The effect of oxygen concentration on the
regiospecificity of the soybean lipoxygenase-1 dioxygenation reaction
was studied. At low oxygen concentrations (<5 µM),
a dramatic change in the regiospecificity of the enzyme was observed
with the hydroperoxy-octadecadienoic acid (HPOD) 13:9 ratio closer to
50:50 instead of the generally reported 95:5. This alteration of
regiospecificity is not an isolated phenomenon, since it occurs during
a reaction carried out under "classical" conditions,
i.e. in a buffer saturated with air before the reaction.
-carotene bleaching and electronic paramagnetic resonance findings
provided evidence that substrate-derived free radical species are
released from the enzyme. The kinetic scheme proposed by Schilstra
et al. (Schilstra, M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1994) Biochemistry 33, 3974-3979) was thus expanded to account for the observed variations in
specificity. The equations describing the branched scheme show two
different kinetic pathways: a fully enzymatic one leading to a
regio-isomeric composition of 13-HPOD:9-HPOD = 95:5, and a
semienzymatic one leading to a regio-isomeric composition of
13-HPOD:9-HPOD = 50:50. The ratio between the two different
pathways depends on oxygen concentration, which thus determines the
overall specificity of the reaction.
 |
INTRODUCTION |
Lipoxygenases (EC 1.13.11.12) are widely distributed in both the
animal and plant kingdoms. They catalyze the dioxygenation of
unsaturated fatty acids containing one or more (1Z,4Z)-pentadiene systems into the corresponding conjugated hydroperoxides. In animal tissues, the most frequently encountered substrate is arachidonic acid
(C20:4), which is dioxygenated by lipoxygenases into
precursors of products involved in inflammatory processes (2), cell
membrane maturation (3), or cancer metastasis (4). The role of plant lipoxygenases, whose main substrates are linoleic (C18:2)
and linolenic (C18:3) acids, is not yet fully elucidated,
although they are implied in processes such as senescence or plant
response to wounding (5).
A single non-heme iron is present in each enzyme and can exist in two
oxidation states: Fe(II) and Fe(III) (6). According to the current
working mechanism (1, 6, 7), the native enzyme, as obtained when
purified, is inactive and in the Fe(II) form. When treated with an
equimolar amount of product, the iron is oxidized to the Fe(III) form,
resulting in an active enzyme. This ferric form can then catalyze the
abstraction of a hydrogen from the bis-allylic carbon atom of the
substrate (S) in a stereo-specific manner, yielding a pentadienyl
radical (S·) complexed with the ferrous enzyme. Bimolecular
oxygen is then added to the pentadienyl radical, either at the C-1 or
the C-5 of the pentadiene system, which leads to the formation of the hydroperoxide product (P) and the reoxidation of the
cofactor to the ferric form (see Scheme
1, upper part).

View larger version (15K):
[in this window]
[in a new window]
|
Scheme 1.
Completed reaction mechanism for soybean-1
lipoxygenase. E, Fe(II)-lipoxygenase; E*,
Fe(III)-lipoxygenase; S, substrate; P, fully enzymatically
formed hydroperoxide product; Q, semienzymatically formed
hydroperoxide product; S·, pentadienyl radical; SO 2,
peroxyl radical; P(Q)O·, alkoxyl radical
formed with P or Q, respectively;
k1, k2,
k4, monomolecular rate constants;
k3, k5,
k6, k7, bimolecular rate constants; KmS, KiS,
KmP, KiP, equilibrium
(dissociation) constants.
|
|
This cycling between the ferric and ferrous forms thus plays a crucial
role in catalysis. The existence of product activation of the enzyme
explains the lag time observed in kinetics occurring under certain
conditions, especially with a high initial [Substrate]/[Product] ratio (1). During the reaction, a small fraction of the complex formed
by the ferrous enzyme and the pentadienyl radical can also dissociate,
regenerating the inactive ferrous enzyme form. A steady-state level of
Fe(II) enzyme is gradually approached.
