Abstract
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 (o) 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-HETE, and 15-HETE formation were each different (5.5, 12.1, and 19.4 microM, respectively); similarly, the K(m) values for PGG(2) and 15-HETE formation by V349L oPGHS-1 were different (11 and 5 microM, 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. IC(50) 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 microM for oPGHS-1 and 15 microM 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.
Highlights
PGH 2: the committed step in the formation of prostanoids (prostaglandins, thromboxane A2)
Our results indicate that arachidonate can be bound in the cyclooxygenase active site of oPGHS-1 in at least three different, catalytically competent arrangements that lead to prostaglandin G2 (PGG2), 11R-HETE, and 15-HETE, respectively, and that these three arrangements of arachidonate occur subsequent to its entry into the cyclooxygenase active site
With 2 M arachidonate and 5 cyclooxygenase units of either partially purified oPGHS-1 or broken cell preparations of COS-1 cells expressing oPGHS-1, 95% of the products were derived from PGG2, 3% was 11HETE, and 2% was 15-HETE; with 2 M arachidonate and broken cell preparations of COS-1 cells expressing V349L oPGHS-1, 70% of the product was PGG2, Ͻ0.5% was 11-HETE, and 30% was 15-HETE
Summary
Materials—Arachidonic acid was purchased from Cayman Chemical Co. (Ann Arbor, MI). [1-14C]Arachidonic acid (40 – 60 mCi/mmol) was purchased from NEN Life Science Products. Fractions from the DEAE-cellulose column were assayed for peroxidase activity and protein concentration. Characterization of Arachidonate-derived Products—Forty hours following transfection, COS-1 cells were collected, sonicated, and resuspended in 0.1 M Tris-HCl, pH 7.5. Aliquots of the cell suspension (75 g of protein) or 5 cyclooxygenase activity units of partially purified oPGHS-1 were incubated for 1 min at 37 °C with various concentrations of [1-14C]arachidonic acid with and without 200 M flurbiprofen. Reverse Phase (RP)-HPLC—Native or V349L oPGHS-1 (75 g of cell protein) prepared from transfected COS cells or semipurified oPGHS-1 (5 cyclooxygenase activity units) were incubated with 2–100 M arachidonic acid for 1 min at 37 °C. Reactions were initiated by adding approximately 250 g of microsomal protein prepared from COS-1 cells or partially purified oPGHS-1 in a volume of 20 –50 l to the assay mixture. Statistical significance was determined using a two-sample t test assuming equal variances
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