From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
Received for publication, October 13, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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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 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.
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 (1-3).
PGHS-1 (or COX-1 (for cyclooxygenase-1)) is a constitutive enzyme,
whereas PGHS-2 (COX-2) is the inducible isoform (1-12).
PGHSs catalyze two separate reactions including a cyclooxygenase
(bisoxygenase) reaction in which AA is converted to PGG2 and a peroxidase reaction in which PGG2 undergoes a
two-electron reduction to PGH2 (1-3). 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 (13, 14). 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 (15-17) thereby inhibiting cyclooxygenase activity
but not peroxidase activity (18-20).
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 (15-17). More recently we determined the structure of AA
bound within the cyclooxygenase active site of ovine (o) PGHS-1 (21).
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 (22), only three of these
conformations are catalytically competent (23); 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-1202 is important for
high affinity binding of AA to PGHS-1 (24, 25) (but not PGHS-2 (26,
27)), that Tyr-385 is involved as a tyrosyl radical in abstracting the
13-pro-S-hydrogen from AA (28, 29), and that Ser-530 and
Ile-523 are determinants of inhibitor specificity (30-34). 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 (31, 35). 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.
Materials--
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 (36), 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.
Preparation of oPGHS-1 Mutants--
Mutants were prepared either
starting with M13mp19-PGHSov, which contains a 2.3-kilobase
SalI fragment coding for native oPGHS-1 and employing the
Bio-Rad Muta-Gene kit and the protocol as described by the manufacturer
(36), or by site-directed mutagenesis of oPGHS-1 in the pSVT7 vector
(28), 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.
Transfection of COS-1 Cells with oPGHS-1 Constructs--
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 (36). Forty hours following
transfection, cells were harvested in ice-cold phosphate-buffered
saline, collected by centrifugation, and resuspended in 0.1 M Tris-HCl, pH 7.5. The cells were disrupted by sonication,
and microsomal membrane fractions were prepared at 0-4 °C, as
described previously (36). Membranes were isolated from
sham-transfected cells in an identical manner. Protein concentrations were determined using the method of Bradford (37) with bovine serum
albumin as the standard. Microsomal preparations were used for Western
blotting and for cyclooxygenase and peroxidase assays.
Cyclooxygenase and Peroxidase Assays--
Cyclooxygenase assays
were performed at 37 °C by monitoring the initial rate of
O2 uptake using an oxygen electrode (23, 38). 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 Vmax
values. 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 with
N,N,N',N'-tetramethylphenylenediamine (TMPD) as the reducing
cosubstrate (39). The reaction mixture contained 0.1 M
Tris-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 mM
H2O2, and the absorbance at 610 nm was
monitored with time.
Inhibition of Cyclooxygenase Activity--
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 t1/2 were
determined from plots of the logarithm of activity versus time.
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.
Western Blot Analysis--
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 (40) 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.
Characterization of Arachidonic Acid-derived Products--
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 (23). 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.
Molecular Modeling and Statistical Analyses--
Mutations were
modeled and analyzed in the program CHAIN (41) 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.
Overview
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 (21). The
carboxylate group of AA interacts with Arg-120; the
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
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
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 (21).
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Fig. 1.
Schematic representation of interactions
between arachidonic acid and amino acid residues lining the
cyclooxygenase active site channel. All dashed lines
represent interactions of 4.0 Å between specific side chain atoms of
the protein and carbon or oxygen atoms of AA (21). The distances
between various carbons of AA and interacting side chain residues are
listed in Tables I and II.
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. 1 and Table I (21)). 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 and II). In analyzing the various mutants, we identified the AA oxygenation products, determined kinetic constants for AA oxygenation, and measured peroxidase activity (Table III). 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 (23). 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.
