From the Department of Chemistry, University of Alberta, Edmonton,
Alberta T6G 2G2, Canada
The pre-steady-state phase of the oxygenase
reaction of prostaglandin endoperoxide synthase with
cis,cis-eicosa-11,14-dienoic acid has been studied using
stopped flow techniques. Because some intermediate forms of
prostaglandin endoperoxide synthase are spectrally
indistinguishable, the enzyme and substrate transformations were
monitored in parallel to simplify the interpretation of the kinetics.
Over a wide range of conditions, the formation of the enzyme
intermediate II, the form of compound I containing the tyrosyl radical,
precedes substrate oxidation. This result supports the occurrence of a
unimolecular conversion of compound I into intermediate II.
Furthermore, the rate of intermediate II formation was stimulated by
increased concentration of dienoic acid, perhaps because of increased
occupation of the fatty acid binding site. The importance of the
unimolecular formation of intermediate II was confirmed by simulated
kinetics of the oxygenase reaction. These results provide evidence that
intermediate II is the primary oxidant in the reaction of prostaglandin
synthase with the dienoic acid, as it is with arachidonic acid.
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INTRODUCTION |
Prostaglandin H endoperoxide synthase (PGH
synthase)1 catalyzes the
first step in the biosynthesis of prostaglandins. This step consists of
two reactions: the conversion of arachidonic acid into prostaglandin
G2 (cyclooxygenase reaction) and the reduction of
prostaglandin G2 to prostaglandin H2
(peroxidase reaction) (1-3). Extensive investigation of these
reactions has revealed enzymatic intermediates analogous to the classic
peroxidase compounds I and II as well as free radical forms of the
enzyme (4-6). Two reaction mechanisms have been proposed for this
system, differing first of all in relation to the initial events of the
cyclooxygenase reaction. The branched chain mechanism considers the
tyrosyl radical of PGH synthase, formed from compound I by the
intramolecular electron transfer process
(Porph·+ FeIV
O
TyrOH
Porph FeIV
O TyrO· + H+), as the primary oxidant that abstracts a hydrogen atom
from the fatty acid substrate (6, 7). The species Porph
FeIV
O TyrO·is referred to as intermediate
II (6). An alternative tightly coupled mechanism has, in the past,
assigned this role to a conventional compound I (8, 9). The choice
between these possibilities is complicated by the fact that
intermediate II and compound II have the same oxidation state of the
heme and, consequently, the same optical spectra. EPR spectroscopy,
which in principle can detect the free radical intermediate, is unable
to follow the reaction on a millisecond time scale, typical for enzyme
conversion. In such a situation, parallel kinetic monitoring of both
enzyme and substrate behavior could be useful.
cis,cis-Eicosa-11,14-dienoic acid (20:2) has been used in
previous studies because it is the only PGH synthase substrate
producing easily measurable conjugated diene products that are readily
detected spectrophotometrically (10-15). Another advantage of this
substrate is that the reaction is relatively slow and can be measured
using micromolar concentrations of enzyme. This provides a rare
opportunity to observe the enzyme and substrate transformations in
parallel, which could give additional information about the PGH
synthase reaction mechanism, compared with results obtained with its
natural substrate arachidonic acid.
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EXPERIMENTAL PROCEDURES |
Materials were purchased from the following sources.
cis,cis-Eicosa-11,14-dienoic acid, hematin,
diethyldithiocarbamate were from Sigma; phenol was from British Drug
House; DE-53 ion exchange chromatography gel was from Whatman; and
Tween 20 was from J. T. Baker Chemical Co.
