Solid State Nuclear Magnetic Resonance Studies of TIM

TIM 的固态核磁共振研究

• 批准号: 6785219
• 负责人: ANN E MCDERMOTT
• 金额: $ 19.92万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6785219
• 关键词: Raman spectrometry; X ray crystallography; active sites; aminoacid; catalyst; chemical kinetics; compression; conformation; enzyme complex; fungal proteins; gene mutation; infrared spectrometry; nuclear magnetic resonance spectroscopy; protein purification; solid state; temperature; triose phosphate isomerase

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 .

描述（由申请人提供）：磷酸三糖异构酶是一种催化艰苦反应的异构酶，为稳定和探测米氏复合物的关键动力学和结构特征提供了绝佳的机会。该提案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。米氏络合物 1.2 埃处的 X 射线衍射显示出高度压缩的活性位点，具有异常短的氢键和意想不到的基底扭转状态。最近的数据，利用三种光谱方法，表明环打开和产物释放是酶促通量的速率决定因素，并提出这样的假设：环打开可能是由酶促反应的完成触发的。现在解决以下问题的时机已经成熟。 (1)其中通用碱谷氨酸165和通用酸组氨酸95被替代氢键配偶体取代的突变体是催化失活的。活性位点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) 米氏络合物中有哪些化学物质，它们的浓度如何取决于温度？所提出的烯二醇化学中间体是否具有显着的群体，正如根据同位素洗脱所预期的那样？底物虽然通过氢键“固定”到活性位点，但似乎在碳中心是可移动的，这表明存在主动重排。正如最小的初级同位素效应所表明的那样，化学反应速率是否比环的运动更快？ (3) 米氏复合物的蛋白质活性位点发生了哪些构象和电离变化，以及沿着化学反应坐标的进展伴随着哪些变化？一般碱基E165在反应初期是如何定位的？ (4) 配体的变化或高度保守的环残基的突变会对环开率和催化产生什么影响？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）来解决。