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INTRODUCTION |
Prostaglandins have roles in a variety of pathophysiological
processes, including inflammation, hemostasis, gastric cytoprotection, and pain sensation (1). Prostaglandin H synthase
(PGHS)1 catalyzes the first
committed step in prostaglandin biosynthesis, the conversion of
arachidonic acid to PGG2, called the cyclooxygenase reaction (2). PGHS also has a heme-dependent peroxidase
activity that reduces the C15 hydroperoxide of PGG2 to a
hydroxyl, producing prostaglandin H2, the precursor of all
other prostanoids (2). Two PGHS isoforms have been identified, termed
PGHS-1 and PGHS-2 (3). PGHS-1 is present at essentially constitutive
levels in a wide variety of cells, whereas PGHS-2 is undetectable in
most quiescent cells but can be strongly induced in some cells by
cytokines and mitogens (3). This very different control of expression of the two distinct PGHS genes has led to the concept that the isoforms
serve distinct physiological functions, with PGHS-1 assigned a
housekeeping role and PGHS-2 implicated in cytokine-mediated events
(3). Cellular prostaglandin synthesis is also tightly regulated at the
level of cyclooxygenase catalysis (4). This catalytic regulation is
particularly interesting because it appears to be different for the two
isoforms. In many cells containing both isoforms, the PGHS-2
cyclooxygenase has been found to be catalytically active at the same
time that the PGHS-1 cyclooxygenase remains latent (4). One possible
biochemical explanation for such differential cellular catalytic
control of the two isoforms is their different hydroperoxide activator
requirements, with the PGHS-2 cyclooxygenase activated at lower
hydroperoxide levels than the PGHS-1 cyclooxygenase (5, 6). Because of
this potential to influence cellular control of prostanoid synthesis,
it is important to define the mechanistic basis for the difference in
activator efficiency between the two PGHS isoforms.
Conversion of latent cyclooxygenase to catalytically competent enzyme
is believed to involve formation of a key catalytic component, a free
radical located on Tyr385 (Tyr371 in PGHS-2) in
the upper part of the cyclooxygenase channel (7-12). In this mechanism
(depicted in Scheme I), generation of the active tyrosyl radical
involves an initial reaction of a hydroperoxide with the peroxidase
site heme to form Intermediate I, analogous to Compound I in
horseradish peroxidase. Intermediate I is oxidized by two equivalents
compared with the resting ferric enzyme. An intramolecular one-electron
transfer from Tyr385 to the heme subsequently produces the
tyrosyl radical (Intermediate II), which has one oxidizing equivalent
on Tyr385 and one on the ferryl heme. Intermediate II, with
its tyrosyl radical, is thought to be the initial oxidant in
cyclooxygenase catalysis (7). In the present studies, we have used
stopped flow spectroscopic measurements to characterize the kinetics of the reactions of PGHS-1 and -2 with several peroxides, with a focus on
the steps leading to formation of Intermediate II. The results show
that formation of this crucial cyclooxygenase intermediate is
considerably faster for PGHS-2 than for PGHS-1, accounting in part for
the more efficient cyclooxygenase activation in PGHS-2.
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MATERIALS AND METHODS |
Glutathione, GSP, glycerol, hemin chloride, and Pharmalytes (pH
5-8) were from Sigma. PD-10 desalting columns were from Supelco. EtOOH
was from Polysciences, and PPHP was purchased from Cayman Chemicals.
Arachidonic acid was obtained from NuChek Preps and was routinely used
without borohydride treatment. Tween 20 was obtained as a 10% solution
from Pierce, and octyl-
-D-glucopyranoside was from
Amresco (Solon, OH).
PGHS-1 was purified from ram seminal vesicles (13). Recombinant human
PGHS-2 was expressed in cultured insect cells and purified to
homogeneity as previously reported (6). Protein concentrations were
determined as described by Peterson (14). The holoenzymes were
reconstituted by addition of heme (1.4 mol/mol subunit), followed by
treatment with DEAE-cellulose and gel filtration chromatography on a
PD-10 column to remove excess heme and to exchange buffer (15). The
holoenzyme concentrations were determined from their absorbance at 410 nm (165 mM
1 cm
1). 15-HPETE was
prepared from arachidonic acid by reaction with soybean lipoxygenase
(16) and purified by high pressure liquid chromatography (17). The
purity of the 15-HPETE was assessed chromatographically, and the
concentration was quantitated from the oxidation of
N,N,N',N'-tetramethyl-p-phenylenediamine
in the presence of excess PGHS-1 (18).