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Based on various simplifying assumptions, we have categorized cyclooxygenase active site residues into five functional groups as follows: (a) residues directly involved in hydrogen abstraction from C-13 of AA (Tyr-385); (b) residues essential for positioning C-13 of AA for hydrogen abstraction (Gly-533 and Tyr-348); (c) residues critical for high affinity AA binding (Arg-120); (d) residues critical for positioning AA in a conformation such that when hydrogen abstraction does occur the AA is appropriately arranged to yield PGG2 (Val-349, Trp-387, and Leu-534); and (e) all other active site residues, which individually make lesser but measurable contributions to optimizing catalytic efficiency. The simplifying assumptions that serve as the basis for this categorization are as follows. First, residues, which when altered yield mutant enzymes without cyclooxygenase activity, are considered to be critical for positioning C-13 for hydrogen abstraction by Tyr-385. Second, residues, which when mutated yield mutant enzymes with substantially increased Km values (>100-fold), are considered to be involved in high affinity AA binding as changes in Km values are reasonable approximations of substrate affinity. And finally, AA can exist in three distinct, catalytically competent conformations in native oPGHS-1 which yield PGG2 (95%), 11-HPETE (2.5%), and (15R,15S)-HPETE (2.5%), respectively (23); as a first approximation, amino acids residues, mutation of which causes a major change in the composition of the oxygenation products, are considered to be those critical for positioning AA in the conformation that yields PGG2. These categories are meant to draw attention to those residues that exhibit the most dramatic effects on catalytic constants and product profiles. Mutations of other residues have similar effects, but to lesser degrees, and are discussed below. We also emphasize that in interpreting the results of the mutational analyses, we have in most instances made the simplifying assumption that amino acid substitutions alter only interactions with the AA chain and have largely ignored potential effects on interactions among adjoining residues.3 This is clearly an oversimplification that can only be justified broadly by pointing out that most of the mutant enzymes retain both peroxidase and cyclooxygenase activities; however, subtle changes in inter-residue interactions brought about by various mutations certainly may have important effects on kinetic properties and product compositions. Future studies on the crystal structures of AA complexed with mutant forms of oPGHS-1 will be necessary to help resolve the relative influences of mutations on interactions with AA versus those with neighboring amino acid residues.
Residues Directly Involved in Hydrogen Abstraction from C-13 of AA (Tyr-385)
As reported previously mutation of Tyr-385 to a phenylalanine eliminates cyclooxygenase activity without eliminating peroxidase activity; moreover, Tyr-385 is nitrated by tetranitromethane when the cyclooxygenase site is unoccupied, but this nitration is prevented in the presence of inhibitors that occupy the cyclooxygenase site (28). Tyr-385 is located adjacent to C-13 of AA in the AA/Co3+-heme oPGHS-1 co-crystal structure (21). There is now considerable evidence that this residue is involved as a tyrosyl radical in abstracting the 13-pro-S-hydrogen from AA in the rate-determining step in cyclooxygenase catalysis (reviewed in Ref. 1).
Residues Essential for Positioning C-13 of AA for Hydrogen Abstraction (Tyr-348 and Gly-533)
Tyrosine 348-- Examination of the crystal structures of oPGHS-1 complexed with nonsteroidal anti-inflammatory drugs (15, 42) and the Co3+-heme oPGHS-1/AA complex (Fig. 1 (21)) suggests that Tyr-348 may be involved in the appropriate positioning of Tyr-385 and/or AA. The distance between the phenolic oxygens of Tyr-348 and Tyr-385 suggests that there is a hydrogen bond between these two atoms that is important for positioning Tyr-385; there are no other contacts between Tyr-348 and Ty385. The CE2 phenyl ring carbon of Tyr-348 is within van der Waals distance of C-12, C-13, and C-14 of AA suggesting that hydrophobic interactions between Tyr-348 and substrate could be important in positioning C-13 of AA. Our results indicate that these latter contacts between Tyr-348 and AA are essential in positioning C-13 of AA but that a hydrogen bond between Tyr-348 and Tyr-385, if it does exist, is not important for cyclooxygenase catalysis.
Results that argue against a hydrogen bonding interaction between Tyr-348 and Tyr-385 that is important for catalysis are as follows. Previous studies have established that substitution of Tyr-348 with phenylalanine has little effect on either the cyclooxygenase or peroxidase activity of the enzyme (28, 29, 43). A more detailed kinetic analysis of Y348F oPGHS-1 performed here indicates that the Vmax/Km is 60% of that of native oPGHS-1 and that there are no significant differences in product formation between native and Y348F oPGHS-1 (Table III). Additionally, Y348F oPGHS-1 forms a tyrosyl radical with properties similar to that of native oPGHS-1 (29, 43).
The concept that Tyr-348 is essential for positioning AA comes from comparing results obtained with native oPGHS-1 and the Y348F and Y348L mutants (Table III). Y348F oPGHS-1 has properties similar to those of native enzyme. In contrast, Y348L oPGHS-1 lacks cyclooxygenase activity while retaining 9% native peroxidase activity; no cyclooxygenase products were detected with Y348L oPGHS-1 even with a sensitive radio thin layer chromatographic assay using [1-14C]arachidonic acid as the substrate. Examination of the Co3+-heme oPGHS-1·AA complex (Fig. 1 (21)) suggests that substituting leucine at position 348 would permit C-13 of AA to move away from Tyr-385 increasing the distance between Tyr-385 and C-13 such that hydrogen abstraction could not occur. The drop in peroxidase activity for this mutant also suggests the possibility of structural changes affecting the peroxidase as well as the cyclooxygenase active site. Tyr-348 lies directly below Tyr-385, which resides in one of the helices contacting the heme group of the peroxidase active site. Substitution of Tyr-348 with a leucine could create a space, which might allow movement of this helix and perturbation of the peroxidase active site. Thus, we conclude that the phenyl ring of Tyr-348 is essential in positioning C-13 of AA with respect to Tyr-385 but could play a role in the preservation of the structural integrity of the peroxidase site as well.
