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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们利用Amber和Glycam力场模拟了黑曲霉的内聚半乳糖醛酸II与细胞壁上的聚半乳糖醛酸（果胶）的结合。在这项研究中，有重要的氨基酸和碳水化合物残基在结合、构象变化和催化中起关键作用。利用MMPBSA方法的模拟后分析，我们评估了溶液中的底物结合自由能。利用计算丙氨酸扫描，将允许我们突变各种残基，并观察它们的重要性，因为它们的缺失是基于电荷、体积和疏水性的。未来的目标是将计算得到的溶剂可接近表面与质谱和氘交换联系起来。氘交换将使我们了解重要的残留物相互作用，以及在存在和不存在聚半乳糖醛酸底物的情况下溶剂可接近的表面。其他目标包括研究PGIP抑制剂存在时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.