Moreover, the position at which dioxygen is inserted defines the
regiospecificity of the enzyme. Under most conditions, soybean lipoxygenase-1 is highly specific for the insertion of dioxygen on the
C-13 atom of linoleic acid (yielding
13-HPOD1) or on the C-15 atom
of arachidonic acid yielding 15-hydroperoxyeicosatetraenoic acid (10,
11). However, this marked specificity can be modulated by certain
factors especially pH (12, 13) or substrate structure in the reaction
medium (14, 15). Almost every attempt to explain the observed
variations in specificity has been based on modifications of the
substrate (charge of the carboxylate group, for example) or of the
enzyme. A kinetic model in which the position of dioxygen insertion
proceeds through two different enzymatic pathways, the overall
specificity being a function of the
KM(O2) for each of the two
pathways, has been proposed (16). Nevertheless, this model fails to
explain the specificity modifications observed with varying pH.
In the present study, we tried to determine and explain the influence
of oxygen concentration on soybean lipoxygenase-1 specificity, in
keeping with the current kinetic model. The oxygen concentration in a
reaction medium at any given time is a function of two parameters: the
initial oxygen concentration and the rate of oxygen consumption by the
reaction itself. Thus we varied initial and continuous oxygenation
conditions (N2, O2 or air bubbling). We also
used sorbitol, a polyol which acts as soluble cosolvent. In previous studies, we have shown that such a cosolvent enhances the dioxygenation rate of soybean lipoxygenase-1 (17), and it also decreases oxygen concentration by altering its solubility. The dramatic specificity modifications observed here are discussed in terms of an expanded kinetic model.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Soybean lipoxygenase-1 was purified according to
the procedure of Axelrod et al. (18) as modified by Galey
et al. (19) and stored at
20 °C under N2.
From a nonlinear least squares fit at [LA] = 50-400 µM
of v = (Vm × [LA])/(KM + [LA]), KM and
Vm values of 39 and 144 µM
min
1, respectively, were determined with 9 nM
enzyme, corresponding to a
kcat/KM value of 6.8 × 106 M
1 s
1.
Linoleic acid (Sigma, 99%) was stored at
20 °C as a 100 mM solution in 9 × 10
3 N
NaOH + 0.7% (v/v) Tween 20 (Sigma) and prepared at 4 °C under anaerobic conditions. The concentration of hydroperoxides in this solution was estimated to be less than 0.6% of the linoleic acid concentration, as obtained by measurement of absorbance at 234 nm (see
below).
13-HPOD was prepared as described by Gibian et al. (20) but
purified with a 0.7-ml C18 Sep-Pak® Cartridge (Waters
Associates, Millipore) and eluted with methanol. The product was stored
at
20 °C as a 10 mM solution in methanol.
3-(2-bromoacetamido)-PROXYL, free radical, was from Aldrich.
-Carotene (Sigma) was prepared as described in Ref. 21 and stored at
20 °C as a 0.23 mM stock solution, assuming
460 nm = 1.39 × 105
M
1 s
1 (22).
Kinetic Measurements--
Dioxygenation reactions were performed
at 25 °C in 0.1 M
Na4P2O7, pH 9.0, buffer containing
3.2 × 10
3 % (v/v) Tween 20 and the desired
substrate concentration. The buffer was saturated by bubbling with
either air, O2, or N2. Oxygen concentration in
the buffer was respectively 228, 1140, and <5 µM (Clark
electrode detection limit). The reaction was followed either by a
polarographic method suitable for measuring pO2
variations even in buffers containing 2 M sorbitol (23) or
by a spectrophotometric method.
pO2 measurements were made with a Clark
electrode covered with a propylene membrane (Radiometer, Denmark) in a
hermetic glass reaction vessel purchased from Tacussel. Reaction buffer
volume was 20 ml. Spectrophotometric measurements were performed by
following A234 nm, using an
of 25,000 M
1 cm
1. In both cases, the
final soybean lipoxygenase-1 concentration was 9.3 nM
unless otherwise specified.
EPR Spectroscopy--
EPR spectra were obtained at room
temperature using a Bruker EMX spectrometer with a Bruker ER041 XG
Microwave Bridge (X-Band). Spectra were collected and analyzed on a
personal computer using WinEPR software (Bruker). The conditions were
as follows: microwave frequency, 9.79 GHz; microwave power, 10.06 milliwatts; modulation amplitude, 0.40 G; receiver gain, 6.32 × 103.
