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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 病原真菌和植物的细胞表面含有丰富多样的多糖。令人惊讶的是，很少有人知道有关的生化过程中涉及多糖的合成和组装。总体目标是了解参与合成在病原体如新型隐球菌中发现的必要多糖的途径和动力学。 NMR可以在酶促反应的实时分析中检测中间体和产物。动力学和底物，中间体和产品的相对浓度的定量测量可以支持在生理条件下的酶的详细模型。 通过外推，还可以研究多酶系统，并且数据可以用于模拟代谢途径。由于这些反应混合物的复杂性和对高灵敏度的需要，正在使用非常高的场NMR光谱仪（800-900 MHz）。 第一阶段是通过测量含有酶和活化糖的组合的NMR管中的底物和产物通量来建立动力学模型。 第二阶段是检查类似的流量在细胞器的准备工作，如高尔基体，或整个细胞。C13标记底物将用于简化光谱数据。 正在研究两个测试系统; 1）在植物（例如豌豆）中由核苷酸-糖形成细胞壁多糖和2）在Tremella mesenterica中形成胞外多糖。此外，纯化的酶系统正在研究，以测试的方法。 植物系统是有用的，因为数量和容易制备的细胞器级分。银耳属真菌是一种非致病性单细胞真核生物，其合成大量的多糖，其结构与致病性隐球菌中的多糖非常相似。 实验将包括在不同的孵育时间期间鉴定酶中间体，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用特定细胞隔室鉴定的生长多糖的结构信息相结合，可以鉴定合成中涉及的步骤。可以检查影响和控制前体的摄取、多糖的伸长和向外壁的输出的因素。