Solid State Nuclear Magnetic Resonance Studies of TIM

描述（由申请人提供）：磷酸丙糖异构酶是一种催化上坡反应的异构酶，提供了稳定和探测米氏复合物的关键动力学和结构特征的主要机会。该建议利用了我们最近在稳定米氏络合物方面的进展，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。在1.2埃的米氏络合物的X-射线衍射显示了一个高度压缩的活性位点与异常短的氢键和一个意想不到的基板扭转状态。最近的数据，利用三种光谱方法，表明开环和产物释放是速率决定酶的吞吐量，并邀请环开放可能是由酶促反应的完成触发的假设。以下问题的处理时机现已成熟。(1)其中通用碱基谷氨酸165和通用酸性组氨酸95被替代氢键配偶体取代的突变体是无催化活性的。活性位点氨基酸的选择如何影响米氏络合物的压缩和底物的极化？(2)米氏络合物中的化学物质是什么，它们的浓度如何取决于温度？拟定的烯二醇盐化学中间体是否具有显著的群体，如基于同位素洗脱所预期的那样？该基板，而“钉扎”通过氢键的活性位点，似乎是移动的在碳中心，表明一个积极的重排。化学反应速率是否比环的运动快，就像最小的主要同位素效应所暗示的那样？(3)米氏复合物蛋白质活性位点的构象和电离变化是什么，以及沿着化学反应坐标的进展伴随着什么变化？如何将通用底座E165定位于反应的初始阶段？(4)配体的变化或高度保守的环残基的突变对环开放速率和催化作用有什么影响？这些问题将通过固态核磁共振，振动光谱和X射线衍射实验，静态和动态的追求。

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 .