Secreted phospholipase A2 (PLA2) is an interfacial enzyme that catalyzes the calcium-dependent hydrolysis of glycerophospholipids to free fatty acids and lyso-phospholipid, which are further converted to eicosanoids and platelet activating factor with broad biological activities. PLA2 is inactive in solution, but undergoes interfacial activation upon binding to biological membranes. Despite extensive studies on secreted PLA2s, the structural basis for interfacial activation and the effects of site-directed mutations remain largely uncharacterized. Two mutants of human group IIA PLA2, with tryptophans incorporated at the 3rd or 5th position in the N-terminal helix, display dramatic differences in activity compared to the wildtype enzyme. This project analyzes the distinct structural changes that occur in PLA2 and two Trp-mutants during interfacial activation, which are responsible for the observed disparities in activity. Additionally, the thermal stability of both mutants was determined in order to explore possible correlations between resistance to thermal denaturation and enzymatic activity. The V3W mutant shows enhanced activity and a higher optimal temperature compared to the wildtype, which may be promoted at least partially by the high affinity of tryptophan for the lipid-aqueous interface. Contrastingly, the FSW enzyme, which has a tryptophan within the substrate-binding pocket, displays greatly diminished activity compared to both the wild-type and V3W mutant, suggesting inefficient loading of substrate. Circular dichroism and fluorescence studies reveal that the differences in activity of the mutants result from distinct structural changes upon activation. Furthermore, thermal denaturation of V3W was partially reversible, whereas F5W showed no recovery of secondary structure following decrease of temperature. Thus, tryptophan incorporation at two close positions modulates the activity of PLA2 in strikingly different ways, which are associated with defined changes in the secondary structure and the thermal stability of the enzyme. Our results may find industrial or pharmaceutical applications, such as production of fatty acids or development of antibacterial agents.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses1990-2015-2041 |
| Date | 01 January 2010 |
| Creators | Reilly, Christopher Reid |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | HIM 1990-2015 |
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