From the Inflammatory Diseases Unit, Roche Bioscience,
Palo Alto, California 94304
Prostaglandin H synthases (PGHSs) catalyze the
conversion of arachidonic acid to prostaglandins. In this report, we
describe the effect of a PGHS2 Y355F mutation on the dynamics of PGHS2 catalysis and inhibition. Tyr355 is part of a
hydrogen-bonding network located at the entrance to the cyclooxygenase
active site. The Y355F mutant exhibited allosteric activation kinetics
in the presence of arachidonic acid that was defined by a curved
Eadie-Scatchard plot and a Hill coefficient of 1.36 ± 0.05. Arachidonic acid-induced allosteric activation has not been directly
observed with wild type PGHS2. The mutation also decreased the observed
time-dependent inhibition by indomethacin, flurbiprofen,
RS-57067, and SC-57666. Detailed kinetic analysis showed that the Y355F
mutation decreased the transition state energy associated with
slow-binding inhibition (EI
) relative to the
energy associated with catalysis (ES
) by 1.33, 0.67, and 1.06 kcal/mol, respectively, for indomethacin, flurbiprofen,
and RS-57067. These observations show Tyr355 to be involved
in the molecular mechanism of time-dependent inhibition. We
interpret these results to indicate that slow binding inhibitors and
the Y355F mutant slow the rate and unmask intrinsic,
dynamic events associated with product formation. We hypothesize that the dynamic events are the equilibrium between relaxed and tightened organizations of the hydrogen-bonding network at the entrance to the
cyclooxygenase active site. It is these rearrangements that control the
rate of substrate binding and ultimately the rate of prostaglandin
formation.
 |
INTRODUCTION |
Prostaglandins are formed from arachidonic acid by constitutive
prostaglandin H synthase 1 (PGHS1)1 and inducible
prostaglandin H synthase 2 (PGHS2) (1). They are important cellular
mediators of many biological functions, including inflammation,
pyresis, and algesia (2, 3). Recent evidence suggests that
prostaglandins formed by PGHS2 mediate inflammation (3, 4). PGHS2 is
also implicated in the pathology of Alzheimer's disease and colon
cancer (5-7).
The latest methodologies in structure-based drug discovery are being
used to identify PGHS2-selective medicines. Inhibitor-bound structures
of PGHS1 and PGHS2 have been solved (8-11). The structures and
additional mutagenesis data have identified a number of important features (12-17) (Fig. 1). The
inhibitors bind in a long channel whose entrance is flanked by three
residues capable of creating a hydrogen-bonding network,
Arg120,2
Glu524, and Tyr355. Arg120 is
required for binding the carboxylic acid moiety of fatty acid substrates and nonsteroidal anti-inflammatory drugs (12, 13). Tyr355 is proposed to be a determinant of specificity in
the 2-phenylproprionic class of inhibitors; inhibition of the PGHS1
phenylalanine mutant by ibuprofen produced a change in the
stereochemical specificity but not potency (13). The channel ends at
residue Tyr385, a residue required for cyclooxygenase
catalytic activity (14). The channel is bordered by Ser530,
the site of aspirin acetylation and a side pocket that nonsteroidal anti-inflammatory drugs can occupy in PGHS2 but not PGHS1. This pocket
is the result of a change in PGHS1I523 to PGHS2V523 and PGHS1H513 to
PGHS2R513 and is important for the specificity of some PGHS2-selective
inhibitors (15-17).

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Fig. 1.
Structure of the active sites of PGHS1
(A) and PGHS2 (B) with key amino acids
highlighted. The PGHS1 structure is bound with flurbiprofen (8),
and the PGHS2 structure is bound with RS-57067 (10). These two views
show two possible arrangements of the hydrogen bonding network: one
involving Arg120, Glu524, Tyr355
(PGHS1) and the other Glu524, Tyr355, and
Arg513 (PGHS2).
