Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O 2 , and two electrons to prostaglandin endoperoxide H 2 (PGH 2 ). Formation of PGH 2 involves an initial oxygenation of arachidonate to yield PGG 2 catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG 2 to form PGH 2 catalyzed by the peroxidase activity. The cyclooxygenase active site is a hydrophobic channel that protrudes from the membrane binding domain into the core of the globular domain of PGHS. In the crystal structure of Co 3؉ -heme ovine PGHS-1 complexed with arachidonic acid, 19 cyclooxygenase active site residues are predicted to make a total of 50 contacts with the substrate (Malkowski, M. G, Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937); two of these are hydrophilic, and 48 involve hydrophobic interactions. We performed mutational analyses to determine the roles of 14 of these residues and 4 other closely neighboring residues in arachidonate binding and oxygenation. Mutants were analyzed for peroxidase and cyclooxygenase activity, and the products formed by various mutants were characterized. Overall, the results indicate that cyclooxygenase active site residues of PGHS-1 fall into five functional categories as follows: (a) residues directly involved in hydrogen abstraction from C-13 of arachidonate (Tyr-385); (b) residues essential for positioning C-13 of arachidonate for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity arachidonate binding (Arg-120); (d) residues critical for positioning arachidonate in a conformation so that when hydrogen abstraction does occur the molecule is optimally arranged to yield PGG 2 versus monohydroperoxy acid products (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make less but measurable contributions to optimal catalytic efficiency.

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 M, respectively); similarly, the K m values for PGG 2 and 15-HETE formation by V349L oPGHS-1 were different (11 and 5 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. 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 M for oPGHS-1 and 15 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.Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) 1 catalyze the conversion of arachidonic acid and O 2 to PGH 2 : the committed step in the formation of prostanoids (prostaglandins, thromboxane A 2 (see Refs. 1-6)). PGHS-1 (or COX-1) is often referred to as the constitutive enzyme whereas PGHS-2 (COX-2) is known as the inducible isoform (7-15). Apart from their important biological roles and their functions as targets of nonsteroidal anti-inflammatory drugs (4, 5), PGHSs are of considerable interest in the context of the structural biology and enzymology of membrane proteins. These enzymes are homodimeric (ϳ72 kDa/subunit), heme-containing, glycoproteins with two catalytic sites; moreover, PGHSs represent a prototype of a new class of integral membrane proteins that appear to be anchored to one leaflet of the lipid bilayer through the hydrophobic surfaces of amphipathic helices and not through more typical transmembrane domains (3, 16 -18). PGHSs catalyze two separate reactions: a cyclooxygenase (bis-oxygen...

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18 -22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co 3؉ protoporphyrin IX-reconstituted ovine PGHS-1 (Co 3؉ -oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and LA bind in the active site with orientations similar to those seen previously with AA and dihomo-␥-linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a "strained" conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. Prostaglandin endoperoxide H synthase (PGHS)1 converts arachidonic acid (AA) to prostaglandin H 2 in the committed step of prostaglandin and thromboxane biosynthesis (1, 2). Two isoforms of PGHS exist in mammalian tissues. PGHS-1 is constitutively expressed and generates prostaglandins in response to hormone stimulation, whereas PGHS-2 is an inducible enzyme that is expressed in response to growth factors, tumor promotors, or cytokines (1-11). Both isoforms are quite similar structurally (12-15) and mechanistically (1, 2, 16), with only subtle kinetic differences in substrate (17, 18) and inhibitor (19, 20) specificities, and hydroperoxide activator requirements (21-23).Both PGHS-1 and -2 catalyze two different reactions: a cyclooxygenase reaction and a peroxidase reaction. Structural studies indicate that the active sites are spatially distinct from each other and separated by a heme group (12-15). The cyclooxygenase reaction occurs within a hydrophobic channel that extends from the membrane binding domain of the enzyme into the core of the globular domain. The fatty acid substrate is positioned within this site in an extended L-shaped conformation (15,24). Cyclooxygenase catalysis begins with abstraction of the 13-pro-S hydrogen from AA by a tyrosyl radical centered on Tyr-385 in the rate-determining step to generate an arachidonyl radical (25-27). Two molecules of O 2 are then sequentially added to the arachidonyl radical to form the bicyclic hydroperoxide prostaglandin G 2 (PGG 2 ). This intermediate diffuses to the peroxidase active site where the 15-hydroperoxyl group undergoes a two-electron reduction to the alcohol prostaglandin H 2 . Although the peroxidase activity can function independently of the cyclooxygenase activity (28), activation of the cyclooxygenase requires a functional peroxidase (1,2,23).AA is the preferred substrate for PGHS-1 and PGHS-2 (17). Both isoforms will, however, oxygenate a variety of n-3 and n-6 18 -22 carbon fatty acids albeit with reduced catalytic efficiencies (17, 29 -36). Most of these alternative fatty acid substrates have somewhat higher K m...

