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• W2023302278 abstract "Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O2, and two electrons to prostaglandin endoperoxide H2 (PGH2). Formation of PGH2 involves an initial oxygenation of arachidonate to yield PGG2 catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG2 to form PGH2 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 Co3+-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 PGG2 versusmonohydroperoxy 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. Prostaglandin endoperoxide H synthases (PGHSs) catalyze the committed step in the biosynthesis of prostaglandins and thromboxane, the conversion of arachidonic acid, two molecules of O2, and two electrons to prostaglandin endoperoxide H2 (PGH2). Formation of PGH2 involves an initial oxygenation of arachidonate to yield PGG2 catalyzed by the cyclooxygenase activity of the enzyme and then a reduction of the 15-hydroperoxyl group of PGG2 to form PGH2 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 Co3+-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 PGG2 versusmonohydroperoxy 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. prostaglandin endoperoxide H synthases prostaglandin nonsteroidal anti-inflammatory drugs human PGHS-2 ovine PGHS-1 arachidonic acid 11-hydroxy(5Z,8Z,12E,14Z)-eicosatetraenoic acid 11-hydroperoxy(5Z,8Z,12E,14Z)-eicosatetraenoic acid 15-hydroxy(5Z,8Z,11Z,13E)-eicosatetraenoic acid 15-hydroperoxy(5Z,8Z,11Z,13E)-eicosatetraenoic acid N,N,N′,N′-tetramethylphenylenediamine reverse phase-high pressure liquid chromatography Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2)1 catalyze the conversion of arachidonic acid (AA), two molecules of O2, and two electrons to PGH2. This is the committed step in the formation of prostaglandins and thromboxane A2 (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 149-182Crossref Scopus (2477) Google Scholar, 2Smith W.L. DeWitt D.L. Dixon F.J. Advances in Immunology. 62. Academic Press, San Diego1996: 167-215Google Scholar, 3Marnett L.J. Rowlinson S.W. Goodwin D.C. Kalgutkar A.S. Lanzo C.A. J. Biol. Chem. 1999; 274: 22903-22906Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). PGHS-1 (or COX-1 (for cyclooxygenase-1)) is a constitutive enzyme, whereas PGHS-2 (COX-2) is the inducible isoform (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 149-182Crossref Scopus (2477) Google Scholar, 2Smith W.L. DeWitt D.L. Dixon F.J. Advances in Immunology. 62. Academic Press, San Diego1996: 167-215Google Scholar, 3Marnett L.J. Rowlinson S.W. Goodwin D.C. Kalgutkar A.S. Lanzo C.A. J. Biol. Chem. 1999; 274: 22903-22906Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 4Raz A. Wyche A. Jiyi F. Seibert K. Needleman P. Samuelsson B. Advances in Prostaglandin, Thromboxane, and Leukotriene Research. 20. Raven Press, Ltd, New York1990: 22-27Google Scholar, 5Jones D.A. Carlton D.P. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1993; 268: 9049-9054Abstract Full Text PDF PubMed Google Scholar, 6Hulkower K.I. Wertheimer S.J. Levin W. Coffey J.W. Anderson C.M. Chen T. DeWitt D.L. Crowl R.M. Hope W.C. Morgan D.W. Arthritis & Rheum. 1994; 37: 653-661Crossref PubMed Scopus (117) Google Scholar, 7Guan Z. Buckman S.Y. Miller B.W. Springer L.D. Morrison A.R. J. Biol. Chem. 1998; 273: 28670-28676Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 8Foegh M.L. Watkins W.D. Prostaglandins in Clinical Practice. Raven Press, Ltd, New York1989: 131-140Google Scholar, 9Evett G.E. Xie W. Chipman J.G. Robertson D.L. Simmons D.L. Arch. Biochem. Biophys. 1993; 306: 169-177Crossref PubMed Scopus (120) Google Scholar, 10Xie W. Herschman H.R. J. Biol. Chem. 1995; 270: 27622-27628Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 11Xie W. Herschman H.R. J. Biol. Chem. 1996; 271: 31742-31748Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 12Kujubu D.A. Fletcher B.S. Varnum B.C. Lim R.W. Herschman H.R. J. Biol. Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar). PGHSs catalyze two separate reactions including a cyclooxygenase (bisoxygenase) reaction in which AA is converted to PGG2and a peroxidase reaction in which PGG2 undergoes a two-electron reduction to PGH2 (1Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 149-182Crossref Scopus (2477) Google Scholar, 2Smith W.L. DeWitt D.L. Dixon F.J. Advances in Immunology. 62. Academic Press, San Diego1996: 167-215Google Scholar, 3Marnett L.J. Rowlinson S.W. Goodwin D.C. Kalgutkar A.S. Lanzo C.A. J. Biol. Chem. 1999; 274: 22903-22906Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). These reactions occur at physically distinct but interactive sites within the cyclooxygenase structure. The cyclooxygenase reaction begins with a rate-limiting abstraction of the 13-pro-S-hydrogen from AA to yield an arachidonyl radical (13Hamberg M. Samuelsson B. J. Biol. Chem. 1967; 242: 5336-5343Abstract Full Text PDF PubMed Google Scholar, 14Tsai A. Kulmacz R.J. Palmer G. J. Biol. Chem. 1995; 270: 10503-10508Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). This is followed by sequential oxygen additions at C-11 and C-15 producing PGG2. NSAIDs compete directly with AA for binding to the cyclooxygenase site (15Picot D. Loll P.J. Garavito M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1154) Google Scholar, 16Kurumbail R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1613) Google Scholar, 17Luong C. Miler A. Barnett J. Chow J. Ramesha C. Browner M.F. Nat. Struct. Biol. 1996; 3: 927-933Crossref PubMed Scopus (562) Google Scholar) thereby inhibiting cyclooxygenase activity but not peroxidase activity (18Mizuno K. Yamamoto S. Lands W.E.M. Prostaglandins. 1982; 23: 743-757PubMed Google Scholar, 19Rome L.H. Lands W.E.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4863-4865Crossref PubMed Scopus (264) Google Scholar, 20Marshall P.J. Kulmacz R.J. Arch. Biochem. Biophys. 1988; 266: 162-170Crossref PubMed Scopus (52) Google Scholar). Crystallographic studies of enzyme-inhibitor complexes have suggested that the cyclooxygenase active site exists in the form of a hydrophobic channel that protrudes into the body of the major globular domain of the protein (15Picot D. Loll P.J. Garavito M. Nature. 1994; 367: 243-249Crossref PubMed Scopus (1154) Google Scholar, 16Kurumbail R.G. Stevens A.M. Gierse J.K. McDonald J.J. Stegeman R.A. Pak J.Y. Gildehaus D. Miyashiro J.M. Penning T.D. Seibert K. Isakson P.C. Stallings W.C. Nature. 1996; 384: 644-648Crossref PubMed Scopus (1613) Google Scholar, 17Luong C. Miler A. Barnett J. Chow J. Ramesha C. Browner M.F. Nat. Struct. Biol. 1996; 3: 927-933Crossref PubMed Scopus (562) Google Scholar). More recently we determined the structure of AA bound within the cyclooxygenase active site of ovine (o) PGHS-1 (21Malkowski M.G. Ginell S. Smith W.L. Garavito R.M. Science. 2000; 289: 1933-1937Crossref PubMed Scopus (260) Google Scholar). AA is bound in an extended L-shaped conformation and makes a total of 48 hydrophobic contacts (i.e. 2.5–4.0 Å) and two hydrophilic contacts with the protein, involving a total of 19 different residues (see Fig. 1 below). Additionally, there are several amino acids that are in the first shell of the cyclooxygenase hydrophobic tunnel and contact other first shell amino acids but lie outside of van der Waals distance to AA. Although AA can assume some 107 low energy conformations (22Gund P. Shen T.Y. J. Med. Chem. 1977; 9: 1146-1152Crossref Scopus (112) Google Scholar), only three of these conformations are catalytically competent (23Thuresson E.D. Lakkides K.M. Smith W.L. J. Biol. Chem. 2000; 275: 8501-8507Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar); one conformation leads to PGG2; one leads to (11R)-HPETE; and the other leads to (15R)- plus (15S)-HPETE. Previous mutational studies have demonstrated that the guanidinium group of Arg-120 2The numbering of residues in ovine PGHS-1 is based on numbering the N-terminal methionine of the signal peptide as residue number 1. is important for high affinity binding of AA to PGHS-1 (24Mancini J.A. Riendeau D. Falgueyret J.P. Vickers P.J. O'Neill G.P. J. Biol. Chem. 1995; 270: 29372-29377Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 25Bhattacharyya D.K. Lecomte M. Rieke C.J. Garavito M. Smith W.L. J. Biol. Chem. 1996; 271: 2179-2184Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) (but not PGHS-2 (26Greig G.M. Francis D.A. Falgueyret J.P. Ouellet M. Percival M.D. Roy P. Bayly C. Mancini J.A. O'Neill G.P. Mol. Pharmacol. 