ENZYMES OF THE PROSTAGLANDIN CASCADE

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The pathological manifestations of sickle cell anemia arises from the polymerization of HbS into long fibers which distort the shape of the red blood cell. The resulting abnormal shape and increased rigidity of the red blood cells impedes blood flow through the capillaries causing the vasoocclusive complications that characterize sickle cell anemia. Cerebral vascular disease is a common and one of many catastrophic complications in children suffering from sickle cell anemia. We have started a long-term investigation into the structure and functional of enzymes in the prostaglandin synthesis cascade. Most of these enzymes are membrane bound enzymes and thus require special handing to prepare them for biophysical analysis. As we have developed methods for crystallizing membrane proteins, they now can be made amendable to X-ray diffraction analysis. Using X- ray crystallography, electron paramagnetic resonance, UV-Vis absorption spectroscopy and fluorine-NMR, we intend to study the enzymes that participate in the synthesis of vasoregulatory prostaglandins. We are now investigating enzyme-drug and enzyme- substrate analog complexes of prostagland H synthase (cyclo- oxygenase), both in solution and in crystals. The products of prostaglandin H synthase, PGH1 and PGH2, serve as precursors for synthesis of prostaglandins which affect platelet aggression and vasodilation. We intend to use the methods we have developed for the study prostaglandin synthase to purify, crystalize and investigate and investigate PGH-PGE isomerase and PGI2 synthase which synthesize powerful vasodilators and inhibitors of platelet aggregation. Our long term goal is to elucidate the structural mechanisms of substrate, product and inhibitor binding to 1) understand better the molecular events occurring during catalysis and 2) to provide the necessary structural information for the design of drugs to control the synthesis of specific prostaglandin. Having inhibitors or activators of the synthesis of specific prostaglandins could allow the development of rational drug therapy for vasoocclusive diseases like sickle cell anemia.