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Dynamics have been shown to play critical roles in the mechanism and
inhibition of PGHSs. Many inhibitors of both PGHS isoforms are
time-dependent (18-21), and the enzymes are allosterically activated by arachidonic acid (22). Selective PGHS2 inhibition in many
cases is correlated with the time-dependent inhibition of
PGHS2 and a lack of time-dependent PGHS1 inhibition (20, 21). A loss of PGHS1 binding affinity coinciding with conformational changes induced by the allosteric activation of PGHS1 also results in
an increase in PGHS2 selectivity (22). Additionally, the allosteric
regulation is proposed to contribute to regulation and selectivity of
prostaglandin formation (22). The factors that relate these dynamic
changes to protein structure are unknown.
The kinetic mechanism of many time-dependent PGHS
inhibitors is reversible slow binding inhibition. The inhibitors bind
to form an initial enzyme-inhibitor complex (EI) that is in
slow equilibrium with another enzyme-inhibitor complex (EI*)
(Scheme 1) (23, 24). Intrinsic to this kinetic mechanism are two
competing equilibria: an intermolecular equilibrium between the
inhibitor and substrate for enzyme and an intramolecular equilibrium
between the two enzyme-inhibitor binding complexes. It is the rates of these equilibria relative to the rate of product formation that will
determine if these equilibria can be kinetically observed.
We have previously described a method to determine the kinetic
constants associated with the reversible slow binding inhibition of
PGHS (24). The method enabled us to determine the transition state
energy between the two enzyme inhibitor complexes, EI and EI*. The transition state energies for indomethacin and
flurbiprofen were both approximately 1.4 kcal/mol lower for PGHS1 than
PGHS2.
We have used this methodology to evaluate the influence of structure on
the functional dynamics of PGHS2 inhibition. The dynamics associated
with inhibition by flurbiprofen, indomethacin, RS-57067, and SC-57666
and the kinetics associated with allosteric activation by arachidonic
acid were evaluated with the PGHS2 Y355F mutant. The
time-dependent inhibition and allosteric activation are
shown to involve the phenolic group of Tyr355. These
dynamics are postulated to be kinetically detectable due to the
unmasking of an equilibrium between a relaxed and tightened organization of the hydrogen-bonding network located at the entrance to
the cyclooxygenase binding pocket.
 |
EXPERIMENTAL PROCEDURES |
Materials--
[3H]Arachidonic acid,
[3H]anadamide, and [3H]prostaglandin
E2 were purchased from NEN Life Science Products; unlabeled
arachidonic acid was from Nu-Chek-Prep, Inc. (Elysian, MN); and
unlabeled anadamide, indomethacin, and flurbiprofen were from Cayman
Chemical Co. (Ann Arbor, MI). RS-57067 and SC-57666 were synthesized at Roche Bioscience. Hemin and tyloxapol were obtained from Sigma. All
other chemicals were of the highest grade available.
Rate Measurements--
Prostaglandin formation was determined in
reactions that contained [3H]arachidonic acid (50 nM to 20 µM; 0.5 µCi) or
[3H]anadamide, tyloxapol (0.05%), phenol (1.2 mM), hemin (0.6 µM), and potassium phosphate
buffer (100 mM, pH 8.0) in a total volume of 0.1 ml unless
otherwise stated. All reactions were incubated at 30 °C. The
reactions containing arachidonic acid were incubated for 30 s (3 min for anadamide) and stopped with 0.4 ml of ice-cold ethanol and
placed on ice. After approximately 30 min on ice, the reactions were
evaporated to dryness and reconstituted in 0.2 ml of 50:50:1
water/methanol/acetic acid, and 0.1 ml was injected onto the HPLC for
analysis. The substrate solution was prepared by combining unlabeled
arachidonic acid in ethanol, [3H]arachidonic acid in
ethanol, and 10 µl of a 0.5% solution of tyloxapol in acetone and
evaporating to dryness. This was reconstituted in 0.05 ml of water. The
reactions were initiated by adding the detergent-solubilized substrate
solution to the enzyme mix containing buffer, phenol, and hemin, which
had been preincubated at room temperature for 1-5 min.