A Y348W mutation disrupted both peroxidase and cyclooxygenase functions; addition of (15S)-HPETE to a reaction mixture containing AA and Y348W oPGHS-1 failed to activate cyclooxygenase activity. The lack of peroxidase activity observed with the Y348W mutant suggests that this substitution has an effect on the peroxidase active site. We speculate that when a tryptophan group is inserted at position 348, it causes a change in the position of Trp-387 and/or Tyr-385 which, in turn, leads to movement of His-388, the proximal heme ligand, and results in a change in the binding of the heme group so that peroxidase catalysis does not occur.
Glycine 533--
Gly-533 lies at the distal end of the
cyclooxygenase active site channel with the C of Gly-533 within van
der Waals distance of C-20 of AA (Fig. 1 and Table I (21)). Previous
studies had shown that substitution of Gly-533 with alanine eliminates
cyclooxygenase but not peroxidase activity (Table III (31)). This
contrasts with observations made with murine PGHS-2 where the
homologous mutant retains activity toward AA (35). Accordingly, we
prepared and analyzed a G533A/I523V oPGHS-1 mutant to determine whether cross-substitution of the one-core residue in the cyclooxygenase active
site which is different between the two isoforms would permit activity
with a G533A mutation in oPGHS-1. The G533A/I523V oPGHS-1 retained 54%
of the peroxidase activity of native oPGHS-1 but, again, was unable to
oxygenate AA at a substrate concentration of 100 µM
(Table III).
Residues Critical for High Affinity AA Binding (Arg-120)
The NH1 guanidinium nitrogen atom of Arg-120 contacts the carboxylate group of AA in the AA/Co3+-heme PGHS-1 co-crystal structure (Fig. 1 and Table I (21)). There is considerable evidence indicating that interactions between Arg-120 and the carboxylate carbons of substrates and inhibitors are important in cyclooxygenase catalysis and inhibition (Table III (24, 25)). Most notably, replacement of Arg-120 with a neutral glutamine causes a 1000-fold increase in the Km for AA indicating that this residue is essential for high affinity AA binding to the cyclooxygenase site (24, 25) and that this involves an ionic linkage between Arg-120 and the carboxylate of AA. In contrast, an ionic interaction between Arg-120 and AA is not important with PGHS-2 where an R120Q mutation has no detectable effect on either the Vmax or the Km for AA oxygenation, although this mutation does cause a small increase in the relative amount of 11-HPETE formed (27).
Residues Critical for Positioning AA in a Conformation Such That When Hydrogen Abstraction Does Occur the AA Is Appropriately Arranged to Yield PGG2 (Val-349, Trp-387, and Leu-534)
Valine 349-- One of the methyl groups (CG1) of Val-349 lies within van der Waals distance of both C-3 (3.36 Å) and C-4 (3.14 Å) of AA in the AA/Co3+-heme oPGHS-1 crystal structure (Fig. 1 (21)). To assess the role of Val-349 in cyclooxygenase catalysis, we substituted this residue with alanine, serine, threonine, and leucine and evaluated these mutants. All of the mutants retained oxygenase and peroxidase activity. The catalytic efficiencies (Vmax/Km values) for the oxygenase reaction ranged from 6 to 64% of native oPGHS-1 (Table III).
Lipid-soluble products synthesized from
[1-14C]arachidonic acid by sham-transfected COS-1 cells
and COS-1 cells transfected with native oPGHS-1 or various oPGHS-1
mutants were separated by thin layer chromatography (Fig.
2) or RP-HPLC and the amounts of each
product quantified (Table III). Mutants in which Val-349 was replaced
with a smaller residue (i.e. V349A, V349S, and V349T oPGHS-1) all produced an abundance of 11-HETE versus
PGG2-derived products and little or no detectable 15-HETE;
the 11-HETE:PGG2 ratio for these mutants increased as the
size of the group at position 349 was decreased. In contrast, as
reported previously (23), replacing Val-349 with a larger leucine
residue (V349L oPGHS-1) led to the generation of a relative abundance
of 15-HETE and no detectable 11-HETE. Sham-transfected COS-1 cells did
not transform [1-14C]arachidonic acid to products.
