From the Division of Hematology, Department of Internal Medicine, University of Texas, Houston, Texas 77030
Received for publication, October 9, 2000, and in revised form, December 19, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Thromboxane synthase
(TXAS) is a "non-classical" cytochrome P450. Without any need for
an external electron donor, or for a reductase or molecular oxygen, it
uses prostaglandin H2 (PGH2) to catalyze
either an isomerization reaction to form thromboxane A2
(TXA2) or a fragmentation reaction to form
12-L-hydroxy-5,8,10-heptadecatrienoic acid and
malondialdehyde (MDA) at a ratio of 1:1:1
(TXA2:heptadecatrienoic acid:MDA). We report here kinetics
of TXAS with heme ligands in binding study and with PGH2 in
enzymatic study. We determined that 1) binding of U44069, an
oxygen-based ligand, is a two-step process; U44069 first binds TXAS,
then ligates the heme-iron with a maximal rate constant of 105-130
s Thromboxane A2
(TXA2)1 is a
potent inducer of vasoconstriction and platelet aggregation. It is
believed to be a crucial factor contributing to a variety of
cardiovascular and pulmonary diseases such as atherosclerosis,
myocardial infarction, and primary pulmonary hypertension (1, 2).
TXA2 is rather labile, being hydrolyzed to the biologically
inactive thromboxane B2 (TXB2) with a half-life of about 30 s in aqueous solution at 37 °C (3). Its
biosynthesis is accomplished by thromboxane synthase (TXAS) using
prostaglandin H2 (PGH2) as the substrate.
Notably, the formation of TXA2 is accompanied by those of
12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and
malondialdehyde (MDA) at a ratio of 1:1:1 (4) (Scheme
1). The biological functions of MDA and
HHT are unclear. However, MDA can form adducts with lysine residues of
proteins or with amine head groups of phospholipids and its adducts
have been detected in atherosclerotic lesions of human aorta (5). It
was also shown to participate in the formation of an important endogenous DNA adduct which may contribute to the etiology of human
genetic disease and cancer (6).
1; 2) binding of cyanide, a carbon-based ligand, is a
one-step process with kon of 2.4 M
1 s
1 and
koff of 0.112 s
1; and 3) both
imidazole and clotrimazole (nitrogen-based ligands) bind TXAS in a
two-step process; an initial binding to the heme-iron with on-rate
constants of 8.4 × 104 M
1
s
1 and 1.5 × 105
M
1 s
1 for imidazole and
clotrimazole, respectively, followed by a slow conformational change
with off-rate constants of 8.8 s
1 and 0.53 s
1, respectively. The results of our binding study
indicate that the TXAS active site is hydrophobic and spacious. In
addition, steady-state kinetic study revealed that TXAS consumed
PGH2 at a rate of 3,800 min
1 and that the
kcat/Km for
PGH2 consumption was 3 × 106
M
1 s
1. Based on these data,
TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan
stopped-flow experiments revealed marginal absorbance changes upon
mixing TXAS with PGH2, indicating minimal accumulation of
any heme-derived intermediates. Freeze-quench EPR measurements for the
same reaction showed minimal change of heme redox state. Further
kinetic analysis using a combination of rapid-mixing chemical quench
and computer simulation showed that the kinetic parameters of
TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of
1.2-2.0 × 107 M
1
s
1; the rate of catalytic conversion of PGH2
to TXA2 or MDA was at least 15,000 s
1 and the
lower limit of the rates for products release was 4,000-6,000 s
1. Given that the cellular PGH2
concentration is quite low, we concluded that under physiological
conditions, the substrate-binding step is the rate-limiting step of the
TXAS-catalyzed reaction, in sharp contrast with "classical" P450 enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (13K):
[in a new window]
Scheme 1.
TXAS is a member of the cytochrome P450 superfamily and is associated
with endoplasmic reticulum (4, 7). Human TXAS was assigned as CYP 5A1.
Unlike other microsomal P450s that require the ubiquitous P450
reductase to shuttle electrons for the mono-oxygenation reaction, TXAS
undergoes an isomerization reaction without reductase or molecular
oxygen. A "classical" P450 accepts an electron from the reductase
and converts heme iron to the Fe(II) state, followed by binding of
molecular oxygen and another one-electron reduction. The subsequent
steps, including oxygen bond cleavage and oxidation of the substrate,
occur rapidly and have been difficult to resolve (8). However, for
P450cam, the rate constants of oxygen binding and substrate
hydroxylation were determined to be 4.1 × 105
M1 s
1 and 34 s
1,
respectively (9). The intermediates of the later steps in the
P450cam reaction were well characterized in a recent
time-resolved x-ray crystallographic study in which the presence of an
oxyferryl intermediate after breakdown of molecular oxygen was
identified (10).
