(Received for publication, September 7, 1995; and in revised form, November 17, 1995)
From the
The reactions of native prostaglandin endoperoxide synthase with
structurally different hydroperoxides have been investigated by using
kinetic spectrophotometric scan and conventional and sequential mixing
stopped-flow experiments. The second order rate constants for compound
I formation are (5.9 ± 0.1) 10
M
s
using t-butyl
hydroperoxide as the oxidant, (2.5 ± 0.1)
10
M
s
for ethyl hydroperoxide
and (5.1 ± 0.6)
10
M
s
for m-chloroperoxybenzoic acid at pH 7.0,
6.7 ± 0.2 °C, and ionic strength 0.1 M. Sequential
mixing, transient state experiments show for the first time that all
hydroperoxides reduce compound I in a bimolecular reaction. Ethyl
hydroperoxide, t-butyl hydroperoxide, and m-chloroperoxybenzoic acid react directly with compound I. The
natural substrate prostaglandin G
forms a transient complex
with compound I before the reduction step occurs. Therefore, compound I
initially transforms to compound II, not to the compound I-tyrosyl
radical. Second order rate constants for the reactions of compound I
are (2.9 ± 0.2)
10
for t-butyl
hydroperoxide, (3.5 ± 0.5)
10
for hydrogen
peroxide, (4.2 ± 0.2)
10
for ethyl
hydroperoxide, and (4.2 ± 0.3)
10
for m-chloroperoxybenzoic acid, all in units of M
s
and same conditions as for compound I
formation. The rate of reaction of prostaglandin G
with
compound I, calculated from the ratio of k
to K
obtained from the saturation curve, is
(1.0 ± 0.2)
10
M
s
at 3.0 ± 0.2 °C. Results are
discussed in the context of the current state of knowledge of the
mechanisms of the cyclooxygenase and peroxidase reactions of
prostaglandin endoperoxide synthase.
Prostaglandin endoperoxide synthase (PGH synthase) ()catalyzes the first committed step of the biosynthesis of
prostaglandins and thromboxanes from arachidonic acid (for recent
reviews, see (1, 2, 3) ). Two distinct
isoforms of PGH synthase are found that are differently expressed in
specific
tissues(4, 5, 6, 7, 8) .
The cDNA isolation and the elucidation of mechanisms by which the PGH
synthase gene transcription is regulated are the basic objectives of
the most recent molecular biological
studies(9, 10, 11, 12, 13, 14) .
Substantial information has become available from recent x-ray
crystallographic(15) , site-directed
mutagenesis-EPR(16, 17) , and kinetic (18, 19) studies. Despite these efforts, progress has
been limited in clarifying the role of the higher oxidation states of
the enzyme and the role and the number of tyrosyl radicals implicated
in the catalytic mechanism.
Upon reaction with organic
hydroperoxides, two distinct spectral intermediates of PGH synthase are
formed resembling compound I and compound II peroxidase
intermediates(20) . The heme Fe(III) of the native enzyme is
oxidized by the peroxide, and an Fe(IV)=O porphyrin cation
radical (compound I) is formed. Compound I is readily converted to a
second intermediate, which we shall call intermediate II. The oxidation
state of intermediate II could be assigned either to an Fe(IV)=O
species with no enzymatic free radical (compound II) (21) or to
a Fe(IV)=O-tyrosyl radical species (compound I-tyrosyl radical)
as in compound I of cytochrome c peroxidase(22) . Both
Fe(IV)=O species could be formed under certain conditions, but
how they are related to each other is a crucial and as yet unresolved
problem in the PGH synthase catalytic mechanism.
The detection of
tyrosyl radicals has been considered to be key evidence for a
postulated reaction mechanism in which the compound I-tyrosyl radical
species abstracts a hydrogen atom from arachidonic acid, which
initiates the oxygenation reaction and regenerates the tyrosyl
residue(23) . If the oxygenated arachidonic acid radical, the
PGG radical, abstracts a hydrogen atom from the tyrosyl
residue to become PGG
, then the tyrosyl radical is
regenerated and a chain reaction could occur. For every compound I
converted to the Fe(IV)=O-tyrosyl radical another chain could be
started, so this mechanism is properly called a branched-chain
mechanism. The branched-chain mechanism could lead to the accumulation
of large amounts of PGG
, not PGH
, which is the
precursor of all other prostaglandins.