1997; 52: 829-838Crossref PubMed Scopus (75) Google Scholar,27Rieke C.J. Mulichak A.M. Garavito R.M. Smith W.L. J. Biol. Chem. 1999; 274: 17109-17114Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar)), that Tyr-385 is involved as a tyrosyl radical in abstracting the 13-pro-S-hydrogen from AA (28Shimokawa T. Kulmacz R.J. DeWitt D.L. Smith W.L. J. Biol. Chem. 1990; 265: 20073-20076Abstract Full Text PDF PubMed Google Scholar, 29Tsai A. Hsi L.C. Kulmacz R.J. Palmer G. Smith W.L. J. Biol. Chem. 1994; 269: 5085-5091Abstract Full Text PDF PubMed Google Scholar), and that Ser-530 and Ile-523 are determinants of inhibitor specificity (30DeWitt D.L. El-Harith E.A. Kraemer S.A. Andrews M.J. Yao E.F. Armstrong R.L. Smith W.L. J. Biol. Chem. 1990; 265: 5192-5198Abstract Full Text PDF PubMed Google Scholar, 31Shimokawa T. Smith W.L. J. Biol. Chem. 1992; 267: 12387-12392Abstract Full Text PDF PubMed Google Scholar, 32Guo Q. Wang L.H. Ruan K.H. Kulmacz R.J. J. Biol. Chem. 1996; 271: 19134-19139Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 33Wong E. Bayly C. Waterman H.L. Riendeau D. Mancini J.A. J. Biol. Chem. 1997; 272: 9280-9286Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 34Gierse J.K. McDonald J.J. Hauser S.D. Rangwala S.H. Koboldt C.M. Seibert K. J. Biol. Chem. 1996; 271: 15810-15814Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Other than for Arg-120 and Tyr-385 relatively little is known about the functions of residues located in the core of the hydrophobic cyclooxygenase tunnel (31Shimokawa T. Smith W.L. J. Biol. Chem. 1992; 267: 12387-12392Abstract Full Text PDF PubMed Google Scholar, 35Rowlinson S.W. Crews B.C. Lanzo C.A. Marnett L.J. J. Biol. Chem. 1999; 274: 23305-23310Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In the study reported here we have performed mutational analyses of a number of the residues that line the hydrophobic active site channel to determine their functional importance in arachidonate binding and oxygenation. Our results suggest that individually and collectively these residues function primarily to position arachidonate in a specific conformation that optimizes its conversion to PGG2. Fatty acids were purchased from Cayman Chemical Co., Ann Arbor, MI. [1-14C]Arachidonic acid (40–60 mCi/mmol) was purchased from PerkinElmer Life Sciences. Flurbiprofen was purchased from Sigma. Diazald® (N-methyl-N-nitroso-p-toluenesulfonamide) was from Aldrich. Restriction enzymes and Dulbecco's modified Eagle's medium were purchased from Life Technologies, Inc. Calf serum and fetal bovine serum were purchased from HyClone. Primary antibodies used for Western blotting were raised in rabbits against purified oPGHS-1 and purified as an IgG fraction (36Spencer A.G. Thuresson E.A. Otto J.C. Song I. Smith T. DeWitt D.L. Garavito R.M. Smith W.L. J. Biol. Chem. 1999; 274: 32936-32942Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and goat anti-rabbit IgG horseradish peroxidase conjugate was purchased from Bio-Rad. Oligonucleotides used as primers for mutagenesis were prepared by the Michigan State University Macromolecular Structure and Sequencing Facility. All other reagents were from common commercial sources. Mutants were prepared either starting with M13mp19-PGHSov, which contains a 2.3-kilobaseSalI fragment coding for native oPGHS-1 and employing the Bio-Rad Muta-Gene kit and the protocol as described by the manufacturer (36Spencer A.G. Thuresson E.A. Otto J.C. Song I. Smith T. DeWitt D.L. Garavito R.M. Smith W.L. J. Biol. Chem. 1999; 274: 32936-32942Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), or by site-directed mutagenesis of oPGHS-1 in the pSVT7 vector (28Shimokawa