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Chiral HPLC analysis of 11-HETEs formed from arachidonic acid by V349A,
V349S, and V349T oPGHS-1 mutants established that the product was
exclusively (11R)-HETE. A representative chromatographic profile for the chiral analysis of the 11-HETE formed by the V349A oPGHS-1 mutant is presented in Fig. 3. As
reported previously, the V349L oPGHS-1 mutant formed a 65:35 mixture of
(15S,15R)-HETE, the same enantiomeric profile
obtained with native oPGHS-1 (23).
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We also examined the interaction of various cyclooxygenase inhibitors with the V349A and V349L mutants. IC50 values for cyclooxygenase inhibition were determined for docosahexaenoic acid (22:6(n-3)), flurbiprofen, and flufenamic acid. Inhibition was studied using the O2 electrode assay with 50 µM AA as substrate. The V349A mutant was more sensitive than native oPGHS-1 to inhibition by both flurbiprofen (IC50 values of 0.28 and 2.9 µM, respectively) and flufenamic acid (IC50 values of 0.9 and 6.0 µM, respectively); the substrate analogue docosahexaenoic acid was a less potent inhibitor of the V349A mutant than the native enzyme (IC50 values of 350 versus 100 µM, respectively). The V349A and V349L oPGHS-1 mutants were inactivated significantly more slowly (t1/2 = 93 and >160 min, respectively) by 0.1 mM aspirin than native oPGHS-1 (t1/2 = 29 min).
Flurbiprofen and flufenamate have similar structures, including two phenyl rings and a carboxylate moiety. The carboxyl groups of flurbiprofen and other carboxylate-containing NSAIDs such as iodosuprofen and iodoindomethacin all form salt bridges with Arg-120 at the mouth of the cyclooxygenase active site (15, 25), and the structurally similar flufenamate is thought to be bound in the same manner. Examination of the flurbiprofen-bound crystal structure of oPGHS-1 shows extensive van der Waals contacts between the CG1 of Val-349 and the lower phenyl ring of flurbiprofen (15). Substitution of Val-349 with an alanine results in more potent inhibition by both flurbiprofen and flufenamate, suggesting that the contacts made by Val-349 are preventing the binding of these inhibitors in the most thermodynamically stable conformation. Thus, the additional space created by the V349A mutation seems to create an active site that can better accommodate the extra bulk of the 2-phenylpropionic acid inhibitors. In contrast, docosahexaenoic acid (22:6(n-3)) is structurally similar to the substrate, arachidonic acid, and is a slightly less potent inhibitor of the V349A oPGHS-1 mutant compared with the native enzyme. This substrate analogue is elongated and contains two extra double bonds when compared with arachidonic acid. It is therefore much more rigid than arachidonate and may only have one effective binding orientation. Computer modeling of 22:6 in the oPGHS-1 cyclooxygenase active site suggests that there would likely be van der Waals interactions between Val-349 and one or more carbons near the carboxyl end of 22:6. Removal of these contacts creates a larger active site that appears to somewhat hamper tight binding of 22:6. Similarly, 22:6 is a less potent inhibitor of PGHS-2, which has a larger and more accommodating active site than PGHS-1 (1, 3, 27).
We reported previously that AA can assume at least three catalytically
competent arrangements in the cyclooxygenase active site of oPGHS-1
(23). These arrangements, occurring at the time of hydrogen
abstraction, lead to different products, PGG2,
(11R)-HPETE, or (15R/S)-HPETE. With native
oPGHS-1 the kinetically most favorable arrangement of AA is that which
yields PGG2. With V349A oPGHS-1 the arrangements yielding
PGG2 and 11-HPETE appear to be equally favorable. 11-HPETE
would be expected to be formed from an arrangement of AA in which C-9
and C-11 were slightly misoriented such that the endoperoxide
bridge between these carbons cannot be formed. The kinetics of both
PGG2 and 11-HPETE formation were found to be similar to one
another with the V349A mutant (Fig. 4),
and the Vmax:Km ratios are similar for
V349A and native oPGHS-1 (Table III). These observations suggest that
the valine to alanine substitution does not appreciably influence the
positioning of C-13 with respect to Tyr-385; moreover, formation of
both PGG2 and 11-HPETE presumably proceeds in the same way
via abstraction of the 13-pro-S-hydrogen and formation of an
11-hydroperoxyl radical (44). Accordingly, we suggest that Val-349
plays a major role in positioning the carboxyl half of AA and
especially in positioning C-9 without appreciably affecting the
location of the half of AA within the cyclooxygenase site.
