Mutational and X-ray Crystallographic Analysis of the Interaction of Dihomo-gamma -linolenic Acid with Prostaglandin Endoperoxide H Synthases*

Elizabeth D. ThuressonDagger §, Michael G. MalkowskiDagger §, Karen M. LakkidesDagger , Caroline Jill RiekeDagger , Anne M. MulichakDagger , Stephen L. Ginell||, R. Michael GaravitoDagger , and William L. SmithDagger **

From the Dagger  Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and the || Structural Biology Center, Argonne National Laboratory, Argonne, Illinois 60439

Received for publication, October 13, 2000, and in revised form, December 11, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

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-gamma -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 L-shaped 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 Vmax/Km 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 omega -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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Prostaglandin endoperoxide H synthases (PGHSs)1 catalyze the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2) (1-3). This is the committed step in the biosynthesis of prostaglandins and thromboxanes. These compounds are local hormones that act at or near their sites of synthesis to coordinate intercellular responses evoked by circulating hormones and perhaps intracellular events associated with cell replication and differentiation (3). There are two PGHS isozymes designated PGHS-1 and PGHS-2 (or cyclooxygenase-1 and -2 (COX-1 and -2)). PGHS-1 and -2 are often referred to as the constitutive and inducible forms of the enzyme, respectively.

PGHS-1 and -2 are closely related structurally (4-7) and mechanistically (2, 3, 8) although there are some subtle kinetic differences between the two isoforms with respect to hydroperoxide activator requirements (9-11) and substrate (12, 13) and inhibitor (14, 15) specificities. Both PGHS isozymes catalyze two different reactions as follows: a cyclooxygenase reaction in which AA is converted to an intermediate prostaglandin endoperoxide PGG2, and a peroxidase reaction in which the 15-hydroperoxyl group of PGG2 is reduced to an alcohol yielding PGH2 (1-3). Although the peroxidase activity can function independently of the cyclooxygenase activity (16), activation of the cyclooxygenase requires a functional peroxidase (2, 3, 11).

X-ray crystallographic studies indicate that the cyclooxygenase reaction occurs in a hydrophobic channel that extends from the membrane binding domain of the enzyme into the core of the globular domain (7). The fatty acid substrate is positioned in this site in an extended L-shaped conformation (7). Cyclooxygenase catalysis begins with abstraction of the 13-pro-S-hydrogen from AA in the rate determining step to generate an AA radical (17, 18). A tyrosyl radical positioned on Tyr-3852 abstracts this hydrogen from the substrate fatty acid (2, 3, 18, 19). The tyrosyl radical is formed as a result of oxidation of the heme group at the peroxidase site of the enzyme (2, 3, 8).

Most studies of the cyclooxygenase activities of PGHS-1 and -2 have utilized AA as the substrate. Although AA is the preferred substrate, both enzymes will oxygenate n-3 and n-6 C18, C20, and C22 fatty acids in vitro with catalytic efficiencies in the range of 0.05-0.7 to that of AA (12). At least some of these alternative substrates including 9,12-octadecadienoate (linoleate; 18:2(n-6)), 8,11,14-eicosatrienoate (dihomo-gamma -linolenate; 20:3(n-6)), and 5,8,11,14,17-eicosapentaenoate (20:5(n-3)) are also oxygenated via cyclooxygenase activity when added exogenously to intact cells (21, 22) or when mobilized from cellular phosphoglycerides (23-27). In tissues such as vesicular gland which have low levels of Delta 5-desaturase activity and thus low levels of AA, DHLA is converted efficiently to 1-series prostanoid products that are found in abundance in semen (25). Other less common fatty acids can also serve as cyclooxygenase substrates including adrenic acid (22:4(n-6)) (28), the Mead acid (5Z,8Z,11Z-eicosatrienoic acid) (29), columbinic acid (5E,9Z,12Z-octadecatrienoic acid) (30, 31), and 5,6-oxido-eicosatrienoic acid (32, 33). Substrates other than AA typically have somewhat higher Km values than AA but can compete with AA for the cyclooxygenase active site thereby inhibiting formation of 2-series prostanoids. This was first documented by Lands and co-workers in vitro (34), but this form of inhibition appears to occur in vivo as well (35-37).

A combination of crystallographic (7) and mutagenic (38, 39) analyses of the interaction of AA within the cyclooxygenase active site of ovine (o) PGHS-1 led us to assign active site residues to five functional categories for AA oxygenation (39) as follows: (a) residues directly involved in abstraction of the 13-pro-S-hydrogen (Tyr-385); (b) residues essential for positioning C-13 for hydrogen abstraction (Tyr-348 and Gly-533); (c) residues essential for high affinity binding of AA (Arg-120); and (d) residues critical for positioning AA such that following abstraction of the 13-pro-S-hydrogen the arachidonyl radical is converted to PGG2 instead of 11- or 15-monohydroperoxy acid products (Val-349, Trp-387, and Leu-534). Here we report studies of oPGHS-1 that were designed (a) to determine how dihomo-gamma -linoleic acid (20:3(n-6); DHLA), a close homolog of AA, binds within the cyclooxygenase active site and (b) to identify active site residues that are determinants of cyclooxygenase fatty acid substrate specificity.

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Materials-- Fatty acids were purchased from Cayman Chemical Co., Ann Arbor, MI. [1-14C]Arachidonic acid (40-60 mCi/mmol) and [1-14C]dihomo-gamma -linolenic (8, 11, 14-eicosatrienoic acid) (50-60 mCi/mmol) were from PerkinElmer Life Sciences. Flurbiprofen was from Sigma. Restriction enzymes and Dulbecco's modified Eagle's medium were purchased from Life Technologies, Inc. Calf serum and fetal bovine serum were from HyClone. Primary antibodies used for Western blotting were raised in rabbits against purified oPGHS-1 or human (h) PGHS-2 and purified as IgG fractions (40). 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, Synthesis, and Sequencing Facility. All other reagents were from common commercial sources.