T. Kulmacz R.J. DeWitt D.L. Smith W.L. J. Biol. Chem. 1990; 265: 20073-20076Abstract Full Text PDF PubMed Google Scholar), employing the Stratagene QuikChange mutagenesis kit and the protocol of the manufacturer. Oligonucleotides used in the preparation of various mutants are summarized in the supplemental table. Plasmids used for transfections were purified by CsCl gradient ultracentrifugation, and mutations were reconfirmed by double-stranded sequencing of the pSVT7 constructs using Sequenase (version 2.0, U. S. Biochemical Corp.) and the protocol described by the manufacturer. COS-1 cells (ATTC CRL-1650) were grown in Dulbecco's modified Eagle's medium containing 8% calf serum and 2% fetal bovine serum until near confluence (∼3 × 106 cells/10 cm dish). Cells were then transfected with pSVT7 plasmids containing cDNAs coding for native oPGHS-1 or mutant oPGHS-1 using the DEAE dextran/chloroquine transfection method as reported previously (36Spencer A.G. Thuresson E.A. Otto J.C. Song I. Smith T. DeWitt D.L. Garavito R.M. Smith W.L. J. Biol. Chem. 1999; 274: 32936-32942Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Forty hours following transfection, cells were harvested in ice-cold phosphate-buffered saline, collected by centrifugation, and resuspended in 0.1m Tris-HCl, pH 7.5. The cells were disrupted by sonication, and microsomal membrane fractions were prepared at 0–4 °C, as described previously (36Spencer A.G. Thuresson E.A. Otto J.C. Song I. Smith T. DeWitt D.L. Garavito R.M. Smith W.L. J. Biol. Chem. 1999; 274: 32936-32942Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Membranes were isolated from sham-transfected cells in an identical manner. Protein concentrations were determined using the method of Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with bovine serum albumin as the standard. Microsomal preparations were used for Western blotting and for cyclooxygenase and peroxidase assays. Cyclooxygenase assays were performed at 37 °C by monitoring the initial rate of O2 uptake using an oxygen electrode (23Thuresson E.D. Lakkides K.M. Smith W.L. J. Biol. Chem. 2000; 275: 8501-8507Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 38Shimokawa T. Smith W.L. J. Biol. Chem. 1991; 266: 6168-6173Abstract Full Text PDF PubMed Google Scholar). Each standard assay mixture contained 3.0 ml of 0.1 m Tris-HCl, pH 8.0, 1 mm phenol, 85 μg of bovine hemoglobin, and 100 μm arachidonic acid. Reactions were initiated by adding ∼250 μg of microsomal protein in a volume of 20–50 μl to the assay chamber. Km values were measured using 0.5–500 μm arachidonate. The shareware program “Hyper” copyrighted in 1993 by J. S. Easterly was used to perform a hyperbolic regression analysis of the enzyme kinetic data to generate Km and Vmaxvalues. Inhibition of cyclooxygenase activity was measured by adding aliquots of microsomal suspensions to assay mixtures containing 100 μm arachidonate and 200 μm flurbiprofen. Peroxidase activity was measured spectrophotometrically withN,N,N′,N′-tetramethylphenylenediamine (TMPD) as the reducing cosubstrate (39Kulmacz R.J. Prostaglandins. 1987; 34: 225-240Crossref PubMed Scopus (50) Google Scholar). The reaction mixture contained 0.1 mTris-HCl, pH 8.0, 0.1 mm TMPD, ∼100 μg of microsomal protein, and 1.7 μm hematin in a total volume of 3 ml. Reactions were initiated by adding 100 ml of 0.3 mmH2O2, and the absorbance at 610 nm was monitored with time. To determine the rates of inactivation of native and mutant oPGHS-1 by aspirin, microsomal enzyme samples were incubated with or without 0.1 mm acetylsalicylate at 37 °C; aliquots were removed at 0, 10, 20, 30, 40, 60, and 90 min, and cyclooxygenase activity was measured as described above. Values for t12 were determined from plots of the logarithm of activity versustime. For determination of the stereoselectivity of Tyr-355 mutant enzymes with R versus S-ibuprofen, these inhibitors were added to the O2 electrode assay chamber at various concentrations prior to the addition of microsomal enzyme preparations. IC50 values for