Oxygen consumption was measured with a YSI model 5300 Biological Oxygen
Monitor equipped with a Clark-type micro oxygen electrode. Data was
collected and transformed using an external connection to a SLM-Aminco
DW2000 data system. Enzyme (20-50 nM) was mixed with hemin
(0.8 µM) and phenol (2 mM) to establish a
base line. The reaction was initiated with arachidonic acid (200 µM) after preincubation with either inhibitor or vehicle.
Arachidonic acid was added in tyloxapol; the final concentration of
tyloxapol was not greater than 0.08%. The maximum velocity was
determined from a first derivative transformation of the reaction
profile. The system was calibrated using the
catalase-dependent oxidation of hydrogen peroxide.
HPLC Conditions--
The products of arachidonic acid and
anadamide oxidation were separated by reverse phase HPLC using a 25-cm,
5 µ, Jones Chromatography apex octadecyl column and detected using a
Packard Flo-one A-500 radioflow detector with a scintillant
mixture:HPLC eluant ratio of 3:1. The strong component of the mobile
phase was 0.1% acetic acid/ammonium hydroxide buffer (pH 6.1), and the
eluting solvent was methanol. The flow rate was 1 ml/min. The following
elution profile was used: 0-5 min 20% methanol; 6-15 min
7 convex
gradient to 60% methanol; 16-20 min 60% methanol; 21-30 min
7
convex gradient to 80% methanol; 31-35 min 80% methanol; 36-45 min
100% methanol. The prostaglandins eluted around 25 min, arachidonic
acid at 44 min, anadamide-derived prostaglandins at 22 min, and
anadamide at 38 min (25).
Mathematical Methods--
A mathematical model was developed,
resulting in a final three-parameter equation describing the initial
maximum velocity of the enzyme-substrate reaction as a function of the
enzyme-inhibitor preincubation time (24). Time-velocity curves were
obtained for various inhibitor concentrations, and the three parameters and their asymptotic standard errors were estimated by simultaneously fitting the time-velocity curves for all inhibitor concentrations to the final equation. The Nelder-Mead simplex algorithm in an adaptation of the software package PCNONLIN was used to estimate the
parameters,
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(Eq. 1)
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where
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(Eq. 2)
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and
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(Eq. 3)
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where T is the preincubation time,
Ki the binding constant associated with
EI formation, k2 the forward
isomerization rate, and k
2 the reverse
isomerization rate.
Mutagenesis, Expression, and Purification of Y355F--
The
baculovirus plasmid vector pSyn XIV VI+ X3 containing the human PGHS2
gene, named pPGHS-2 (26) was mutagenized following the USE protocol
(Pharmacia Biotech Inc.). The selecting primer 3909 (5'-TTTCACACCGGATATCGTGCACTCTCAG-3') mutates a unique NdeI site into EcoRV. The mutagenic primer,
5'-GTGCAACACTTAAGTGGCT(T/C)TCACTTCA-3' (changed nucleotides in boldface type), was synthesized containing restriction endonuclease sites useful for selecting the desired mutants. pPGHS-2 (25 fmol) was heat-denatured in the presence of 25 pmol of primer 3909 and the phosphorylated mutagenic primers. Following
an annealing step, the mutant DNA strand was synthesized by incubating
for 1 h at 37 °C with a reaction mix containing dNTPs, ATP, T4
ligase, T4 DNA polymerase, and T4 gene 32 protein. The final
mutagenesis reactions were digested in a 50-µl reaction with 5-10
units of NdeI to linearize wild type homoduplex DNA. Ten
µl of digest were used to transform 40 µl of competent
BMH71-18mutS Escherichia coli cells (a repair-defective
strain; CLONTECH, Palo Alto, CA). Transformants
were incubated in 3 ml of growth medium with 100 µg of ampicillin/ml
at 37 °C, overnight. Plasmid DNAs were miniprepped and digested
again with NdeI, prior to transformation of competent
DH5
E. coli cells (from Life Technologies, Inc.). These
transformations were plated on LB agar with ampicillin to obtain single
colonies. Plasmid DNAs from a handful of colonies were miniprepped and
screened for the selecting restriction site. Sequencing of the
mutagenized target region (using a Sequenase DNA sequencing kit from
U.S. Biochemical Corp.) confirmed the presence of the desired codon
change. Transfection of Sf9 insect cells with the mutated,
transplacement vector was carried out as described previously (27).
