还原为5,6,7,8-四氢叶酸（THF），在维持细胞内四氢叶酸水平中起核心作用。四氢呋喃是正常细胞代谢所必需的，因为它是几种代谢物合成的前体。因此，DHFR作为快速增殖疾病（如癌症和细菌感染）的重要药物靶点。在整个复杂的催化循环中，中间步骤的详细结构图为将结构与动力学联系起来提供了独特的基础，从而提供了对催化循环的更深层次的理解。核磁共振研究将用于研究各种DHFR复合物的相对慢分子运动（us-ms）。高度发达的carr - purceli - meiboomm - gill （CPMG）核磁共振实验非常适合研究毫秒时间尺度的运动，并将这样实施。在这个范围内发生的分子运动被认为是最重要的，因为这是发生临界氢化物转移步骤的速率（~ 950/s）。在建立了酶的主链动力学之后，还将尝试通过核磁共振同位素标记来研究底物和辅因子的运动。