PGH synthase was isolated from ram seminal vesicles microsomes using
the method of Marnett et al. (16), modified by MacDonald and
Dunford (17). Cyclooxygenase activity was measured with a Clark-type
polarographic electrode by monitoring oxygen consumption in the
reaction of arachidonic acid with PGH synthase in 0.1 M phosphate buffer, pH 8.0, containing 200 µM arachidonic
acid, 1 µM hematin, and 1 mM phenol. Oxygen
calibration was performed according to Robinson and Cooper (18), and
protein content was measured by the Bio-Rad protein assay using bovine
serum albumin as a standard. Preparations of PGH synthase contained
30-50% holoenzyme and consumed 20-40 µmol of oxygen/min/mg of
protein; for kinetic measurements, they were reconstituted with the
appropriate amount of hematin (19). The kinetic experiments were
conducted on an Applied Photophysics SX.17MV (Micro-Volume) stopped
flow reaction analyzer. Parallel kinetic runs at 234 nm (absorption
peak of conjugated diene products), 414 nm (isosbestic point between
compound I and intermediate II), and 426 nm (isosbestic point between
compound I and resting enzyme) were performed at 25 °C in 0.1 M phosphate buffer, pH 8.0, to monitor oxidation of 20:2
and formation of compound I and compound I-tyrosyl radical
plus compound II. The amount of 20:2 hydroperoxide that
always contaminates 20:2 preparations and initiates the oxygenase
reaction was regulated either by reduction with immobilized borohydride
(20) or by oxidation of 20:2 with oxygen (12) and measured by
absorption at 234 nm (21). Molar absorptivities of conjugated diene
products, PGH synthase, and its intermediates were taken from earlier
studies (21-23). Removal of diethyldithiocarbamate (DDC), used in the
enzyme isolation as a stabilizer, was performed, when necessary, by
passing the enzyme solution through Sephadex G-25 (medium)
preequilibrated with 0.1 M phosphate buffer, pH 8.0, containing 30% glycerol (24). Data were analyzed using a stopped flow
reaction analyser kinetic software and a pro Fit data analysis program
(Cherwell Scientific, UK).
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RESULTS |
The spontaneous oxygenase reaction was studied using 0.75 µM PGH synthase and 10-170 µM 20:2.
Typical kinetic traces of the initial phase at different concentrations
of 20:2 and 20:2 hydroperoxide are shown in Fig.
1. The common feature of all these
experiments is the almost simultaneous formation of compound I and
intermediate II and considerable delay in appearance of conjugated
diene products. Because addition of oxygen to the fatty acid radical is
very fast, formation of the conjugated diene products reflects the rate
of hydrogen atom abstraction from 20:2 by PGH synthase.

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Fig. 1.
Kinetics of interaction of PGH synthase (0.75 µM) and 20:2 (44 µM, panel A
and 172 µM, panel B). Formation of
reaction products was monitored at 234 nm (absorption peak of
conjugated dienes), formation of compound I at 414 nm (isosbestic point
between compound I and intermediate II), and formation of intermediate II at 426 nm (isosbestic point between resting enzyme and compound I).
Dotted lines represent original kinetic traces, and
solid lines represent traces averaged over five successive
points (total number of recorded points = 400).
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The formation of intermediate II and oxidation of 20:2 at different
concentrations of 20:2 and constant concentration of 20:2 hydroperoxide
is shown in Fig. 2. An increase in 20:2
concentration stimulates both formation of intermediate II and
oxidation of 20:2, but again, transformation of substrate noticeably
lags the enzyme conversion.

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Fig. 2.
Influence of [20:2] on the formation of
intermediate II (solid lines) and oxidation of 20:2
(dotted lines). The reaction mixture contained 0.75 µM PGH synthase, 1.9 µM 20:2 hydroperoxide, and the following concentrations of 20:2: 44.7 µM
(traces marked 1), 88.4 µM (traces
marked 2), and 131.5 µM (traces marked
3). Kinetic traces consist of averages of each successive five
points (total number of recorded points = 400).
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The reaction of PGH synthase with 20:2 was performed under single
turnover conditions using an approximately equimolar amount of 20:2
hydroperoxide to prevent the reaction of excess hydroperoxide with the
compound I to produce compound II (19, 25). Kinetic traces for the
system containing 0.77 µM PGH synthase, 0.83 µM 20:2 hydroperoxide, and 9 µM 20:2 are
shown in Fig. 3. The results demonstrate
that reaction of equimolar amounts of PGH synthase and hydroperoxide
leads to formation of intermediate II before the appearance of newly
formed hydroperoxide. The molar absorptivity of conjugated dienes at
234 nm is 24 mM
1 cm
1 (21), and
the difference in molar absorptivity between intermediate II and
resting enzyme at 426 nm is about 17 mM
1
cm
1 (23). Therefore, the absorbance traces can be
compared on an approximately equimolar basis.

