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 cycloox ygenase activities of prostaglandin endoperoxide H synthases (PGHSs) 1 and 2, The initial, rate-limiting step is abstraction of the 13-proS hydrogen f rom 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 accompan iment to ongoing structural studies designed to determine the orientation o f 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 valu es for formation of each product. Native forms of oPGHS-1 produced primaril y PGG(2) but also several monohydroxy acids, which, in order of abundance, were 11R-hydroxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11R-HETE), 15S-hydrox y-5Z, 8Z,11Z,13E-eicosatetraenoic acid (15S-HETE), and 15R-HETE, V349L oPGH S-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 oPG HS-1 were different (11 and 5 mu M, respectively). These results establish that arachidonate can assume at least three catalytically productive arrang ements within the cyclooxygenase site of oPGHS-1 leading to PGG(2), 11R-HET E, and 15S-HETE and/or 15R-HETE, respectively. IC50 values for inhibition o f formation of the individual products by the competitive inhibitor, ibupro fen, 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 cyclo oxygenase active site.