(Received for publication, October 17, 1995; and in revised form, November 21, 1995)
From the
Examination of the crystal structure of the ovine prostaglandin
endoperoxide synthase-1 (PGHS-1)/S-flurbiprofen complex
(Picot, D., Loll, P. J., and Garavito, R. M.(1994) Nature 367,
243-2491) suggests (a) that the carboxyl group of arachidonic
acid interacts with the arginino group of Arg; (b) that Arg
forms an important salt bridge with
Glu
; and (c) that Tyr
, which is in
close proximity to Arg
, could determine the
stereochemical specificity of PGHS-1 toward 2-phenylpropionic acid
inhibitors. To test these concepts, we used site-directed mutagenesis
to prepare ovine PGHS-1 mutants having modifications of Arg
(R120K, R120Q, R120E), Glu
(E524D, E524Q, E524K),
and Tyr
(Y355F) and examined the properties of the mutant
enzymes expressed in COS-1 cells. All of the mutants retained at least
part of their cyclooxygenase and peroxidase activities except the R120E
mutant, which had no detectable activity. The K
values of the R120K and R120Q mutants with arachidonic acid
were 87 and 3300 µM, respectively, versus 4
µM for native PGHS-1. The R120Q mutant failed to undergo
suicide inactivation during catalysis or time-dependent inhibition by
flurbiprofen. These results are consistent with Arg
binding the carboxylate group of arachidonate and suggest that
interaction of the carboxylate group of substrates and inhibitors with
Arg
is necessary for suicide inactivation and
time-dependent inhibition, respectively. The K
values for the E524D, E524Q, and E524K mutants were not
significantly different from values obtained for the native PGHS-1,
suggesting that this residue is not importantly involved in catalysis
or substrate binding. The effect of modifications of Arg
and Tyr
on the stereospecificity of inhibitor
binding were determined. Ratios of IC
values for
cyclooxygenase inhibition by D- and L-ibuprofen, a
competitive cyclooxygenase inhibitor, were 32, 67, and 7.1 for native
PGHS-1, R120Q PGHS-1, and Y355F PGHS-1, respectively. The decreased
stereochemical specificity observed with the Y355F PGHS-1 mutant
suggests that Tyr
is a determinant of the
stereospecificity of PGHS-1 toward inhibitors of the 2-phenylpropionic
acid class.
Prostaglandin endoperoxide H synthase-1 (PGHS-1) ()catalyzes the committed step in the conversion of
arachidonic acid to prostaglandins and thromboxanes (1) . The
processed form of ovine PGHS-1 is an N-glycosylated
hemoprotein having 576 amino acids and an apparent subunit molecular
mass of 72
kDa(2, 3, 4, 5, 6) . PGHS-1
exhibits both a cyclooxygenase activity involved in converting
arachidonate to PGG
and a hydroperoxidase activity that
reduces the hydroperoxyl group of PGG
to
PGH
(7, 8, 9) . Although a single
heme molecule is essential for both activities, the cyclooxygenase and
peroxidase substrate binding sites are functionally and spatially
distinct(10, 11, 12, 13, 14, 15) .
Examination of the crystal structure of the ovine
PGHS-1/S-flurbiprofen inhibitor complex indicates that the
cyclooxygenase active site is a long hydrophobic channel extending from
the putative membrane binding domain into the center of the globular
catalytic domain (Fig. 1)(10) . In the crystal
structure, the arginino group of Arg located near the
channel opening is complexed with the carboxylate group of
flurbiprofen. The carboxylate group of Glu
appears to
form a salt bridge with the arginino group of Arg
.