Preparation of oPGHS-1 and hPGHS-2 Mutants-- Mutants were prepared by site-directed mutagenesis of oPGHS-1 in the pSVT7 vector employing the Stratagene QuikChange mutagenesis kit and the protocol of the manufacturer (39, 40). Oligonucleotides used in the preparation of various mutants were reported previously (39) except for two oPGHS-1 mutants I434V, 5'-1375GCCAGCCTGCAGGCCGGGTTGGTGGGGGTAGG-3', and H513R, 5'-1617CTTGAGAAGTGTCGACCGAACTCCATCTTTGG-3', used for the construction of a quadruple mutant V349A/I523V/I434V/H513R and two hPGHS-2 mutants (41) V349A, 5'-1082GAAGATTATGCCCAACACTTG-3', and V523I, 5'-1602CCATCTTTGGTGAAACGATGATAGAAGTTGGAGCACC-3'. Plasmids used for transfections were purified by CsCl gradient ultracentrifugation, and mutations were reconfirmed by double-stranded sequencing of the pSVT7 (oPGHS-1) or pOSML (hPGHS-2) constructs using Sequenase (version 2.0, U. S. Biochemical Corp.) and the protocol described by the manufacturer as described previously (39).

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 and transfected with pSVT7 (oPGHS-1) or pOSML (hPGHS-2) plasmid constructs containing cDNAs encoding native or mutant enzymes using the DEAE dextran/chloroquine transfection method (39, 41). Forty hours following transfection, cells were harvested, and microsomal membrane fractions were prepared as described previously (39, 40). Protein concentrations were determined using the method of Bradford (42) with bovine serum albumin as the standard. Microsomal preparations were used for Western blotting and for cyclooxygenase and peroxidase assays. All mutant enzymes described in this report were expressed at protein levels similar to that of the native enzyme and retained >= 50% the peroxidase activity of native oPGHS-1 (39).

Cyclooxygenase and Peroxidase Assays-- Cyclooxygenase assays were performed at 37 °C by monitoring the initial rate of O2 uptake using an oxygen electrode (39). Reactions were initiated by adding ~250 µg of microsomal protein in a volume of 20-50 µl to the assay chamber. The oxygenation of AA and DHLA by native and mutant oPGHSs-1 or hPGHSs-2 was in all cases completely inhibited by the addition of 0.2 mM flurbiprofen to the reaction mixture. All data were normalized to the relative levels of PGHS protein expression as determined by Western blotting and densitometry. Fatty acid substrate concentrations of 100 µM were used to estimate maximal rates. Km values were measured using concentrations of fatty acid substrates between 0.5 and 500 µM as described previously (39). Peroxidase activities were measured spectrophotometrically with N,N,N',N'-tetramethylphenylenediamine (TMPD) as the reducing cosubstrate (43) as reported previously (38). Reactions were initiated by adding 100 µl of 0.3 mM H2O2, and the absorbance at 610 nm was monitored with time.

Western Blot Analysis-- Microsomal samples (~5 µg of protein) were resolved by one-dimensional SDS-PAGE 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 or hPGHS-2 (40, 44) 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 Fatty Acid Oxygenation 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 by adding [1-14C]AA or [1-14C]DHLA (35 µM final concentration) 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.

Purification and Crystallization-- Apo-oPGHS-1 was purified and reconstituted with Co3+-protoporphyrin IX as described previously (7, 45). The Co3+-heme oPGHS-1/DHLA complex was formed by the addition of a 5-fold molar excess of DHLA prior to crystallization. Crystals were grown in sitting-drops by combining the Co3+-heme oPGHS-1·DHLA complex with buffer consisting of 0.64 M sodium citrate, 0.3-0.6 M LiCl, 1 mM NaN3, 0.33% (w/v) n-octyl-beta -D-glucopyranoside, pH 6.5, and equilibrating over reservoir solutions containing 0.64-0.84 M sodium citrate, 0.3-0.6 M LiCl, and 1 mM NaN3 at 20 °C. The crystals were harvested and transferred to stabilization buffer (0.9 M sodium citrate, pH 6.5, 1.0 M LiCl, 0.15% (w/v) n-octyl-beta -D-glucopyranoside), followed by a single step transfer to stabilization buffer with 24% (w/v) sucrose as a cryoprotectant. The crystals were immediately frozen in liquid propane at -165 °C.

Data Collection-- The Co3+-heme oPGHS-1/DHLA data set was assembled from two crystals. Data were collected at -165 °C on beamline 19-ID of the Structural Biology Center (Advanced Photon Source, Argonne, IL) at a wavelength of 1.03321 Å. Each scan was processed individually using HKL2000 (46) and then scaled together using SCALEPACK (46). Details of the data collection and reduction are summarized in Table I.

                              
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Table I
Data collection and refinement statistics for Co3+-heme oPGHS-1/DHLA

