I. Phospholipase A2. Medicinal interest in phospholipase A2 has escalated in recent years since it controls the release of arachidonic acid from the phospholipid pool for the biosynthesis of prostaglandins and leukotrienes. Inhibitors of phospholipase A2 may function as antiinflammatory agents. We would like to continue our studies on the rational design of phospholipase A2 inhibitors. The role of the calcium in the catalysis has not been established. Suitably designed inhibitors will be prepared to test the idea that the calcium is involved in polarizing the carbonyl group of the ester substrate and in the stabilization of the tetrahedral intermediate that forms in the reaction. Additional kinetic studies will be carried out in an attempt to learn why the enzyme is much more active toward aggregated rather than monomeric substrates. Solvent and carbon-13 kinetic isotope effects on the reaction will be studied in order to probe the rate-limiting step for the hydrolysis. Finally, we will explore the possibility of determining the conformation of inhibitors bound to the enzyme using a paramagnetic probe NMR technique. II. Inhibitors of Enzymes in the Diaminopimelate Pathway. A number of fluorinated analogues of diaminopimelic acid will be prepared and tested as antibacterial agents and as inhibitors of the enzymes in the diaminopimelate pathway. A general synthetic approach to beta,beta- difluorinated amino acids will be developed. III. Dynamics of Enzyme-Bound Inhibitors. We have recently prepared tow tripeptide tight-binding inhibitors of the aspartic protease penicillopepsin. These peptides contain the novel amino acids difluorostatine and difluorostatone. X-ray analysis of the protease inhibitor complexes shows that the two structures are essentially identical. We will prepare a series of peptide inhibitors labelled with 15N in the amide positions. We will then carry out isotope-edited NMR experiments to measure the rate of amide proto-exchange with solvent. These studies should be useful in determining whether the hydrogen exchange is controlled by solvent accessibility or local denaturation of the complex. We will also attempt to use isotope-edited NMR to determine the conformation of the bound inhibitor.
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