Obviously, the effect of Val-349 on the position of C-9 must occur
indirectly because Val-349 contacts only C-3 and C-4 of the substrate
(Fig. 1). Placing smaller amino acids at position 349 apparently
permits the carboxyl half of AA greater flexibility, which in turn
translates into a small shift in the orientation of C-9 with respect to
C-11.
|
Tryptophan 387--
As shown in Fig. 1 Trp-387 resides near the
apex of the cyclooxygenase channel and is positioned with its CH2
indole ring carbon 3.38 Å from C-11 and 3.65 Å from C-12 of AA;
additionally, the CZ2 phenyl ring carbon of Trp-387 is 3.95 Å from
C-11 of AA (Fig. 1 and Table I (21)). Trp-387 also makes a van der
Waals contact with Tyr-385 (21). Previous studies of W387R, W387F, and
W387S oPGHS-1 had indicated that W387R and W387S oPGHS-1 lack cyclooxygenase activity; W387S oPGHS-1 had about 5% of the peroxidase activity of native enzyme (Table III (45)). In contrast, W387F exhibits
both cyclooxygenase and peroxidase activities and generates a tyrosyl
radical signal upon incubation with hydroperoxide (45). We prepared and
characterized two other mutants for the present study, W387A and W387L
oPGHS-1, and we performed additional kinetic studies and product
analyses with the W387F mutant. W387A lacked cyclooxygenase activity
but retained substantial peroxidase activity (Table III). W387L oPGHS-1
had about 10% of the cyclooxygenase activity of native enzyme. The
Vmax value for W387F oPGHS-1 was about 45% of
that observed with native enzyme although there was about a 10-fold
increase in the overall Km. W387F and W387L oPGHS-1
formed relatively large amounts of 11-HETE (>30% of total products)
from AA (Table III); in each case, as expected (46), the 11-HETE was
found by chiral HPLC analyses to be exclusively (11R)-HETE
(data not shown). As was found for native oPGHS-1 and V349L oPGHS-1
(23), the Km values for the formation of
PGG2, 11-HPETE, and 15-HPETE were different from one
another (Fig. 5).
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The results of the product analyses with W387F and W387L oPGHS-1 are similar to those observed with V349A oPGHS-1 in that all of these mutants form large amounts of 11-HETE. Again, the likely explanation is that the arrangement of AA, which leads to 11-HPETE, is relatively stabilized with these mutants such that the orientations of C-11 and C-9 do not permit formation of the endoperoxide. In the case of the W387F and W387L oPGHS-1 mutants, there is additional space at position 387. This, in turn, allows AA additional flexibility about C-11 where there are normally van der Waals interactions between this carbon and the CH2 and CZ2 carbons of Trp-387. As a consequence AA can more readily assume a conformation that leads to 11-HPETE formation.
Leucine 534--
The CD1 and CD2 methyl groups of Leu-534 are
within van der Waals distance of C-15 (3.54 Å), C-16 (3.6 Å), and
C-18 (3.91 Å) of AA in the AA/Co3+-heme oPGHS-1 crystal
structure (21). Interestingly, substitution of Leu-534 with either
alanine or valine yields mutant enzymes that produce large amounts of
15-HETE (Table III), and the 15-HETE that is produced is almost
exclusively (95%) (15S)-HETE (Fig. 6); in contrast, with native oPGHS-1
(15S)-HETE is composed of 65% of the total 15-HETE.
Apparently, having a hydrophobic residue smaller than leucine at
position 534 enlarges the cyclooxygenase active site near C-15
providing for relatively greater access of O2 antarafacial
to the site of hydrogen abstraction. Thus, Leu-534 is important in
facilitating formation of PGG2 versus 15-HPETE.
