The goal of the proposed research is to obtain a detailed understanding of the structure and mechanism of action of the enzyme lipoxygenase. This is important because the enzyme plays a pivotal role in the metabolism of polyunsaturated fatty acids in a way that impacts a wide variety of medically relevant physiological processes. In the lipoxygenase pathway arachidonic acid is converted into several families of metabolites including the leukotrienes. That these compounds are mediators of allergic and inflammatory reactions is now firmly established. Additionally the metabolites of lipoxygenase catalysis have been associated with numerous health problems including vascular disease, heart disease, and lung disease. The proposed research is to create novel compounds to act as ligands for lipoxygenases. In particular studies will build on previous success in the design and synthesis of substrate and product analogs. We will focus on unsaturated and polyunsaturated fatty acids containing sulfur substitution for carbon in the aliphatic chain. The characterization of complexes between the synthetic ligands and the enzyme will be carried out using uv-visible, EPR, and Mossbauer spectroscopies. Any covalent modification of lipoxygenase, e.g. affinity labeling, will be identified in terms of the point of attachment to the polypeptide chain. The three dimensional structure of the enzyme will also be investigated. The structure of the native enzyme will be obtained on crystals now available in this laboratory and will rely on the techniques of isomorphous replacement using heavy atom derivatives. The cocrystallization of the synthetic ligands with the enzyme will also be investigated. Once a good model of the lipoxygenase molecule has been established, it will be possible to use difference Fourier or molecular replacement techniques to solve the structures of the complexes and related isoenzymes. Visualization of the active site of lipoxygenase will provide a template for the rational design of new inhibitors.
