9632095 Biltonen The long term objective of this proposal is to provide a comprehensive framework within which to formulate a precise definition of phospholipase A2 activation in thermodynamic, kinetic, and structural terms. The proposed work will focus on developing an understanding of the putative structural changes leading to full activation. This work will include definition of the enzyme-substrate interaction locus and putative dimer interfacial region 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 interactions and calcium binding in aqueous solution and on the membrane surface. Recent work has suggested that domain formation on the lipid surface could lead to protein clustering which may be related to activation. Whether this potential clustering is related to protein dimerization is still an open question, however, and will be pursued. Such characterization is necessary to design and interpret kinetic experiments aimed at defining the temporal sequence of events of the activation process. Finally, a structural description of these enzyme systems will be provided by multidimension nuclear magnetic resonance studies. Specifically, changes in the dynamics and structure of the enzyme upon binding to lipid surfaces will be investigated. This project will focus on the properties of two naturally occurring enzymes, phospholipase A2 from porcine pancreas and the monomeric form from the water moccasin. Particular attention will be given to the latter enzyme since it has been expressed in E. coli and specific mutations can be engineered. %%% A detailed investigation of the structural and energetic changes associated with the binding to and activation of the enzyme phospholipase A2 on lipid membranes will be undertaken. Specific questions to be addressed include what protein sites are involved in the interaction, does protein-protein interaction play a role in activation and what structural changes in the enzyme occur upon binding. A variety of spectroscopic and calorimetric techniques will be used. In addition protein engineering will be used to produce interesting variants of the wild type enzyme. This information will prove useful in developing an understanding of the functioning and regulation of this enzyme specifically and the nature of catalysis on membrane surfaces, generally. ***