(15S)-HETE is the only 15-HETE formed by native human PGHS-2
(46), and the cyclooxygenase site of PGHS-2 is somewhat larger and more
accommodating than that of PGHS-1 (1).
|
Residues That Optimize Cyclooxygenase Catalysis (Phe-205, Phe-209, Phe-381, Ser-353, Tyr-355, Ile-377, Leu-384, Phe-518, Met-522, Ser-530, and Leu-531)
Phenylalanine 205, Phenylalanine 209, and Phenylalanine 381-- Various phenyl ring carbons of Phe-205, Phe-209, and Phe-381 lie within van der Waals distance of C-14 through C-20 of AA (Fig. 1 and Table I (21)). Leucine substitutions of Phe-209, Phe-205, and Phe-381 reduce the Vmax for AA oxygenation somewhat but have little effect on the product distribution (Table III). It is possible that the leucine substitutions are able to maintain some of the original phenylalanine contacts. Alanine substitutions at positions 205, 209, and 381 have relatively little impact on Km values but reduce the Vmax for AA oxygenation to a greater degree than observed for leucine substitutions, particularly for F381A which has a Vmax of only 4% of the native enzyme, and furthermore cause a modest change in the product profile (i.e. relatively small increases in 11-HPETE formation). Examination of the crystal structure indicates that the edge of the phenyl ring of Phe-205 makes four van der Waals contacts with C-14, C-15, and C-18 and that one face of the phenyl ring of Phe-209 makes six contacts with C-17, C-18, and C-19. However, removal of these contacts has only modest effects on PGHS catalysis (Table III). The crystal structure predicts that C-14, C-15, C-17, and C-18 of AA are each contacted by at least one other active site residue (Val-344, Tyr-348, and/or Leu-534 (Table I)). Apparently, these latter interactions are (a) relatively more important functionally than the contacts made by either Phe-205 or Phe-209 and/or (b) compensate for the loss of these contacts in the mutant proteins containing substitutions of either Phe-205 or Phe-209. For instance, as described above, Tyr-348 makes van der Waals contacts with C-14 of AA, and elimination of this contact with a leucine substitution results in a complete loss of cyclooxygenase activity. Thus, the Tyr-348 contact with C-14 appears to be a major contributor to substrate positioning and stabilization at this point in the AA molecule, whereas the C-14 contact made by Phe-205 is relatively unimportant. Also, there are 34 contacts between active site residues and the methyl half of AA compared with 16 contacts with the carboxyl end; thus, eliminating a contact with the methyl end of AA may be relatively less important than eliminating a contact with the carboxyl half of AA. The positioning of AA seems to be most critically dependent on having enough space at the methyl end of the substrate (e.g. Gly-533) to permit the correct positioning of the 13-pro-S-hydrogen with respect to Tyr-385.
Serine 353--
The C carbon of Ser-353 makes a van der Waals
contact with C-3 of AA at the side opposite the C-3 and C-4 contacts
made by Val-349 (Fig. 1 and Table I (21)). When this residue was
mutated to glycine, alanine, or threonine, all of the mutants retained substantial cyclooxygenase activity. The products formed by the S353G
and S353A oPGHS-1 mutants were the same as those formed by native
oPGHS-1; S353T oPGHS-1 formed larger amounts of both 11- and 15-HETE. A
V349A/S353T double mutant, designed to remove all contacts between C-3
and C-4, had only 3% of the cyclooxygenase activity of the native
enzyme compared with the single mutants that had 55 (V349A) and 42%
(S353T) of native activity. This suggests that a C-3 contact is
particularly important, albeit indirectly, in positioning C-13 with
respect to Tyr-385.
Tyrosine 355-- Tyr-355 resides near the mouth of the cyclooxygenase active site channel directly across from Arg-120. The phenolic group of Tyr-355 is located 3.09 Å from one of the carboxylate oxygens of AA, and one of the meta carbon positions of Tyr-355 (CE1) is within van der Waals distance (3.78 Å) of C-1 of AA. The Y355A, Y355L, and Y355F oPGHS-1 mutants exhibited peroxidase activities comparable to native oPGHS-1, but the maximal cyclooxygenase activities were only 10-20% that of native enzyme (Table III). However, the Km values for AA of the Tyr-355 mutants were only 2-5-fold higher than that of native oPGHS-1. Tyr-355 mutants formed 2-3 times more 11-HETE than native enzyme but comparable amounts of 15-HETE. These results are consistent with a subtle role for Tyr-355 in helping both to align the carboxyl half of AA, particularly C-9 with respect to C-11, at the time of endoperoxide formation and to position C-13 with respect to Tyr-385.
There are differences between the abilities of native oPGHS-1 and Tyr-355 mutants to interact with R- and S-isomers of ibuprofen (Table IV). As the size of the side chain at position 355 is decreased, there is a corresponding decrease in the ability of the mutants to discriminate between R- and S-isomers of ibuprofen. This finding is consistent with our previous results (25). Finally, there was a difference in the ability of flurbiprofen to inhibit Tyr-355 mutants. The IC50 value for instantaneous inhibition of native oPGHS-1 was determined to be 1 versus 50 µM for Y355F oPGHS-1. With 50 µM the t1/2 for time-dependent inhibition of native oPGHS-1 was 1 min, whereas the t1/2 for Y355F oPGHS-1 was between 10 and 20 min. A role for Tyr-355 in time-dependent inhibition of human PGHS-2 has been demonstrated previously (47, 48).
|
Isoleucine 377--
Ile-377 lies at the distal end of the
cyclooxygenase active site channel with one of its carbons
within 3.7 Å of C-20 of arachidonate (Fig. 1 and Table I (21)).