It has been shown that the branched-chain mechanism cannot explain coupled cyclooxygenase and peroxidase activities when an excess of a substrate electron donor is present; yet, the chain reaction cannot be excluded under conditions when the peroxidase reaction is rate-limiting, i.e. at low concentration or in the absence of a peroxidase electron donor(18) . Under such conditions, however, the inactivation of the enzyme is severe, and the tyrosyl radical(s) could be participating in these processes.
An alternative non-chain mechanism involves tyrosyl radical formation at a later stage in the reaction(18, 19) . The present study was undertaken to test the occurrence of the critical elementary reaction in the tyrosyl radical chain reaction model(23) , the spontaneous formation of the compound I-tyrosyl radical from compound I. Evidence will be shown that when a hydroperoxide is present, the compound I-tyrosyl radical is not formed from compound I. Compound I rather abstracts a hydrogen atom from the hydroperoxide to produce compound II and hydroperoxyl radical, as can occur with myeloperoxidase(24) . The compound I-tyrosyl radical could then be formed from compound II and hydroperoxyl radical as previously suggested (18, 19).
The
concentration of horseradish peroxidase was determined
spectrophotometrically at 403 nm using a molar absorptivity of 1.02
10
M
cm
(25) . The concentration of
hydroperoxide stock solutions was determined by the horseradish
peroxidase assay(26) .
PGH synthase, isolated from sheep
seminal vesicles(27, 28) , consisted of approximately
a 50/50 mixture of apo- and holoenzyme and had a specific activity of
40 µmol of arachidonic acid (mg of protein/min) in the presence of 1 µM hematin and 1 mM phenol. Protein content was determined by the Bio-Rad protein
assay. The concentration of the enzyme heme was determined at 410 nm
using an
of 123 mM
cm
(29) . For spectral and kinetic
measurements, a 1-ml aliquot of the 50/50 apo-/holoenzyme stock
solution was reconstituted by adding an amount of hematin equal to the
amount of apoenzyme. The final heme concentration of the enzyme,
determined after keeping the enzyme sample on ice for 10-15 min,
was usually 23-25 µM.
The kinetic traces at 414 nm were single-exponential, and the
pseudo-first order rate constants k were
linear functions of [ROOH] (the plot and a kinetic trace for
EtOOH are shown in Fig. 1). The bimolecular rate constants k
were calculated from the slopes of the
straight-line plots of k
versus [ROOH]. Resulting values for different hydroperoxides
are presented in Table 1, together with a published value for
PGG
(23) . The results indicate that the aliphatic
hydroperoxides H
O
, EtOOH, and t-BuOOH
are significantly poorer substrates for native enzyme than the aromatic
hydroperoxide m-Cl PBA. The rate constants for compound I
formation decrease in the order m-Cl PBA
EtOOH >
H
O
> t-BuOOH. For m-Cl
PBA, k
was two to three orders of magnitude higher
(5
10
M
s
) than for other hydroperoxides and similar
to the value obtained with the natural substrate PGG
(Table 1).
Figure 1:
Plot of
observed pseudo first order rate constant for the formation of PGH
synthase compound I (k) versus the
concentration of ethyl hydroperoxide. 0.5 µM solution of
PGH synthase in 0.1 M phosphate buffer, pH 7.03, was mixed
with different [EtOOH] at 6.7 ± 0.2 °C. Inset, typical time course trace and exponential curve fit
obtained at 414 nm. Mixture contained 0.5 µM enzyme and
55.4 µM EtOOH; k
= 177
± 3 s
, and total
A =
0.011.
Figure 2:
Plot
of kversus [EtOOH] and
[m-Cl PBA] for PGH synthase intermediate II
formation under pre-steady state conditions. The slopes of the linear
plots are the second order rate constant in units of
mM
s
.
, purified m-Cl PBA;
, unpurified m-Cl PBA;
,
EtOOH. Inset, the relative increase in absorbance at 426 nm
and the curve fit for the reaction of 0.5 µM enzyme and 60
µMm-Cl PBA; k
=
36.2 ± 0.4 s
, and total
A = 0.009.
