Different catalytically competent arrangements of arachidonic acid within the cyclooxygenase active site of prostaglandin endoperoxide H synthase-1 lead to the formation of different oxygenated products

Arachidonic acid is converted to prostaglandin G(2) (PGG(2)) by the cyclooxygenase activities of prostaglandin endoperoxide H synthases (PGHSs) 1 and 2, The initial, rate-limiting step is abstraction of the 13-proS hydrogen from arachidonate which, for PGG(2) formation, is followed by insertion of O-2 at C-11, cyclization, and a second O-2 insertion at C-15, As an accompaniment to ongoing structural studies designed to determine the orientation of arachidonate in the cyclooxygenase site, we analyzed the products formed from arachidonate by (a solubilized, partially purified ovine to) PGHS-1; (b) membrane-associated, recombinant oPGHS-1; and (c) a membrane-associated, recombinant active site mutant (V349L oPGHS-1) and determined kinetic values for formation of each product. Native forms of oPGHS-1 produced primarily PGG(2) but also several monohydroxy acids, which, in order of abundance, were 11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11R-HETE), 15S-hydroxy-5Z, 8Z,11Z,13E-eicosatetraenoic acid (15S-HETE), and 15R-HETE, V349L oPGHS-1 formed primarily PGG(2), 15S-HETE, and 15R-HETE but only trace amounts of 11R-HETE, With native enzyme, the K-m values for PGG(2), 11-METE, and 15-METE formation were each different (5.5, 12.1, and 19.4 mu M, respectively); similarly, the K-m, values for PGG(2) and 15-METE formation by V349L oPGHS-1 were different (11 and 5 mu M, respectively). These results establish that arachidonate can assume at least three catalytically productive arrangements within the cyclooxygenase site of oPGHS-1 leading to PGG(2), 11R-HETE, and 15S-HETE and/or 15R-HETE, respectively. IC50 values for inhibition of formation of the individual products by the competitive inhibitor, ibuprofen, were determined and found to be the same for a given enzyme form (i.e. 175 mu M for oPGHS-1 and 15 mu M for V349L oPGHS-1), These latter results are most simply rationalized by a kinetic model in which arachidonate forms various catalytically competent arrangements only after entering the cyclooxygenase active site.