each of the stereoisomers of ibuprofen were determined, and the R/S ratios for inhibition of each of the Tyr-355 mutant enzymes and native oPGHS-1 were calculated. For time-dependent inhibition studies, flurbiprofen at an appropriate concentration (e.g. 50 μm) was preincubated with the enzyme (250 μg of microsomal protein) at 37 °C for various times; cyclooxygenase measurements were then performed upon adding enzyme-inhibitor complex to the assay chamber. Microsomal samples (∼5 μg of protein) were resolved by one-dimensional SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose membranes using a Hoeffer Scientific Semi-dry Transfer apparatus. Membranes were blocked for 12 h in 3% nonfat dry milk, 0.1% Tween 20, and Tris-buffered saline, followed by a 2-h incubation with a peptide-directed antibody against oPGHS-1 (40Otto J.C. DeWitt D.L. Smith W.L. J. Biol. Chem. 1993; 268: 18234-18242Abstract Full Text PDF PubMed Google Scholar) in 1% dry milk, 0.1% Tween 20, and Tris-buffered saline at room temperature. Membranes were washed and incubated for 1 h with a 1:2000 dilution of goat anti-rabbit IgG-horseradish peroxidase, after which they were incubated with Amersham Pharmacia Biotech ECL reagents and exposed to film for chemiluminescence. A general protocol for product analysis is as follows. 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 (100–250 μg of protein) were incubated for 1–10 min at 37 °C in 0.1 m Tris-HCl, pH 7.5, containing 1 mm phenol and 6.8 μg of bovine hemoglobin in a total volume of 200 μl. Reactions were initiated with 35 μm[1-14C]arachidonic acid and were performed with or without 200 μm flurbiprofen and stopped by adding 1.4 ml of CHCl3:MeOH (1:1; v/v). Insoluble cell debris was removed by centrifugation, and 0.6 ml of CHCl3 and 0.32 ml of 0.88% formic acid were added to the resulting supernatant. The organic phase was collected, dried under N2, redissolved in 50 μl of CHCl3, and spotted on a Silica Gel 60 thin layer chromatography plate; the lipid products were chromatographed for 1 h in benzene:dioxane:formic acid:acetic acid (82:14:1:1, v/v). Products were visualized by autoradiography and quantified by liquid scintillation counting. Negative control values from samples incubated with 200 μm flurbiprofen were subtracted from the experimental values observed for each sample in the absence of flurbiprofen. For RP-HPLC analyses of products, native or mutant oPGHS-1 (1 mg of microsomal protein) was reacted with 100 μm arachidonic acid for 30 min at 37 °C, and products were collected as described previously (23Thuresson E.D. Lakkides K.M. Smith W.L. J. Biol. Chem. 2000; 275: 8501-8507Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Products were dried under N2 and resuspended in HPLC buffers (1:1, v/v). 15- and 11-HETEs were separated by reverse phase-HPLC using a C18 column (Vydac); the Waters model 600 HPLC was equipped with a 990 photo diode array detector set to 200 and 234 nm. The strong component of the mobile phase was 0.1% acetic acid, and the eluting solvent was acetonitrile. The flow rate was 1 ml/min. The following elution profile was used. Initial conditions were 30% acetonitrile, increased linearly over 30 min to 50% acetonitrile, then increased linearly from 30 to 100 min to 75% acetonitrile, then increased linearly from 100 to 125 min to 100% acetonitrile and held for 5 min at 100% before returning to initial conditions. The retention times of 15-HETE and 11-HETE averaged 36 and 38 min, respectively. Material obtained by RP-HPLC was esterified by treatment with excess diazomethane and subjected to chiral-phase HPLC. Chiral-phase HPLC separations of the methyl esters of 11- and 15-HETEs were performed with a Chiralcel OC column (250 × 4.6 mm; Daicel Chemical Industries, Osaka, Japan) using hexane/2-propanol (90:10, v/v) as the solvent and a flow rate of 0.5 ml/min. Diazomethane was prepared from Diazald® and distilled in ether per the Aldrich Technical Bulletin AL-180. Mutations were modeled and analyzed in the program CHAIN (41Sack J.S. J. Mol. Graphics. 