100 ml of 0.1 M
Na4P2O7, pH 9.0, buffer containing
3.2 × 10
3 % (v/v) Tween 20; 29 µM
spin label (3-(2-bromoacetamido)-PROXYL) and 625 µM
linoleic acid were saturated by bubbling with N2 for 30 min. The reaction was initiated by adding soybean lipoxygenase-1 (20 nM final concentration) and carried out under
N2 bubbling.
-Carotene Cooxidation--
100 ml of 0.1 M
Na4P2O7, pH 9.0, buffer containing
3.2 × 10
3 % (v/v) Tween 20, the chosen substrate
concentration, and 7.5 nM soybean lipoxygenase-1 were
loaded with a Masterflex® pump (Cole-Parmer) at minimum
flow on a tangential ultrafiltration device (Amicon H1P10-43).
Retentate (>10 kDa, i.e. enzyme) was recycled into the
substrate and buffer reactor, and filtrate (<10 kDa, i.e.
substrate, product, and derived species) was directly flowed into a
-carotene solution circulating as a closed circuit into a
spectrophotometer with a Gilson Minipuls 2 pump at maximal flow.
Absorbance variations caused by
-carotene cooxidation or dilution
were thus recorded continuously at 460 nm without contact between
enzyme and
-carotene.
Absorbance variations caused by dilution by the filtrate of the
-carotene solution were calculated as
Adilution = (A0 × V0)/(V0 + (X × t)) with A0,
initial absorbance; V0, initial
-carotene volume (ml); and X, filtrate flow into the
-carotene tank
(ml/min). Variations caused by
-carotene cooxidation were calculated
as
A
car = Ameasured + (A0
Adilution).
Hydroperoxide Characterization--
An isocratic, normal-phase
high performance liquid chromatography method was applied using a
µPorasil column (Waters, 3.9 × 300 mm). Elution solvent
consisted of hexane/diethylether/acetic acid (980/20/1) (v/v). The
injection volume was 20 µl with a flow rate of 1 ml/min. Detection
was performed at 234 nm with a Waters 481 Lambda-Max
spectrophotometer.
Numerical Simulations--
Numerical simulations and parameter
estimations were performed with Matlab® software (Math
Works Inc.) on a 670 MP Sun server (Division Mathematiques Appliquées, Université de Compiègne, France). The set
of differential equations describing the time-dependent
variations of the various species was integrated using a Gear
algorithm, suitable for integrating stiff equations. Optimization of
the parameter values was achieved by minimizing (spline method) a
"cost" function, defined as the sum of the squared differences
between experimental observations and model predictions.
Both O2 and hydroperoxide time-dependent
concentrations were recorded for each experiment (and simultaneously
taken into account for the cost function calculation), except for
experiments carried out under high hydroperoxide initial
concentrations, where initial A234 nm values
were too high to allow spectrophotometric assays. Nine experimental
conditions differing in terms of initial product and substrate
concentration were chosen. Soybean lipoxygenase-1 concentrations ranged
from 7.7 to 8.0 nM, and the first minute of reaction was
recorded. Each experiment was performed in triplicate. For further
details concerning parameter estimations, see Refs. 1, 7, and 31. The
complete mathematical treatment of parameter estimations and numerical
simulations is also available upon request to the authors.
 |
RESULTS |
Regiospecificity Variations During the Reaction--
When initial
oxygen concentrations are smaller than substrate concentrations, the
reaction of soybean lipoxygenase-1 shows three distinct phases (Fig.
