The formation of bioactive eicosanoids is initiated by stereoselective oxygenation of the substrate, arachidonic acid, by lipoxygenase (LOX) and cyclooxygenase (COX) enzymes. The products mediate important physiological activities and are key modulators of the inflammatory response. Individual enzymes of the LOX family catalyze oxygenation of arachidonate with distinct positional specificity and stereo specificity. In an earlier cycle of this grant we discovered novel human LOX enzymes with 15 S-LOX (named I 5-LOX-2) and 1 2R-LOX specificities. We will use these and related LOX enzymes to examine the structural basis for their differences in reaction with arachidonic acid. This is a practically important goal for the future rational design of specific inhibitors. Currently a 5-LOX inhibitor is used therapeutically to treat asthma, but other LOX inhibitors are too non-specific for practical applications. The COX enzymes are the targets of aspirin and other widely used antiinflammatory medications, and to fully understand their mechanism of action will require an understanding of how the selective oxygenation reactions with arachidonic acid are controlled. We propose a novel hypothesis that will challenge current ideas of oxygen channeling within the cyclooxygenase active site and lead, we believe, to a new concept that will be applicable also to lipoxygenase catalysis. Our hypothesis is that enzyme-induced localization of the substrate free radical will, by well precedented chemistry, direct the targeted reactions with molecular oxygen. To test this idea with COX enzymes we will examine the stereo fidelity of the C-13 hydrogen abstraction in the conversion of arachidonic acid to distinct products. We will also examine the control of oxygenation at the C-Il position using novel fatty acid substrates, and at C-15 by changing the specificity of the enzyme. As a third arm of this project we will investigate the physiological roles of I 5-LOX-2, the enzyme we discovered and that we have shown to have highly selected expression in certain epithelial tissues. In addition to characterizing the expression of human LOX-2, we aim to develop new animal models of 15-LOX-2 physiology in tissues of the rat. This is prompted by our finding that the mouse homologue of l5-LOX-2, an 8-LOX, has completely different reaction specificity and patterns of expression. We have preliminary evidence that the rat enzyme is a closer structural relative to human l5-LOX-2 will be a suitable animal model for physiological studies. Finally, in the theme of novel oxygenations we will characterize novel lipoxygenase activities in animal models of vascular tone and cell proliferation.