Individual baculovirus isolates, selected by assaying cyclooxygenase or
peroxidase activity, underwent three rounds of amplification for
scale-up production of human PGHS2 Y355F. Mutated PGHS2 was purified
from Sf9 insect cells by a modification of the procedure of
Barnett and co-workers (26). The cells were lysed in lysis buffer
containing 1 mM diethylthiocarbamate (from Sigma) and
centrifuged at 2,000 × g for 20 min. The supernatant was added to a lectin column in which the mobile phase contained 0.1%
(w/v) dodecyl maltoside instead of octyl glucoside. Fractions containing PGHS2 from the anion exchange HPLC columns and the lentil
lectin-Sepharose 4B columns were identified by the
N,N,N',N'-tetramethylphenylenediamine coupled peroxidase assay (28). Final protein samples were essentially single bands as determined by SDS-polyacrylamide gel electrophoresis and Western blotting. Enzyme concentrations were determined from the
absorbance at 411 nm using an extinction coefficient of 123 mM
1 cm
1 (29).
Reaction Coordinates--
The Gibbs free energies used to create
the reaction coordinates were determined as follows: the free energy of
the unbound enzyme was normalized to zero; the energy of EI
was determined from the equation,
|
(Eq. 4)
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where R is the gas constant and T is the
absolute temperature; and the activation energy for the transition
state between EI and EI* was calculated from the
Eyring equation,
|
(Eq. 5)
|
where h is Plank's constant and k is the
Boltzman constant. The activation energy for
ES
was determined from
kcat/Km, which represents the
change in free energy between the transition state complex
(ES
) and the free enzyme and substrate
(E + S).
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(Eq. 6)
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The free energy of ES was calculated by subtracting
the free energy associated with kcat, determined
by the Eyring equation, from the free energy associated with
ES
. The activation energies between free
enzyme and EI and ES were assumed to be small
relative to those associated with ES
and
EI
.
 |
RESULTS |
Substrate Oxidation Kinetics--
The apparent
Km and kcat values associated
with prostaglandin formation from arachidonic acid and its ethanolamide analog, anadamide (25), catalyzed by purified recombinant human PGHS2
and the site-directed mutant PGHS2 Y355F are shown in Table I. The fit of the data to the
Michaelis-Menton equation showed the Y355F mutation to decrease the
kcat associated with arachidonic acid oxidation
by 2-fold, without a large effect on the apparent Km. Anadamide oxidation by the wild type enzyme
showed a 6-fold increase in apparent Km and a 3-fold
decrease in kcat as compare with arachidonic
acid. The Y355F mutation decreased the apparent Km
associated with anadamide 2-fold relative to wild type.
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Table I
The kinetic constants associated with the oxidation of arachidonic acid
and anadamide by PGHS2 and PGHS2 Y355F
The enzymes were incubated with arachidonic acid for 30 s or
anadamide for 3 min, and the amount of prostaglandins formed was
determined by HPLC. The statistics are the S.E. determined from best
fit of a single representative experiment.
mut G T = RTln[(kcat/Km)wt/(kcat/Km)mut]; R G T = RTln[(kcat/Km)AA/(kcat/Km)anad].