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Fig. 3.
Kinetics of interaction of PGH synthase and
20:2 at stoichiometric level of 20:2 hydroperoxide. PGH synthase,
0.77 µM; hydroperoxide of 20:2, 0.83 µM;
20:2, 9 µM. Formation of reaction products was monitored
at 234 nm (absorption peak of conjugated dienes), formation of compound
I at 414 nm (isosbestic point between compound I and intermediate II),
and formation of intermediate II at 426 nm (isosbestic point between
resting enzyme and compound I). Dotted lines represent
original kinetic traces, and solid lines represent traces
averaged over five successive points.
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DDC is commonly used as a stabilizer during purification and storage of
PGH synthase. Under our conditions, it was present in the final mixture
in micromolar concentrations, and its reducing action could result in
formation of compound II (24). This possibility was tested by
performing the reaction at different levels of DDC. The kinetics of
appearance of compoundII/intermediate II were fitted to a
single exponential function, and kobs values
obtained in this way were plotted against [DDC] (Fig.
4). This plot indicates that both
DDC-dependent and -independent reactions contribute to the
observed reactions. The DDC-dependent part corresponds to
reduction of compound I to compound II, whereas the DDC-independent part corresponds to the spontaneous conversion of compound I to intermediate II.

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Fig. 4.
Effect of DDC on intermediate II formation.
kobs values were found from exponential fits of
kinetic traces of intermediate II formation at 426 nm. Reaction
conditions: PGH synthase, 0.73 µM; 20:2 hydroperoxide,
0.86 µM; 20:2, 4.2 µM; DDC 3.1, 6.8, and 11.0 µM.
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To achieve greater clarity on the role of DDC, it was replaced by
glycerol as stabilizing agent. This resulted in a considerable decrease
of the rate of change in absorbance. However, the lag between the
kinetics of appearance of intermediate II and oxidation of 20:2
remained. As a typical example Fig. 5
shows kinetic traces for the reaction of PGH synthase with 1.3 equivalents of 20:2 hydroperoxide and 5 equivalents of 20:2 in a
DDC-free medium.

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Fig. 5.
Kinetics of interaction of PGH synthase and
20:2 in DDC-free medium at stoichiometric level of 20:2
hydroperoxide. PGH synthase, 0.76 µM; hydroperoxide
of 20:2, 0.97 µM; 20:2, 4 µM. Formation of
reaction products was monitored at 234 nm (absorption peak of
conjugated dienes), formation of compound I at 414 nm (isosbestic point
between compound I and intermediate II), and formation of intermediate
II at 426 nm (isosbestic point between resting enzyme and compound I).
Dotted lines represent original kinetic traces, and
solid lines represent traces averaged over five successive
points.
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DISCUSSION |
A published mechanism of the oxygenase reaction of PGH synthase
(E) with 20:2 (AH) includes the following initial steps up to the
rate-limiting formation of eicosadienoyl radical (12-14),
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(Reaction 1)
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(Reaction 2)
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where E-I is compound I and E-II is compound II. The initiation
role of compound I in fatty acid oxidation, proposed by this scheme,
disagrees with the observed asynchronous oxidation AH and formation of
E-II. Possible alternatives, based on the unimolecular transformation
of E-I into intermediate II, E-I-Tyr·, which contains the same
number of oxidizing equivalents as compound I (6), or reaction of E-I
with a second molecule of hydroperoxide (19), are shown below.
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(Reaction 1)
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(Reaction 3)
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(Reaction 4)
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(Reaction 1)
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(Reaction 5)
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(Reaction 4)
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Reaction 5 in Scheme 3 is the sum of the following two consecutive
reactions.