Stopped flow kinetic studies were conducted at 4 °C on a
Bio-Sequential DX-18MV stopped flow instrument (Applied Photophysics, Leatherhead, Surrey, UK) as described previously (19), using equal
volume mixing. The Soret maximum of resting enzyme (410 nm for PGHS-1
and 408 nm for PGHS-2) was used to monitor the formation of
Intermediate I. Formation of Intermediate II was monitored for both
isoforms at 424 nm, near the isosbestic point between resting enzyme
and Intermediate I in PGHS-1 (7, 19). Reaction rates were obtained by
fitting averaged kinetic data from at least three replicate runs to an
exponential function. Both holoenzyme and peroxide were generally
diluted in a 0.1 M potassium phosphate, pH 7.3, containing
10% glycerol, 0.1% Tween 20, and 0.1%
octyl-
-D-glucopyranoside.
Computer simulations of cyclooxygenase kinetics in the presence of
added GSP were carried out by numerical integration using the SCoP
program (Simulation Resources, Redlands, CA). Calculations were based
on the PGHS mechanism shown in Scheme II, with separate provisions for
GSP activity. The mechanism is based on the branched chain tyrosyl
radical mechanism proposed by Dietz et al. (7), with
addition of two enzyme intermediates (E(III)/PPIX/Tyr* and E(IV)/PPIX*/Tyr*) to allow continued peroxidase activity at the heme
site after formation of the tyrosyl radical in the cyclooxygenase site.
The rate equations used in the SCoP program were as follows.
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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(Eq. 6)
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(Eq. 7)
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(Eq. 8)
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(Eq. 10)
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(Eq. 11)
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In this model, cyclooxygenase activity is a simple, saturable
function (the Km value for arachidonate is
KmAA) reflecting the rate constant
(k5), the arachidonate concentration ([AA]),
and the total concentration of enzyme forms with activated cyclooxygenase (E(III)/PPIX/Tyr*, E(IV)/PPIX*/Tyr*, and
E(IV)/PPIX/Tyr*). The term for cyclooxygenase activity appears in
Equations 8 and 9. Similarly, the glutathione peroxidase activity is a
simple, saturable function reflecting the peroxide level ([ROOH]),
the Km value for hydroperoxide, PGG2
(KmGSP), and the GSP activity
(calculated from the ratio of added GSP units to added cyclooxygenase
units, RGC), the cyclooxygenase specific activity (k5), and the initial PGHS
concentration [E(III)/PPIX/Tyr0]). The term for GSP
activity appears in Equations 9 and 10. The initial concentrations and
parameter values for Equations 1-11 are shown in Table
I. The value of k1
was based on measured values for the rate of Intermediate I formation
with lipid hydroperoxides (19). A range of k2
values was tested, with the lower bound being the observed value for
conversion of Intermediate I to Intermediate II in PGHS-1
("Results" and Ref. 19). The rates for reduction of higher
oxidation states of heme (k3 and
k4) were consistent with the measured value for
the overall rate of return to ferric heme with phenolic reducing
cosubstrates (20). The value of k5 represents
the turnover number calculated from a PGHS-1 cyclooxygenase specific
activity of 100 µmol O2/min/mg protein. A range of
k6 values were tested, as described under
"Results." The value of k7 was set at
10
3 times that of k5 to fit the
typical observation of about 1000 cyclooxygenase catalytic events
before self-inactivation of PGHS-1 (21). Measured values were used for
the cyclooxygenase Km for arachidonate
(KmAA) and for the GSP
Km for PGG2
(KmGSP) (21, 22).