Arachidonate is postulated to bind with its
-methyl group
extending into the core of the channel and its carboxylate group
complexed to Arg
. The phenolic side chain of Tyr
is present near the mouth of the channel opposite
Arg
. This bulky group places a constriction on one side
of the channel which may determine the stereoselectivity of PGHS-1 for
the S-isomers of 2-phenylpropionic acid
inhibitors(16) , such as ibuprofen and flurbiprofen. Finally,
some PGHS-1 and PGHS-2 inhibitors, including flurbiprofen, cause a
time-dependent, slowly reversible inhibition of cyclooxygenase
activity(17, 18, 19, 20, 21) ;
however, the methyl esters of these types of compounds are freely
reversible (although less potent) inhibitors(17) . This latter
observation suggests that modification of the amino acid involved in
binding the carboxylate group of time-dependent inhibitors such as
flurbiprofen would affect the mechanism of inhibition. To begin testing
the functions of Arg
, Glu
, and Tyr
in the binding of fatty acid substrates and 2-phenylpropionic
acid inhibitors, we prepared appropriate mutants of ovine PGHS-1 and
examined their kinetic properties.
Figure 1:
Stereo view of the
amino acid side chains which create the cyclooxygenase active site
channel in ovine PGHS-1. The active site lies between the heme group
(in red) at the top and the channel mouth. Bound
within the cyclooxygenase active site is the NSAID flurbiprofen (yellow) which lies near Ser (orange),
the site of aspirin acetylation. The putative radical donor,
Tyr
(blue), lies between the heme and
flurbiprofen. The carboxylate of flurbiprofen ligands Arg
(green) and Tyr
(blue).
Glu
(red) may also interact with arginine
120.
Assays of prostaglandin formation
by transfected cells were performed essentially as described
previously(26) . Approximately 40 h post-transfections, the
cells were removed from culture dishes using a rubber policeman,
collected by centrifugation at 1000 g for 10 min, and
resuspended in 0.5 ml of Dulbecco's modified Eagle's
medium/three 100-mm culture plates. The resuspended cells were
incubated with 60 µM [1-
C]arachidonic acid or 10 µM [1-
C]
-linolenic acid for 15 min at 37
°C. To terminate the reactions, the cells were centrifuged for 5
min at 1000
g, the medium was removed, and 4 volumes
of ice-cold acetone were added to the medium. After removal of
denatured protein by centrifugation, the supernatant containing the
radioactive prostaglandin products and unreacted arachidonic acid was
acidified with 0.5 volume of 0.2 M HCl and extracted with 6
volumes of chloroform. The organic phase was evaporated to dryness
under a stream of nitrogen, resuspended in chloroform, and applied to a
silica gel 60 thin-layer chromatographic plate. The lipid products were
separated by chromatography twice in benzene:dioxane:acetic acid:formic
acid (82:14:1:1, v/v/v/v), and the thin-layer chromatography plates
were then exposed to Kodak XAR-5 film, typically for 40 h.
Autoradiographic bands were quantified by densitometry using a Visage
110 Image Analyzer. The percentage of radioactive arachidonate
converted to prostaglandins was calculated by dividing the density
attributable to PGF
plus PGE
plus
PGD
by the total density of all bands and multiplying by
100.
COS-1 cells expressing human PGHS-1(18) , human PGHS-2,
ovine PGHS-1 (18) , or ovine R120Q PGHS-1 were incubated with
10 µM [1-C]
-linolenic acid for
15 min at 37 °C and the products extracted and separated by
radio-thin-layer chromatography essentially as described above.
Products chromatographing as 12-hydroxy-9,13,15-octadecatrienoic acid
(12-HOTrE) were quantified by densitometry(27) .
IC values for instantaneous inhibition
of the cyclooxygenase activities of microsomal preparations of ovine
PGHS-1, R120Q PGHS-1, and Y355F PGHS-1 by ibuprofen or flurbiprofen
were determined by measuring cyclooxygenase activity in the presence of
various concentrations of inhibitor added directly to the assay mixture
prior to the addition of enzyme(18) . To measure time-dependent
inhibition of cyclooxygenase activity by flurbiprofen, microsomal
preparations of PGHS-1 or R120Q PGHS-1 were incubated at 37 °C for
0-20 min in the presence and absence of various concentrations of
flurbiprofen. Aliquots of these preincubation mixtures were then
assayed for cyclooxygenase activity.
