APPLICATION OF NMR TO THE STUDY OF ENZYME SYSTEMS

核磁共振在酶系统研究中的应用

• 批准号: 8168858
• 负责人: JOHN GLUSHKA
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-10 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168858
• 关键词: Biochemical Process; Biochemical Reaction; Cell Wall; Cell surface; Cells; Computer Retrieval of Information on Scientific Projects Database; Cryptococcus neoformans; Data; Enzyme Kinetics; Enzymes; Eukaryota; Funding; Goals; Golgi Apparatus; Grant; Institution; Kinetics; Label; Measurement; Measures; Metabolic Pathway; Methodology; Modeling; Organelles; Pathway interactions; Physiological; Pisum sativum; Plants; Polysaccharides; Preparation; Process; Reaction; Relative (related person); Research; Research Personnel; Resources; Source; Staging; Structure; System; Testing; Time; Tube; United States National Institutes of Health; enzyme model; fungus; pathogen; plant fungi; research study; sugar; sugar nucleotide; uptake

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pathogenic fungi and plants have abundant and diverse types of polysaccharides (PS) decorating their cell surfaces. Surprisingly, little is known regarding the biochemical process involved in polysaccharide synthesis and their assembly. The overall goal is to understand the pathways and kinetics involved in synthesizing necessary polysaccharides found in pathogens such as Cryptococcus neoformans. NMR can detect intermediates as well as products in real-time analysis of enzymatic reactions. A quantitative measurement of kinetics and relative concentrations of substrates, intermediates and products can support a detailed model of the enzyme under physiological conditions. By extrapolation, multi-enzymatic systems can also be studied and the data can be used to model metabolic pathways. Very high-field NMR spectrometers (800-900 MHz) are being used due to the complexity of these reaction mixtures and the need for high sensitivity. The first stage is to build kinetic models by measuring substrate and product fluxes in the NMR tube containing combinations of enzymes and activated sugars. The second stage is to examine similar fluxes in organelle preparations such as Golgi, or whole cells. C13-labeled substrates will be used to simplify the spectral data. Two test systems are being investigated; 1) formation of cell-wall polysaccharides from nucleotide-sugars in plants (e.g. pea) and 2) formation of exo-polysaccharides in Tremella mesenterica. In addition, purified enzyme systems are being studied to test the methodology. The plant systems are useful because of the quantity and ease of preparation of the organelle fractions. The Tremella fungus is a non-pathogenic single cell eukaryote that synthesizes large quantities of a polysaccharide very similar in structure to polysaccharides in the pathogenic Cryptoccoccus. The experiments will include identifying the enzymatic intermediates during different incubation times, determining enzyme kinetics of these reactions, and following formation of polysaccharide products. By combining analyses of dynamic processes with structural information of the growing polysaccharide identified with particular cell compartments, the steps involved in the synthesis can be identified. Factors influencing and controlling the uptake of precursors, the elongation of the polysaccharide and the export to the outer wall can be examined.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 病原真菌和植物的细胞表面装饰着丰富多样的多糖（PS）。令人惊讶的是，人们对多糖合成及其组装所涉及的生化过程知之甚少。总体目标是了解合成新型隐球菌等病原体中必需多糖所涉及的途径和动力学。 NMR 可以在酶反应的实时分析中检测中间体和产物。底物、中间体和产物的动力学和相对浓度的定量测量可以支持生理条件下酶的详细模型。 通过外推，还可以研究多酶系统，并且数据可用于模拟代谢途径。由于这些反应混合物的复杂性和对高灵敏度的需求，正在使用极高场核磁共振波谱仪 (800-900 MHz)。 第一阶段是通过测量含有酶和活性糖组合的 NMR 管中的底物和产物通量来建立动力学模型。 第二阶段是检查细胞器制剂（例如高尔基体或全细胞）中的类似通量。 C13 标记的底物将用于简化光谱数据。 正在研究两个测试系统； 1) 从植物（例如豌豆）中的核苷酸糖形成细胞壁多糖，以及 2) 在银耳中形成外多糖。此外，正在研究纯化的酶系统以测试该方法。 由于细胞器级分的数量和制备的容易性，植物系统是有用的。银耳真菌是一种非致病性单细胞真核生物，可合成大量多糖，其结构与致病性隐球菌中的多糖非常相似。 实验将包括鉴定不同孵育时间期间的酶中间体、确定这些反应的酶动力学以及多糖产物的形成。通过将动态过程的分析与特定细胞区室识别的生长多糖的结构信息相结合，可以识别合成中涉及的步骤。可以检查影响和控制前体的吸收、多糖的伸长和向外壁的输出的因素。