Structural Fluctuations in the Catalytic Cycle of DHFR

描述（由申请人提供）：二氢叶酸还原酶（DHFR）通过NADPH依赖性还原7，8-二氢叶酸（DHF）为5，6，7，8-四氢叶酸（THF），在维持细胞内四氢叶酸水平方面发挥重要作用。THF是正常细胞代谢所必需的，因为它在几种代谢物的合成中充当前体。因此，DHFR作为一个重要的药物靶点，用于快速增殖的疾病，如癌症和细菌感染。一个详细的结构图片存在的中间步骤，在整个复杂的催化循环提供了一个独特的基础，结构与动力学相关联，从而提供了一个更深入的了解催化循环。将实施NMR研究以研究各种DHFR复合物的相对慢的分子运动（us-ms）。高度发达的Carr-PurcelI-Meiboom-Gill（CPMG）NMR实验非常适合于研究毫秒时间尺度的运动，并将这样实施。在此范围内发生的分子运动被认为是最重要的，因为这是临界氢化物转移步骤发生的速率（~ 950/s）。在建立了酶的骨架动力学之后，还将尝试通过同位素标记来观察底物和辅因子的运动，以通过NMR进行研究。

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.