Figure 2:
Prostaglandin formation by COS-1 cells
expressing native ovine PGHS-1 or ovine PGHSs-1 having mutations at
Arg or Glu
. COS-1 cells transfected with
pSVT7 constructs encoding native ovine PGHS-1 or the indicated mutant
PGHSs-1 were assayed for prostaglandin biosynthetic activity using 60
µM [1-
C]arachidonate as detailed in
the text. Percent prostaglandin formation was calculated from
densitometric analyses of autoradiographic data as described in the
text. The percentage of density in regions corresponding to
prostaglandin products (sum of the densities attributable to
PGE
, PGF
, and PGD
) formed by
the mutants was divided by the percentage of radioactivity in
prostaglandin bands determined for native PGHS-1 and multiplied by 100.
The data represent the mean ± S.E. of three separate
experiments, each involving a separate
transfection.
Ovine native PGHS-1 and the various PGHS-1 mutants
were expressed transiently in COS-1 cells, and microsomal membranes
prepared from the cells were assayed for cyclooxygenase activity, using
100 µM arachidonate, and peroxidase activity, using
HO
and guaiacol as the cosubstrates (Fig. 3). Again, the R120E mutant lacked detectable
cyclooxygenase and peroxidase activities. The R120K mutant exhibited
only 10% of cyclooxygenase activity of native PGHS-1, but increased
levels of peroxidase activity. The R120Q mutant had only 5% of the
cyclooxygenase activity of the native enzyme but 60% of native
peroxidase activity. Mutations of Glu
had little effect
on either the cyclooxygenase or peroxidase activities of PGHS-1.
Figure 3:
Cyclooxygenase and peroxidase activities
of native ovine PGHS-1 or ovine PGHSs-1 having mutations at Arg or Glu
. Microsomal membranes prepared from COS-1
cells transfected with pSVT7 constructs encoding native ovine PGHS-1 or
the indicated mutant were assayed for cyclooxygenase (COX) and
peroxidase (POX) activities as described in the text.
Activities are presented as a percentage of native ovine PGHS-1
activities and represent the average of duplicate determinations from
two separate transfections. The average cyclooxygenase activity of
native ovine PGHS-1 was 117 nmol of O
/min/mg of microsomal
protein, and the average peroxidase activity of native ovine PGHS-1 was
93 nmol of H
O
/min/mg of microsomal
protein.
The
rate of oxygenation by native PGHS-1 falls by greater than 90% within 2
min of adding enzyme to the assay chamber (Fig. 4); this is due
to suicide inactivation of native ovine PGHS-1(1) . In
contrast, the rate of oxygenation of arachidonate by the R120Q mutant
remained relatively constant for several minutes after mixing enzyme
and substrates. The t values for suicide
inactivation of native PGHS-1 and R120Q PGHS-1 were estimated by
plotting the log of activity at different times after initiation of
oxygenation versus time and found to be approximately 15 and
330 s, respectively (Fig. 4). Clearly, R120Q PGHS-1 undergoes
suicide inactivation at a much slower rate than native enzyme; this
difference between rates of suicide inactivation accounts for the
rather small (2-fold) difference in prostaglandin product formation
observed after a 15-min incubation (Fig. 2) versus the
rather large (20-fold) difference in initial velocities of oxygenation (Fig. 3) when comparing native PGHS-1 and R120Q PGHS-1.
Figure 4:
Time-dependent inactivation of native
ovine PGHS-1 and R120Q PGHS-1. Microsomes prepared from COS-1 cells
expressing either native PGHS-1 or R120Q PGHS-1 were added to a
standard oxygen electrode assay mixture containing 100 µM arachidonic acid and 200 µM O. The rates
of oxygen consumption were determined at the indicated times after
initiation of oxygenation. Values for t
were
estimated to be 15 s for PGHS-1 and 330 s for R120Q PGHS-1 in two
separate experiments.
The K values determined for arachidonic acid with
native PGHS-1 and various mutant PGHSs-1 are presented in Table 2. Native ovine PGHS-1 had a K
value
of 4 µM with arachidonate. In the case of the R120K
mutant, this value increased about 20-fold to 87 µM. An
even more dramatic increase of roughly 1000-fold was observed for the
R120Q mutant. In contrast, the E524K, E524Q, and E524D mutants of ovine
PGHS-1 exhibited K
values in the low micromolar
range, similar to that of native PGHS-1.
