The enzyme prostaglandin H (PGH) synthase catalyzes the oxidation of arachidonic acid to the hydroperoxy endoperoxide, PGG2, and the reduction of PGG2 to the hydroxy endoperoxide, PGH2. The enzyme has an important role in numerous physiological and pathophysiological processes including inflammation, thrombosis, carcinogenesis, and metastasis. Recent results suggest it is induced in response to treatment of cells with tumor promoters. The hydroperoxide intermediate, PGG2, triggers the oxidative inactivation of PGH synthase, which limits its ability to make bioactive arachidonic acid metabolites. Reducing substrates for the peroxidase activity of PHG synthase lower the steady-state level of PGG2, protect the enzyme from inactivation, and enhance the conversion of arachidonate to PGH2. This may account for the ability of certain drugs to stimulate biosynthesis of the antithrombotic and antimetastatic agent, prostacyclin, by vascular endothelium. Relatively little is known of the structure of PGH synthase and its relation to function. We propose a series of investigations to determine the primary structure of PGH synthase, elucidate the biochemical basis of hydroperoxide-dependent inactivation of PGH synthase, isolate the active site domain of PGH synthase, and design maximally effective peroxidase reducing substrates. Recombinant DNA technology will be used to determine the DNA sequence of a molecular clone of cDNA for ram seminal vesicle PGH synthase. Exhaustive tryptic digestion, mapping, and sequencing will be employed to detect peptides containing amino acid residues oxidized as a result of self-inactivation. The location of oxidized residues in the sequence should be useful in establishing the location of the active site(s) of the cyclooxygenase and peroxidase of PGH synthase. A series of aralkyl sulfides will be constructed that possess high peroxidase reducing substrate activity in order to maximize pharmacological protection of PGH synthase from oxidative inactivation. Literature precedents suggest that such compounds should be effective antithrombotic and antimetastatic agents. Controlled proteolysis of PGH synthase appears to generate a membrane- binding domain and an active site domain. Techniques will be developed to purify the active site domain in order to characterize its cyclooxygenase and peroxidase activities for comparison to the intact enzyme. The proposed experiments should significantly advance our knowledge of structure-function relationships for PGH synthase, reveal the biochemical basis for self-catalyzed inactivation, and provide new strategies for the development of antithrombotic and antimetastatic agents.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Wayne State University
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