Co3+-Heme oPGHS-1/DHLA Structural Solution and Refinement-- Crystals of the Co3+-heme oPGHS-1·DHLA complex are isomorphous with those of Co3+-heme oPGHS-1 complexed with AA (7). Therefore, all ligands, detergent, and carbohydrate molecules were removed from the Co3+-heme oPGHS-1·AA complex (Protein Data Bank code 1DIY), and the protein portion (residues 32-584) was used as the starting model. Refinement was performed using CNS version 0.9a (47), employing the maximum likelihood target using amplitudes, a bulk solvent correction, and an overall temperature (B factor) correction, utilizing 2sigma data from 20.0 to 3.0 Å. Initial rigid-body refinement, followed by cycles of positional and group B factor refinement (main chain/side chain), resulted in initial R and free R values of 25.8 and 28.8%, respectively. The 2Fo - Fc and Fo - Fc electron density maps calculated at this stage clearly showed the position for Co3+-protoporphyrin IX and 9 carbohydrate residues. These features are readily observable in the Co3+-heme oPGHS-1·AA complex (7) and have not been included in the initial refinement protocol and map calculations, thus providing a good check of model quality. In addition, strong electron density was observed for DHLA bound in the cyclooxygenase active site channel as well as for three n-octyl-beta -D-glucopyranoside detergent molecules bound to the membrane binding domain. These molecules were built into their corresponding electron density, followed by cycles of positional refinement, group B factor refinement, and model inspection. A total of 60 water molecules was then added at positions that were within 2.4-3.6 Å of a hydrogen bond donor or acceptor and had electron density peaks greater than 2.5sigma in Fo - Fc maps. The final model has R and free R values of 23.7 and 27.7%, respectively (Table I). All residues lie within the most favored or allowed regions of the Ramachandran plot, and the parameters evaluated by PROCHECK (48) are well within the bounds established from well refined structures at equivalent resolution. The mean positional error in the structure was calculated to be 0.41 Å (Table I).

Structural Analysis-- van der Waals and hydrogen bond interactions were calculated using the program CHAIN (49). The criteria for a hydrogen bond are that the three angles C=O-H, O-H-N, and H-N-C be greater than 90° and the NO distance not exceed 3.6 Å. The upper limit on distance for consideration as a van der Waals contact is 4.0 Å. Superposition of coordinates between structures was done using the program ALIGN (50) and the coordinates for all calcium atoms unless otherwise stated. The coordinates for Co3+-heme oPGHS-1/DHLA have been deposited at the Protein Data Bank (code 1FE2). Mutations were modeled and analyzed in the program CHAIN (49).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Structure of Dihomo-gamma -linolenic Acid (DHLA) Bound in the Cyclooxygenase Active Site-- We crystallized a complex of the catalytically inert Co3+-heme oPGHS-1 with DHLA (4). The crystal structure of the Co3+-heme oPGHS-1·DHLA complex reveals all of the gross features expected of this dimeric, heme-containing enzyme (4, 7). The tertiary structures of the N-terminal epidermal growth factor-like domain, the membrane binding domain, and the catalytic domain were well resolved; in addition, interpretable electron density was observed for Co3+-protoporphyrin IX, carbohydrate moieties at the three N-linked glycosylation sites, and three beta -octyl glucoside detergent molecules bound to the membrane binding domain (data not shown).

DHLA binds in the cyclooxygenase active site channel in an extended L-shaped conformation grossly similar to that seen for AA (7) (Figs. 1 and 2). The carboxylate group is positioned to interact with the side chain of Arg-120, a known determinant of substrate binding to PGHS-1 (7, 39, 51), whereas the omega -end of DHLA binds in a hydrophobic groove located between Ser-530 and Gly-533 along helix 17 (residues 520-535). C-7 through C-14 are threaded around the side chain of Ser-530, the residue that can be acetylated by aspirin via an S-shaped kink which, in turn, positions C-13 below Tyr-385 for abstraction of the 13-pro-S-hydrogen. Furthermore, C-11 is positioned such that it is accessible to O2 addition to the antarafacial side of the substrate.


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Fig. 1.   Stereo views of DHLA bound in the cyclooxygenase active site in the Co3+-heme oPGHS-1·DHLA complex. A, the initial positive Fo - Fc electron density (purple) within the cyclooxygenase channel, contoured at 2.0sigma , is shown with the final refined model of DHLA (red). The electron density for the carboxylate moiety and carbons C-2 to C-18 of DHLA was always strong; weaker density was observed for C-19 and C-20. Side chain atoms for all residues that contact the substrate at the carboxylate, C-2 through C-12, and C-14 through C-20 are colored yellow, orange, and green, respectively. Ser-530 (light green), which is acetylated by aspirin, lies below Tyr-385 (blue), the likely radical donor during catalysis. Residues Val-228 and Phe-518 have been omitted for clarity. B, the view of the cyclooxygenase active site rotated 90o about the vertical axis (using the same color scheme as in A). The positive Fo - Fc omit electron density (light blue) contoured at 2.5sigma for DHLA is shown. All figures were created fully or in part using the program SETOR (57).


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Fig. 2.   Comparison of the binding of AA and DHLA within the cyclooxygenase active site. A stereo view of DHLA (red) and AA (light blue) (7) bound within in the cyclooxygenase active site channel of oPGHS-1. Active site residues are colored as in Fig. 1. The absence of the C5/C6 double bond in DHLA allows for greater conformational flexibility in the carboxyl half of the substrates as compared with AA. This is reflected in the 1.1-Å r.m.s. deviation between carbon positions in DHLA versus AA for C-1 to C-10. Additionally, the position of the Cgamma -2 atom of Ile-523 (orange in DHLA versus blue in AA) and the Ogamma atom on Ser-530 (light green in DHLA versus magenta in AA) move to accommodate DHLA in the active site.

Despite their similar L-shaped conformations in the cyclooxygenase active site, the carboxyl halves of the DHLA and AA molecules differ quite significantly in their respective positions (Fig. 2 and Table II). As the crystallographic analysis was based on medium resolution diffraction data, it was important to verify that the differences observed between the two substrates were significant and not due to crystallographic artifacts or over-interpretation. To test this hypothesis, we calculated Fo - Fc difference electron density maps using the observed structure factors from the Co3+-heme oPGHS-1·DHLA complex but with phases calculated from the refined Co3+-heme oPGHS-1 protein model and the refined model of AA from the Co3+-heme oPGHS-1/AA crystal structure (7) placed in the cyclooxygenase active site. As the root mean square deviation in the carboxylate region (C-2 through C-10) between AA and DHLA is greater than 1 Å, we would expect to observe the appropriate positive and negative difference density peaks in the Fo - Fc difference electron density maps if the data contained sufficient information to detect these conformational differences. Analysis of these maps contoured at 3sigma showed two positive peaks corresponding to the correct location for C-3 and C-4 and C-6 through C-8 (data not shown); these are the two regions in DHLA whose positions show the largest deviations from AA (Fig. 2). Similarly, appropriate negative difference peaks corresponding to misplacement of the above carbons were also observed. Therefore, we can conclude that the diffraction data are of sufficient quality and resolution to discern the differences in conformation between DHLA and AA in the cyclooxygenase active site and that the current model accurately represents those differences at this resolution. Moreover, no constraints on substrate binding arising from the cyclooxygenase catalytic mechanism were used in building the DHLA model and no alternate models could be built for DHLA given its stereochemical constraints.