Figure 3:
Plot of kversus [H
O
] and
[t-BuOOH] for the formation of PGH intermediate II
under the same conditions as in Fig. 2. The saturation kinetic
parameters are as follows: k
= 45
± 5 s
and K
= 1.3 ± 0.2 mM for
H
O
(
); k
=
62 ± 6 s
and K
= 1.7 ± 0.2 mM for t-BuOOH
(
). Inset, double-exponential kinetic trace obtained at
426 nm after mixing native enzyme (0.5 µM) and
H
O
(150 µM) at 4.2 ± 0.2
°C. k
= 4.75 ± 0.35 (fast
phase); rate constant for the slower phase, 0.19 ± 0.05
s
; and total
A = 0.010. It is k
for the fast phase plotted versus [H
O
] in the main part of the
figure. For most other experiments, the slower phase was not
reproducible.
Figure 4:
The pH dependence for the faster phase in
the formation of PGH synthase intermediate II using
HO
as substrate. The experiments were performed
under pre-steady state conditions. The faster rates, k
, measured at several pH, were plotted as a
function of [H
O
] as shown in Fig. 3; the values of k
and K
were obtained from the curve fits to
the rectangular hyperbolae; A = k
or K
.
Superoxide dismutase slightly increased the
first, fast phase in intermediate II formation but did not eliminate
the second, slow phase even at 10 µM concentration. Bovine
serum albumin showed a similar effect to that of superoxide dismutase
(data not shown). Because of poor reproducibility, the functional
relationship between the rate of the slower phase and
[HO
] could not be ascertained. At
some pH values, this relationship was linear, but at many other pH
values, the relationship could not be established.
Figure 5: Spectral changes observed during the reaction of stoichiometric amounts of PGH synthase and m-Cl PBA. Kinetic scans were recorded in the 350-450-nm region (380-450-nm region is shown) at 6.7 ± 0.2 °C. The final concentrations were 0.50 µM enzyme and 0.65 µMm-Cl PBA. Broad arrows indicate the direction of the absorbance change with increasing time. A, spectra 0-80 ms after mixing, indicating that only compound I was formed; B, spectra 0-80 and 80-200 ms after mixing showing formation of the second intermediate. Isosbestic points between native enzyme and compound I (422 nm) and between compound I and the second intermediate (413 nm) are indicated.
The kinetic trace at 414 nm obtained with m-Cl PBA for the above conditions had two distinct transient phases separated by a steady state phase (Fig. 6). The fast decrease in absorbance is the formation of compound I, and slow increase in absorbance at longer times indicates the conversion of compound I to intermediate II. There was a short steady state period of 30 ms when compound I was stable, and the time within that range, designated by an arrow in Fig. 6(60 ms after the start of the reaction), was selected as an optimal time for studying the reactions of compound I by using the sequential mixing technique.
Figure 6: Kinetic trace at 414 nm showing the time delay between the formation and spontaneous decay of PGH synthase compound I. PGH synthase compound I was formed from the stoichiometric amounts of enzyme and m-Cl PBA as described in Fig. 5. The arrow indicates the time (60 ms) selected as a pre-set delay time in the sequential mixing experiments.
Fig. 7shows the linear plots of the observed pseudo-first
order rate constant kversus [ROOH] for the reactions of compound I with EtOOH, t-BuOOH, and m-Cl PBA under transient state
conditions. A noticeable difference between the transient state and
pre-steady state results was obtained only with t-BuOOH. The
plot of k
versus [t-BuOOH] from the transient state results is
linear (Fig. 7), not curved as was observed in the pre-steady
state (Fig. 3).
Figure 7:
Transient state kinetics for the PGH
synthase compound I reduction to compound II obtained in the sequential
mixing experiments. Compound I was formed from the stoichiometric
amounts of enzyme and m-Cl PBA, and a hydroperoxide was
introduced after 55-60 ms. The reaction rates were measured at
426 nm as described in Fig. 2. Second order rate constants
determined from the slopes are collected in Table 2. The curves
have a common intercept at 15 s;
, EtOOH;
, m-Cl PBA; and
, t-BuOOH. Inset, curve a, change in absorbance at 426 nm
obtained after mixing 0.5 µM enzyme with 0.65 µMm-Cl PBA. Curve b, same conditions as for curve a but with 1.3 mM EtOOH also present. For a, k
= 14.1 ± 0.2
s
and
A = 0.007; for b, k
= 68 ± 1
s
and
A =
0.011.