1988; 6: 224-225Crossref Google Scholar) using the coordinates from the crystal structure of Co3+-oPGHS-1 complexed with AA (Protein Data Bank code 1DIY) and Fe3+-oPGHS-1 complexed with flurbiprofen (Protein Data Bank code 1CQE). Statistical significance of the kinetic data (Fig. 5) was determined using a two-sample t test assuming equal variances. As illustrated in Fig. 1, arachidonic acid (AA) is bound in an extended L-conformation in the AA/Co3+-heme oPGHS-1 co-crystal structure (21Malkowski M.G. Ginell S. Smith W.L. Garavito R.M. Science. 2000; 289: 1933-1937Crossref PubMed Scopus (260) Google Scholar). The carboxylate group of AA interacts with Arg-120; the ω end abuts Ile-377 and Gly-533; the 13-pro-S-hydrogen is appropriately aligned with Tyr-385, and there is ample space for the first O2 insertion at C-11 and facile bridging of the incipient 11-hydroperoxyl radical to C-9 to form the endoperoxide. Formation of the cyclopentane ring is proposed to involve rotation about the C-10/C-11 bond and consequent movement of the ω terminus so that C-12 can react with the C-8 radical that is produced upon formation of the endoperoxide group; this movement, in turn, positions C-15 adjacent to Tyr-385 for a second antarafacial O2 insertion and a one-electron reduction of the 15-hydroperoxyl radical by Tyr-385 to regenerate the Tyr-385 radical (21Malkowski M.G. Ginell S. Smith W.L. Garavito R.M. Science. 2000; 289: 1933-1937Crossref PubMed Scopus (260) Google Scholar). In the AA/Co3+-heme oPGHS-1 co-crystal structure AA makes two hydrophilic and 48 hydrophobic contacts involving a total of 19 residues in the first shell of the cyclooxygenase active site (Fig. 1and Table I (21Malkowski M.G. Ginell S. Smith W.L. Garavito R.M. Science. 2000; 289: 1933-1937Crossref PubMed Scopus (260) Google Scholar)). We have now mutated a total of 14 of the residues that are putatively involved in direct interactions with AA as well as four other residues (Leu-384, Phe-518, Met-522, and Leu-531) that are in the first shell of the active site but do not directly contact the substrate (Fig. 1; Tables I andII). In analyzing the various mutants, we identified the AA oxygenation products, determined kinetic constants for AA oxygenation, and measured peroxidase activity (TableIII). Cyclooxygenase and peroxidase activities for native and all mutant enzymes were normalized to levels of protein expression determined from Western blot analysis and densitometric quantitation (23Thuresson E.D. Lakkides K.M. Smith W.L. J. Biol. Chem. 2000; 275: 8501-8507Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The results of Western transfer blotting indicated that the native enzyme as well as all mutants tested were expressed at similar levels by COS-1 cells (data not shown). A discussion of the implications of the results is presented below for individual residues.Table IContacts between AA and cyclooxygenase active site residuesvan der Waals and hydrogen bond interactions were calculated using CHAIN (41Sack J.S. J. Mol. Graphics. 1988; 6: 224-225Crossref Google Scholar). van der Waals contacts within 4 Å are listed as well as two hydrophilic interactions including a salt bridge between the AA carboxylate and the NH1 atom of Arg-120 (angle = 143 °) and a hydrogen bond to the OH group of Tyr-355 (angle = 115°).Table IIInteractions between residues within the cyclooxygenase active site that do not contact arachidonic acidTable IIInteractions between residues within the cyclooxygenase active site that do not contact arachidonic acidvan der Waals and hydrogen bond interactions were calculated using CHAIN (41Sack J.S. J. Mol. Graphics. 