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RESULTS |
Reaction of PGHS-1 and -2 with EtOOH--
Reaction of PGHS-1 or
PGHS-2 with the small hydrophilic peroxide, EtOOH, produced an initial
rapid decrease in the Soret absorbance peaks of the resting holoenzymes
(data not shown), reflecting the formation of Intermediate I (Scheme
I). The observed rate for Intermediate I
formation increased linearly with the EtOOH concentration for both
PGHS-1 and -2 (Fig. 1A). The
slopes of the lines fitted to the data in Fig. 1A were used
to estimate k1 values of 3.4 × 106 M
1 s
1 for
PGHS-1 and 0.5 × 106 M
1
s
1 for PGHS-2. Thus, the initial reaction of PGHS-1 with
EtOOH was approximately 7-fold faster than the corresponding reaction
of PGHS-2.

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Scheme I.
Branched chain radical mechanism for PGHS
peroxidase and cyclooxygenase catalysis based on a proposal by Ruf and
colleagues (7). The letters to the right
side of each enzyme intermediate indicate the redox state of the
heme iron (III or IV), whether the porphyrin is
in ground (PPIX) or free radical (PPIX*) state,
and whether the cyclooxygenase active site tyrosine residue
(Tyr385 in PGHS-1 and Tyr371 in PGHS-2) is in
ground state (Tyr) or has a tyrosyl radical present
(Tyr*). Einact represents
self-inactivated enzyme, ROOH and ROH are
hydroperoxide and the corresponding alcohol, e
is an electron donor (reducing cosubstrate), and AA is
arachidonic acid.
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Fig. 1.
Kinetics of PGHS-1 and -2 peroxidase
reactions with EtOOH at 3.9 °C. A, effects of EtOOH
concentration on the rate of Intermediate I formation in PGHS-1 ( )
and PGHS-2 ( ). Changes in resting enzyme levels were monitored at
either 410 nm (PGHS-1) or 408 nm (PGHS-2). The enzyme concentrations
were 0.5 µM heme after mixing. B, effects of
EtOOH concentration on the rate of formation of Intermediate II for
PGHS-1 ( ) and PGHS-2 ( ). Conditions were the same as those
described above for A, except that the reactions were
monitored at 424 nm. Details are described under "Materials and
Methods."
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Observations of the reactions with EtOOH at 424 nm, which reflect
formation of Intermediate II (Scheme I), revealed different patterns
for the two isoforms (Fig. 1B). With PGHS-1, the observed rate for Intermediate II formation initially increased with the EtOOH
concentration, indicating that step 1 in the mechanism shown in Scheme
I was rate-limiting. The observed rate leveled off above 100 µM EtOOH, indicating that step 2 in Scheme I became
rate-limiting at higher peroxide levels. The plateau value estimated
from fitting the data to a hyperbolic equation, 80 s
1,
provides an estimate for the first order rate constant
(k2) with PGHS-1. In contrast, with PGHS-2 the
observed rate of Intermediate II formation increased linearly as the
EtOOH level was raised, without any indication of a plateau, even at
observed rates approaching 300 s
1 (Fig. 1B).
Thus, the value of k2 for PGHS-2 in reaction
with EtOOH must be well above 300 s
1, and conversion of
Intermediate I to Intermediate II is clearly much faster for PGHS-2
than for PGHS-1.
Reaction of PGHS-1 and -2 with 15-HPETE--
Reaction of PGHS-1
and -2 with the fatty acid hydroperoxide, 15-HPETE, led to rapid
formation of Intermediate I, as indicated by the decrease in the Soret
absorbance (data not shown). The observed rate for Intermediate I
formation increased linearly with 15-HPETE concentration for both PGHS
isoforms (Fig. 2A). The second
order rate constant (k1) estimated from the data
was 2.3 × 107 M
1
s
1 for PGHS-1 and 2.5 × 107
M
1 s
1 for PGHS-2, indicating
that the two isoforms have very similar reactivity with this lipid
hydroperoxide. On the other hand, observations of the kinetics of
Intermediate II formation revealed divergent behavior for PGHS-1 and
-2. For PGHS-1, the observed rate for Intermediate II formation
increased linearly at lower 15-HPETE concentrations but began to level
off at peroxide concentrations above 100 µM, indicating
that the second step in Scheme I was becoming rate-limiting (Fig.
