Solid State Nuclear Magnetic Resonance Studies of TIM

描述（由申请人提供）：三磷酸异构酶是一种催化上坡反应的异构酶，为稳定和探测Michaelis复合物的关键动力学和结构特征提供了绝佳的机会。该建议利用了我们最近在稳定Michaelis复合物方面的进展，以及我们对该系统的一系列生物物理探针的优化，以便可以研究单个动力学事件和中间体。Michaelis配合物的1.2埃x射线衍射显示了一个高度压缩的活性位点，具有异常短的氢键和意想不到的底物扭转态。最近的数据，利用三种光谱方法，表明环打开和产物释放是决定酶通量的速率，并提出环打开可能由酶反应完成触发的假设。以下问题现在已经成熟。(1)一般碱基谷氨酸165和一般酸组氨酸95被替代的氢键伴侣取代的突变体催化活性不高。活性位点氨基酸的选择如何影响米切里斯复合物的压缩和底物的极化？(2) Michaelis复合体中有哪些化学物质？它们的浓度如何随温度变化？所提出的烯二酮类化学中间体是否具有显著的种群，正如根据同位素冲刷所预期的那样？底物虽然通过氢键“固定”在活性位点上，但在碳中心似乎是可移动的，这表明有活性重排。化学反应速率是否比环的运动快，正如最小的初级同位素效应所暗示的那样？(3) Michaelis复合物蛋白活性位点的构象和电离变化是什么？沿着化学反应坐标的进展有什么变化？在反应的初始阶段，一般碱E165是如何定位的？(4)配体的变化或强保守环残基的突变对环开率和催化有什么影响？这些问题将通过静态和动态的固态核磁共振、振动光谱和x射线衍射实验来解决。

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 .