Solid State Nuclear Magnetic Resonance Studies of TIM

描述(申请人提供)：磷酸丙糖异构酶是一种催化上坡反应的异构酶，为稳定和探索米氏复合体的关键动力学和结构特征提供了绝佳的机会。这一建议利用了我们在稳定米氏复合体方面的最新进展，以及我们对该系统的一系列生物物理探测器的优化，以便可以研究单个动力学事件和中间体(S)。米氏络合物1.2埃的X射线衍射谱显示出一个高度压缩的活性中心，具有异常短的氢键和意想不到的衬底扭转状态。最近的数据，利用三种光谱方法，表明环的打开和产物的释放是酶吞吐量的速率决定因素，并提出了环的打开可能是由酶反应完成触发的假设。以下问题现在已经成熟，可以继续研究了。(1)将普通碱性谷氨酸165和普通酸性组氨酸95替换为替代氢键配对的突变体是催化失活的。活性部位氨基酸的选择如何影响米氏复合体的压缩和底物的极化？(2)米氏复合体中的化学物种是什么，它们的浓度如何依赖于温度？根据同位素洗脱的结果，所提议的黑云母化学中间体是否有大量的种群？底物虽然通过氢键固定在活性中心，但似乎在碳中心是可移动的，这暗示着一种活跃的重排。根据最小的初级同位素效应，化学反应速度是否快于环的运动？(3)米氏复合体中蛋白质活性部位的构象和电离变化是什么，以及伴随着化学反应坐标的变化是什么？总碱基E165在反应的初始阶段是如何定位的？(4)配体的变化或高度保守的环残基的突变对开环率和催化有什么影响？这些问题将通过固体核磁共振、振动光谱和X射线衍射实验来探索，包括静态和动态实验。

DESCRIPTION (provided by applicant): Triosephosphate isomerase, an isomerase catalyzing an uphill reaction, offers a prime opportunity to stabilize and probe key kinetic and structural features of a Michaelis complex. This proposal capitalizes on our recent advances in stabilizing the Michaelis complex, and our optimization of a range of biophysical probes for the system, so that individual kinetic events and intermediate(s) can be studied. X-ray diffraction at 1.2Angstroms of the Michaelis complex shows a highly compressed active site with unusually short hydrogen bonds and an unexpected substrate torsional state. Recent data, utilizing three spectroscopic methods, indicate that loop opening and product release is rate determining for enzymatic throughput, and invite the hypothesis that loop opening may be triggered by the completion of the enzymatic reaction. The following issues are now ripe for pursuit. (1) Mutants in which the general base glutamic acid 165 and the general acid histidine 95 are replaced with alternative hydrogen bond partners are catalytically inactive. How does selection of the active site amino acids influence compression in the Michaelis complex and polarization of the substrate? (2) What are the chemical species in the Michaelis complex, and how do their concentrations depend upon temperature? Does the proposed enediolate chemical intermediate have a significant population, as might be expected based upon isotope washout? The substrate, while "pinned" through hydrogen bonds to the active site, appears to be mobile at the carbon centers, suggestive of an active rearrangement. Are the chemical reaction rates faster than the loop's motion, as suggested by the minimal primary isotope effects? (3) What are the conformational and ionization changes in the active site of the protein for the Michaelis complex, and what changes accompany the progress along the chemical reaction coordinate? How is the general base E165 positioned for the initial stage of the reaction? (4) What implications would changes in the ligand or mutations in the strongly conserved loop residues have for loop opening rates and for catalysis? These questions would be pursued through solid state NMR, vibrational spectroscopy and X-ray diffraction experiments, both static and dynamic .