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: 8361798
• 负责人: JOHN GLUSHKA
• 金额: $ 0.18万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361798
• 关键词: Biochemical Process; Biochemical Reaction; Cell Wall; Cell surface; Cells; Cryptococcus neoformans; Data; Enzyme Kinetics; Enzymes; Eukaryota; Funding; Goals; Golgi Apparatus; Grant; Kinetics; Label; Measurement; Measures; Metabolic Pathway; Methodology; Modeling; National Center for Research Resources; Organelles; Pathway interactions; Physiological; Pisum sativum; Plants; Polysaccharides; Preparation; Principal Investigator; Process; Reaction; Relative (related person); Research; Research Infrastructure; Resources; Source; Staging; Structure; System; Testing; Time; Tube; United States National Institutes of Health; cost; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 致病真菌和植物具有装饰其细胞表面的丰富多种多样的多糖（PS）。令人惊讶的是，关于多糖合成及其组装的生化过程知之甚少。总体目的是了解在病原体（例如新虫类球菌）中发现的必要多糖所涉及的途径和动力学。 NMR可以在实时分析酶促反应中检测中间体和产品。对底物，中间体和产品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，并且数据可用于建模代谢途径。由于这些反应混合物的复杂性以及对高灵敏度的需求，使用了非常高的NMR光谱仪（800-900 MHz）。 第一阶段是通过在含有酶和活化糖组合的NMR管中测量底物和产品通量来构建动力学模型。 第二阶段是检查细胞器制剂中的类似通量，例如高尔基体或整个细胞。 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）中的核苷酸糖中形成细胞壁多糖，（2）在Tremella Mesenterica中形成外核酸糖。另外，正在研究纯化的酶系统以测试方法。 由于细胞器分数的数量和易于制备，植物系统之所以有用。螺丝菌真菌是一种非致病的单细胞真核生物，它合成了大量的多糖在结构上与致病性隐孢子虫中多糖非常相似的多糖。 这些实验将包括在不同的孵育时间内识别酶促中间体，确定这些反应的酶动力学，并在形成多糖产物之后。通过将动态过程的分析与特定细胞室鉴定的生长多糖的结构信息结合在一起，可以识别合成中所涉及的步骤。可以检查影响和控制前体吸收的因素，多糖的伸长和向外壁的出口。