2B). A plateau was not reached, due to dead time limitations
of the stopped flow instrument, but fitting of the data to a hyperbolic
equation indicated a k2 value of approximately 900 s
1. With PGHS-1, the observed rate of Intermediate II
formation was slower than that for Intermediate I at all 15-HPETE
levels. For PGHS-2, the observed rate of Intermediate II formation was indistinguishable from that for Intermediate I formation throughout the
15-HPETE concentration range tested (Fig. 2B,
inset), indicating that the first step was always
rate-limiting and precluding estimation of a k2
value. However, Intermediate II formation was so much faster for PGHS-2
than for PGHS-1 at all 15-HPETE levels used (Fig. 2B), so
the k2 value must be much greater for PGHS-2
than PGHS-1.

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Fig. 2.
Kinetics of PGHS-1 and -2 peroxidase
reactions with 15-HPETE at 3.9 °C. A, effects of 15-HPETE
concentration on the rate of Intermediate I formation in PGHS-1 ( )
and PGHS-2 ( ). B, effects of 15-HPETE concentration on
the rate of formation of Intermediate II for PGHS-1 ( ) and PGHS-2
( ). Inset, comparison of PGHS-2 data at 408 nm ( ) and
424 nm ( ). Conditions were as described in the legend to Fig.
1.
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Reaction of PGHS-2 with PPHP--
The peroxidase
intermediate kinetics of PGHS-2 were also examined with a second
hydrophobic hydroperoxide, PPHP. The observed rate for formation of
Intermediate I was essentially the same as that for Intermediate II at
each of the PPHP levels tested (Fig. 3).
This is the same result found for 15-HPETE (Fig. 2) and again indicates
that the first step in Scheme I remained rate-limiting and that the
value of k2 for PGHS-2 is quite large with both
PPHP and 15-HPETE. The value of k1 for PGHS-2
with PPHP estimated from the data in Fig. 3 is 1 × 107 M
1 s
1, quite
comparable with the value obtained with 15-HPETE above.

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Fig. 3.
Kinetics of PGHS-2 peroxidase reaction with
PPHP at 4 °C. Stopped flow kinetics were monitored at 406 nm
( ) and at 424 nm ( ). Details are as described under "Materials
and Methods."
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Kinetic scan experiments were carried out for the reaction of PGHS-2
with PPHP to examine the spectral changes in more detail (Fig.
4). The Soret peak was found to
simultaneously decrease in intensity and shift to longer wavelengths as
the reaction proceeded, with one isosbestic point near 414 nm. This is
in marked contrast to the behavior of PGHS-1 where the decrease in
Soret intensity, which reflects conversion of resting enzyme to
Intermediate I, occurred before the red shift, reflecting conversion of
Intermediate I to Intermediate II (7, 19, 23). Further, in PGHS-1 there is an isosbestic point between resting enzyme and Intermediate I near
424 nm in reactions with lipid hydroperoxides (7, 19, 23), quite
distinct from the isosbestic point observed at 414 nm for PGHS-2 (Fig.
4). The coordinated diminution and red shift of the Soret band observed
during reaction of PGHS-2 with PPHP suggests that resting enzyme is
converting to Intermediate II without significant transient
accumulation of Intermediate I. This prominence of resting enzyme and
Intermediate II as the principal species during reaction of PGHS-2 with
PPHP is entirely consistent with the observation that the first step is
rate-limiting for both hydrophobic hydroperoxides in the single
wavelength stopped flow experiments (Figs. 2 and 3).

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Fig. 4.
Spectral changes in the Soret region during
reaction of PGHS-2 (0.6 µM) with
PPHP (50 µM) at 4.2 °C.
Stopped flow observations of the reaction kinetics were performed at 2 nm increments between 390 and 450 nm. The data were collated to obtain
absorption spectra at 2-ms intervals during the reaction. The
arrows indicate the direction of the absorbance changes as
the reaction progressed. Details are as described under "Materials
and Methods."
