DYNAMICS AND CATALYTIC PROPERTIES RELATIONSHIP OF MODEL ENZYME STUDIED BY ESR

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。 在这个项目中，我们试图建立模型酶(枯草杆菌毒素Carslberg)溶解在有机溶剂中的动力学和催化性质之间的关系。我们用几种有机溶剂悬浮的活性部位TEMPO自旋标记丝氨酸蛋白酶Carlsberg获得的初步EPR数据显示，在酶暴露在有机介质中的时间内，双组分光谱发生变化。此外，使用FRET和荧光光谱进行的类似观察导致我们假设，在长时间暴露于有机介质中，活性部位环境极性的变化会导致底物(和活性部位抑制剂，如TEMPO)采用不同的结合方向。我们目前正在使用MOMD拟合程序NLSL.MOMD来评估我们的模型酶在四种不同的有机溶剂中在不同的溶剂暴露时间下获得的多个EPR数据。用旋转扩散速率和各组分的取向电势表征了随曝光时间变化的双组分EPR谱。我们预计这个项目会有一份手稿。

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.