1): an oxygen consumption phase (OC) due
to the enzymatic reaction, followed by a pseudo-stationary phase (SP)
where apparent [O2]
0, and a headspace oxygen
dissolution phase (DP). The last phase starts when the enzymatic
reaction is finished and is due to the dissolution in the reaction
medium of oxygen present in the headspace of the reaction vessel. At the end of the first phase OC (Table I,
panel A), specificity is quite similar to that usually
described (13-HPOD:9-HPOD = 95:5), but at the end of the
pseudo-stationary phase (SP), the 9-HPOD percentage relative to
([9-HPOD] + [13-HPOD]) slightly increases (13-HPOD:9-HPOD = 89:11). This variation in specificity is more pronounced when 2 M sorbitol is added to the buffer (13-HPOD:9-HPOD = 82:18 at the end of the SP, Table I, panel A).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Typical course of a dioxygenation reaction
initiated by soybean lipoxygenase-1 (LOX), under oxygen concentrations
limiting relative to substrate as followed by a Clark electrode.
[LA] = 400 µM. The three phases are OC, SP, and
DP.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Soybean lipoxygenase-1 specificity with air (panels A and B,
[O2]initial = 228 µM) or oxygen (panel
C, [O2]initial = 1140 µM) bubbling
before the beginning of the reaction
Panel A, [LA]initial = 300 µM; panel B,
[LA]initial = 190 µM; panel C,
[LA]initial = 800 µM. The column labeled
"Reaction phase" refers to the reaction phase at the end of which
the 13:9 ratio has been determined.
|
|
When oxygen concentrations are not limiting relative to substrate
concentrations (i.e. [O2]initial > [S]initial), this specificity variation is not
observed (Table I, panels B and C). Under these conditions, specificity is independent of the reaction phase or the
presence of sorbitol. Fig. 2 shows that
the higher the [S]/[O2] ratio, the greater the change
in specificity toward 9-HPOD formation at the end of the SP; under high
oxygen concentrations (continuous oxygen bubbling throughout the
reaction), the specificity is not altered (Fig. 2,
). Moreover, the
specificity varies with substrate concentration; the 9-HPOD percentage
increases with increasing linoleic acid concentrations (Fig. 2).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Regiospecificity of the dioxygenation
reaction at the end of the pseudo-stationary phase (SP) without
bubbling ( ) or with continuous bubbling of the buffer throughout the
reaction with either air ( ) or oxygen ( ). 9-HPOD(%) refers
to 9-HPOD percentage relative to ([13-HPOD] + [9-HPOD]).
|
|
Incubation of a 13-HPOD:9-HPOD mixture in an initial 95:5 ratio,
without substrate and with or without enzyme, does not change the
isomeric composition of the product (data not shown). Thus the
regiospecificity modification is not caused by an
enzyme-dependent or -independent isomerization of the
product. Neither can it be due to linoleic acid autoxidation, since a
2-h incubation of buffer containing substrate but no enzyme failed to
give rise to HPOD amounts comparable to those observed here (data not
shown).
Hence, the observed variation of specificity occurs when oxygen
concentration is limiting relative to substrate. Assuming a
stoichiometry of 1:1 during the OC phase between oxygen and substrate
enzymatic consumption, some untransformed substrate remains at the
beginning of the SP when [O2]initial < [S]initial. Thus, the specificity variation is related to
the presence of residual substrate at the beginning of the second phase
(SP).
Furthermore, when N2 is flushed in the reactor headspace at
the beginning of SP, this specificity variation is not observed (Fig.
3). This implies that the specificity
alteration occurs when the conditions are not completely anaerobic
during SP. During this phase, the apparent oxygen concentration
represents the difference between the oxygen dissolution rate and the
enzyme activity rate. As shown in Table
II, except for pure oxygen bubbling in
buffer, oxygen dissolution from the reactor headspace into the buffer is always slower than its consumption by the reaction, resulting in
apparently null oxygen concentrations (Fig. 1). This implies that the
oxygen concentration during the SP allows some dioxygenation to occur.