TXAS, which uses an endoperoxide as the substrate, apparently undergoes
a different mechanism. Hecker and Ullrich (11) proposed a mechanistic
model for TXAS involving a homolytic scission of PGH2 that
led to the formation of heme-Fe(IV) and alkoxy radical intermediates
for enzyme and substrate species, respectively. In the present study,
we attempted to characterize the intermediates of the TXAS reaction
employing optical and EPR spectroscopies. EPR studies failed to
identify any radical species. Stopped-flow spectroscopy revealed no
significant absorbance changes at the heme center. Further kinetic
analysis using the rapid-mixing chemical quench and computer simulation
showed that the substrate-binding step is the rate-limiting step of
TXAS-catalyzed reaction, and that the catalytic conversion of
PGH2 to TXA2 or MDA by TXAS was remarkably fast.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals-- Arachidonic acid was obtained from NuChek Prep (Elysian, MN). U44069 (15-hydroxy-9,11-(epoxymethano)prosta-5,13-dienoic acid) was from Cayman Chemical (Ann Arbor, MI). Isopropyl alcohol used for PGH2 preparation was first treated with crystals of sodium sulfate, followed with 12-mesh t.h.e. desiccant (EM Science, Gibbstown, NJ). [1-14C]Arachidonic acid (55 mCi/mmol) was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). 4,6-Dihydroxy-2-mercaptopyrimidine, also known as thiobarbituric acid (TBA), was purchased from Fisher Scientific.
Preparation of PGH2--
PGH2 was
prepared following published procedures with some modifications (12).
Briefly, 1 mg of arachidonic acid was incubated with 8 ml of 0.1 M potassium Pi, pH 7.2 (pre-saturated with
oxygen), containing 0.5 mM phenol and 1 mM
hemin. The reaction was initiated by adding 300 µl of prostaglandin
synthase-1 (about 6-8 mg of protein/ml) obtained from the supernatant
fraction of Tween 20-treated ram seminal microsomes. The reaction
mixture was then incubated, with stirring, for 2 min at 23 °C, and
then acidified with 0.8 ml of 2 M citric acid.
PGH2 was immediately extracted twice with 10 ml of
ether:hexane mixture (v/v, 5:1). The combined organic layers were dried
over crystals of sodium sulfate, concentrated by purging with nitrogen
gas and replaced with the HPLC solvent (hexane:isopropyl
alcohol:acetonitrile:acetic acid, 95:5:0.05:0.05) to a final volume of
0.5 ml. The extracts were then filtrated through a 0.5-µm
Millex-LCR4 filter (Millipore, Bedford, MA), concentrated
to 0.2-ml and applied to a straight-phase silica HPLC column (Waters
Spherisorb S5W, 10 mm × 250 mm). The HPLC system was interfaced
to a Waters Millennium32 Program (Waters, Milford,
MA). The column was eluted isocratically with the HPLC solvent at a
flow rate of 1 ml/min and the eluent was simultaneously monitored at
200 and 234 nm with a Waters 2487 dual wavelength absorbance detector.
PGH2 eluted as a discrete band with a retention time of
about 16 min, as indicated by the appearance of a 200-nm peak, which
was not seen when monitored at 234 nm. Fractions were collected,
concentrated under nitrogen gas and stored at 70 °C.
PGH2 was stable under this condition for at least 6 months.
In preparing PGH2 for stopped-flow and EPR experiments,
HPLC solvent was evaporated under nitrogen gas prior to use and
immediately replaced with isopropyl alcohol.
Quantitation of PGH2 was carried out by including 0.5 µCi of [1-14C]arachidonic acid (~0.00273 mg) in 1 mg of cold arachidonic acid for preparation of PGH2. After being purified by HPLC, [1-14C]PGH2 was quantitatively determined by scintillation counting. To avoid handling the radioactive materials in the latter experiments, samples of [1-14C]PGH2 in various concentrations were subjected to HPLC separation under the aforementioned conditions and areas within PGH2 peaks (monitored at 200 nm) were determined. Plots of peak areas versus PGH2 concentrations were used as the standard curves for determinations of non-radioactive PGH2 concentrations.
Protein Purification and Activity Assay--
A recombinant human
TXAS, with a histidine-tag at the carboxyl terminus, was expressed in
Escherichia coli and purified by nickel and octyl column
chromatography, as previously described for the wild-type TXAS (13).
TXAS was concentrated by ultrafiltration and dialyzed against buffer
containing 50 mM potassium Pi, pH 7.4, 10% glycerol, and 0.005% Emulgen 913. TXAS activity was measured by
spectroscopic assay for the formation of MDA. TXAS was added to 300 µl of 50 mM potassium Pi, pH 7.4, and 0.1%
Lubrol. The reaction was initiated by adding 40 µM
PGH2 and MDA formation was followed by absorbance increase
at 268 nm at 23 °C using a Shimadzu UV-2401PC Spectrophotometer.
TXAS concentration was determined by absorbance at 418 nm using the
extinction coefficient of 100 mM1
cm
1 (13). Alternatively, TXAS activity was measured by a
coupled assay as previously described (13), using arachidonic acid and ovine prostaglandin H synthase-1 to generate PGH2 in
situ. Quantitation of TXB2, the stable hydration
product of TXA2, was carried out by radioimmunoassay
(14).