-Linolenic acid is a
C
fatty acid that can be oxidized to 12-HOTrE by either
human PGHS-1 or PGHS-2(27) ; however,
-linolenic acid
exhibits a particularly low K
/K
value with human PGHS-1(27) . Shown in Fig. 5is a
comparison of 12-HOTre formation from
[1-
C]linolenic acid by COS-1 cells expressing
human PGHS-1, human PGHS-2, ovine PGHS-1, or the R120Q mutant of ovine
PGHS-1. Although ovine PGHS-1 formed product from
-linolenic acid,
the R120Q mutant failed to form any product. Thus, modification of
Arg
dramatically affects the oxygenation of this C
fatty acid substrate as well as arachidonate. To determine
whether elimination of the positively charged Arg
group
permits PGHS-1 to use fatty acid methyl esters as substrates, we also
incubated microsomal preparations of COS-1 cells expressing native
PGHS-1 and the R120Q PGHS-1 mutant with methyl arachidonate. However,
no oxygenation of the methyl ester was observed with either enzyme
preparation as determined by O
electrode assays.
Figure 5:
Formation of
12-hydroxy-9,11,15-octadecatrienoic acid (12-HOTre) from
[1-C]
-linolenic acid by COS-1 cells
expressing human PGHS-1, human PGHS-2, ovine PGHS-1, or R120Q PGHS-1.
The experiment was performed as described in the legend to Fig. 1, except that the transfected COS-1 cells were incubated
with 10 µM [1-
C]
-linolenic
acid instead of arachidonic acid. Product comigrating with 13-HODE (and
corresponding to 12-13-hydroxy-(9Z,
11E)-octadecadienoic acid (27) ) was quantified by
densitometry, and the densitometric data are plotted here as described
in the legend to Fig. 1.
Figure 6:
Dose-response curves for inhibition by D- and L-ibuprofen of the cyclooxygenase activities
of native ovine PGHS-1, Y355F PGHS-1, and R120Q PGHS-1. The
cyclooxygenase activities of microsomal membranes prepared from COS-1
cells expressing native ovine PGHS-1 (A), R120Q PGHS-1 (B), or Y355F PGHS-1 (C) were measured using an
O electrode assay in the presence of 100 µM arachidonate and the indicated concentrations of D- or L-ibuprofen (IBP) as described in the text. Enzyme
was added to the assay mixture containing substrates and inhibitor to
determine instantaneous inhibition. The maximal activities in the
absence of inhibitor (100%) were 140, 12, and 21 nmol of arachidonate
consumed per min/mg of microsomal protein for native ovine PGHS-1,
R120Q PGHS-1, or Y355F PGHS-1, respectively. The experiments depicted
in this figure were performed twice with similar
results.
Figure 7:
Time course for inhibition of ovine PGHS-1
and R120Q PGHS-1 by flurbiprofen. Microsomal membranes prepared from
COS-1 cells expressing R120Q PGHS-1 were incubated for the indicated
times at 37 °C with 10M flurbiprofen
and then assayed for cyclooxygenase activity using an O
electrode. The experiments depicted in this figure were performed
three times with similar results.
The studies reported here were designed to examine postulated
roles for Arg, Glu
, and Tyr
(10) in the binding of fatty acids and nonsteroidal
anti-inflammatory drugs to the cyclooxygenase active site of ovine
PGHS-1. Our results support the concept that Arg
is
involved in binding the carboxylate moieties of arachidonate and two
different 2-phenylpropionate inhibitors, ibuprofen and flurbiprofen,
but that Glu
is not involved in either catalysis or
substrate binding (Fig. 1). Placing a negative charge in the
form of the side chain of a glutamate at residue 120 reduced
cyclooxygenase activity to undetectable levels. However, this loss of
cyclooxygenase activity was accompanied by a corresponding loss of
peroxidase activity, suggesting that the R120E mutant does not fold
properly(2, 13) ; also consistent with this conclusion
was the observation of substantially diminished expression of the R120E
mutant compared with all the catalytically active PGHS-1 mutants
examined in this study. When Arg
was replaced with a
glutamine residue having a neutral side chain, the R120Q PGHS-1 mutant
retained detectable levels of cyclooxygenase activity (5%) and
peroxidase activity (approximately 60%), but the K
value for arachidonate was about 1000-fold higher than that seen
for the native enzyme. The lack of activity of the R120Q PGHS-1 toward
-linolenic acid suggests that Arg
is also required
for binding this fatty acid in the cyclooxygenase active site. Overall,
the studies of the R120Q mutant PGHS-1 indicate that Arg
is not essential for catalysis, but that this residue affects the
affinity of the enzyme for arachidonate. Taken in the context of the
crystal structure of PGHS-1 (10) , we interpret these results
as indicating that the arginino group of Arg
binds to the
carboxylate group of arachidonate.