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Effects of Intermediate II Formation Rate on Cyclooxygenase
Kinetics--
Kinetic simulations were used to predict the effect of
changes in the rate of Intermediate II formation on the overall
cyclooxygenase kinetics, in particular the requirement of the
cyclooxygenase for hydroperoxide activator. Experimentally, the
hydroperoxide activator requirements for PGHS-1 and -2 are estimated
from the sensitivities of the two cyclooxygenase activities to
inhibition by added hydroperoxide scavenger enzyme, GSP (6). In this
process, the cyclooxygenase velocity achieved by a fixed amount of PGHS is measured in the presence of increasing amounts of GSP. The ratio of
added GSP activity to control cyclooxygenase activity (RGC) needed for complete cyclooxygenase
suppression is used as an empirical measure of the efficiency of
cyclooxygenase activation by hydroperoxide. The value of this end point
RGC was found to be about 75 for PGHS-1 and 700 in PGHS-2 (6). Simulations of the cyclooxygenase kinetics were carried
out by numerical integration of rate equations derived from a
mechanistic model (Scheme II) as
described under "Materials and Methods." Experimentally based estimates are available for each of the rate constants in the model
except for k6, the rate of tyrosyl radical
quenching by reducing cosubstrate.

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Scheme II.
Branched chain radical mechanism used for
computer simulation of PGHS reaction kinetics. This mechanism is
adapted from that shown in Scheme I as described under "Materials and
Methods". E(III)PPIX/Tyr is resting enzyme,
E(IV)PPIX*/Tyr is Intermediate I (equivalent to Compound I),
E(IV)/PPIX/Tyr is Compound II, E(IV)/PPIX/Tyr* is
Intermediate II, E(III)/PPIX/Tyr* has a ferric heme and
tyrosyl radical (not included in Scheme I), E(IV)PPIX*/Tyr*
has a ferryl heme and both porphyrin and tyrosyl radicals (not included
in Scheme I), and E(inact) is totally inactivated enzyme.
The three enzyme intermediates with tyrosyl radical (Tyr*)
are assumed to be competent for cyclooxygenase catalysis. AA
is arachidonic acid, ROOH represents hydroperoxide
(PGG2), and AH is reducing cosubstrate.
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The sensitivity of the system to the value of k6
was explored using values of the other parameters appropriate for
PGHS-1, including a value of 350 s
1 for
k2. The simulations predicted that the
cyclooxygenase activity becomes more easily suppressed by GSP as the
k6 value increases, with the end point
RGC value decreasing from about 130 for
k6 = 1000 M
1
s
1 to about 7 for k6 = 2 × 104 M
1 s
1 (Fig.
5). The inverse relationship between the
k6 value and the end point
RGC is readily apparent from the
inset in Fig. 5. A k6 value of 2000 M
1 s
1 predicted an end point
RGC close to the experimentally observed value
of 75 for PGHS-1 (6). The sensitivity of the system to the value of
k2 then was explored with several values of
k6 (Fig. 6). The
end point RGC was predicted to increase as the
k2 value was increased, with most of the change
occurring between k2 values of 350 and 2000 s
1. Regardless of the k6 value
chosen, at saturating k2 values the end point
RGC reached a plateau about 50% over the value
predicted for a k2 of 350 s
1 (Fig.
6).

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Fig. 5.
Influence of k6
value on the sensitivity of cyclooxygenase activity to inhibition by
added GSP. The cyclooxygenase kinetics were predicted by computer
simulation for a fixed amount of PGHS in the presence of the varying
amounts of GSP (indicated by the ratio RGC) with
a k2 value of 350 s 1 and a
k6 value of 1000 M 1
s 1 ( ), 2000 M 1
s 1 ( ), 4000 M 1
s 1 ( ), 8000 M 1
s 1 ( ), or 20000 M 1
s 1 (×). Details of the simulation procedure
are as described under "Materials and Methods." Lines
fitted to the points are extrapolated to the x axis to
obtain the end point RGC values, which are
presented in the inset as a function of the
k6 value used.
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Fig. 6.
Influence of k2
value on the sensitivity of cyclooxygenase activity to inhibition by
added GSP. End point RGC values were
predicted for the indicated k2 values by
computer simulation of cyclooxygenase kinetics for a fixed amount of
PGHS in the presence of the varying amounts of GSP, with
k6 values of 250 M 1
s 1 ( ), 750 M 1
s 1 ( ), 2000 M 1
s 1 ( ), or 4000 M 1
s 1 ( ). Details of the simulation procedure are as
described in the legend to Fig. 5 and under "Materials and
Methods."