                              
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Table II
Distances between carbon atoms of arachidonic acid and dihomo-gamma -linolenic acid in Co3+-heme oPGHS-1 co-crystal structures
The crystal structures of Co3+-heme oPGHS-1 complexed with AA and DHLA were superimposed using the program ALIGN (50), and the distance between carbon atoms of the substrates were measured using the program CHAIN (49). The mean positional error of the Co3+-heme oPGHS-1/DHLA structure is 0.41 Å.

Comparison of DHLA and AA Binding in the Cyclooxygenase Active Site-- The r.m.s. deviation between the crystal structures of Co3+-heme oPGHS-1/AA and Co3+-heme oPGHS-1/DHLA is 0.19 Å (550 out of 554 Calpha atoms) indicating that the overall tertiary makeup of both structures is conserved. DHLA makes a total of 62 contacts with 19 residues lining the cyclooxygenase channel (Fig. 3). Only 3 of the 62 contacts are hydrophilic, consisting of a salt bridge between the carboxylate and the guanidinium group of Arg-120 and two hydrogen bonds between the carboxylate and the side chains of Arg-120 and Tyr-355. These contacts are key to stabilizing the carboxylate group in what is otherwise a predominantly hydrophobic active site channel. AA also makes contacts with 19 active site residues in the active center although with four significant differences as follows: Ser-353 and Ile-377 make contacts with AA but not DHLA, whereas Val-228 and Phe518 make contacts with DHLA but not AA. Mutational analysis of both Ser-353 and Ile-377 (39), which contact C-3 and C-20 of AA, respectively (7), have indicated that neither of these residues is especially important either for binding the substrate or for positioning C-13 with respect to Tyr-385 for cyclooxygenase catalysis (39).


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Fig. 3.   Interactions between DHLA and cyclooxygenase active site residues. A schematic diagram of the interactions between DHLA and residues within the cyclooxygenase channel. Every other carbon atom of DHLA is labeled, and the hydrogens for C-13 have been modeled. All dashed lines represent interactions within 4.0 Å between the specific side chain atom of the protein and DHLA. Only 3 of the 62 contacts between DHLA and cyclooxygenase channel residues are hydrophilic.

As mentioned above, the carboxyl halves of the DHLA and AA molecules differ quite significantly in their respective conformations (Fig. 2 and Table II). Specifically, the relative positions of C-2 through C-10 are very different between AA and DHLA due to the absence of the C-5/C-6 double bond in DHLA. However, C-1, whose position is critical for the formation of carboxylate interactions with Arg-120, and carbons C-11 through C-20, which are involved in the alignment of C-13 for hydrogen abstraction and for the initial oxygen insertion, are in the same positions for both substrates. This is further reflected in the r.m.s. deviation between carbon positions in DHLA versus AA calculated for C-1 through C-10 and C-11 through C-20 (1.07 and 0.45 Å, respectively). The absence of a C-5/C-6 double bond in DHLA permits greater conformational flexibility of the carboxyl half of this fatty acid versus AA. C-2 through C-10 take on a more compact, coiled structure that allows DHLA to make the required carboxylate interaction with Arg-120 and to properly align C-13 below Tyr-385 for hydrogen abstraction (Fig. 2). As a consequence of this binding arrangement, the carboxyl half of DHLA is positioned closer to helix 17, where it makes 15 contacts with active site residues. Conversely, there are only four contacts with helix 6 (Figs. 1 and 3). Moreover, the CG2 atom of Ile-523 and Ogamma of Ser-530 move to accommodate the substrate. These atoms move 0.9 and 1.1 Å, respectively, relative to their positions in the Co3+-heme oPGHS-1·AA complex (Fig. 2). Despite the different positions for C-8 and C-9 in DHLA versus AA, there are no stereochemical or geometrical constraints introduced that would prevent endoperoxide bridge formation and subsequent cyclopentane ring closure via the same mechanism proposed for AA (7).

Determinants of Substrate Specificity-- To learn more about the structural basis for the substrate specificities of cyclooxygenases, we first compared effects of active site substitutions on the rates of oxygenation of DHLA and AA focusing on hydrophobic residues that contact the alkyl chains of these fatty acids (Figs. 2 and 3). All of the hydrophobic residues examined, when mutated, decreased the rates of oxygenation of AA and DHLA when tested with relatively high (100 µM) substrate concentrations (Table III). For most substitutions, the rate of oxygenation of DHLA was somewhat less than the rate of oxygenation of AA, presumably because DHLA is a slightly poorer substrate for the native enzyme. However, substitutions of most hydrophobic active site residues caused approximately parallel changes in the rates of oxygenation of both DHLA and AA (Table III). The obvious exceptions were Val-349 and Ser-530.

                              
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Table III
Comparison of oxygenase rates for oPGHS-1 cyclooxygenase active site mutants with arachidonic and dihomo-gamma -linolenic acids
Oxygenase activities were measured with an oxygen electrode as described in the text using 100 µM AA or DHLA. Values are calculated for fatty acid turnover and where applicable are corrected for the percentage of mono- and bisoxygenated products formed. A value of 100% is assigned for the oxygenase activity of native oPGHS-1 with AA. Rate measurements and Km values represent the means from a minimum of four separate determinations with standard deviations within 10% of all values reported.