Substitution of Ile-377 with a smaller valine residue yields a mutant
enzyme with kinetic properties very similar to native oPGHS-1.
Substitution of a valine at this position will result in the removal of
the
carbons and increase the distance between this residue
and C-20 of AA minimally to 5.2 Å, a distance too great to support van
der Waals interactions. This indicates that the contacts made by
Ile-377 make little contribution to either substrate binding or
positioning of C-13 with respect to Tyr-385 but does not address the
possible necessity for a space-filling residue at this position. I377V
oPGHS-1 forms somewhat more 11-HETE than native oPGHS-1 (Table III),
suggesting that this residue does play a small role in orienting C-11
with respect to C-9.
Leucine 384, Phenylalanine 518, and Methionine 522-- Leu-384, Phe-518, and Met-522 adjoin one another and form a portion of the first shell of the cyclooxygenase hydrophobic channel. These residues neighbor but are outside of van der Waals contact distance of the C-6 to C-9 segment of AA (Fig. 1). Leu-384 contacts several other active site residues including Trp-387, Met-522, and Gly-526 (Table II); Phe-518 interacts with Met-522 and Ile-523; Met-522 contacts Trp-387 and Gly-526. Leu-384, Phe-518, and Met-522 mutants all retained appreciable peroxidase and cyclooxygenase activities. Substitutions of Phe-518 and Met-522 tended to increase slightly the production of 11-HETE relative to other oxygenase products in most cases without appreciable effects on the Km values for AA. Substitutions of Leu-384 had no effect on the distribution of oxygenated products formed from [1-14C]arachidonic acid. Km values were not determined for these latter mutants.
Serine 530--
Ser-530 is the site of acetylation of oPGHS-1 by
aspirin (30, 42), and acetylation of oPGHS-1 by aspirin eliminates
cyclooxygenase activity. In the AA/Co3+-heme oPGHS-1
crystal structure the C and C
carbons of Ser-530 make van der
Waals contacts with C-10 and C-16 (Fig. 1 (21)); these contacts are on
the side of AA directly opposite that Tyr-385. Earlier studies had
shown that substitution of Ser-530 with alanine has relatively little
effect on AA oxygenation, that replacement of Ser-530 with threonine
causes a 95% drop in catalytic efficiency, and that replacement of
Ser-530 with glutamine prevents catalysis apparently by blocking
substrate access to the Tyr-385 radical (30, 31). Here we have extended
these findings showing (a) that a S530V mutant is
catalytically inactive (Table III) and (b) that the S530T
mutant forms significant amounts of (15R)-HETE but not
(15S)-HETE (Fig. 7). Recent
studies have indicated that aspirin-acetylated PGHS-2 forms exclusively
(15R)-HPETE (46, 49) and that formation of this product
involves removal of the 13-pro-S-hydrogen (44). Assuming
that (15R)-HPETE synthesis by S530T oPGHS-1 occurs in a
similar manner, this would imply that (15R)-HPETE formation
involves suprafacial addition of O2 to C-15. This is
reasonably rationalized by observation of Fig. 1 where addition of a
residue slightly larger than serine at position 530 might be expected
to block antarafacial O2 addition without preventing
abstraction of the 13-pro-S-hydrogen. Moreover, a slightly larger valine residue would be expected to narrow the cyclooxygenase channel so that C-13 would be mispositioned with respect to Tyr-385 such that hydrogen abstraction could not occur.
|
Leucine 531--
Leu-531 is present in the first shell of the
cyclooxygenase site in close proximity to Arg- 120 on the side opposite
the carboxylate group of AA and outside of van der Waals contact
distance from the carbon skeleton of AA (Fig. 1). Substitutions of
Leu-531 with similarly sized hydrophilic residues (Asp and Asn)
decreased the Vmax values for AA but with little
effect on the Km values (31). In contrast,
replacement of Leu-531 with alanine caused a 25-fold increase in the
Km. The relative amounts of oxygenated products
formed with Leu-531 mutants are similar to those formed with the native
enzyme (Table III). Apparently, replacement of Leu-531 with hydrophilic
residues results in stable but improper liganding of the substrate
carboxylate to the enzyme, resulting in a decreased
Vmax and slight mispositioning of C-13 of
arachidonate with respect to Tyr-385. Reduction of the residue size at
position 531, however, is damaging to high affinity substrate binding
of the substrate carboxylate to the enzyme, supporting the notion that
proper AA binding to Arg-120 is critical for PGHS-1 activity, and even
modest structural changes in the immediate environment are not tolerated.