Stopped-flow and EPR Spectroscopy-- Stopped-flow spectroscopy was performed, under the conditions as described in the figure legends, on an Applied Photophysics model SX-18MV. In the ligand binding studies, TXAS and ligands prepared in 50 mM potassium Pi, pH 7.4, 10% glycerol, and 0.005% Emulgen 913 were reacted by equal mixing mode at 23 °C. In the TXAS-catalyzed reaction, TXAS was kept in the aforementioned buffer but PGH2 was dissolved in isopropyl alcohol prior to the experiments. A 10:1 (TXAS:PGH2) mixing was used. An independent experiment showed that up to 10% of isopropyl alcohol had no effect on TXAS activity. Kinetic data were collected either at the single wavelength mode or by photodiode array detector to record spectra over 300-700 nm.
For EPR measurements, TXAS and PGH2 were mixed using a System 1000 rapid quench apparatus (Update Instrument Inc., Madison, WI). About 12 µM TXAS was loaded in two 7.2-ml syringes and PGH2 was loaded in a 0.5-ml syringe to give a 29:1 mixing ratio. Reactants once passed via a Wiskind 4-grid mixer were directly injected into a pre-chilled EPR tube and immediately dropped into a dry ice-acetone bath. EPR spectra were recorded at liquid helium temperature on a Varian E-6 spectrometer.
Rapid-mixing Chemical Quench Studies-- Rapid quench kinetic studies were also carried out using a System 1000 rapid quench apparatus. TXAS was in 50 mM potassium Pi, pH 7.4, 10% glycerol, and 0.005% Emulgen 913 whereas PGH2 was in the HPLC solvent. TXAS was top-loaded into a 7.2-ml syringe and PGH2 was loaded into a 0.5-ml syringe, thus giving a TXAS:PGH2 mixing ratio of 14.4:1. Reaction time was controlled either by using aging tubes of various lengths (for reactions of less than 150 ms) or a programmed push-push mechanism. Reactions were carried out at room temperature (21-23 °C). The concentration of TXAS, after mixing, was kept at 5 µM and those of PGH2 were at 5, 15, and 50 µM. After the indicated time points, the reactions were quenched by mixing through the second mixer, with an equal volume (~200 µl) of a solution containing 10% trichloroacetic acid and 0.3 M HCl loaded in a second 7.2-ml syringe. The ram velocity of the instrument was operated at 0.8 cm/s for both rapid-mixing and chemical quench steps.
Quantitation of MDA thus formed was determined using the TBA assay as
previously described (15). Briefly, 200 µl of the quenched mixture
was centrifuged at 12,000 × g for 5 min to remove denatured proteins, and 180 µl of the supernatant was added to 180 µl of TBA solution (0.53% TBA in 50 mM potassium
Pi, pH 7.4). The sample was heated at 70 °C for 30 min
and then cooled at room temperature for 10 min before measuring the
absorbance at 532 nm. The concentration of MDA-TBA adduct was
calculated based on 532 = 156 mM
1 cm
1 (16).
Computer Simulation--
The ScoP Program (Simulation Resources
Inc., Redlands, CA) was used for kinetic simulation of the proposed
model shown in Table I. The assignments of rate constants were tested
by comparing with the experimental data and will be detailed under
"Results."
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Binding of U44069 to TXAS--
U44069 is a stable TXAS
substrate analog with the C-11 oxygen of PGH2 replaced by a
carbon atom whereas the C-9 oxygen remains intact. It was shown
previously that U44069 interacted with TXAS by replacing the initial
heme ligand (most likely water) with the C-9 oxygen atom (11). Binding
of U44069 to TXAS induced a blue shift in the Soret peak and
equilibrium dissociation constant was determined to be 28 µM from the difference spectra that had a peak at 409 nm
and a trough at 426 nm (13). To investigate the mechanism of substrate
binding to TXAS, we carried out stopped-flow spectroscopy using U44069
as the ligand and monitored the absorbance changes at 409 and 426 nm.
Fig. 1A described stopped-flow
traces observed upon mixing 2 µM TXAS with 10-60
µM U44069. A control sample, where TXAS was mixed with
buffer alone, resulted in no significant absorbance changes (data not
shown). For all concentrations of U44069 used in these kinetic
measurements, a single exponential absorbance change was observed for
both wavelengths. The pseudo-first order rate constant,
kobs, was obtained by an exponential fitting algorithm. Plots of kobs monitored at 409 and
426 nm versus U44069 concentrations followed
Michaelis-Menten-type kinetics (Fig. 1B), suggesting that
binding of U44069 to TXAS is a two-step binding process,
|
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
Binding of Cyanide, Imidazole, and Clotrimazole to TXAS--
To
compare the binding nature of other heme ligands, we chose cyanide
(C-based ligand), imidazole (small N-based ligand), and clotrimazole
(large N-based ligand) for detailed kinetic binding studies. Addition
of CN to TXAS induced a red shift in the Soret peak from
418 to 447 nm. Under pseudo first-order conditions,
kobs were obtained for each cyanide
concentration from the exponential time courses at 447 nm. Fig.
