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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。我们正在模拟结合的内切多聚半乳糖醛酸酶II从尼日尔曲霉的多聚半乳糖醛酸（果胶）从细胞壁使用琥珀和Glycam力场。在这项研究中，有重要的氨基酸和碳水化合物残基，发挥关键作用的结合，构象变化和催化。采用后模拟分析与MMPBSA的方法，我们正在评估在溶液中的底物结合自由能。采用计算丙氨酸扫描，将使我们能够突变各种残基，并观察它们的重要性，由于它们的缺乏基于电荷，体积和疏水性。未来的目标是将溶剂可及表面与质谱和氘交换计算相关联。氘交换将使我们了解重要的残基相互作用，以及在存在和不存在的聚半乳糖醛酸底物的溶剂可及表面。其他目的包括研究在PGIP抑制剂存在下果胶底物与酶的结合以及抑制剂对酶/底物复合物的影响。

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.