Phospholipase A2 is a superfamily of enzymes that play a major role in cellular homeostasis and disease development. This class of enzymes acts on a diverse set of phospholipids substrates, often releasing products that have a role in inflammatory pathways. Because phospholipases A2 play such a significant role in disease progression, understanding their mechanisms of action is vital in order to develop inhibitors for therapeutic interventions. In this thesis, the pathways of activation and inhibition are studied to learn about phospholipase’s structural dynamics and kinetic activity.
A computational study grounded in hydrogen deuterium exchange data reveals the binding mode of lipoprotein-associated phospholipase A2. Docking and molecular dynamic simulations were carried out using several substrates and a potent and selective inhibitor developed by GlaxoSmithKline. These results reveal a binding mode of lipoprotein-associated phospholipase A2 and how the enzyme is regulated by membrane allosterism. Membrane association causes a conformational change to occur, making it easier for substrates and inhibitors to enter the binding pocket. As ligands bind, lipoprotein-associated phospholipase A2 adjusts the binding pocket to fit around the ligand, ensuring a hydrophobic pocket forms so that catalytic hydrolysis may take place.
Activity assays were performed on calcium-independent phospholipase A 2 to observe enzyme regulation by ATP binding. This short study confirms that this enzyme has preferential cleavage toward certain head groups of substrates. It also demonstrates that concentrations of ATP up to 10µM activate calcium-independent phospholipase A2 when being analyzed using a high throughput LC-MS assay.
Finally, the hydrolytic activity of cytosolic phospholipase A2, calcium-independent phospholipase A2, and secreted phospholipase A2 toward phosphatidylinositols and phosphatidylinositol 4,5-bisphosphates was looked at. These preliminary studies reveal all three enzymes do not hydrolyze phosphatidylinositol 4,5-bisphosphates. Surprisingly, all thee enzymes act on phosphatidylinositols, with preferential cleavage of species that contained 16 and 18 carbon length chains.
|Advisor:||Dennis, Edward A.|
|Commitee:||McCammon, Andrew J., Muller, Ulrich|
|School:||University of California, San Diego|
|School Location:||United States -- California|
|Source:||MAI 57/01M(E), Masters Abstracts International|
|Keywords:||ATP, Activation, Computational, Inhibitor, PIP2, Phospholipase A2|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be