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DISCUSSION |
The cyclooxygenase activity of PGHS-1 has long been known to
require hydroperoxides for initiation (24). This requirement for a
hydroperoxide activator leads to a strong positive feedback loop
because the cyclooxygenase product, PGG2, is itself a
hydroperoxide (Scheme I). The feedback loop is thought to be comprised
of the hydroperoxide-dependent generation of a tyrosyl
radical (steps 1 and 2 in Scheme I) and the formation of additional
hydroperoxide in cyclooxygenase catalysis itself (step 5 in Scheme I);
the overall pattern is that of an autocatalytic branched chain reaction
(25). More recently, it has been established that cyclooxygenase
activity of PGHS-2 also requires a hydroperoxide activator (5, 6). The
observation that the PGHS-2 cyclooxygenase is activated at hydroperoxide levels approximately 10-fold lower than those needed for
PGHS-1 (6) indicates that the positive feedback loop is more efficient
in PGHS-2 than in PGHS-1. This difference could conceivably originate
at any of the steps in the feedback loop. Given the similar
cyclooxygenase specific activities observed for the two human isoforms
expressed in the same system (26), however, it seems unlikely that the
more efficient activation in PGHS-2 is due to more efficient
cyclooxygenase propagation (step 5 in Scheme I). Rather, attention
focuses on differences in the formation (steps 1 and 2 in Scheme I) or
dissipation (step 6 in Scheme I) of the catalytically active tyrosyl
radicals on Tyr385 in PGHS-1 and on Tyr371 in
PGHS-2.
Formation of the key tyrosyl radical is proposed to involve oxidized
enzyme intermediates in the peroxidase cycle (7). The initial reaction
with hydroperoxide at the heme site leads to a two-electron oxidation
of resting enzyme to form Intermediate I (step 1 in Scheme I). This
species is analogous to Compound I in horseradish peroxidase and
carries one oxidizing equivalent on the ferryl iron and the other as a
porphyrin free radical (23). The next step in the postulated activation
process (step 2 in Scheme I) is an intramolecular electron transfer
from a tyrosine residue in the cyclooxygenase site (Tyr385
of PGHS-1 and Tyr371 of PGHS-2) to the porphyrin, forming a
tyrosyl radical and bringing the heme to one oxidizing equivalent above
the resting state (7). This intramolecular electron transfer
mechanistically links the cyclooxygenase and peroxidase catalytic
cycles and distinguishes the PGHS isoforms from other
heme-dependent peroxidases that do not have an oxygenase
activity (27, 28).
The results presented here show that the rates of formation of
Intermediate I during reaction of the two PGHS isoforms with the fatty
acid hydroperoxide, 15-HPETE, are essentially the same (Fig. 2).
Therefore, it is reasonable to expect similar behavior for
PGG2, the relevant fatty acid hydroperoxide formed during cyclooxygenase catalysis with arachidonate. PGHS-1 does have a higher
Intermediate I formation rate than PGHS-2 for reaction with EtOOH (Fig.
1), presumably reflecting structural differences between the isoforms
at the peroxidase active site that favor interactions of PGHS-1 with
small hydrophilic peroxides. The major kinetic difference between the
two isoforms found in the present study was in the rate of formation of
Intermediate II. The rate was much faster for PGHS-2 than for PGHS-1
with both hydrophilic and hydrophobic hydroperoxides (Figs. 1 and 2).
For PGHS-1, formation of Intermediate I was rate-limiting at low
hydroperoxide levels, whereas conversion of Intermediate I to
Intermediate II was rate-limiting at higher peroxide levels. For
PGHS-2, interconversion of Intermediate I to II was so fast that
formation of Intermediate I was rate-limiting at all hydroperoxide
levels tested, and there was no appreciable accumulation of
Intermediate I (Fig. 4). Thus, it is clear that the final step in the
process of cyclooxygenase activation by lipid hydroperoxide is
distinctly faster in PGHS-2 than in PGHS-1.
Although PGHS-1 and -2 share 60% overall amino acid identity, the
conservation is much higher in the regions around the peroxidase and
cyclooxygenase active sites (2). The three-dimensional structures are
also well conserved in the active sites, with differences in backbone
positions of the two isoforms averaging less than 0.4 angstrom (8-10).