Some cyclooxygenase active site mutants (i.e. V349A, V349L, W387F, W387L, and S530T oPGHS-1) have previously been shown to alter rather dramatically the distribution of oxygenated products formed from AA by oPGHS-1 (39). Accordingly, we analyzed the oxygenation products formed from [1-14C]DHLA by these mutants (data not shown). All of these mutants, with the exception of S530T oPGHS-1, produced an excess of hydroxy acid products (HETrEs) from DHLA relative to the primary product PGG1 but in significantly reduced amounts compared with AA. For example, when compared with native enzyme V349A oPGHS-1 produced a 20-fold increase in 11-HETE from AA (39) but less than a 2-fold increase in HETrE products from DHLA. W387F and W387L oPGHS-1 produced a 13-fold increase in HETE products from AA (39) and a 6-fold increase in HETrE products from DHLA. S530T oPGHS-1, which relative to native enzyme produces an abundance of 15-HETE from AA (39), formed products in ratios identical to the native enzyme when incubated with DHLA.

We interpret the rate data (Table III) and the results of the product analyses broadly to indicate that residues other than Val-349 and Ser-530 perform similar functions in the oxygenation of both AA and DHLA (39). As discussed below substitutions of Val-349 and Ser-530 were examined in greater detail as possible determinants of substrate specificity.

Val-349 as a Determinant of Fatty Acid Substrate Specificity of oPGHS-1-- A V349A substitution in oPGHS-1 caused a dramatic decrease in the Vmax/Km values for DHLA relative to the change seen with AA (Table IV). Although V349A oPGHS-1 oxygenates AA with a catalytic efficiency that is 65% that of native oPGHS-1 (39), the Vmax/Km value for the V349A oPGHS-1 mutant was decreased more than 800-fold with DHLA (Table IV). Because DHLA was such an inefficient substrate for V349A oPGHS-1, we were able to test DHLA as an inhibitor of AA oxygenation. DHLA was a competitive inhibitor of AA oxygenation with a KI value of 2 µM (Table IV). The similarity between the Km value of native oPGHS-1 for DHLA and the KI value for DHLA inhibition of AA oxygenation by V349A oPGHS-1 suggests that DHLA can bind to the V349A oPGHS-1 mutant with approximately the same affinity as DHLA binds native oPGHS-1; however, DHLA must bind V349A oPGHS-1 in a catalytically unproductive conformation such that abstraction of the 13-pro-S-hydrogen cannot occur efficiently.

                              
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Table IV
Kinetic properties for oxygenation of arachidonic acid and dihomo-gamma -linolenic acid by native and mutant oPGHS-1 and hPGHS-2
Oxygenase activity was measured with an oxygen electrode as described in the text using either arachidonate or dihomo-gamma -linolenate as the substrate. Values are calculated for fatty acid turnover and are corrected for the percentage of mono- and bisoxygenated products formed. A value of 100% is assigned for the oxygenase activity of native PGHS-1 and -2 with arachidonic acid. Vmax and Km values represent the means from a minimum of four separate determinations with standard deviations 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 or DHLA. ND, not determined.

While substitution of Val-349 with alanine has a relatively large impact on the ability of oPGHS-1 to oxygenate DHLA, substitution with a larger residue, leucine, has a much more modest effect relative to the decrease in catalytic efficiency observed with AA (Table IV). The Vmax/Km value for V349L is decreased about 6-fold with AA and about 19-fold with DHLA. The difference between the efficiency of AA and DHLA oxygenation by V349L is largely due to the increased Km value for oxygenation of DHLA.

The greater importance of Val-349 in the efficiency of oxygenation of DHLA versus AA by oPGHS-1 can be rationalized by comparing the co-crystal structures of these two homologous fatty acids in complexes with oPGHS-1 (Fig. 2 and Table II). Val-349 is positioned slightly differently in the two structures. Additionally, Val-349 makes a single van der Waals contact with DHLA involving C-5 of the substrate and the CG1 atom of Val-349; in contrast, AA makes two contacts with Val-349 involving C-3 and C-4. The C-3 through C-5 segment of DHLA is positioned on one side by interactions involving Ala527, Gly-526, and Ile-523, whereas Val-349 acts as a structural bumper on the other side of this fatty acid.

Computer modeling indicates that substitution of Val-349 with alanine opens up a substantial pocket into which the C-3 to C-5 segment of the DHLA molecule could move. This could occur because of the flexibility of DHLA in the C-3 to C-5 region. This movement would lead to a significant change in the position of C-13 of DHLA with respect to Tyr-385 such that abstraction of the 13-pro-S-hydrogen could not occur efficiently. The structure of AA is more constrained because of the presence of its C-5/C-6 double bond and thus would be less affected by the V349A substitution. The movement that does occur with AA affects the position of C-9 with respect to C-11 leading to increased 11-HPETE formation but does not affect the positioning of C-13 with respect to Tyr-385 (39).

Computer modeling of a leucine at position 349 in the Co3+-heme oPGHS-1·AA and Co3+-heme oPGHS-1·DHLA complexes suggests that leucine would crowd the carboxyl end of both AA and DHLA with the net effect of moving C-13 slightly away from Tyr-385. However, the extra rigidity of AA compared with DHLA apparently makes AA slightly more resistant to the intrusion of the larger leucine at position 349. Again however, compared with V349A oPGHS-1, V349L oPGHS-1 has relatively similar activities with both AA and DHLA.

Ile-523 also contacts C-5 of DHLA (Figs. 1-3 and Table II), but elimination of the C-5 contact with Ile-523 by replacement of this residue with an alanine is relatively unimportant functionally apparently because of compensatory interactions with Ala-527 and Gly-526 (Fig. 2 and Tables II-IV).

