INTERFACIAL CATALYSIS BY PHOSPHOLIPASE A2

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Several processes are regulated by interfacial catalysis on the membrane surfaces. Thus phospholipase A2 (PLA2) are interesting not only as prototypes for interfacial catalysis in general, but also because they mobilize precursors for the biosynthesis of eicosanoids and platelet activating factor, which ultimately control a wide range of secretory and inflammatory processes. The long range objective of our studies on interfacial catalysis is to gain detailed kinetic, biophysical, and molecular knowledge about PLA2 at the interface. An understanding of the mechanism would also help in the design, assay and characterization of specific inhibitors, which could be useful in establishing and controlling the biological functions of PLA2. During the next five years we plan to use the kinetic and equilibrium methods developed recently in our laboratory for pig PLA2 (type I) to achieve the following objectives: (a) to obtain the equilibrium and kinetic parameters for other PLA2, such as the cloned type II enzyme from synovial fluid, the type III PLA2 from bee venom, and the arachidonate-specific 89 KD enzyme from the cytoplasm of a human cell line; (b) to characterize the amino acid residues exposed to the aqueous phase on the enzyme bound to the interface; (c) to spectroscopically characterize the E, E*, and E*L forms of PLA2; (d) to establish if the catalytic or the product release step is rate-limiting during the catalytic turnover; (e) to investigate the kinetics of hydrolysis of short chain substrates under the conditions where they are dispersed as solitary monomers, so as to obtain insights into the possibility of the formation of a solitary ES complex; (f) to characterize the kinetics of hydrolysis of long chain phospholipids codispersed with bile salts to examine if on small aggregates the rate of hydrolysis is limited by the rate of replenishment of the substrate. By integrating the information obtained from such studies, we hope to obtain a general and complete kinetic description of interfacial catalysis by PLA2 from several sources. These studies would provide physical and molecular insights into the processes that occur not only on the binding of the enzyme to the interface, but also those that occur on the binding of inhibitors and products to PLA2 at the interface. Specific mutants of PLA2 will also be used to identify the residues involved in the binding of the enzyme to the interface and the residues involved in the binding of ligands to the catalytic active site region.