The linear plots had a common intercept at 15
s (Fig. 7). This value represents the rate
for the spontaneous decay of compound I. The spontaneous rate was also
determined in separate experiments in a multi-mixing assay in which a
second peroxide was not added (curve a, inset to Fig. 7). Thus, only the 1:1.3 ratio of enzyme and m-Cl
PBA were reacted. The results were in good agreement with the intercept
value obtained in Fig. 7. The term ``spontaneous'' can
be explained by the reduction of PGH synthase compound I by the
stabilizing agent DDC to compound II (31, 32, 33) . Small amounts of DDC are
required for enzyme stability.
The linear kinetics and finite intercept for the reaction of PGH synthase compound I with EtOOH, t-BuOOH, and m-Cl PBA in the presence of DDC can be explained by a single mechanism in which both the hydroperoxide and DDC serve as hydrogen atom donors and reduce PGH synthase compound I to compound II (E-II) as in and and :
where k is the second-order spontaneous
rate constant for the reduction of compound I by DDC and k
is the corresponding first order rate constant
(the intercept of the plots in Fig. 7. is in full
agreement with the experimental data depicted in Fig. 7.
The
bimolecular rate constants for compound II formation (k) obtained from the transient state
experiments were calculated from the slopes of the straight-line plots
of k
versus [ROOH], and the
results are shown in Table 2. For comparison, the values obtained
form the pre-steady state method are also shown in Table 2. Thus,
for two different kinetic methods, the rate constants for compound II
formation with EtOOH and m-Cl PBA are remarkably similar. For t-BuOOH, however, the rate constant k
was slightly higher from the classical stopped-flow experiments
than from the sequential mixing experiments (Table 2).
Figure 8:
Saturation dependence of the observed
rates k for the PGH synthase compound I
reaction with PGG
. In the transient state experiments,
compound I was formed from the stoichiometric amounts of native enzyme
and m-Cl PBA. K
= 67
± 11 µM, and k
= 69
± 6 s
. Inset, single exponential
trace at 426 nm showing that compound I reacts with PGG
.
Compound I prepared in 57 ms was then reacted with 35 µM PGG
. The pre-mixing time does not appear on the
kinetic trace scale; the time scale shown (0-500 ms) is for the
events after mixing compound I and PGG
. k
= 19.4 ± 0.2
s
, and total
A =
0.012.
The parameters k (69 ± 6
s
) and K
(70 ± 10
µM) were obtained from the plot of k
versus [PGG
] in Fig. 8. No
corrections for the DDC spontaneous reaction (k
)
were made. At 3 °C, k
was 4
s
. Since an excellent fit of the theoretical curve
to the experimental data was obtained, it appears that the formation of
the E-I-PGG
complex () prevented the reaction
of compound I with the small amount of DDC.
For
[PGG]
K
, the k
value approximates the pseudo-first order
rate constant for the bimolecular reaction of compound I and PGG
as in and :
The apparent second order rate constant for the reaction of
compound I and PGG, k
, obtained
from , is (1.0 ± 0.2)
10
M
s
(Table 2). As shown in Table 2, the k
values decreased in the order PGG
> m-Cl PBA > EtOOH > H
O
> t-BuOOH.
Figure 9:
Spectral and kinetic data showing
different behavior of the PGH synthase intermediate II at low and high
hydroperoxide concentrations. A, at the 1:1.3 ratio of enzyme
(0.5 µM) and m-Cl PBA (0.65 µM), the
second intermediate is completely reconverted to native enzyme. In the
presence of an excess (50 µM) of EtOOH, a steady state
spectrum of the intermediate II, which coincided with spectrum
a, was present. B, decay of the steady state intermediate
is slow (k = 0.08 ± 0.02
s
) and zero-order with respect to
[EtOOH].
Hydroperoxides are initiators of
tyrosyl radical formation, and the oxygenation of arachidonic acid for
which PGH synthase compound I formation is a prerequisite
step(1, 2, 3) . On the other hand,
hydroperoxides inactivate the enzyme in the absence of peroxidase
electron donors(37, 38) . Our work is an extensive
pre-steady state and transient state kinetic study with a series of
structurally different hydroperoxides and PGG, in which the
primary goal was to deduce the role of hydroperoxides and compound I in
tyrosyl radical formation.
