Thesis advisor: Mary F. Roberts / The phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes (a monomer in solution) shows unusual kinetic properties compared to other well-studied phospholipases: (i) increased specific activity with decreasing protein concentration, (ii) activation of the phosphotransferase step by salts, and (iii) activation of both the interfacial phosphotransferase and water-soluble phosphodiesterase steps by zwitterionic and neutral amphiphiles. A variety of biophysical studies (fluorescence, NMR, monolayer, vesicle binding) of enzyme/lipid complexes coupled with kinetics have allowed us to propose a model that accounts for these features. The enzyme binds tightly to anionic surfaces and much more weakly to a zwitterionic interface. The tight binding can be reduced by adding KCl at concentrations that activate the enzyme. In the crystal structure of the enzyme, many basic residues are clustered on the sides and bottom of TIM-barrel far away from the opening to the active site. These cause the enzyme to adopt a non-productive orientation on negatively charged membranes that leads to a reversible clustering of anionic lipids and vesicle aggregation. An increased surface concentration of zwitterionic / neutral amphiphiles along with the salt disperses the anionic substrate, shields charges on the protein, and enhances productive encounters of the protein with substrate molecules. This model has been tested by examining the behavior of enzyme with citraconylated lysines and mutants of neutral surface residues at the rim of the active site. The unusual kinetic behavior of this PI-PLC also appears to contribute to the escape of L. monocytogenes from vacuoles during infection. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_101327 |
| Date | January 2008 |
| Creators | Chen, Wei |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
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