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

关于它的结构、动力学和催化机理的问题 丝氨酸蛋白酶将使用氮-15和碳13核磁共振进行处理。在……里面 一般，特别标记的酶衍生物，通过以下途径获得 将采用生物合成富集化技术，以便于 检测和解析所需的信号，浓缩的酶将 在溶液和固体两种状态下都要研究。固态研究将会 包括记录粉末光谱和通过 幻角样本旋转(质量)。冻干粉和冻干粉 我们将检查酶的结晶形态。这一行动的具体目标 建议包括：(1)表征活性中心的性质 官能团，特别注意评价 这些性质对催化的意义。这些属性包括 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。(2) 结晶学中各元素之间的对应关系的测定 确定的结构和溶液中平衡结构的结构。 (3)缓蚀剂结合对其性能影响的测定 活动站点功能组。(4)发展使用方法 核磁共振直接研究酶-底物复合体。

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.