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CONCLUSION |
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Nineteen residues in the cyclooxygenase channel of oPGHS-1 are predicted to make contacts with arachidonic acid (Fig. 1 and Table I (21)). Seven of these residues are most critical in permitting oPGHS-1 to convert arachidonic acid to PGG2. Tyr-385 provides the tyrosyl radical essential for initiating the rate-limiting step of cyclooxygenase catalysis, abstraction of the 13-pro-S-hydrogen; Tyr-348 and Gly-533 are involved in hydrophobic interactions with AA that are necessary to position C-13 appropriately with respect to Tyr-385; Arg-120 provides an ionic interaction with the carboxyl end of AA that is the key element in high affinity substrate binding. Without these latter interactions, catalysis cannot occur at all (except in the case of R120Q oPGHS-1 where the catalytic efficiency is decreased by 70,000-fold). Val-349, Trp-387, and Leu-534 provide hydrophobic interactions with AA, which contribute not primarily to initiation of catalysis but to positioning AA such that when hydrogen abstraction occurs the fatty acid molecule is optimally aligned to yield PGG2 rather than monohydroperoxide products. Val-349 stabilizes the carboxyl half of AA to promote proper positioning of C-9 with respect to C-11 for efficient endoperoxide formation; Trp-387 contributes to efficient endoperoxide formation through its stabilizing interactions with C-11 of AA, and Leu-534 provides steric hindrance that blocks premature oxygenation at C-15. The remaining residues comprising the cyclooxygenase active site channel provide measurable but lesser contributions to optimizing catalysis.
Related studies on the interaction between PGHS-1 and
dihomo--linolenic acid are described in an accompanying article
(51).
![]() |
ACKNOWLEDGEMENTS |
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We thank to Dr. Honggao Yan for providing expertise in enzyme kinetics and Joseph Leykam of the MSU Macromolecular Structure Facility for assistance with HPLC.
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FOOTNOTES |
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* This work was supported in part by Grants P01 GM57323 (to W. L. S. and R. M. G.), R01 HL56773 (to R. M. G.), and NRS HL10170 (to M. G. M.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Table I.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, 513 Biochemistry Bldg., Michigan State
University, East Lansing, MI 48824. Tel.: 517-355-1604; Fax: 517- 353-9334; E-mail: smithww@pilot.msu.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009377200
2 The numbering of residues in ovine PGHS-1 is based on numbering the N-terminal methionine of the signal peptide as residue number 1.
3 Although modeling specific local perturbations in the arachidonate-protein complex is straightforward, extrapolation of any modeling effort to deal with possible long range structural disruptions arising from a mutation is problematic and unsupported because little is known about the dynamic behavior of the PGHS active site. X-ray crystallographic work on PGHS-1 and PGHS-2 indicates that the PGHS structure is extraordinarily rigid when crystallized and that the structures of PGHS-1 and -2 are very similar in the presence and absence of ligands and between the isoforms. This is not an artifact of crystallization but rather due to the existence of a low-free energy conformer of the protein. Thus, only one conformational state of the enzyme is known, and modeling long range conformational changes is moot because no criteria can be developed to assess the validity of possible changes in conformation.
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ABBREVIATIONS |
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The abbreviations used are: PGHSs, prostaglandin endoperoxide H synthases; PG, prostaglandin; NSAIDs, nonsteroidal anti-inflammatory drugs; hPGHS-2, human PGHS-2; oPGHS-1, ovine PGHS-1; AA, arachidonic acid; 11-HETE, 11-hydroxy(5Z,8Z,12E,14Z)-eicosatetraenoic acid; 11-HPETE, 11-hydroperoxy(5Z,8Z,12E,14Z)-eicosatetraenoic acid; 15-HETE, 15-hydroxy(5Z,8Z,11Z,13E)-eicosatetraenoic acid; 15-HPETE, 15-hydroperoxy(5Z,8Z,11Z,13E)-eicosatetraenoic acid; TMPD, N,N,N',N'-tetramethylphenylenediamine; RP-HPLC, reverse phase-high pressure liquid chromatography.
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