We found that m-Cl PBA could be utilized for the quantitative formation of compound I and that compound I thus formed was sufficiently stable to study its reactions with other substrates using the sequential mixing technique ( Fig. 5and Fig. 6).
The transient state results clearly establish that m-Cl PBA, EtOOH, and t-BuOOH exhibit bimolecular kinetics in their reactions with compound I and confirm results from the pre-steady state experiments that the initial product obtained is compound II. The second order rate constants for compound II formation obtained by the two different kinetic methods were the same for m-Cl PBA and EtOOH. For m-Cl PBA and EtOOH, the formation of compound I is two orders of magnitude faster than the formation of compound II ( Table 1and Table 2) so that the two reactions are cleanly separated.
Similar rate constants for compound I and compound II formation were obtained with t-BuOOH ( Table 1and Table 2). Thus, the saturation kinetics obtained by the pre-steady state method is a function of two sequential reactions (Fig. 3). The linear kinetics obtained by the transient state method (Fig. 7) is the accurate description for this reaction, showing that the t-BuOOH reaction mechanism is the same as for EtOOH and m-Cl PBA.
For HO
, the faster phase
exhibited saturation kinetics under pre-steady state conditions (Fig. 4), but with pre-formed compound I in the transient state
the results were not reproducible. The slower phase of the
H
O
reaction was not reproducible for all
experimental conditions. Deleterious effects of H
O
and/or superoxide appear to be occurring.
For the reaction of
PGG with compound I under transient state conditions, the
saturation kinetics are very similar to those obtained with the
pre-steady state experiments(23) . If the intramolecular
electron transfer mechanism were occurring, then compound I should form
the compound I-tyrosyl radical at a rate independent of the
concentration of PGG
, which is not the case. The transient
state results prove that PGG
is a reductant of compound I
as are other hydroperoxides. The reason that saturation, not linear,
kinetics is obtained is that the reaction occurs through the formation
of a transient complex between compound I and PGG
. For the
previously observed saturation kinetics obtained under pre-steady state
conditions, the possibility of complex formation was not taken into
consideration, and direct reduction of compound I was erroneously
excluded(23) .
This reaction for tyrosyl radical formation was originally proposed as part of both the branched-chain tyrosyl radical mechanism (23) and the non-chain mechanism(18, 19) . Thus, the hydroperoxyl radical could abstract a hydrogen atom from a tyrosyl residue to form the compound I-tyrosyl radical. However, in the branched-chain mechanism, both a unimolecular pathway (no reaction with hydroperoxide) and bimolecular pathway (reaction with a hydroperoxyl radical) for compound I-tyrosyl radical formation were proposed(23) . The sum of and is the following unimolecular reaction:
However, this work excludes the unimolecular pathway in , i.e. direct intramolecular electron transfer
from the tyrosyl residue to the porphyrin cation radical.
The
compound I-tyrosyl radical cannot be distinguished spectroscopically
from compound II since they have the same porphyrin-iron(IV)
chromophore. Thus, the measured rate constant k cannot be an intrinsic but only an apparent second order rate
constant for the reaction of compound I with hydroperoxides. At one
extreme, the formation of compound II could be rate-controlling (), as we assumed for the sake of simplicity in the
interpretation of the single-exponential kinetic results. At another
extreme, the second step, the formation of the compound I-tyrosyl
radical in , could be rate-controlling. A combination of
the two might explain the biphasic kinetics obtained with
H
O
.
Further support that compound II is
formed before the compound I-tyrosyl radical is obtained from the
spectral and kinetic studies on intermediate II (Fig. 9).
Intermediate II returned to native enzyme when no excess of
hydroperoxide was present, indicating that it is compound II reacting
with DDC. When a hydroperoxide was present in high excess, the steady
state spectrum of intermediate II was very stable. Intermediate II
decayed slowly and independently from the hydroperoxide concentration.
This indicates that when DDC was consumed, compound II did not react
with neutral hydroperoxide but reacted with the hydroperoxyl radical,
causing the formation of the compound I-tyrosyl radical. In the
opposite situation, if the hydroperoxides reacted with compound II, the k would be a function of the hydroperoxide
concentration. The above results are in agreement with EPR studies,
which proved that tyrosyl radicals are formed under similar
conditions(16, 17, 34, 35, 36) .