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A more complete evaluation of the data showed a curved Eadie-Scatchard
plot for oxidation of arachidonic acid by the Y355F mutant (Fig.
2). The data was replotted to the Hill
equation (Fig. 2). The Hill plot appeared to be biphasic, with
cooperativity observed below 0.5 µM arachidonic acid
(n = 1.36 ± 0.05, r2 = 0.994). No cooperativity was observed above 0.5 µM
arachidonic acid (n = 1.01 ± 0.03, r2 = 0.994). Fitting the Hill equation to the
entire data set (n = 1.10 ± 0.03, r2 = 0.996) resulted in a nonrandom distribution
of the residuals as compared with the biphasic fit (Fig. 2, upper
left panel). These results are consistent with positive
cooperative activation of PGHS2 Y355F by arachidonic acid. Arachidonic
acid has been directly observed to induce allosteric activation of
PGHS1, not PGHS2. However, fluorescent quenching experiments indirectly
indicated that PGHS2 was activated at very low arachidonic acid
concentrations (22). These results suggest that the Y355F mutant is
less responsive to the arachidonic acid-induced activation than the
wild type enzyme. No direct evidence for allosteric activation was
observed with the Y355F mutant using anadamide as substrate or with any of the other PGHS2 site-directed mutants that we have investigated (R120Q, R513H, E524Q, V523I, and the double mutant R513H/V523I) (data
not shown).

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Fig. 2.
Cooperative activation of PGHS2 Y355F by
arachidonic acid. The data associated with PGHS2 Y355F was fitted
to the Hill equation in a mono- and biphasic manner. The
Eadie-Scatchard plot of PGHS2 ( ) and PGHS2 Y355F ( ) are shown in
the lower right. In the upper left panels are the
residuals associated with the fit of the data to the Hill equation. The
left is the fit to the entire data set, the
middle is below 0.5 µM arachidonic acid, and
the right is above 0.5 µM. The scale of the
y axis residuals is ±0.015.
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Changes in transition state energy
(
G
T) due to changes in an R
group or a mutation can be calculated from Equation 7 (30).

G
T represents the change in
free energy required to reach the transition state complex
(ES
) from free enzyme and substrate
(E + S).
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(Eq. 7)
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The Y355F mutation decreased the transition state energy for
prostaglandin formation from arachidonic acid by 0.3 kcal/mol; no
change was observed with anadamide. This suggests that the phenolic
group of the Tyr355 does not contribute to the
rate-limiting step in catalysis. The change of the carboxylic acid
(arachidonic acid) to the ethanolamide (anadamide) results in a 1.80 and 1.47 kcal/mol decrease in the respective transition state energies
for wild type and mutant. The carboxylic acid to ethanolamide
modification slows the rate-limiting step in catalysis independent of
Tyr355.
Time-dependent Inhibition--
We investigated the
impact of the PGHS2 Y355F mutation on time-dependent
inhibition with four time-dependent cyclooxygenase inhibitors (Fig. 3); the inhibitors were
incubated for various times prior to the addition of 200 µM arachidonic acid. The maximum initial velocities were
determined from the first derivative of the oxygen consumption profile.
It is important to realize that the extent of inhibition observed under
these experimental conditions represents only the amount of enzyme
sequestered in the EI* complex. All enzyme complexes that
are in rapid equilibrium with free enzyme should react with the
supersaturating concentrations of substrate (24).3 Visual inspection of
the inhibition profiles shows that the inhibitors achieve maximal
inhibition much more rapidly with the PGHS2 Y355F mutant (only the
indomethacin data is shown; Fig. 4).

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Fig. 4.
The PGHS2 Y355F mutation decreases the time
to steady state for time-dependent inhibition of PGHS2 by
indomethacin. PGHS2 ( ) and PGHS2Y355F ( ) were incubated with
2.5 µM indomethacin, and rates were determined using the
oxygen consumption assay as described under "Experimental
Procedures."