In fact, oxygen concentration during the SP is actually stationary as
the enzymatic reaction is not really over before the beginning of the
DP. For this reason, the duration of the SP increases with substrate
concentration (data not shown).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Regiospecificity of the dioxygenation
reaction at the end of the oxygen consumption phase OC ( ) or at the
end of the stationary phase SP with N2 flushing the reactor
headspace at the beginning of SP ( ). [LA]initial = 300 µM; [O2]initial = 230 µM.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Initial rates of oxygen dissolution and enzyme reaction in air- or
oxygen-saturated buffer with or without 2 M sorbitol as determined by pO2 measurements (Clark electrode)
|
|
Table II also shows that oxygen dissolution rates are approximately 10 times lower when 2 M sorbitol is added to the buffer. As
the enzymatic reaction rate with added sorbitol is higher (17), the
available oxygen concentration during the pseudo-stationary phase is
decreased in the presence of this polyol. This would account for the
greater specificity variation at the end of SP, when 2 M
sorbitol is added in the buffer.
Lipoxygenase Specificity at Low Oxygen Concentrations--
When
the reaction buffer is bubbled with N2 before the reaction
(i.e. [O2]initial < 5 µM), soybean lipoxygenase-1 catalyzes the oxygenation
reaction with a specificity dramatically different from that observed
with higher oxygen concentrations (Fig.
4); the enzyme produces almost equal
amounts of 13- and 9-HPOD throughout the reaction course (13:9
ratio
55:45). This ratio is similar to that observed in the
auto-oxidation reaction (24).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Regiospecificity of the dioxygenation
reaction initiated at low initial oxygen concentration (initial
N2 bubbling) in buffer ( ) or 2 M sorbitol
containing buffer ( ). [LA]initial = 300 µM; [O2]initial < 5 µM.
|
|
Assuming that all the substrate has been transformed at the beginning
of the headspace oxygen dissolution phase (DP) and that the substrate
is transformed during the first phase (OC) with a 13:9 ratio = 95:5, the specificity occurring during the SP phase of a reaction
initiated at oxygen concentrations limiting relative to substrate, has
been estimated at
|
(Eq. 1a)
|
where (%9-HPOD)SP is the calculated 9-HPOD percentage
relative to ([13-HPOD] + [9-HPOD]) occurring during the
pseudo-stationary phase SP; [S]i is the initial linoleic acid
concentration; (%9-HPOD)DP is the 9-HPOD percentage
relative to ([13-HPOD] + [9-HPOD]) observed at the beginning of the
headspace oxygen dissolution phase DP (end of the reaction)
[O2]i is the initial oxygen concentration.
Fig. 5 shows that the calculated
(%9-HPOD)SP, i.e. the calculated specificity
occurring during the SP phase of a reaction initiated at limiting
oxygen concentrations relative to substrate, is approximately the same
as the 9-HPOD percentage observed during a reaction carried out under
low initial oxygen concentration (Fig. 4). During SP, soybean
lipoxygenase-1 would catalyze the dioxygenation reaction with a
ratio
50:50.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Observed specificity at low initial oxygen
concentration (<5 µM) ( ) or calculated specificity
during the reaction phase SP of a reaction initiated at
[O2] = 230 µM ( ). The calculated % 9-HPOD ((%9-HPOD)SP) is estimated on the basis of the
specificity at the end of the reaction as described under
"Results."
|
|
Detection of a Free Radical Released from the Enzyme at Low Oxygen
Concentration--
According to the most widely accepted mechanism (1,
6, 7), presented in the upper part of Scheme 1, it has been
hypothesized that the ES· complex could dissociate instead of
associating with O2, leading to regeneration of the ferrous
enzyme (E). Together with this regeneration, a pentadienyl radical
(S·) would be released in the reaction medium (1, 7). This hypothesis has recently been strengthened by the demonstration that
hydrogen abstraction from the substrate at the enzyme catalytic site
occurs before molecular oxygen enters the reaction (25). We thus sought
the presence of a substrate-based free radical species, released from
the enzyme at low oxygen concentration.
