Prostaglandin H synthase: Distinct binding sites for cyclooxygenase and peroxidase substrates

Abstract

Prostaglandin H synthase has two distinct catalytic activities: a cyclooxygenase activity that forms prostaglandin G 2 from arachidonic acid; and a peroxidase activity that reduces prostaglandin G 2 to prostaglandin H 2. Lipid hydroperoxides, such as prostaglandin G 2, also initiate the cyclooxygenase reaction, probably via peroxidase reaction cycle enzyme intermediates. The relation between the binding sites for lipid substrates of the two activities was investigated with an analysis of the effects of arachidonic and docosahexaenoic acids on the reaction kinetics of the peroxidase activity, and their effects on the ability of a lipid hydroperoxide to initiate the cyclooxygenase reaction. The cyclooxygenase activity of pure ovine synthase was found to have an apparent K m value for arachidonate of 5.3 μ m and a K i value (competetive inhibitor) for docosahexaenoate of 5.2 μ m. When present at 20 μ m neither fatty acid had a significant effect on the apparent K m value of the peroxidase for 15-hydroperoxyeicosatetraenoic acid: the values were 7.6 μ m in the absence of docosahexaenoic acid and 5.9 μ m in its presence, and (using aspirin-treated synthase) 13.7 μ m in the absence of arachidonic acid and 15.7 μ m in its presence. Over a range of 1 to 110 μ m the level of arachidonate had no significant effect on the initiation of the cyclooxygenase reaction by 15-hydroperoxyeicosatetraenoic acid. The inability of either arachidonic acid or docosahexaenoic acid to interfere with the interaction between the peroxidase and lipid hydroperoxides indicates that the cyclooxygenase and peroxidase activities of prostaglandin H synthase have distinct binding sites for their lipid substrates.