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-␥-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall Lshaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 Å. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V max /K m for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the -half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40-and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.

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Prostaglandin endoperoxide H synthase-1 (PGHS-1) is expressed constitutively in murine NIH 3T3 cells and RAW 264.7 cells. PGHS-2 is inducibly expressed in these cells following stimulation with serum or bacterial lipopolysaccharide (LPS), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis established that a variety of G protein-linked and peroxisomal proliferator-activated prostanoid receptors are expressed in both of these cell types. The levels of the EP2 and EP4 prostaglandin E 2 (PGE 2 ) receptors and the prostaglandin I 2 receptor were changed in these cells by serum or LPS stimulation. Quantitative RT-PCR indicated that the mRNA for the murine EP4 receptor, the butaprost-insensitive PGE 2 receptor that couples to G s , increases 1.5-3-fold in response to serum (NIH 3T3) or LPS (RAW 264.7) with a time course approximating the induction of PGHS-2 expression. To study expression of the EP4 receptor we isolated the mouse EP4 receptor gene; the gene is 10 kilobase pairs (kb) in length and, like other known prostanoid receptor genes, contains three exons and two introns. The first intron is 0.5 kb and is located 16 base pairs (bp) downstream of the translational start site. This is a different location than that of the first introns of other prostanoid receptor genes. The second intron is located immediately following the sixth transmembrane domain at the same position as the second intron of the thromboxane A 2 receptor, prostaglandin D 2 receptor, prostaglandin I 2 receptor, and one of the PGE 2 (EP1) receptor genes. A major transcriptional start was detected at ؊142 bp upstream of the translational start. There are a variety of putative cis-acting elements within 1.5 kb upstream of the translational start site and within the first intron. Promoter analyses of the EP4 receptor gene promoter in RAW 264.7 cells indicated that there is a constitutive negative regulatory region between ؊992 and ؊928 bp, a constitutive positive region between ؊928 and ؊554 bp, and an LPS/serum-responsive region between ؊554 and ؊116 bp.Prostaglandins and thromboxanes are biologically active metabolites of arachidonic acid formed by the sequential actions of prostaglandin endoperoxide H synthase-1 and -2 (PGHS-1 and -2) 1 and specific prostaglandin and thromboxane synthases (1). Prostanoids cause a variety of physiological actions including contraction and relaxation of smooth muscle, inhibition and stimulation of neurotransmitter release, inhibition of gastric acid secretion, inhibition of inflammatory mediator release, regulation of platelet aggregation, and control of water and salt reabsorption in the kidney (1). Prostanoids synthesized on the endoplasmic reticulum via the PGHS-1 biosynthetic system (2, 3) are transported to the outside of cells through a prostanoid transporter(s) (4). Following their exit from cells, these newly formed prostanoids mediate their effects through cell surface, G protein-linked receptors (5, 6). Prostanoids are also synthesized in association with the nuclear envelope, ...

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