Abstract
Prostaglandin endoperoxide H synthases 1 and 2, also known as cyclooxygenases (COXs) 1 and 2, convert arachidonic acid (AA) to prostaglandin endoperoxide H(2). Prostaglandin endoperoxide H synthases are targets of nonspecific nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors called coxibs. PGHS-2 is a sequence homodimer. Each monomer has a peroxidase and a COX active site. We find that human PGHS-2 functions as a conformational heterodimer having a catalytic monomer (E(cat)) and an allosteric monomer (E(allo)). Heme binds tightly only to the peroxidase site of E(cat), whereas substrates, as well as certain inhibitors (e.g. celecoxib), bind the COX site of E(cat). E(cat) is regulated by E(allo) in a manner dependent on what ligand is bound to E(allo). Substrate and nonsubstrate fatty acids (FAs) and some COX inhibitors (e.g. naproxen) preferentially bind to the COX site of E(allo). AA can bind to E(cat) and E(allo), but the affinity of AA for E(allo) is 25 times that for E(cat). Palmitic acid, an efficacious stimulator of human PGHS-2, binds only E(allo) in palmitic acid/murine PGHS-2 co-crystals. Nonsubstrate FAs can potentiate or attenuate actions of COX inhibitors depending on the FA and whether the inhibitor binds E(cat) or E(allo). Our studies suggest that the concentration and composition of the free FA pool in the environment in which PGHS-2 functions in cells, the FA tone, is a key factor regulating PGHS-2 activity and its responses to COX inhibitors. We suggest that differences in FA tone occurring with different diets will likely affect both base-line prostanoid synthesis and responses to COX inhibitors.
Highlights
Donic acid (AA) to prostaglandin H2 (PGH2) in the committed step of prostanoid biosynthesis [1,2,3,4,5,6]
Heme titrations of huPGHS-2 were performed in the presence of 20 M palmitic acid (PA), 25 M celecoxib, 50 M naproxen, a combination of 20 M palmitic acid and 25 M celecoxib, and 1.0 and 2.5 mM aspirin
We find that huPGHS-2 binds only one heme per dimer with high affinity (Kd ϭ 88 nM) and that maximal COX activity occurs with a stoichiometry of one heme per subunit
Summary
Materials—Arachidonic acid (AA), oleic acid (18:19), palmitoleic acid (16:17), elaidic acid (18:19t), 15-hydroxyeicosatetraenoic acid, and 12-hydroxyheptadecaenoic acid were purchased from Cayman Chemical Co. Reactions were initiated by adding enzyme to the assay chamber unless otherwise indicated. In experiments involving COX inhibitors, the enzyme preparations were typically preincubated with the inhibitor plus or minus a FA, and an aliquot was added to the reaction chamber. COX assays performed at high enzyme/substrate ratios were conducted in a 100-l reaction mixture containing 1 M [1-14C]AA, 0.10 –2.0 M huPGHS-2, 5 M hematin, and 1 mM phenol in 0.1 M Tris-HCl, pH 8.0, at 37 °C. Aliquots of a 200 M hematin solution in 20 mM Tris-HCl, pH 8.3, containing 40 mM KCl, 0.1% C10E6, and 2% DMSO were added to a quartz cuvette containing 200 l of 5 M apohuPGHS-2 in the same buffer solution without DMSO at room temperature [26, 27]. The crystals were transferred to a solution of 30% polyacrylic acid 5100, 100 mM HEPES, pH 7.5, 20 mM MgCl2, and 0.6% (w/v) -OG supplemented with 10% ethylene
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