2A shows the traces of
absorbance changes for complex formation displaying monophasic
exponential character. The inset of Fig. 2A shows
that kobs is linearly related to
CN
concentrations, suggesting a simple, one-step binding
reaction.
|
![]() |
(Eq. 3) |
Previous work has shown that imidazole binds TXAS and causes the shift
of the Soret peak to 434 nm with Kd of 33 µM (13). In the present stopped-flow studies, absorbance
changes at 434 nm were followed after mixing TXAS with excess imidazole (Fig. 2B). In contrast to U44069 and cyanide, all the
stopped-flow traces from the imidazole binding kinetics were
best-fitted using two exponential terms. The amplitude of the first
phase (fast phase) accounted for most of the total absorbance changes
(62-81%) and increased as the concentration of imidazole increased,
whereas the amplitude of the second phase (slow phase) was essentially unchanged over the range of 50-1000 µM imidazole. When
plotted against the concentrations of imidazole,
kobs of the fast phase reveals a linear
dependence whereas kobs of the slow phase is independent of imidazole concentrations (inset of Fig.
2B). These results indicated a two-step binding reaction:
the first step requires that the original heme ligand of TXAS be
replaced by imidazole, and this step is also accompanied by a larger
absorbance change, whereas the second step is presumably a slow
conformational change in which heme-bound imidazole is re-oriented to
reach the final conformation. Linear regression analyses of the fast
phase yielded the slope, 8.4 × 104
M1 s
1, as an estimate for
kon, and the y intercept, 8.84 s
1 for koff. The rate constants of
slow phase were in the range of 0.84-1.40 s
1.
Clotrimazole is an imidazole derivative heme ligand. The carbon atom at
the imidazole N-1 position is bonded to three phenyl rings with
vertical and horizontal dimensions of ~8.5 and 9.5 Å, as compared
with 2.8 and 4.2 Å for imidazole. We have shown that the
clotrimazole-induced difference spectra had a peak and a trough at 431 and 410 nm, respectively (13). Stopped-flow analysis following the
absorbance changes at 431 nm showed that the kinetics of clotrimazole
binding to TXAS was very similar in character to that achieved by
imidazole. Two kinetic phases were observed upon binding (Fig.
2C), and kobs of the first phase (fast phase) was linearly dependent on clotrimazole over the
concentrations of 3.1-18.7 µM (inset of Fig.
3C). Higher clotrimazole
concentrations were unachievable due to the low solubility of the
ligand in the reaction buffer. The amplitude of the fast phase
accounted for 63-72% of the total observed amplitude. The
second-order rate constant and the intercept obtained from the plot of
kobs versus ligand concentrations
were 1.51 × 105 M1
s
1 and 0.53 s
1, respectively. Similar to
imidazole, the amplitudes and rate constants of the clotrimazole-TXAS
binding slow phase (i.e. ~0.2-0.4 s
1) were
concentration independent. In a similar experimental set-up where
absorbance changes were monitored at 410 nm, stopped-flow traces were
nearly mirror image to those monitored at 431 nm and kinetic parameters
obtained from both wavelengths were almost identical (data not
shown).
|
Steady-state Kinetic Analysis of TXAS--
We reported previously
that the recombinant TXAS had a Km of 20 µM PGH2 and a Vmax of
12 µmol of TXA2 formed/min/mg of protein, as compared
with the native platelet TXAS which has a Km of 22 µM PGH2 and a Vmax of
24 µmol of TXA2 or MDA formed/min/mg of protein (4, 18).
It should be noted that these kinetic parameters were determined by
different assay systems. We used the coupled assay that included
prostaglandin H synthase-1 and arachidonic acid, whereas the other
groups added PGH2 directly to the assay mixture instead. In
the present study, we measured the TXAS activity by following the MDA
formation at 268 nm (268 = 31.5 mM
1 cm
1) after adding
PGH2 to the assay buffer. The initial rates were calculated
from the slope of the first 10-20 s of the reaction. Nonlinear
least-square analysis of TXAS activity versus
PGH2 concentrations (1.5 to 240 µM), as shown
in Fig. 3, gave the recombinant TXAS a Km of
21.3 ± 2.2 µM and a Vmax of
32.6 ± 0.8 µmol of MDA/min/mg of protein, equivalent to a
turnover number, kcat, of 1960 min
1.
Stopped-flow and Freeze-quench EPR Spectroscopic
Studies--
Although steady-state kinetic analysis can provide rate
parameters such as Km and
kcat, it cannot be used to dissect each step in
the catalytic cycle. To test whether heme chemistry is the
rate-limiting step of TXAS reaction, transient kinetic measurement were
conducted. Our initial attempt to study the role of heme iron involved
in TXAS catalysis was carried out by following the absorbance changes
at 418 nm in a stopped-flow apparatus. Several conditions were tested:
TXAS concentrations (1-4 µM), PGH2
concentrations (1-10-fold molar excess of TXAS) and temperature (4 °C and room temperature). Nevertheless, no significant absorbance changes were observed in all the conditions we tested (data not shown).