The nature of the steady state species present when an excess of
hydroperoxide is added could also be resolved by determining whether a
one-electron (for compound II) or a two-electron donor (for compound
I-Tyr) converts the species to the native enzyme. We tried to
utilize reduced cytochrome c for this purpose. The attempt was
unsuccessful because of the instability of the reduced cytochrome c and the high reactivity of the enzyme. The results were not
reproducible.
The present paper shows for the first time
that disappearance of compound I is accelerated by hydroperoxides and
that the likely first product is compound II. In addition, the very
large rate constant for the reaction of compound I of PGH synthase with
phenol was measured and found to be (8.3 ± 0.2) 10
M
s
at 6.7 °C (Table 2).
Recent experiments and pre-steady state models have
been published on the reactions of arachidonic and eicosadienoic acids
with native enzyme (19) . Under pre-steady state conditions,
the rate of formation of intermediate II was found to be a function of
fatty acid concentration. The rate of reaction of arachidonic acid with
compound I (1.2 10
M
s
, 30 °C; (19) ) is significantly
slower than the reaction with PGG
(1.0
10
M
s
, 3 °C; Table 2), which means that arachidonic acid probably could not
compete with PGG
for compound I at higher PGG
concentrations. For arachidonic acid, the model that fit the
experimental data was one in which the products of the compound
I-arachidonic acid reaction are compound II and the carbon radical of
arachidonic acid. The latter reacts rapidly with molecular oxygen to
form the PGG
radical, which abstracts the phenolic hydrogen
atom from Tyr
to form PGG
and a compound
I-tyrosyl radical. The observed second order kinetics with both
eicosadienoic and arachidonic acids could be due to fast formation of
the corresponding hydroperoxides, which do react with compound I. The
experiments with fatty acids were performed under aerobic conditions so
that the newly formed hydroperoxides participated in the overall
reaction. However, the reduction of compound I by hydroperoxides was
not included in the pre-steady state model because at that time no data
were available indicating that such a reaction occurs. Thus, the
pre-steady state models for reactions of fatty acids (19) did
not directly prove that the fatty acids react with compound I but did
provide evidence against the spontaneous formation of a tyrosyl radical
from compound I. The results could not be fit with the branched-chain
mechanism. Only future transient state kinetic studies with arachidonic
acid and compound I in the absence of oxygen will give a definitive
answer as to whether compound I or the compound I-tyrosyl radical
formed later in the cycle is the species that initiates the fatty acid
oxygenation.
Two papers describe the kinetics of the oxygenase
reaction of PGH synthase with cis,cis-eicosa-11,14-dienoic acid (39) and
the effect of Trolox C on the reaction(40) . A surprising 2:1
ratio occurs in the maximum rate of dienoic acid reaction compared to
the maximum rate of molecular oxygen consumption, whereas the expected
1:1 ratio is obtained when the enzyme is soybean lipoxygenase. Trolox C
accelerates the PGH synthase-catalyzed reaction at low concentration,
and the accelerating effect decreases for higher Trolox C
concentration. For all concentrations of Trolox C, from 0 to 250
µM, the 2:1 ratio is maintained. The results are explained
in terms of participation of two tyrosyl radicals on the enzyme, one
which participates in the reaction cycle (Tyr) and the
other which leads to enzyme inactivation. There is no intrinsic reason
why a tyrosyl radical cannot also be found on compound II, and the
existence of such a radical is a necessary part of the proposed
inactivation mechanism. The effect of Trolox C must be on the
inactivation pathways; otherwise, it would change the 2:1
stoichiometry.
In an elegant EPR study, direct proof of arachidonic acid oxidation by a tyrosyl radical was obtained and of coupling between the peroxidase and cyclooxygenase pathways(41) . The existence of other tyrosyl radicals is also discussed. Therefore, it is no longer possible to discuss a non-tyrosyl radical reaction mechanism(42) .
The original steady state study of the effect of ferulic acid provided evidence against the branched-chain mechanism for the part of the cyclooxygenase reaction, which was stimulated by ferulic acid(18) . Based on the fixed 1:2 stoichiometry between arachidonic and ferulic acids, it was proposed that arachidonic acid reacts with compound I and the peroxidase electron donor ferulic acid with the compound I-tyrosyl radical and compound II, thus preventing enzyme inactivation(18) .
