UC San Diego

The phospholipase A₂ (PLA₂) superfamily consists of many different groups of enzymes that catalyze the hydrolysis of the sn-2 ester bond in a variety of different phospholipids. The products of this reaction, a free fatty acid, and lysophospholipid have many different important physiological roles. This reaction occurs on a two dimensional lipid surface. The mechanism of this reaction has important consequences for not only phospholipid hydrolysis, but all of lipid enzymology. To better understand the mechanism of different phospholipase A₂ enzymes we have employed deuterium exchange mass spectrometry (DXMS). This technique has the ability to determine solvent accessibility and conformational changes not seen with x-ray crystallography or other structural techniques. This methodology has proven particularly useful to determine lipid binding sites that would be impossible to characterize with standard NMR or x-ray methodologies. This thesis first discusses the PLA₂ superfamily of enzymes and the different roles they have been shown to play in inflammatory diseases, as well as the deuterium exchange mass spectrometry methodology. With this background we were able to study the GIA cobra venom PLA₂ which has acted as a model lipid enzyme for over a hundred years, and determine the effects of binding both metal ions and phospholipid substrate. We also examined the GIVA human cPLA₂, which has been implicated in numerous diseases, and determined inter-domain contacts not seen in x-ray crystallography, as well as examining metal binding and phospholipid surface binding. The movement of the lid region was discovered using DXMS methods, that has until now only been a hypothesis. Finally we studied the binding of different inhibitors and used a combination of molecular modeling and DXMS towards the design of better GIVA PLA₂ inhibitors