Lipoxygenase catalysis is an important feature of polyunsaturated fatty acid metabolism. The end products of the lipoxygenase initiated metabolism of arachidonic acid in humans are potent mediators in a number of medically relevant physiological processes. The leukotrienes, for example, play an important role in diseases where there is involvement of the inflammatory response. The long term goal of this research is to provide a fundamental understanding of the role of lipoxygenase catalysis in polyunsaturated fatty acid metabolism. With this kind of information, it will be possible to more readily control this aspect of lipid metabolism, and thereby to ameliorate the symptoms of numerous human diseases. A number of important discoveries have recently occurred in this laboratory: the three dimensional structure of soybean lipoxygenase-3 was determined by X-ray crystallography, a number of small molecule inhibitors were synthesized, and the cDNA for the enzyme was cloned and site directed mutagenesis was conducted on the active protein as expressed in E. coli. These developments provide us with a unique opportunity to probe for the dynamic mechanistic details of lipoxygenase catalysis. It is our hypothesis that the reaction takes place in the channels connecting the cofactor iron site with the surface of the molecule by a mechanism that involves free radical intermediates.
The specific aims of the present proposal are: 1. To solve the three dimensional structure of lipoxygenase-3 in the active or iron(III) form. 2. To elucidate the three dimensional structures of complexes of the active form of lipoxygenase-3 with inhibitors and small molecule ligands related to the substrate and product of the reaction. 3. To explore the dynamic solution properties of the protein and its interactions with small molecules using isothermal titration calorimetry and differential scanning calorimetry, and to exploit these discoveries to gain a better understanding of lipoxygenase catalysis, e.g. cofactor iron extraction, reconstitution and substitution. 4. To conduct recombinant modifications of lipoxygenase-3 in order to determine domain interactions, as well as to identify specific amino acid side chains that are necessary for substrate recognition and catalysis.
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