MODELING THE MECHANISM OF ENZYME ACTIVITY IN THE EPG/PGIP ACID SYSTEM

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。我们正在使用琥珀和Glycam力场模拟来自黑曲霉的内切多聚半乳糖醛酸酶II与其来自细胞壁的多聚半乳糖醛酸(果胶)的结合。在本研究中，有一些重要的氨基酸和碳水化合物残基在结合、构象变化和催化方面起着关键作用。使用MMPBSA方法的后模拟分析，我们正在计算溶液中的底物结合自由能。使用计算丙氨酸扫描，将允许我们突变各种残基，并观察它们的重要性，因为它们基于电荷、体积和疏水性而不存在。未来的目标是将通过计算发现的溶剂可及表面与质谱学和氘交换相关联。氢交换将使我们深入了解重要的残基相互作用，以及在存在和不存在多聚半乳糖醛酸底物的情况下溶剂可访问的表面。其他目标包括研究在PGIP抑制剂存在的情况下果胶底物与酶的结合，以及抑制剂对酶/底物复合体的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are modeling the binding of endopolygalacturonase II from Aspergillus niger to its polygalacturonic acid (pectin) from cell walls using Amber and Glycam forcefields. In this investigation, there are important amino acid and carbohydrate residues that play a key role in binding, conformational change and catalysis. Employing post-simulational analysis with the MMPBSA approach, we are evaluating substrate binding free energies in solution. Employing computational alanine-scanning, will allow us to mutate various residues and observe their importance due to their absence based on charge, bulkiness and hydrophobicity. Future objectives are to correlate the solvent accessible surface found computationally with mass spectrometry and deuterium exchange. Deuterium exchange will give us insight on important residue interactions as well as a solvent accessible surface in the presence and absence of the polygalacturonic acid substrate. Other objectives include investigating the binding of the pectin substrate to enzyme in the presence of the PGIP inhibitor and the effects of the inhibitor on the enzyme/substrate complex.