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The impact of the Y355F mutation on the kinetic parameters associated
with slow binding inhibition by flurbiprofen, indomethacin, and
RS-57067 is shown in Table II. The
carboxylic acids, flurbiprofen and indomethacin, form the EI
complex with a similar affinity for both enzymes (similar
Ki) but rearranged more rapidly to the
EI* complex with PGHS2 Y355F (increase in
k2) (Table II). The mutation increases the rate
of equilibration between EI and EI* without
increasing the apparent binding affinity. RS-57067, a non-carboxylic
acid inhibitor, bound tighter to EI with PGHS2 Y355F. The
reverse isomerization rate from EI* increased 3-fold with
PGHS2 Y355F, while the forward rate did not change.
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Table II
Kinetic constants associated with inhibition of PGHS2 and PGHS2 Y355F
by indomethacin, flurbiprofen, and RS-57067
The rate constants associated with inhibition of wild type PGHS2 by
indomethacin and flurbiprofen were previously reported by Callan
et al. (24), and those associated with RS-57067 were reported by Swinney et al. (22). %EI* represents
the percentage of inhibitor complexed in EI* at saturation.
k2 and k 2 are expressed
in s 1, Ki in µM.
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In order for the Y355F mutation to influence the isomerization between
EI and EI*, it must lower the transition state
between the two forms of the inhibitor-bound enzyme (EI
).
The extent of the mutation's effect on the transition state is not
directly evident from the magnitude of the individual rate constants;
therefore, we constructed free energy profiles to evaluate the effect
of the mutation on the transition state energies. (The reaction
coordinate for indomethacin is shown in Fig.
5.) Calculation of the influence of the
Y355F mutation on binding free energies (
G) is shown in Tables III and
IV. Indomethacin and flurbiprofen binding
to PGHS2 Y355F had minimal effect on the free energy associated with
the initial enzyme-inhibitor complex (EI); however, the
change lowered the energy associated with the EI to
EI* transition state by 1.63 and 0.97 kcal/mol, respectively
(Table III, Fig. 5). The ground state energy associated with
EI* was stabilized by 0.25 kcal/mol for flurbiprofen and
1.46 kcal/mol for indomethacin. RS-57067 binding to PGHS2
Tyr355 was stabilized by 1.29 and 0.83 kcal/mol in
EI and EI*, respectively, and the transition
state energy was lowered by 1.36 kcal/mol. The mutation decreased the
entire RS-57067 reaction coordinate by the approximately 1 kcal/mol.

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Fig. 5.
The reaction coordinate for
indomethacin. PGHS2 is represented by the solid line;
PGHS2Y355F is shown by the dashed line.
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Table III
Gibbs free energy ( G) associated with the EI to EI* transition
of indomethacin, flurbiprofen, and RS-57067 for PGHS2 and PGHS2
Y355F
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Table IV
Differences in the activation free energy between product formation
(ES ) and slow binding inhibition (EI ) for
wild type PGHS2 and PGHS2 Y355F
Gkcat and
Gk2 represent the change in free energy
between the transition states (ES and
EI ) and free enzyme and substrate
(E + S) or inhibitor (E + I), respectively. GYF represents effect of the Y355F
mutation of the difference in transition state energy between
ES and EI .
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The mutation increased the rate at which the inhibitors approached the
EI/EI* equilibrium by increasing the forward rate
for indomethacin, both the forward and reverse rates for flurbiprofen, and the reverse rates for RS-57067.
