Specialized enzymes have evolved for the catalytic turnover of natural substrates which form organized interfaces in aqueous dispersions. Among these, the phospholipases A2 have attracted attention not only as prototypes for study of interfacial catalysis and models of membrane active proteins. They also comprise a class of promising pharmacological targets because they control a wide range of secretory and inflammatory processes, including cellular defense mechanisms. The pharmacological promise of these targets has not been realized yet, largely because the kinetic protocols for interfacial catalysis are not adequately established. The long-range objective is to develop a detailed understanding of interfacial catalysis and activation by phospholipase A2, and to account for this in protein structural terms. The focus during the 1991-1995 period was dissecting the binding to the interface from catalytic turnover steps in the interface. This allowed an analytical description of the highly processive reaction in terms of interfacial catalytic and equilibrium parameters. To develop structure-function relationships between the interfacial binding and events of the catalytic cycle, the hypothesis is that the binding of secreted phospholipase A2 to the interface requires multiple sites, and that some of these interactions modulate the catalytic parameters through the calcium cofactor. The specific goals are: (a) to establish the role of the cation on the substrate binding and chemical steps; (b) to develop an analytical description of the kinetics of hydrolysis of short chain substrates; (c) to develop a kinetic and structural basis of interfacial binding and activation; (d) to characterize the effects of inhibitors that interfere with the binding of enzyme to the interface; and (e) to map the lipid-exposed region and identify residues that contact the interface. Such studies will extend the kinetic description of interfacial catalysis and ultimately help in the characterization of the activated form of the enzyme. The kinetic and equilibrium parameters will provide physical and molecular correlates for the processes that occur on the binding of the enzyme to the interface, as well as on the binding of calcium, substrate, inhibitors and products to PLA2 at the interface. This information is not accessible from the study of the soluble form of the enzyme in the aqueous phase, or through the usual methods for the determination of macromolecular structure (X-ray or NMR); however, it provides a basis for correlation of function with structural features, which will be pursued further by the use of suitable mutants. The investigators will also continue to search, develop and use specific inhibitors of PLA2 for their effects on cellular processes, which should ultimately help in realizing the pharmacological potential of interfacial enzymes.
Showing the most recent 10 out of 89 publications