Overall, Val-349 appears to provide the one critical interaction with the carboxyl end of DHLA that is responsible for proper positioning of the 13-pro-S-hydrogen for abstraction by Tyr-385.

Val-349 in Human PGHS-2-- Although Val-349 is an important determinant of the substrate specificity for PGHS-1, it is not an important determinant for PGHS-2 (Table IV). V349A human (h) PGHS-2 oxygenates DHLA and AA with comparable efficiencies, approximately half of those observed with the native enzyme (Table IV). V349A hPGHS-2 does behaves like V349A oPGHS-1 in producing large amounts of 11-HETE-30% compared with 5% for native hPGHS-1. However, there were no differences in the distribution of oxygenated products formed from DHLA by V349A hPGHS-2 versus native hPGHS-2. Overall, the key difference between V349A oPGHS-1 and V349A hPGHS-2 is the inability of V349A oPGHS-1 to oxygenate DHLA efficiently (Table IV).

The differences between the specificities of V349A oPGHS-1 and V349A hPGHS-2 for DHLA led us to construct two additional oPGHS-1 mutants (V349A/I523V and V349A/I523V/H513R/I434V oPGHS-1) in an attempt to develop a modified `version of V349A oPGHS-1 that would mimic V349A hPGHS-2 (Table IV). However, both the double (V349A/I523V oPGHS-1) and quadruple (V349A/I523V/H513R/I434V oPGHS-1) V349A mutants exhibited the same profile of oxygenation products as the corresponding single V349A oPGHS-1 and hPGHS-2 mutants, and the double and quadruple mutants showed only small increases in the rates of oxygenation of both AA and DHLA (Table IV). Finally, substituting Val-523 with an isoleucine in V349A hPGHS-2 simply decreased the catalytic efficiencies of oxygenation of both AA and DHLA.

The volume of the cyclooxygenase active site of hPGHS-2 is ~20% larger than that of oPGHS-1 (4-6). This volume difference is the result of three differences in amino acid residues between the isozymes as follows: Ile-523 is a valine; His-513 is an arginine; and Ile-434 is a valine in PGHS-2. Computer modeling of the V349A/I523V/H513R/I434V oPGHS-1 quadruple mutant indicates that this mutant is similar structurally to PGHS-2 (i.e. the cyclooxygenase active site of the quadruple mutant has a 20% greater volume than that of native oPGHS-1). Nonetheless, the V349A/I523V/H513R/I434V oPGHS-1 mutant has a substrate specificity that closely resembles V349A oPGHS-1 and not V349A hPGHS-2. Thus, the reason that V349A hPGHS-2 but not V349A oPGHS-1 can oxygenate DHLA efficiently must be attributable to a factor other than the difference between the volumes of the cyclooxygenase sites of the isozymes. It is possible that differences in the interactions of the carboxylate groups of AA and DHLA with Arg-120 in PGHS-1 versus PGHS-2 (41, 51-52) in some way override the differences in volume.3

Ser-530 as a Determinant of Substrate Specificity-- Ser-530 is the site of acetylation of oPGHS-1 by aspirin (53, 54). Earlier studies with oPGHS-1 involving AA as the substrate 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 or valine prevents catalysis apparently by blocking substrate access to the Tyr-385 radical (39, 53, 55).

We have extended these findings to show that a S530A mutation has a somewhat larger effect on the efficiency of oxygenation of DHLA versus AA due primarily to a greater than 4-fold increase in Km (Table III). Furthermore, there is a 750-fold decrease in the efficiency of oxygenation of DHLA with the S530T oPGHS-1 mutant (Table III). Although S530T oPGHS-1 is relatively inactive with both AA and DHLA, there is both a 5-fold decrease in the Vmax for oxygenation of DHLA versus AA and a 7.5-fold increase in the Km. As discussed above, S530T oPGHS-1 produces a distribution of oxygenated products similar to the native enzyme when incubated with DHLA, whereas this mutant produces a relative abundance of (15R)-HETE when incubated with AA (39). Overall, the data with the Ser-530 mutants indicate that substitutions of this residue with amino acids having either larger or smaller side chains is considerably more important for the oxygenation of DHLA than AA probably due to an effect on the positioning of the 13-pro-S-hydrogen of these substrates for abstraction by Tyr-385.

There are three van der Waals contacts between Ser-530 and AA involving C-10 and C-16 of the substrate (7, 39). There are six van der Waals contacts between Ser-530 and DHLA involving C-10, C-13, C-15, C-16, and C-17 (Figs. 2 and 3 and Table II). In both cases, the contacts are on the side of the substrate directly opposite Tyr-385. Ser-530 is one of only two residues in the cyclooxygenase active site that has a slightly different orientation in the Co3+-heme oPGHS-1/DHLA crystal structure compared with the oPGHS-1-AA structure. In the DHLA structure both the Calpha and Cbeta of Ser-530 are oriented more toward the fatty acid molecule than in the AA structure. Two critical contacts made between Ser-530 and DHLA not observed with AA are contacts at C-13 (the position of hydrogen abstraction) and C-15. Apparently, the greater flexibility of the carboxyl end of DHLA requires that the enzyme provide additional stabilizing influences near C-13 to optimize positioning of the 13-pro-S-hydrogen with respect to Tyr-385. Analysis of the Co3+-heme oPGHS-1/DHLA crystal structure indicates that the mid-section of the fatty acid is folded more tightly against Ser-530 than is that of AA, making the residue at position 530 that much more essential for efficient catalysis in the case of DHLA.