We then expanded our observation using rapid-scan stopped flow
spectroscopy. Stopped flow recording of reaction time from instrument
dead-time to 1 s upon mixing 4 µM TXAS with 1-, 3-, and 10-fold molar excess of PGH2, again, revealed no
significant absorbance changes over 300-700 nm. As described below,
1 s of reaction time was sufficient to convert nearly all the
substrate to products. Fig. 4A
shows that the absorption spectra of TXAS interaction with 3-fold molar
excess of PGH2 from 6 to 210 ms are nearly
indistinguishable.
|
A TXAS reaction mechanism proposed by Hecker and Ullrich (11) involved a homolytic scission of O-O bond of PGH2 to form an alkoxy radical. To test this key step in the postulated TXAS reaction mechanism, we employed freeze-quench EPR spectroscopy in an attempt to establish the chemical structure of alkoxy radical during TXAS catalysis. Reactions were carried out at 23 °C by a rapid-freeze apparatus that mixed 12 µM TXAS with PGH2 (final concentrations of ~50 and 100 µM). The apparatus allowed the reactants to interact for about 10 ms and the reaction mixture was immediately freeze-trapped in a dry ice-acetone bath, which took less than 2 s to freeze the samples. Samples obtained by this method were stored in liquid nitrogen prior to undertaking the EPR spectroscopic study. EPR spectra of PGH2-treated TXAS, as shown in Fig. 4B, revealed no differences from that of the isopropyl alcohol-treated control or from resting TXAS (data not shown).
Since PGH2 is very labile in water having half-life of about 5 min (19), we hence examined whether PGH2 was degraded due to the contamination of trace amounts of water in isopropyl alcohol. PGH2 stock was analyzed before and after the stopped-flow and EPR experiments by following the capacity of TXAS-catalyzed MDA and HHT formation. The degree of PGH2 degradation varied from 10 to 40%; lower PGH2 concentrations were degraded faster. Despite the instability of PGH2 during the experiments, the remaining concentrations of PGH2 were still sufficient to interact with TXAS and thus allow us to observe the signal changes if the proposed reaction mechanism was correct. The only other possibility is that the TXAS-catalyzed reaction takes place so rapidly that it exceeds the stopped-flow spectrophotometer reaction rate limit and therefore cannot be measured.
Rapid-mixing Chemical Quench Studies-- To gain further understanding of the kinetic mechanism by which TXAS converts PGH2 to TXA2 and MDA, we performed rapid-mixing experiments using chemical quench techniques as described under "Experimental Procedures." Durations of reaction were allowed to carry out ranging from 3.2 ms to 1 s. The enzymatic products, i.e. TXB2 and MDA, were quantitatively determined by radioimmunoassay and TBA assay, respectively. Blank TXB2 and MDA were obtained by manually mixing TXAS, HPLC solvent without PGH2, and the quenching solution. PGH2 was kept in the HPLC solvent to avoid degradation, and TXAS activity, in a control experiment, was not affected by up to 10% of HPLC solvent concentration. The remaining PGH2 in the syringe was collected after the rapid quench experiments and was assayed for its capacity to synthesize MDA and HHT by TXAS. Little PGH2 was degraded (<5%) during the entire experimental period.
In all three levels of PGH2 concentration tested,
more than 30% of PGH2 was converted to the products at the
shortest reaction time, 3.2 ms, and nearly all the substrate was
consumed at 0.3 s. Fig. 5 shows that
formation of TXB2, MDA, and combination of both products at
the given reaction time using 1- (panel A), 3- (panel B), and 10-fold (panel C) molar ratio of
PGH2:TXAS. Further kinetic analysis of this TXAS-catalyzed
reaction was conducted by computer simulation of a simplified reaction
mechanism in which PGH2 was converted to
TXA2 and MDA at the same rate
(k2 as shown in Table
I, Scheme
2). Optimal simulations using the ScoP
program were done with the following settings. The
k1 and k1 were varied
while keeping k1/k
1 = 20 µM, comparable to the dissociation constant of TXAS
and U44069. Because the TXAS-catalyzed reaction is thought to be
irreversible, k
2, k
3,
and k
4 were set at 0.01 s
1, an
arbitrarily small number. The values of k1,
k
1, k2, k3, and k4 produced from
the optimal simulation for 1-, 3-, and 10-fold rations of
PGH2:TXAS are summarized in Table I. We also tested this
kinetic mechanism by comparing the simulation results and experimental
data obtained from the stopped-flow experiments that followed the
absorbance changes at 418 nm. For the ScoP simulation program, we
assumed that absorption spectrum of the intermediate, i.e.
