DYNAMICS AND CATALYTIC PROPERTIES RELATIONSHIP OF MODEL ENZYME STUDIED BY ESR

ESR研究模型酶的动力学与催化性能关系

• 批准号: 8364006
• 负责人: ZHICHUN LIANG
• 金额: $ 1.32万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364006
• 关键词: Active Sites; Adopted; Binding; Data; Diffusion; Environment; Enzymes; Exposure to; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Funding; Grant; Lead; Manuscripts; National Center for Research Resources; Organic solvent product; Principal Investigator; Property; Research; Research Infrastructure; Resources; Serine Protease; Solvents; Source; Spin Labels; Subtilisins; Subtilopeptidase A; Technology; Time; United States National Institutes of Health; cost; enzyme model; inhibitor/antagonist; programs

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. In this project we seek to establish the relationship between the dynamics and the catalytic properties of a model enzyme (subtilisin Carslberg) dissolved in organic solvents. Our preliminary EPR data obtained with the active site TEMPO spin-labeled serine protease subtilisin Carlsberg suspended in several organic solvents show a two component spectra that changes during the time the enzyme is exposed to organic media. Furthermore, similar observations made using FRET and Fluorescence spectroscopy lead us to hypothesize that a change in the polarity of the active site environment induces substrates (and active site inhibitors such as TEMPO) to adopt a different binding orientation during prolonged exposure to organic media. We are presently using MOMD fitting program, NLSL.MOMD, to evaluate multiple EPR data obtained with our model enzyme suspended in four different organic solvents during different solvent exposure times. The two-component EPR spectra that change with exposure time are characterized by rotational diffusion rates and orienting potential of each component. We anticipate one manuscript to emerge from this project.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 在这个项目中，我们试图建立溶解在有机溶剂中的模型酶（枯草杆菌蛋白酶 Carslberg）的动力学和催化特性之间的关系。我们通过悬浮在几种有机溶剂中的活性位点 TEMPO 自旋标记丝氨酸蛋白酶枯草杆菌蛋白酶嘉士伯获得的初步 EPR 数据显示，在酶暴露于有机介质期间，两组分光谱发生变化。 此外，使用 FRET 和荧光光谱进行的类似观察使我们推测，活性位点环境极性的变化会导致底物（和活性位点抑制剂，如 TEMPO）在长时间暴露于有机介质期间采用不同的结合方向。我们目前正在使用 MOMD 拟合程序 NLSL.MOMD 来评估在不同溶剂暴露时间内悬浮在四种不同有机溶剂中的模型酶获得的多个 EPR 数据。随曝光时间变化的双组分 EPR 光谱的特征是每个组分的旋转扩散速率和取向势。我们预计该项目将出现一份手稿。