Conclusion-- AA is the preferred substrate for the cyclooxygenase activity of PGHS-1 and PGHS-2. Both isozymes can also oxygenate other C18, C20, and C22 polyunsaturated fatty acids albeit with lesser efficiencies than AA (13, 20, 28-34). DHLA is the closest AA homologue lacking only the the C-5/C-6 double bond of AA and is oxygenated with 50% of the catalytic efficiency of AA by native oPGHS-1 (Table IV). In this study we have shown that DHLA is bound in the cyclooxygenase active site of PGHS-1 in an L-shaped conformation in a manner similar to that of AA but that the regions between C2-and C-10 are positioned quite differently for the two substrates. Additionally, we have observed that among the 19 active site residues that contact DHLA, Val-349 and Ser-530 are particularly important for the efficient oxygenation of DHLA and thus serve as determinants of cyclooxygenase fatty acid specificity.

    ACKNOWLEDGEMENT

The use of the Advanced Photon Source was supported by the United States Department of Energy, Basic Energy Sciences, Office of Energy Research, under Contract W-31-109-Eng-38.

    FOOTNOTES

* This work was supported in part by Program Project Grants P01 GM57323 (to W. L. S. and R. M. G.) and R01 HL56773 (to R. M. G.) 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 atomic coordinates and structure factors (code 1FE2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§ Both authors contributed equally to this work.

Supported by NRS Award F32 HL10170-01 from the National Institutes of Health.

** 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.M009378200

2 The numbering of residues in oPGHS-1 is based on numbering the N-terminal methionine of the signal peptide as residue number 1 (56), and the homologous numbering system is used for human PGHS-2 (5, 6).

3 The differences observed between the oPGHS-1 and hPGHS-2 V349A mutants in their abilities to oxygenate AA versus DHLA may be due indirectly to the reliance of substrate binding to PGHS-1 on an ionic interaction between Arg-120 and the carboxylate of fatty acid substrates (51); PGHS-2 does not need this ionic interaction for effective substrate binding and oxygenation (41). This difference may provide substrates with more freedom of movement in the PGHS-2 active site.

