NITROGEN-15 & CARBON-13 NMR STUDIES OF SERINE PROTEASES

氮15

• 批准号: 3071137
• 负责人: WILLIAM W BACHOVCHIN
• 金额: $ 4.81万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-02-01 至 1988-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3071137
• 关键词: Bacillus; Bacillus subtilis; Streptomyces; aspartate; chemical group; enzyme structure; enzyme substrate complex; histidine; hydrogen bond; hydroxyl group; nuclear magnetic resonance spectroscopy; peptidases; protease inhibitor; protonation; serine; solid state; solutions; subtilisins; tautomer; trypsin

Questions pertaining to the structure, dynamics, and catalytic mechanism of serine proteases will be addressed using nitrogen-15 and carbon 13 NMR. In general, specifically labelled enzyme derivatives, obtained through biosynthetic enrichment techniques, will be employed in order to facilitate detection and resolution of desired signals, and the enriched enzymes will be studied in both solution and solid states. The solid state studies will include recording both powder spectra, and high resolution spectra through magic angle sample spinning (MASS). Both lyophilized powder and crystalline forms of the enzyme will be examined. Specific aims of this proposal include: (1) Characterizing the properties of the active site functional groups, with special attention given to evaluating the significance of these properties for catalysis. These properties include microscopic pka's, hydrogen-bond interactions, tautomeric structure, functional group dynamics, and dynamics of proton exchange. (2) Determination of the correspondance between the crystallographically determined structure and that of the equilibrium structure in solution. (3) Determination of the effect of inhibitor binding on the properties of the active site functional groups. (4) Development of methods for using NMR to directly study enzyme-substrate complexes.

与结构、动力学和催化机制有关的问题 丝氨酸蛋白酶将使用氮-15和碳-13 NMR来处理。 在 一般的，特异性标记的酶衍生物，通过 生物合成富集技术，将采用，以促进 所需信号的检测和解析，并且富集的酶将 可以在溶液和固体状态下进行研究。 固态研究将 包括记录粉末光谱和高分辨率光谱， 魔角试样纺丝（MASS）。 冻干粉和 将检查酶的结晶形式。 具体目标 提出的建议包括：（1）表征活性中心的性质 职能组，特别注意评价 这些性质对催化的重要性。 这些性能包括 微观PKA，氢键相互作用，互变异构结构， 官能团动力学和质子交换动力学。 （二） 晶体学上的对应性测定 确定的结构和溶液中的平衡结构。 (3)测定抑制剂结合对聚合物的性质的影响 活性位点官能团。 (4)制定使用方法 NMR直接研究酶-底物复合物。