APPLICATION OF NMR TO THE STUDY OF ENZYME SYSTEMS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 病原真菌和植物细胞表面含有丰富多样的多糖。令人惊讶的是，对参与多糖合成和组装的生化过程知之甚少。总的目标是了解合成必需多糖的途径和动力学，这些多糖存在于新生隐球菌等病原体中。 在酶反应的实时分析中，核磁共振可以检测中间体和产物。对底物、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，也可以研究多酶系统，数据可以用来对代谢途径进行建模。由于这些反应混合物的复杂性和对高灵敏度的需求，正在使用超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核磁共振试管中的底物和产物通量来建立动力学模型。第二个阶段是检查类似的细胞器制剂中的通量，如高尔基体或整个细胞。将使用C13标记的底物来简化光谱数据。 目前正在研究两个测试系统：(1)在植物(如豌豆)中由核苷酸-糖形成细胞壁多糖；(2)在银耳中形成胞外多糖。此外，正在对纯化的酶系统进行研究，以测试该方法。植物系统是有用的，因为细胞器组分的数量和制备容易。银耳真菌是一种非致病性单细胞真核生物，它能合成大量结构与致病隐球菌中的多糖非常相似的多糖。 这些实验将包括确定不同孵化时间的酶中间体，确定这些反应的酶动力学，并跟踪多糖产品的形成。通过将动态过程的分析与特定细胞隔间中生长的多糖的结构信息相结合，可以识别合成过程中涉及的步骤。可以考察影响和控制前体的摄取、多糖的伸长和向外壁输出的因素。

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.