EP in Scheme 2, was similar to that of the P450 Compound I that had an
extinction coefficient at 418 nm of 60 mM
1
cm
1 (20). The rate constant for substrate-binding,
k1, determined from the simulation program was
1.2-2.0 × 107 M
1
s
1. A k1 value less than
107 M
1 s
1 will
significantly retard the kinetics of overall product formation. Notably, data could only be fitted when k2 was
set near 15,000 s
1. Smaller k2
values would cause a significant accumulation of Compound I
intermediate, as indicated by a large A418
decrease at the early time points. In these experiments, MDA was
produced at higher levels than TXB2 with a molar ratio of
0.6:0.4, in contrast to the previous reports that had a
MDA:TXB2 ratio of 1:1. This may be because TXB2
was collected under very acidic conditions (10% trichloroacetic acid
and 0.3 M HCl) and was slightly degraded. Optimal
simulation gave the rate constants of MDA and TXB2 release as 6,000 s
1 and 4,000 s
1, respectively.
These values are also the lower limits since smaller values shift the
minimum of the A418 kinetic data toward longer time points. Simulation results for changes in concentration of TXAS,
MDA, and TXB2 are in agreement with the experimental data and are presented in Fig. 5. These results clearly indicated that the
reaction intermediates are very short-lived, and the substrate is
rapidly converted to products once it binds TXAS.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TXAS, as a resting enzyme, has a typical low spin P450 heme with an oxygen-based distal ligand (4, 13). Several types of heme ligands including U44069 (oxygen-based ligand), imidazole, clotrimazole (both are nitrogen-based ligands), and cyanide (carbon-based ligand) were studied to determine their kinetic binding characteristics with TXAS using stopped-flow spectroscopy. All these ligands caused Type II spectral changes, indicating that the exogenous ligands formed a low-spin coordinate heme complex. However, their binding kinetics to TXAS were quite different. U44069 and cyanide binding to TXAS exhibited monophasic kinetics, in contrast to the biphasic kinetics found for imidazole and clotrimazole. Analysis of kobs versus ligand concentrations further indicated that cyanide underwent a one-step binding process, whereas U44069, imidazole, and clotrimazole underwent two-step binding processes.
Interestingly, the binding mechanisms of those ligands involved in the
two-step binding processes are distinctly different. U44069 first binds
TXAS, but not heme iron, and then replaces the original heme ligand.
The two nitrogen-based ligands, nonetheless, bind heme iron first and
then undergo a conformational change before they reach equilibrium.
Cyanide, which is charged but is the smallest molecule tested, had the
lowest on-rate constant (2.4 M1
s
1). This low rate constant compared with other
hemoproteins is likely due to the strong electron-donating thiolate
ligand. On the other hand, clotrimazole, although much bulkier than
imidazole, had a higher on-rate constant (8.4 × 104
M
1 s
1 and 1.5 × 105 M
1 s
1 for
imidazole and clotrimazole, respectively). These results indicated that
the TXAS active site is both hydrophobic and spacious, consistent with
our previously reported findings (13).
Classical eukaryotic P450s catalyze hydroxylation reactions at a
very slow rate with kcat of 1-10 mol of product
formed/min/mol of P450 (21). Many prokaryotic P450s, however, have
somewhat higher catalytic activities. For example, P450cam
catalyzes the reaction at a rate of ~1900 mol/min/mol of protein
(22), and so does P450BM-3 (23), a soluble protein in which the
reductase domain is naturally fused to the oxygenase domain. For the
"non-classical" P450s such as allene-oxide synthase and
nitric-oxide reductase that do not need reductase for catalysis, the
turnover numbers seems to be much higher. Allene-oxide synthase, a P450
acting as a dehydrase which converts lipid hydroperoxide to allene
oxide, appears to form product at a rate of >60,000 mol/min/mol of
protein (24). Nitric-oxide reductase, i.e.
P450nor, which catalyzes NO to N2O has a
turnover number of 72,000 mol/min/mol of protein (25). However,
prostacyclin synthase, another non-classical P450 that uses
PGH2 as the natural substrate, has a lower turnover number
(~150 mol/min/mol of protein) (11, 26). TXAS, as observed in this
study, formed MDA or TXA2 at a rate of 1,900 min1. In other words, TXAS consumed PGH2 at a
rate of 3,800 min
1. Furthermore, the ratio of
kcat/Km (an index of
catalytic efficiency) of TXAS for PGH2 consumption is thus
3 × 106 M
1
s
1. Compared with carbonic anhydrase, an extremely
efficient enzyme that has a kcat/Km of
107-108 M
1
s
1 (27), TXAS should be considered as a very efficient
catalyst. It should also be noted that many P450s including
P450cam can act as non-classical P450s and convert
PGH2 to MDA and HHT, but not TXA2, without
reductase, molecular oxygen, or any electron donor (11). Their
"HHT/MDA synthase" activities, nonetheless, are much lower than
TXAS. A recent report showed that four microsomal P450s (P4501A2, 2B1,
2E1, and 3A4) had Kd for U44069 of ~200
µM and when assayed at 50 µM
PGH2, their catalytic activities were 1-10
min
1 (28). It would appear that PGH2 was
readily converted to MDA and HHT by heme in the context of P450 where a
hydrophobic environment is present. A study of chemical models for
heme-catalyzed PGH2 reactions showed that 9% of HHT was
formed in a phosphate buffer, whereas 33% of HHT was formed in
acetonitrile, a less polar solvent (11). TXAS is more efficient in
HHT/MDA synthesis not only because its active site is hydrophobic but
it also has a greater affinity for PGH2. Furthermore,
several TXAS active site amino acid residues, as we have previously
shown (29), may be involved in the reaction. It is intriguing to note
that prostacyclin synthase, although it has a high affinity for
PGH2 (Kd is ~10 µM),
does not catalyze HHT/MDA formation (11). What structural elements determine the HHT/MDA activity of P450 remains a challenging topic.