The production of a free radical under low oxygen concentration has
been demonstrated by EPR spectroscopy. The EPR spectrum of 29 µM spin label in the presence of linoleic acid in a
buffer saturated by bubbling with N2 for 30 min is shown in
Fig. 6A. The reaction was
initiated by adding soybean lipoxygenase-1 and carried out under
N2 bubbling. After a 3 min reaction time, the spin label
EPR signal has totally disappeared, as shown in Fig. 6B. The
EPR signal of the spin label is stable for more than 6 h in the
same environment in the absence of enzyme (data not shown). The
disappearance of the EPR signal can thus only be attributed to spin
label silencing by an enzyme-produced free radical.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
EPR spectra of 3-(2-bromoacetamido)-PROXYL,
29 µM, in 100 ml of 0.1 M
Na4P2O7, pH 9.0, buffer containing
625 µM linoleic acid, saturated by bubbling with
N2 for 30 min before the reaction (A) and at 3 min after initiation of the dioxygenation reaction by 20 nM
soybean lipoxygenase-1 (B). The buffer was
equilibrated by N2 bubbling throughout the reaction.
|
|
This data shows that even at low oxygen concentrations (<5
µM), a radical species is formed by the enzyme. It also
shows that during the soybean lipoxygenase-1 reaction, substrate
deprotonation occurs before oxygen enters the reaction, in agreement
with recently published data (25).
We also used
-carotene bleaching to detect the presence of a free
radical dissociated from the enzyme. Soybean lipoxygenase-1 is known to
catalyze cooxidation (bleaching) of
-carotene in the presence of
linoleic acid (21) resulting in a decrease in
-carotene absorbance
at 460 nm. This bleaching is caused by soybean lipoxygenase-1-produced
free radicals derived from substrate. To separate the enzyme from
-carotene and thus detect the release of free radical species by the
enzyme, the reaction medium containing substrate and enzyme was pumped
into a tangential ultrafiltration device. The retentate (enzyme) was
recycled into the reaction vessel, and the filtrate (substrate,
product, and derived species) was directly flowed into a
-carotene
solution circulating as a closed circuit into a spectrophotometer. Thus
lipoxygenase is never in contact with the pigment, the bleaching of
which can only be ascribed to the presence of free radicals not bound
to the enzyme (see "Experimental Procedures"). Under these
conditions, Fig. 7 shows that at low
oxygen concentrations (N2 bubbling), the absorbance
variation is lower than that caused by
-carotene dilution,
indicating the release by the enzyme of free radical species not
detectable under higher oxygen concentrations (air bubbling) nor in the
absence of enzyme at low oxygen concentrations (data not shown).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
Absorbance variations during -carotene
cooxidation by the filtrate of a tangential ultrafiltration device
separating soybean lipoxygenase-1 (retentate) from free substrate,
product and derived species (filtrate) during the course of the
dioxygenation reaction (see "Experimental Procedures").
Reaction carried out under N2 ( ) or air ( ) bubbling.
[LA]initial = 1 mM;
[ -carotene]initial = 3 µM; [soybean
lipoxygenase-1] = 7.5 nM.
|
|
Construction and Derivation of the Kinetic Model--
According to
the current working kinetic scheme proposed by Schilstra et
al. (1) and presented in the upper part of Scheme 1, soybean
lipoxygenase-1 is obtained when purified as a ferrous form (E). This
form is catalytically inactive but can bind substrate (S;
e.g. linoleic acid), thus leading to the observed substrate inhibition. Upon binding a molecule of product (hydroperoxide), this
ferrous form is activated to the ferric, catalytically active form
(E*). This form is assumed to bind either product (leading to product
inhibition) or substrate leading to an E*S complex. The
enzyme-catalyzed substrate deprotonation then leads to a complex formed
by ferrous enzyme and substrate free radical (probably pentadienyl),
ES·. The position of this deprotonation is tightly regulated by
the enzyme; with substrates presenting several pentadiene systems (e.g. arachidonic acid), one system is preferentially
deprotonated depending on the enzyme isoform. The ES· complex
then binds O2, which is specifically added by the enzyme to
either the C-1 or C-5 carbon of the pentadiene system. This regio- (and
stereo-) specificity of enzyme-catalyzed O2 insertion is
the basis of the enzyme specificity. In the case of soybean lipoxygenase-1, the specificity observed under nonlimiting oxygen concentrations relative to substrate (13:9 ratio = 95:5), reflects the specificity with which the enzyme catalyzes specifically the insertion of O2 on the C-13 carbon. In this case, both the
position of substrate deprotonation and O2 insertion are
enzyme controlled. This pathway will thus be referred to as "fully
enzymatically" controlled.