Tyr
Phe-induced Decrease in Slow Binding Inhibition--
Slow
binding kinetic behavior is proposed to occur when
k2 is slower than kcat
(31). The k2 values determined for inhibition of
PGHS2 by flurbiprofen (0.105 s
1), indomethacin (0.034 s
1), and RS-57067 (0.21 s
1) compared with
the kcat reported for PGHS2 (13 s
1) are consistent with this proposal. The data in Table
IV show quantitatively the reason for the observed decrease in the
time-dependent inhibition associated with the Y355F mutant
and also provide a thermodynamic explanation for the kinetics of slow
binding inhibition. The Y355F mutation decreases the relative
difference between the EI
/ES
transition
state energies by 1.33, 0.67, and 1.06 kcal/mol for indomethacin,
flurbiprofen, and RS-57067, respectively (Table IV). It is important to
distinguish between the equilibrium rate of EI and
EI* and the observation of slow binding inhibition. The
observation of slow binding inhibition occurs when the equilibrium rate
is similar or slower than the rate of catalysis. The equilibrium can
occur in the absence of observable slow binding inhibition if it is
more rapid than the rate of catalysis.
Loss of Apparent Irreversibility with SC-57666--
SC-57666 is a
selective, time-dependent reversible PGHS2 inhibitor (32).
Compounds of this class have been proposed to induce inactivation of
PGHS2 (21). Preincubation of wild type PGHS2 with SC-57666 resulted in
incomplete consumption of oxygen as compared with control (Fig.
6, top). We have interpreted
incomplete oxygen consumption to correlate with the inactivation of
PGHS2 or extremely slow dissociation from PGHS2. Incubation of SC-57666 with PGHS2 Y355F only slightly delays the total oxygen consumption (Fig. 6, bottom). These results suggest that SC-57666
binding to PGHS2 Y355F is rapidly reversible. This is a dramatic
contrast to the extremely slow dissociation observed with wild type
PGHS2.

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Fig. 6.
The effect of SC-57666 on oxygen consumption
by PGHS2 and PGHS2 Y355F. Oxygen consumption was measured
following the addition of arachidonic acid (200 µM) to
PGHS2 (9 nM) and PGHS2 Y355F (27 nM) in the
absence or presence of SC-57666 (1 µM). SC-57666 was
added 1 min prior to the addition of arachidonic acid. One unit of
oxygen is equivalent to 15 nmol. The maximum initial rates of oxygen
consumption determined from the first derivative of the reaction
profile were 15 s 1 for wild type and 11.3 s 1 for Y355F.
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 |
DISCUSSION |
This work describes the effect of the Y355F mutation on the
relative equilibria that comprise the slow binding inhibition of
prostaglandin formation by PGHS2. The slow binding inhibition is the
result of an intramolecular equilibrium between two kinetic states,
EI and EI*, that competes with the rate of
prostaglandin formation. The results show that increasing the rate of
equilibration between EI and EI* relative to the
rate of product formation decreases the likelihood of observing the
slow binding inhibition. The overall impact of the Y355F mutation on
the competition between prostaglandin formation and slow binding
inhibition is to decrease the time it takes to establish the
EI/EI* equilibrium. These results suggest a role
for Tyr355 in the molecular mechanism of
time-dependent inhibition of PGHSs.
This work also describes the effect of the Y355F mutation on the
equilibrium between the allosterically activated and unactivated enzyme
forms. We directly observed arachidonic acid to induce the allosteric
kinetics associated with the PGHS2 Y355F mutant (Fig. 2). As we noted
earlier, allosteric activation by arachidonic acid has been directly
observed with PGHS1, not PGHS2. However, fluorescent quenching
experiments indirectly indicate that PGHS2 is activated at very low
arachidonic acid concentrations (22). One explanation for these results
is that the Y355F mutation slows the rate of activation by arachidonic
acid, thereby allowing it to be kinetically detectable. These results
suggest a role for Tyr355 in the molecular mechanism of
allosteric activation.
To observe a kinetic event, it must be partially rate-limiting. A
practice used to delineate mechanisms of catalysis is to employ
substrate analogs and isotopes to decrease the rates of reaction. In
some instances, this will expose or unmask previously undetectable kinetic steps. We propose that the observations of slow
binding inhibition and allosteric activation associated with the PGHS2
Y355F mutant are kinetically detectable because they unmask kinetic
steps intrinsic to the activation and binding of arachidonic acid to
PGHSs.