1988; 6: 224-225Crossref Google Scholar). van der Waals contacts within 4are listed. Hydrogen bonding was assigned for O–H N, O.H, and HNC bond angles >90 ° and distances not exceeding 3.6Table IIIKinetic properties and product analyses for oPGHS-1 cyclooxygenase active site mutantsTable IIIKinetic properties and product analyses for oPGHS-1 cyclooxygenase active site mutantsPeroxidase activity was measured spectrophotometrically using 0.2 mm H2O2 and 100 μm TMPD as substrates, and oxygenase activity was measured with an oxygen electrode as described in the text. Values are calculated for arachidonic acid turnover and are corrected for the percentage of mono- and bisoxygenated products formed with the mutants. A value of 100% is assigned for peroxidase and oxygenase activity of native oPGHS-1. Standard deviation from the mean is within 10% of all values reported. Relative Vmax values reported for mutant enzymes for which Km values were not determined represent rate measurements performed using 100 μm AA. Values are from a minimum of four separate determinations. ND, not determined. van der Waals and hydrogen bond interactions were calculated using CHAIN (41Sack J.S. J. Mol. Graphics. 1988; 6: 224-225Crossref Google Scholar). van der Waals contacts within 4 Å are listed as well as two hydrophilic interactions including a salt bridge between the AA carboxylate and the NH1 atom of Arg-120 (angle = 143 °) and a hydrogen bond to the OH group of Tyr-355 (angle = 115°). van der Waals and hydrogen bond interactions were calculated using CHAIN (41Sack J.S. J. Mol. Graphics. 1988; 6: 224-225Crossref Google Scholar). van der Waals contacts within 4are listed. Hydrogen bonding was assigned for O–H N, O.H, and HNC bond angles >90 ° and distances not exceeding 3.6 Peroxidase activity was measured spectrophotometrically using 0.2 mm H2O2 and 100 μm TMPD as substrates, and oxygenase activity was measured with an oxygen electrode as described in the text. Values are calculated for arachidonic acid turnover and are corrected for the percentage of mono- and bisoxygenated products formed with the mutants. A value of 100% is assigned for peroxidase and oxygenase activity of native oPGHS-1. Standard deviation from the mean is within 10% of all values reported. Relative Vmax values reporte" @default.
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• W2023302278 date "2001-03-01" @default.
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• W2023302278 title "Prostaglandin Endoperoxide H Synthase-1" @default.
• W2023302278 cites W1485442945 @default.
• W2023302278 cites W1490609633 @default.
• W2023302278 cites W1525772448 @default.
• W2023302278 cites W1527453674 @default.
• W2023302278 cites W1531513729 @default.
• W2023302278 cites W1544157645 @default.
• W2023302278 cites W1586663904 @default.
• W2023302278 cites W1596000517 @default.
• W2023302278 cites W1660326086 @default.
• W2023302278 cites W1674582945 @default.
• W2023302278 cites W1902547891 @default.
• W2023302278 cites W1964665460 @default.
• W2023302278 cites W1965837061 @default.
• W2023302278 cites W1968444137 @default.
• W2023302278 cites W1970287261 @default.
• W2023302278 cites W1976654542 @default.
• W2023302278 cites W1978518126 @default.
• W2023302278 cites W1987212025 @default.
• W2023302278 cites W1991987072 @default.
• W2023302278 cites W1997761684 @default.
• W2023302278 cites W2002840016 @default.
• W2023302278 cites W2007504470 @default.
• W2023302278 cites W2010533134 @default.
• W2023302278 cites W2010967782 @default.
• W2023302278 cites W2013687281 @default.
• W2023302278 cites W2013769810 @default.
• W2023302278 cites W2023200971 @default.
• W2023302278 cites W2024715019 @default.
• W2023302278 cites W2026457555 @default.
• W2023302278 cites W2030850720 @default.
• W2023302278 cites W2034294731 @default.
• W2023302278 cites W2048234380 @default.
• W2023302278 cites W2049771771 @default.
• W2023302278 cites W2059750485 @default.
• W2023302278 cites W2061592031 @default.
• W2023302278 cites W2062641944 @default.
• W2023302278 cites W2066395665 @default.
• W2023302278 cites W2068319865 @default.
• W2023302278 cites W2082522536 @default.
• W2023302278 cites W2088363148 @default.
• W2023302278 cites W2115350007 @default.
• W2023302278 cites W2126853519 @default.
• W2023302278 cites W2138149192 @default.
• W2023302278 cites W2139988779 @default.
• W2023302278 cites W4293247451 @default.
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