The long term objective of this proposal is to provide a precise definition of phospholipase A2 activation in thermodynamic, kinetic, and structural terms. This definition is important in developing an understanding of the functioning and regulation of the enzyme and the nature of catalysis at membrane interfaces. The proposed work will focus on understanding the putative structural changes leading to full activation. This work will include definition of the interfacial regions and enzyme- substrate interaction locus and the evaluation of specific sites of interaction in thermodynamic, kinetic and structural terms. An important aspect of the proposed work will be thermodynamic characterization of protein-protein interaction and calcium binding in aqueous solution. Such characterization is necessary to design and interpret kinetic experiments aimed at defining the temporal sequence of events of the activation process. This project will initially focus on the properties of three naturally occurring enzymes, phospholipase A2 from porcine pancreas, from a rattlesnake, and from the water moccasin. The principle technique to obtain structural information about phospholipases will be nuclear magnetic resonance spectroscopy. Reaction microcalorimetry, differential scanning calorimetry, pH stat techniques, fluorescence spectroscopy, and high pressure liquid chromatography will also be extensively used to characterize these systems. In addition, site specific mutagenesis of synthetic phospholipase genes will be used perturb protein interaction sites.