Tyr355 can potentially participate in the
time-dependent inhibition and allosteric activation through
its interactions with the substrate, the inhibitor, and/or the hydrogen
bonding network. The structures of PGHS2 show at least two possible
hydrogen bonding conformations near the entrance to the cyclooxygenase
binding pocket: one consisting of Arg120,
Glu524, and Tyr355 and another consisting of
Arg513, Glu524, and Tyr355. We
propose that it is the equilibrium between these two hydrogen bonding
arrangements that is responsible for the allosteric activation and that
the disruption of this equilibrium contributes to the slow binding
inhibition. We envision a mechanism whereby the hydrogen bonding
network at the entrance to the cyclooxygenase site is relaxed in the activated enzyme and tightened in
the unactivated and inhibitor bound forms (Fig.
7). The
Arg513/Glu524/Tyr355 hydrogen
bonding network may predominate in the relaxed form, leaving
Arg120 to freely interact with substrate, whereas in the
tightened conformation the
Arg120/Glu524/Tyr355 hydrogen
bonding network locks the substrate into a catalytically competent
conformation (Fig. 7). For PGHS2, the rates of these transitions are
normally too rapid relative to catalysis to be observed; however,
inhibitors or mutations that interact with the reorganization of the
hydrogen bonding network will slow the reorganization rates relative to
catalysis and make the reorganization of the hydrogen bonding network
partially rate-limiting. This hypothesis is consistent with the
proposal by Browner and co-workers (10). They suggested from analysis
of the structural data that it is the ability of the channel to move
between open and closed conformations that allows both substrates and
inhibitors to reach the internal binding site.

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Fig. 7.
Schematic depiction of the proposed model for
allosteric activation and slow binding inhibition of PGHSs.
Substrate activation results in relaxation of the hydrogen bonding
network to expose the binding site. Substrate or inhibitor then rapidly bind to the exposed binding site. The hydrogen bond network will then
rapidly tighten around the substrate-bound enzyme and slowly tighten
around the inhibitor-bound enzyme.
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Anadamide is the ethanolamide derivative of arachidonic acid. Anadamide
was recently shown to be selectively metabolized by PGHS2 to a novel
class of prostaglandins (25). We did not observe allosteric activation
using anadamide as substrate with either wild type PGHS2 or the Y355F
mutant. The free energy required for catalysis is increased by greater
than 1.4 kcal/mol for anadamide relative to arachidonic acid for both
the wild type and PGHS2 Y355F. The slower rate of catalysis for
anadamide relative to arachidonic acid may mask the allosteric
activation. Consistent with this proposal is the observed decrease in
the time-dependent inhibition of PGHS2 when anadamide is
used as substrate.4 We
conclude that the slower rate of anadamide oxidation relative to
arachidonic acid masks the intrinsic equilibria associated with the
allosteric activation and time-dependent inhibition.
In summary, we have used the tools of site-directed mutagenesis and
kinetic analysis to pinpoint an involvement of the phenolic group of
Tyr355 in time-dependent inhibition and
allosteric activation of PGHS2. We propose that these dynamic events
unmask intrinsic changes associated with an equilibrium between relaxed
and tightened forms of the hydrogen bonding network at the entrance to
the cyclooxygenase binding site. The position of the equilibrium
between the relaxed and tightened forms will control substrate binding
and ultimately the rate of prostaglandin formation.
We thank Aaron Miller for the homology
modeling of PGHS2 and the suggestion to study Tyr355; Joan
Chow and Jim Barnett for providing wild type PGHS2; and the chemists of
the COX team led by Eric Sjogren for providing RS-57067 and SC-57666.
We also thank Chakk Ramesha for many thoughtful discussions and
critical review of the manuscript and Michelle Browner for providing
Fig. 1 and comments on the manuscript.