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(Reaction 6)
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(Reaction 7)
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To determine the rate constant for compound I formation (Reaction
1) kinetic traces at 414 nm were obtained over a range of
concentrations of 20:2 hydroperoxide. Initial parts of these curves
were fitted to a single exponential function, and the resultant kobs values were plotted against [20:2
hydroperoxide]. The plot (Fig. 6) gives
kapp = 2.9 × 107
M
1 s
1, which
is close to the rate constants for other
similar hydroperoxides. The rate constants for reactions (Reactions
3) were estimated from the literature (6, 12, 19, 26), and the
following values were used in the simulations: for Scheme 2, k3 = 65 s
1,
k4 = 3 × 105
M
1 s
1; for Scheme 3, k5 = 3 × 106
M
1 s
1,
k4 = 3 × 105
M
1 s
1.

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Fig. 6.
Plot of kobs versus
[20:2-OOH] for compound I formation in DDC (panel A) and
glycerol (panel B) containing medium.
kapp = 3 × 107
M 1 s 1 (panel A) and
2.5 × 107 M 1
s 1 (panel B).
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Fig. 7.
Experimental and simulated (according to
Schemes 2 and 3) kinetics of initial phase of the reaction PGH synthase
and 20:2. The solid line represents formation of
(E-I-Tyr·+ E-II), and the dotted line represents
consumption of 20:2. Initial conditions were as follows: PGH synthase,
0.75 µM; 20:2 and 20:2 hydroperoxide, 19 and 5 µM, respectively (panels A), or 21 and 0.9 µM (panels B).
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The behavior of these reaction schemes in relation to 20:2
consumption and production of intermediate II and compound II was tested with a Monte-Carlo method using a CKS simulation program (IBM).
Fig. 7 demonstrates experimental and simulated kinetics, using the
above rate constants for initiation of the oxygenase reaction with 1.2 or 6.7 eq of 20: 2 hydroperoxide. It shows that the asynchronous
oxidation of substrate and appearance of intermediate II at excess of
hydroperoxide can be qualitatively predicted by both Schemes 2 and 3, but for a stoichiometric level of hydroperoxide, the rate of oxidation
of 20:2 is far too small according to Scheme 3.
Reactions of compound I of PGH synthase are of crucial importance for
understanding the catalytic mechanism of this enzyme. Since compound I
can convert into the spectrally indistinguishable compound I-tyrosyl
radical and compound II, the interpretation of the kinetics of compound
I decay becomes difficult. Originally, the enzyme product of compound I
decay in the oxygenase reaction of PGH synthase with 20:2 was
interpreted as production of compound II, based on the fact that
conversion of compound I was a function of fatty acid concentration,
indicating a bimolecular reaction. This led to the proposal of Reaction
2 (12) with a delayed formation of the tyrosyl radical in Reactions 6 and 7 (19). However, when both participants of the reaction are
monitored, it is apparent that appearance of intermediate II precedes
oxidation of fatty acid, though both reactions are functions of the
concentration of 20:2. This suggests that dependence of intermediate II
production on the concentration of 20:2 is an indirect effect. The
stimulation of intermediate II formation by 20:2 could be the result of
increased occupation of the fatty acid binding site. This could be the
basis of a switching mechanism which would favor initiation of
cyclooxygenase cycle and prevent conversion of compound I to compound
II when peroxidase reducing substrates are present.
In this light, conversion of compound I into compound II/intermediate
II in the reaction of PGH synthase with 20:2 could be caused by one the
following: (a) reduction of compound I by DDC used as stabilizing agent
(24); (b) reaction of compound I with a second molecule of
hydroperoxide (19); or (c) intramolecular electron transfer from
Tyr-385 to the porphyrin
-cation radical (6).
Participation of DDC in intermediate II formation was excluded by the
results obtained in a DDC-free medium. The relative significance of
bimolecular and unimolecular paths for formation of intermediate II
(reasons b and c) apparently depends on the level
of hydroperoxide. However, the presence of a large excess of
hydroperoxide in vivo appears unlikely, which leaves the
intramolecular electron transfer as the most likely route to generate
the tyrosyl radical.
From the crystal structure, the arachidonic acid binding site was shown
to be located on the proximal side of the heme (27). The role of the
Tyr-385 radical in the oxidation of arachidonic acid is now firmly
established (28). From the present results, it would appear that the
initiation of the oxidation of the eicosadienoic acid proceeds by a
similar mechanism.