As a result, there are no readily apparent structural differences in
the vicinity of the heme and Tyr385 (Tyr371) in
the available crystallographic data (8, 10) that explain the observed
differences in the value of k2 in PGHS-1 and -2. It remains possible that the active site structures in the crystals differ from those of the active enzymes in solution or that differences in structural dynamics lead to the observed differences in electron transfer rate.
The potential effects of the faster rate of Intermediate II formation
in PGHS-2 on overall cyclooxygenase kinetics need to be considered in
the context of the cellular environment in which the PGHS isoforms
operate. Most cells have a large excess of peroxide scavenging enzymes,
such as GSP, over peroxide generating enzymes, such as the
cyclooxygenases (21). This preponderance of peroxide scavenging
capacity tends to keep the cellular hydroperoxide level well below
those encountered in vitro and may thereby accentuate the
impact of differences in activation efficiency. Indeed, analyzing the
effects of added peroxide scavengers has revealed features of feedback
activation by hydroperoxide that are not apparent in routine
cyclooxygenase assays (22, 29, 30), and titration with GSP has been
used to quantify the strength of the feedback loops in PGHS-1 and -2 (6). Kinetic modelling of the complex combination of PGHS and GSP is
thus very useful in predicting how differences in individual rate
constants might influence cyclooxygenase catalysis in
vivo.
The kinetic behavior of systems containing both PGHS and GSP can
readily be predicted using numerical integration of equations based on
mechanistic models (6, 22), and so we used this approach to predict the
effect of changes in the k2 value on the sensitivity of the cyclooxygenase to inhibition by hydroperoxide scavenger. The mechanistic model chosen for kinetic simulations (Scheme
II and "Materials and Methods") is based on the branched chain
tyrosyl radical mechanism proposed by Ruf and colleagues (Ref. 7; see
also Scheme I). The mechanism was modified to include two additional
intermediates (E(III)/PPIX/Tyr* and E(IV)/PPIX*/Tyr* in Scheme II) to
permit redox cycle events at the peroxidase site to continue after
generation of the tyrosyl radical in the cyclooxygenase site. A simple
route to self-inactivation from intermediates containing a tyrosyl
radical (E(III)/PPIX/Tyr*, E(IV)/PPIX*/Tyr*, and E(IV)/PPIX/Tyr*in Scheme II) was added. The values of all rate constants in the model, except k6, were based on experimental
observations, and the value of k6 for PGHS-1 was
constrained by measurements from reactions with GSP present (Fig. 5).
Thus, the general features of the kinetic behavior predicted by the
model should be reliable. Earlier simulations of the GSP/PGHS system
(6, 22) were based on a simpler paradigm that did not specify the
individual steps in peroxidase catalysis and thus was not useful in
evaluating the effects of the k2 value on the
kinetic behavior.
The simulation results predict that increases in
k2 above the value of about 350 s
1
observed for PGHS-1 increase the resistance to inhibition by the
peroxide scavenger by up to 50% (Fig. 6). This is less than the 8-fold
difference in resistance to inhibition by GSP actually observed for the
two cyclooxygenase activities (6). It thus appears that the increased
rate of Intermediate II formation (k2) observed
here for PGHS-2 compared with PGHS-1 can account for only part of the
difference in hydroperoxide activation efficiency between the two
isoforms. The simulation results also indicate that the resistance of
the cyclooxygenase activity to inhibition by GSP is quite sensitive to
the stability of the tyrosyl radical in Intermediate II, with the
resistance increasing as the value of the k6
rate constant was decreased (Fig. 5). With a k6
value of 250 M
1 s
1 and a
k2 value of about 2000 s
1, the
predicted end point RGC value was above 600 (Fig. 6), close to the end point RGC value of
650 actually observed for human PGHS-2 (6). The ability of the
mechanistic model to simulate the GSP sensitivity of PGHS-2 once the
k6 value is decreased suggests that the active
site tyrosyl radical in PGHS-2 is less readily quenched by reducing
cosubstrates than the corresponding tyrosyl radical in PGHS-1. Electron
paramagnetic resonance kinetic measurements will be needed to test this
intriguing possibility that differing tyrosyl radical stabilities in
the two PGHS isoforms also contribute to the difference in
hydroperoxide activator efficiency.