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 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic ␣-helices (helices A-D) that include residues 74 -122 in ovine PGHS-1 (oPGHS-1) and residues 59 -108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[ 125 I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74 -140 and 59 -111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn 104 . When this glycosylation site was eliminated (HelB/ N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form.

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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