In this study, the rate constant of each catalytic step,
i.e. PGH2 to TXA2 or MDA/HHT, is
estimated to be ~15,000 s1. The half-life of the
intermediate(s) at 23 °C is therefore much less than the dead-time
of the stopped-flow apparatus (~1.5 ms). Furthermore, the rate
constant of substrate binding is 12-20 × 106
M
1 s
1 and the in
vivo binding constant could be calculated if the cellular PGH2 concentration was known. To the best of our knowledge,
cellular PGH2 concentration has not been reported due in
part to the instability of PGH2 in aqueous solution.
However, concentrations of arachidonic acid, the substrate for
prostaglandin H synthase, in many inflammatory blood cells and lung
tissues were less than 10 µM (30, 31). In pancreatic
islets, cellular un-esterified arachidonate concentrations of 38-75
µM were reported under glucose-induced conditions. But the maximally effective concentration, including exogenous
arachidonic acid, was 30-40 µM (32). Since
arachidonic acid is a substrate for many other pathways in addition to
prostaglandin H synthase, it is probably safe to assume that the
cellular PGH2 concentration is generally less than 40 µM. If that is the case, the substrate-binding rate
constant for TXAS in the physiological conditions would be less than
800 s
1, and this value is much smaller than the other
forward rate constants. We therefore conclude that the
substrate-binding step is the rate-limiting step in the TXAS-catalyzed reaction.
P450s were also classified according to the identity of their electron
donors. Class I P450s require both ferredoxin and ferredoxin reductase
for electron transfer, whereas Class II P450s require only a P450
reductase (33). In both Class I and Class II P450s, the rate-limiting
step of the overall reaction is at the stage of the second electron
transfer to the heme center. Class III P450s that do not require any
reductase, are not well characterized regarding their kinetic
mechanisms. An elegant study on P450nor, a Class III P450,
revealed that binding of NO and NADH substrates were fast to form the
intermediate I, an NO-bound two-electron reduced species (25). The
intermediate I was slowly converted to the products. The rate-limiting
step of P450nor is thus in the chemical steps. In contrast
to other P450s, the rate-limiting step of TXAS is at the step of
substrate binding. To the best of our knowledge, this kinetic mechanism
is the first example of Class III P450s in which substrate binding is
the rate-limiting step.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Graham Palmer at Rice University for generous access to the EPR facility and Dr. Richard J. Kulmacz for valuable suggestions and helpful assistance in preparation and purification of PGH2.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants HL-60625 (to L.-H. W.), GM-44911 and GM-56818 (to A.-L. T.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to the results of this work.
§ To whom correspondence should be addressed: Div. of Hematology, Dept. of Internal Medicine, University of Texas- Houston, 6431 Fannin, Houston, TX 77030. Tel.: 713-500-6794; Fax: 713-500-6810; E-mail: lee-ho.wang@uth.tmc.edu.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M009177200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TXA2, thromboxane A2; TXAS, thromboxane A2 synthase; TXB2, thromboxane B2; PGH2, prostaglandin H2; HHT, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; MDA, malondialdehyde; TBA, thiobarbituric acid; HPLC, high performance liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Samuelsson, B., Goldyne, M., Granstrom, E., Hamberg, M., Hammarstrom, S., and Malmsten, C. (1978) Annu. Rev. Biochem. 47, 997-1029[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Vane, J. R.,
and Botting, R.
(1987)
FASEB J.
1,
89-96 |
3. | Hamberg, M., Svensson, J., and Samuelsson, B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2994-2998[Abstract] |
4. |
Haurand, M.,
and Ullrich, V.
(1985)
J. Biol. Chem.
260,
15059-15067 |
5. | Uchida, K. (1999) Trends Cardiovasc. Med. 9, 109-113[CrossRef][Medline] [Order article via Infotrieve] |
6. | Chaudhary, A. K., Nokubo, M., Reddy, G. R., Yeola, S. N., Morrow, J. D., Blair, I. A., and Marnett, L. J. (1994) Science 265, 1580-1582[Medline] [Order article via Infotrieve] |
7. |
Ruan, K.-H.,
Wang, L.-H.,
Wu, K. K.,
and Kulmacz, R. J.