    ABBREVIATIONS

The abbreviations used are: PGHSs, prostaglandin endoperoxide H synthases; PG, prostaglandin; AA, arachidonic acid; DHLA, dihomo-gamma -linolenic acid; hPGHS-2, human PGHS-2; oPGHS-1, ovine PGHS-1; 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; HETrEs, 11- and 15-hydroxyeicosatrienoic acids; r.m.s., root mean square.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Smith, W. L., Garavito, R. M., and DeWitt, D. L. (1996) J. Biol. Chem. 271, 33157-33160[Free Full Text]
2. Marnett, L. J., Rowlinson, S. W., Goodwin, D. C., Kalgutkar, A. S., and Lanzo, C. A. (1999) J. Biol. Chem. 274, 22903-22906[Free Full Text]
3. Smith, W. L., DeWitt, D. L., and Garavito, R. M. (2000) Annu. Rev. Biochem. 69, 149-182
4. Picot, D., Loll, P. J., and Garavito, M. (1994) Nature 367, 243-249[CrossRef][Medline] [Order article via Infotrieve]
5. Kurumbail, 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., and Stallings, W. C. (1996) Nature 384, 644-648[CrossRef][Medline] [Order article via Infotrieve]
6. Luong, C., Miler, A., Barnett, J., Chow, J., Ramesha, C., and Browner, M. F. (1996) Nat. Struct. Biol. 3, 927-933[Medline] [Order article via Infotrieve]
7. Malkowski, M. G., Ginell, S., Smith, W. L., and Garavito, R. M. (2000) Science 289, 1933-1937[Abstract/Free Full Text]
8. Chen, W., Pawelek, T. R., and Kulmacz, R. J. (1999) J. Biol. Chem. 274, 20301-20306[Abstract/Free Full Text]
9. Capdevila, J. H., Morrow, J. D., Belosludtsev, Y. Y., Beauchamp, D. R., DuBois, R. N., and Falck, J. R. (1995) Biochemistry 34, 3325-3337[Medline] [Order article via Infotrieve]
10. Kulmacz, R. J., and Wang, L. H. (1995) J. Biol. Chem. 270, 24019-24023[Abstract/Free Full Text]
11. Tsai, A., Wu, G., Palmer, G., Bambai, B., Koehn, J. A., Marshall, P. J., and Kulmacz, R. J. (1999) J. Biol. Chem. 274, 21695-21700[Abstract/Free Full Text]
12. Laneuville, O., Breuer, D. K., Xu, N., Huang, Z. H., Gage, D. A., Watson, J. T., Lagarde, M., DeWitt, D. L., and Smith, W. L. (1995) J. Biol. Chem. 270, 19330-19336[Abstract/Free Full Text]
13. Smith, W. L., Rieke, C. J., Thuresson, E. D., Mulichak, A. M., and Garavito, R. M. (2000) in Advances in Eicosanoid Research (Serhan, C. N. , and Perez, H. D., eds), Vol. 31 , pp. 53-65, Springer-Verlag, Berlin
14. DeWitt, D. L. (1999) Mol. Pharmacol. 55, 625-631[Free Full Text]
15. Marnett, L. J., and Kalgutkar, A. S. (1999) Trends Pharmacol. Sci. 20, 465-469[CrossRef][Medline] [Order article via Infotrieve]
16. Koshkin, V., and Dunford, H. B. (1999) Biochim. Biophys. Acta 1430, 341-348[Medline] [Order article via Infotrieve]
17. Hamberg, M., and Samuelsson, B. (1967) J. Biol. Chem. 242, 5336-5343[Abstract/Free Full Text]
18. Tsai, A., Kulmacz, R. J., and Palmer, G. (1995) J. Biol. Chem. 270, 10503-10508[Abstract/Free Full Text]
19. Shimokawa, T., Kulmacz, R. J., DeWitt, D. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 20073-20076[Abstract/Free Full Text]
20. Hsi, L. C., Tsai, A. L., Kulmacz, R. J., English, D. G., Siefker, A. O., Otto, J. C., and Smith, W. L. (1993) J. Lipid Mediators 6, 131-138[Medline] [Order article via Infotrieve]
21. Baer, A. N., Costello, P. B., and Green, F. A. (1991) Biochim. Biophys. Acta 1085, 45-52[Medline] [Order article via Infotrieve]
22. Kaduce, T. L., Figard, P. H., Leifur, R., and Spector, A. A. (1989) J. Biol. Chem. 264, 6823-6830[Abstract/Free Full Text]
23. Baer, A. N., Costello, P. B., and Green, F. A. (1991) J. Lipid Res. 32, 341-347[Abstract]
24. Abeywardena, M. Y., Fischer, S., Schweer, H., and Charnock, J. S. (1989) Biochim. Biophys. Acta 1003, 161-166[Medline] [Order article via Infotrieve]
25. Knapp, H. R. (1990) Prostaglandins 39, 407-423[Medline] [Order article via Infotrieve]
26. Leaver, H. A., Howie, A., and Wilson, N. H. (1991) Prostaglandins Leukot. Essent. Fatty Acids 42, 217-224[Medline] [Order article via Infotrieve]
27. Engels, F., Willems, H., and Nijkamp, F. P. (1986) FEBS Lett. 209, 249-253[CrossRef][Medline] [Order article via Infotrieve]
28. Larsen, L. N., Dahl, E., and Bremer, J. (1996) Biochim. Biophys. Acta 1299, 47-53[Medline] [Order article via Infotrieve]
29. Oliw, E. H., Hornsten, L., Sprecher, H., and Hamberg, M. (1993) Arch. Biochem. Biophys. 305, 288-297[CrossRef][Medline] [Order article via Infotrieve]
30. Elliott, W. J., Morrison, A. R., Sprecher, H. W., and Needleman, P. (1985) J. Biol. Chem. 260, 987-992[Abstract/Free Full Text]
31. Nugteren, D. H., and Christ Hazelhof, E. (1987) Prostaglandins 33, 403-417[CrossRef][Medline] [Order article via Infotrieve]
32. Oliw, E. H. (1984) J. Biol. Chem. 259, 2716-2721[Abstract/Free Full Text]
33. Balazy, M. (1991) J. Biol. Chem. 266, 23561-23567[Abstract/Free Full Text]
34. Lands, W. E. M., LeTellier, P. R., Rome, L. H., and Vanderhoek, J. Y. (1973) Adv. Biosci. 9, 15-28
35. Needleman, P., Whitaker, M. O., Wyche, A., Watters, K., Sprecher, H., and Raz, A. (1980) Prostaglandins 19, 165-181[CrossRef][Medline] [Order article via Infotrieve]
36. Spector, A. A., Kaduce, T. L., Figard, P. H., Norton, K. C., Hoak, J. C., and Czervionke, R. L. (1983) J. Lipid Res. 24, 1595-1604[Abstract]
37. Lagarde, M., Drouot, B., Guichardant, M., and Dechavanne, M. (1984) Biomed. Biochim. Acta 43, 319-322
38. Thuresson, E. D., Lakkides, K. M., and Smith, W. L. (2000) J. Biol. Chem. 275, 8501-8507[Abstract/Free Full Text]
39. Thuresson, E. D., Lakkides, K. M., Rieke, C. J., Sun, Y., Wingerd, B. A., Micielli, R., Mulichak, A. M., Malkowski, M. G., Garavito, R. M., and Smith, W. L. (2000) J. Biol. Chem. 266, 10347-10357
40. Spencer, A. G., Thuresson, E. A., Otto, J. C., Song, I., Smith, T., DeWitt, D. L., Garavito, R. M., and Smith, W. L. (1999) J. Biol. Chem. 274, 32936-32942[Abstract/Free Full Text]
41. Rieke, C. J., Mulichak, A. M., Garavito, R. M., and Smith, W. L. (1999) J. Biol. Chem. 274, 17109-17114[Abstract/Free Full Text]
42. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
43. Kulmacz, R. J. (1987) Prostaglandins 34, 225-240[CrossRef][Medline] [Order article via Infotrieve]
44. Otto, J. C., DeWitt, D. L., and Smith, W. L. (1993) J. Biol. Chem. 268, 18234-18242[Abstract/Free Full Text]
45. Malkowski, M. G., Theisen, M. J., Scharmen, A., and Garavito, R. M. (2000) Arch. Biochem. Biophys. 380, 39-45[CrossRef][Medline] [Order article via Infotrieve]
46. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
47. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse, Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-21[CrossRef][Medline] [Order article via Infotrieve]
48. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
49. Sack, J. S. (1988) J. Mol. Graphics 6, 224-225
50. Satow, Y., Cohen, G. H., Padlan, E. A., and Davies, D. R. (1986) J. Mol. Biol. 190, 593-604[Medline] [Order article via Infotrieve]
51. Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179-2184[Abstract/Free Full Text]
52. Kozak, K. R., Rowlinson, S. W., and Marnett, L. J. (2000) J. Biol. Chem. 275, 33744-33749[Abstract/Free Full Text]
53. DeWitt, D. L., El-Harith, E. A., Kraemer, S. A., Andrews, M. J., Yao, E. F., Armstrong, R. L., and Smith, W. L. (1990) J. Biol. Chem. 265, 5192-5198[Abstract/Free Full Text]
54. Loll, P. J., Picot, D., and Garavito, R. M. (1995) Nat. Struct. Biol. 2, 637-643[Medline] [Order article via Infotrieve]
55. Shimokawa, T., and Smith, W. L. (1992) J. Biol. Chem. 267, 12387-12392[Abstract/Free Full Text]
56. DeWitt, D. L., and Smith, W. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1212-1416
57. Evans, S. V. (1993) J. Mol. Graphics 11, 134-138[CrossRef][Medline] [Order article via Infotrieve]


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