When IC values were
determined using the same concentration of arachidonate, the
concentration of D-ibuprofen required to cause half-maximal
inhibition of R120Q PGHS-1 was 10-20 times higher than that
required to inhibit the native enzyme (Fig. 6). Because the
difference in K
values for arachidonate between
native PGHS-1 and R120Q PGHS-1 differ by about 1000-fold (Table 2), this observation suggests that Arg
is
relatively more important for arachidonate binding than ibuprofen
binding.
Previous studies had shown that Tyr of ovine
PGHS-1 can be nitrated in the presence but not the absence of either
indomethacin or ibuprofen(12, 31) . These findings
indicated that Tyr
could reside in or near the
cyclooxygenase active site of the enzyme; nonetheless, the Y355F PGHS-1
mutant retained appreciable activity, indicating that Tyr
is not essential for catalysis(12) . When the crystal
structure of the ovine PGHS-1/S-flurbiprofen complex was
solved, Tyr
was found to reside near the mouth of the
cyclooxygenase channel and to neighbor the
-methyl group of S-flurbiprofen. Accordingly, Garavito and co-workers (10) proposed that modification of this Tyr
might
alter the stereochemical specificity of PGHS-1 toward inhibitors of the
2-phenylpropionic acid group such as ibuprofen and flurbiprofen.
Indeed, the Y355F PGHS-1 mutant, which has a relatively small decrease
in side chain size at this position, exhibited considerably less
specificity toward D- and L-ibuprofen than did the
native PGHS-1.
Nonsteroidal anti-inflammatory drugs fall into two
major functional groups based on their abilities to cause simple
competitive versus time-dependent, competitive inhibition (19, 21) . Ibuprofen is a freely reversible
competitive inhibitor, whereas flurbiprofen and indomethacin cause a
time-dependent inhibition which is only slowly reversible (17, 18, 19, 20, 32) .
However, the methyl esters of time-dependent inhibitors are freely
reversible, competitive inhibitors(17) . In the case of
flurbiprofen, esterification would be expected to diminish the ability
of the inhibitor to bind Arg. Our results indicate that
the converse is also true because the R120Q mutant of PGHS-1 did not
undergo a time-dependent inhibition even in the presence of high
concentrations (10
M) of flurbiprofen.
Thus, binding of the carboxylate group of inhibitors is one important
characteristic of time-dependent inhibition. We speculate that one
requirement for time-dependent inhibition is that the inhibitor remain
in the cyclooxygenase active site long enough for some secondary
NSAID-induced change in protein structure to occur and that the
occupancy time for binding of nonsteroidal anti-inflammatory drugs such
as flurbiprofen to the active site of the R120Q mutant is too short to
permit such structural changes to occur.
A final unexpected
characteristic of the R120Q PGHS-1 mutant is its incapacity to undergo
suicide inactivation. This feature has been observed with several other
forms of ovine PGHS-1, including the Mn-heme PGHS-1 (33) and several mutants having modification near the active
site tyrosine, notably H386A/Q ovine PGHS-1 (13) and H372A
human PGHS-2. (
)However, unlike these mutants, the R120Q
PGHS-1 mutant exhibits considerable peroxidase activity. One
characteristic of peroxidase-deficient forms of PGHS-1 is that they
fail to form a tyrosyl radical in abundance(33) . It will be of
interest to determine if the same is true of R120Q PGHS-1.