(1993)
J. Biol. Chem.
268,
19483-19490 |
8. | Mueller, E. J., Loida, P. J., and Sligar, S. G. (1995) in Cytochrome P450; Structure, Mechanism and Biochemistry (Ortiz de Montellano, P. R., ed) , pp. 83-124, Plenum Press, New York |
9. |
Gerber, N. C.,
and Sligar, S. G.
(1994)
J. Biol. Chem.
269,
4260-4266 |
10. |
Schlichting, I.,
Berendzen, J.,
Chu, K.,
Stock, A. M.,
Maves, S. A.,
Benson, D. E.,
Sweet, R. M.,
Ringe, D.,
Petsko, G. A.,
and Sligar, S. G.
(2000)
Science
287,
1615-1622 |
11. |
Hecker, M.,
and Ullrich, V.
(1989)
J. Biol. Chem.
264,
141-150 |
12. | Hecker, M., Hatzelmann, A., and Ullrich, V. (1987) Biochem. Pharmacol. 36, 851-855[Medline] [Order article via Infotrieve] |
13. |
Hsu, P.-Y.,
Tsai, A.-L.,
Kulmacz, R. J.,
and Wang, L.-H.
(1999)
J. Biol. Chem.
274,
762-769 |
14. | Wu, K. K., LeBreton, G. C., Tai, H.-H., and Chen, Y. C. (1981) J. Clin. Invest. 67, 1801-1804[Medline] [Order article via Infotrieve] |
15. | Ledergerber, D., and Hartmann, R. W. (1995) J. Enzyme Inhib. 9, 253-261[Medline] [Order article via Infotrieve] |
16. | Janero, D. R. (1990) Free Radic. Biol. Med. 9, 515-540[CrossRef][Medline] [Order article via Infotrieve] |
17. | Fierke, C. A., and Hammes, G. G. (1995) Methods Enzymol. 249, 3-37[Medline] [Order article via Infotrieve] |
18. | Hecker, M., Haurand, M., Ullrich, V., Diczfalusy, and Hammarstrom, S. (1987) Arch. Biochem. Biophys. 254, 124-135[Medline] [Order article via Infotrieve] |
19. | Hamberg, M., Svensson, J., Wakabayashi, T., and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 345-349[Abstract] |
20. | Watanabe, Y., and Groves, J. T. (1992) in The Enzymes (Sigman, D. S., ed), Vol. 20 , pp. 405-452, Academic Press, San Diego, CA |
21. |
Guengerich, F. P.
(1991)
J. Biol. Chem.
266,
10019-10022 |
22. | Peterson, J. A., and Mock, D. M. (1975) in Cytochrome P450 and b5 (Cooper, D. Y. , Rosenthal, O. , Synder, R. , and Witmer, C., eds) , pp. 311-324, Plenum Press, New York |
23. |
Narhi, L. O.,
and Fulco, A. J.
(1986)
J. Biol. Chem.
261,
7160-7169 |
24. | Song, W. C., and Brash, A. R. (1991) Science 253, 781-784[Medline] [Order article via Infotrieve] |
25. |
Shiro, Y.,
Fujii, M.,
Iizuka, T.,
Adachi, S.,
Tsukamoto, K.,
Nakahara, K.,
and Shoun, H.
(1993)
J. Biol. Chem.
270,
1617-1623 |
26. |
Hara, S.,
Miyata, A.,
Yokoyama, C.,
Inoue, H.,
Brugger, R.,
Lottspeich, F.,
Ullrich, V.,
and Tanabe, T.
(1994)
J. Biol. Chem.
269,
19897-19903 |
27. | Lindskog, S., Engberg, P., Forsman, C., Ibrahim, S. A., Johnson, B. H., Simonsson, I., and Tibell, L. (1984) Ann. N. Y. Acad. Sci. 429, 61-75[Abstract] |
28. |
Plastaras, J. P.,
Guengerich, P.,
Nebert, D. W.,
and Marnett, L. J.
(2000)
J. Biol. Chem.
275,
11784-11790 |
29. |
Wang, L.-H.,
Matijevic-Aleksic, N.,
Hsu, P.-Y.,
Ruan, K.-H.,
Wu, K. K.,
and Kulmacz, R. J.
(1996)
J. Biol. Chem.
271,
19970-19975 |
30. | Triggiani, M., Oriente, A., Seeds, M. C., Bass, D. A., Marone, G., and Chilton, F. H. (1995) J. Exp. Med. 182, 1181-1190[Abstract] |
31. | Chilton, F. H., Fonteh, A. N., Surette, M. E., Triggiani, M., and Winkler, J. D. (1996) Biochim. Biophys. Acta 1299, 1-15[Medline] [Order article via Infotrieve] |
32. | Wolf, B. A., Pasquale, S. M., and Turk, J. (1991) Biochemistry 30, 6372-6379[Medline] [Order article via Infotrieve] |
33. |
Graham-Lorence, S.,
and Peterson, J. A.
(1996)
FASEB. J.
10,
206-214 |
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
All ASBMB Journals | Molecular and Cellular Proteomics |
Journal of Lipid Research | Biochemistry and Molecular Biology Education |