In the latest paper, it is
concluded that the branched-chain mechanism is correct(43) .
Reaction mixtures were quenched by addition of cold ether-methanol
mixtures and analyzed for PGG and PGH by thin-layer
chromatography. After 10-20 s, product formation remained
essentially constant with PGH
always in excess of PGG
except for the spontaneous reaction (no added reductant). This
indicates that the enzyme is inactivated with respect to cycloxygenase
activity after 10-20 s. Peroxidase activity, tested by adding
15-hydroperoxyeicosatetraenoic acid to reaction mixtures containing
ferulic acid, also has almost disappeared in the first 20 s.
A major
criticism of the earlier study using ferulic acid as a peroxidase
substrate is that no correction was applied for the lag between oxygen
concentration in bulk solution and in the electrode compartment of the
Clark electrode(43) . Oxygen diffuses across the electrode
membrane via a first order process with a rate constant value typically
of the order of 0.25 s(43, 44) .
The criticism is invalid, because in the earlier study the spontaneous
rate of cyclooxygenase activity was subtracted from the total
cyclooxygenase activity to get the stimulated rate, and the correction
for the diffusion lag period cancels out(18) . The experiments
on the eicosadienoic acid reaction with PGH synthase and soybean
lipoxygenase provide another check on the validity of our measurements
of oxygen uptake(19) . The correct 1:1 stoichiometry between
rate of dienoic acid reaction and rate of oxygen uptake was obtained
for the soybean lipoxygenase reaction and provided a calibration for
the PGH synthase experiments, performed on the same instrument.
Therefore, the oxygen results are not caused by an instrumental
artifact. Ferulic acid is highly reactive, even in buffer solution in
the presence of light(18) , and a large blank correction should
have been applied in the most recent work(43) .
There are discrepancies in rate constants used for modeling studies (19, 43) . Although most of the data in the latest ferulic acid study were obtained under conditions where enzyme inactivation is dominant, no term for inactivation was included in the modeling(43) . Partly because of this there is an erroneous assumption of a fixed, large requirement of hydroperoxide for the tightly coupled mechanism, leading to the conclusion that the tightly coupled mechanism cannot fit the experimental data(43) ; however, it can(19) .
It is autoxidation of arachidonic acid, or presence of endogenous oxidant, that initiates the cyclooxygenase reaction. Autoxidation of reactive hydrocarbons is continually occurring and can occur via a non-enzymatic chain reaction. In the horseradish peroxidase reaction with indole-3-acetic acid, there is such a chain reaction(45) . Although there is great need for a thorough investigation of the initiation of PGH synthase reactions, it will be very difficult to separate reactions of endogenous and exogenous oxidants.
There is another interesting parallel between the horseradish peroxidase reaction with indole-3-acetic acid and the PGH synthase cyclooxygenase reaction. Some workers believed that the peroxidase reaction with indole-3-acetic acid was initiated by reduction of the Fe(III) of the native enzyme to Fe(II). It definitely does not occur at pH 7(45) . It was thought that the same reduction might be required with PGH synthase, which was disproven by the detection of the oxidized peroxidase-like intermediates of PGH synthase, not reduced enzyme(20) . There is no longer any question about compound I and compound II participation in the PGH synthase reactions. The debate is over the precise roles.
Finally, it is claimed that cosubstrate is required for cyclooxygenase activity in the tightly coupled mechanism(43) . Our study of the effect of ferulic acid clearly differentiated between a spontaneous cyclooxygenase reaction and a cyclooxygenase reaction stimulated by ferulic acid. There was never any question of the existence of a spontaneous reaction(18) .
A plausible explanation for many
of the discrepancies in the literature is that many results have been
obtained with enzyme that is appreciably inactivated. Enzyme
inactivation begins with addition of oxidant to the enzyme and is best
minimized by making measurements during the first 5 s of reaction. A
chain reaction involving a tyrosyl radical can explain the accumulation
of PGG, and it is our postulate that this is both a result
of, and a pathway to, enzyme inactivation. An appreciable accumulation
of PGG
in the cold solvent quench experiments could occur
in the dead time of the quenching experiments. A question that needs to
be addressed is that if PGH
is the initial reactant in the
synthesis of other prostaglandins and the thromboxanes, why would
nature desire an accumulation of highly reactive PGG
instead?