PROBING THE LIGAND INDUCED CONFORMATIONAL CHANGE OF BETA-PHOSPHOGLUCOMUTASE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 本文利用小角X射线散射（SAXS）研究了配体诱导β-磷酸葡萄糖变位酶（bPGM）构象变化的机理。 bPGM是磷酸转移酶的卤代烷酸脱卤酶超家族（HADSF）的代表性成员。 该酶已通过X射线晶体学在开放和封闭的配体结合构象中被广泛表征。 虽然构象的变化是有据可查的，这种转变的机制仍然是未知的。 bPGM的配体葡萄糖1，6-（双）磷酸的结合位点含有磷酰基部分和糖环的位点。 所提出的实验将表征的SAXS模式的酶的存在下，占据全部或部分的活性位点的配体的浓度增加。 通过确定天然配体的组成部分的影响，我们希望确定关键的蛋白质-配体相互作用，诱导构象变化。 此外，可以识别任何构象中间体的存在。 给定足够的分辨率，我们将确定每个部分配体的热力学量Kclose，并将这些结果与公开的KI或KM值进行比较。 对酶的初步SAXS数据显示，s=0.1-0.4的动量传递值之间的信号存在显著差异，未配体和抑制剂结合酶之间的Rg差异为1.3 ½，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.