From the 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
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ABSTRACT |
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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- 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- 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- Materials--
Fatty acids were purchased from Cayman Chemical
Co., Ann Arbor, MI. [1-14C]Arachidonic acid (40-60
mCi/mmol) and [1-14C]dihomo- 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 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- Data Collection--
The Co3+-heme oPGHS-1/DHLA data
set was assembled from two crystals. Data were collected at 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 2 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).
Structure of Dihomo-
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
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 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 C
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 O 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.
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.
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 C 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.
-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
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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
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).
-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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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.
50% the peroxidase
activity of native oPGHS-1 (39).
-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-
-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.
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.
Data collection and refinement statistics for Co3+-heme
oPGHS-1/DHLA
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-
-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 2.5
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).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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
-octyl glucoside
detergent molecules bound to the membrane binding domain (data not shown).
-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.
View larger version (63K):
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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 2.0
, 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 2.5
for DHLA is shown.
All figures were created fully or in part using the program SETOR
(57).
View larger version (35K):
[in a new window]
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 C -2 atom of
Ile-523 (orange in DHLA versus blue in AA) and
the O
atom on Ser-530 (light green in DHLA
versus magenta in AA) move to accommodate DHLA in
the active site.
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
3
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.
Distances between carbon atoms of arachidonic acid and
dihomo--linolenic acid in Co3+-heme oPGHS-1 co-crystal
structures
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).
View larger version (31K):
[in a new window]
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.
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).
Comparison of oxygenase rates for oPGHS-1 cyclooxygenase active site
mutants with arachidonic and dihomo--linolenic acids
Kinetic properties for oxygenation of arachidonic acid and
dihomo--linolenic acid by native and mutant oPGHS-1 and hPGHS-2
-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.
and C
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.
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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.
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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.
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ABBREVIATIONS |
---|
The abbreviations used are:
PGHSs, prostaglandin
endoperoxide H synthases;
PG, prostaglandin;
AA, arachidonic acid;
DHLA, dihomo--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.
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