PROBING THE LIGAND INDUCED CONFORMATIONAL CHANGE OF BETA-PHOSPHOGLUCOMUTASE

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。 我们建议用小角X射线散射(SAXS)研究配体诱导β-磷酸葡萄糖变位酶(BPGM)构象变化的机制。BPGM是卤代烷酸脱卤酶超家族(HADSF)中具有代表性的成员。该酶已通过X射线结晶学以开放和封闭的配体结合构象进行了广泛的表征。虽然这种构象变化已经有了很好的记录，但这种转变的机制仍然不清楚。BPGM的配体1，6-(双)磷酸葡萄糖的结合部位同时含有磷酸基和糖环。拟议的实验将表征当占据全部或部分活性部位的配体浓度增加时酶的SAXS图谱。通过确定天然配基的组成部分的影响，我们希望确定导致构象变化的关键蛋白质-配基相互作用。此外，可以识别任何构象中间体的存在。如果有足够的分辨率，我们将确定每个部分配体的热力学数量K_(Close)，并将这些结果与已公布的KI或Km值进行比较。对该酶的SAXS初步数据表明，动量传递值在S=0.10-0.4之间的信号有显著差异，未连接酶和抑制剂结合酶之间的Rg有1.3°的差异，表明该方法具有足够的分辨率来区分这两种状态。

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. We propose to study the mechanism of the ligand induced conformational change in beta-phosphoglucomutase (bPGM) via small angle x-ray scattering (SAXS). bPGM is a representative member of the Haloalkanoate Dehalogenase Superfamily (HADSF) of phosphotransferases. The enzyme has been extensively characterized via x-ray crystallography in open and closed, ligand bound conformation. While the conformational change is well documented, the mechanism of this transition is still unknown. The binding site for the ligand of bPGM, glucose 1,6-(bis)phosphate, contains sites for both phosphoryl moieties and the sugar ring. The proposed experiments will characterize the SAXS pattern of the enzyme in the presence of increasing concentration of ligands that occupy all or part of the active site. By determining the effect of component moieties of the native ligands, we hope to identify the crucial protein-ligand interactions that induce the conformational change. In addition, the presence of any conformational intermediates can be recognized. Given sufficient resolution, we will determine the thermodynamic quantity Kclose for each of the partial ligands and compare these results to published KI or KM values. Preliminary SAXS data on the enzyme shows significant difference in signal between momentum transfer values of s=0.1-0.4 and a 1.3¿ difference in Rg between the unliganded and inhibitor bound enzyme, indicating that this method has sufficient resolution to discern the two states.