To explain the observed change in specificity at low oxygen
concentrations, the current working kinetic model was expanded (Scheme
1). This model is based on the model presented by (1) but assumes that
the ES· complex can dissociate (as a function of the kinetic
constant k4), leading to the release of a
pentadienyl free radical S· in the buffer (Scheme 1, lower
part). Our contribution to the model is to hypothesize that this
highly reactive radical, once released, could combine with oxygen to
form SO2·. This free radical, as in the classical
pentadienyl system auto-oxidation scheme (26, 27), would then associate
with either itself leading to termination products (e.g.
linoleic acid dimers), or with a substrate molecule leading to
S· (which contributes to SO2· regeneration)
and a hydroperoxide product, here designated as Q.
During the formation of the hydroperoxide Q, only substrate
deprotonation is enzyme controlled but not the O2 insertion
step. Oxygen thus is added in a nonspecific manner, and the
13-HPOD:9-HPOD composition of Q is close to 50:50. This
pathway to hydroperoxide production is therefore only
"semienzymatically" controlled. P and Q are
both linoleic acid hydroperoxides but show different regio-isomeric
compositions; they have been artificially differentiated in this scheme
only to account for specificity observations. For purposes of
simplicity, it has furthermore been considered that [Q]
KiP, so that product inhibition by
Q is not taken into account.
The rates expressing Scheme 1 were derived and simulated to ensure that
at high oxygen concentrations the fully enzymatic pathway becomes
dominant, leading to marked specificity for 13-HPOD, whereas under low
oxygen concentrations, the semienzymatic pathway is preferentially
expressed leading to a 13-HPOD:9-HPOD ratio close to 50:50.
Equations 1-10, describing Scheme 1, were obtained with steady-state
assumptions, rapid equilibrium assumptions also being made for some
segments, and derived as described in Ref. 7.
|
(Eq. 2)
|
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
where
|
(Eq. 6)
|
|
(Eq. 7)
|
|
(Eq. 8)
|
|
(Eq. 9)
|
|
(Eq. 10)
|
With the steady state approximation, i.e.
|
(Eq. 11)
|
the system (1-9) simplifies to
|
(Eq. 12)
|
|
(Eq. 13)
|
|
(Eq. 14)
|
where
|
(Eq. 15)
|
|
(Eq. 16)
|
|
(Eq. 17)
|
|
(Eq. 18)
|
and
|
(Eq. 19)
|
Equations 14-17 show four different types of rates; two
"hydroperoxidase" rates (
hP and
hQ)
corresponding to the rate at which P andQ,
respectively, are consumed by the enzyme activation, and two
dioxygenation rates (
OE and
OSE) corresponding,
respectively, to the rate at which the fully enzymatic and
semienzymatic reactions occur. Thus
OE represents the rate
concerning the appearance of P, and
OSE that concerning the
appearance of Q.
To simulate these rates, the kinetic parameters have been estimated.
The value of KmS is based on the value of KM for linoleic acid (see "Experimental Procedures"). The values for
k5 and k7 are based on
those found in the literature for linoleate auto-oxidation (27,28). All
other parameters have been estimated as described under "Experimental
Procedures" and the corresponding values given in Table
III. Except for k2
and k6, the determined values agree well with
previously reported results (see Table III). The values for
k2 and k6 are much higher
than those previously reported (1,29,30,31). The significance of these
high values is commented on under "Discussion." The different
simulations carried out using the estimated values of Table III fit
well to the observed data as shown in
Fig.8. As can be seen from Fig.8,
B and C, the fit of the simulation to the
observed time-dependent [O2] variations is
slightly worse than to [HPOD] variations. Indeed, the polarographic assay used to follow [O2] variations is an accurate
method to determine relative [O2] variations (as rate
measurement) but is less reproducible when measuring absolute
[O2]. This can also be seen from the high standard
deviations concerning [O2]inFig.8, B and
C. The simulation of d[P]/dt and d[Q]/dt
versus [O2]initial clearly shows
that d[Q]/dt is much less dependent on [O2] than d[P]/dt (Fig.9). At high
[O2],P appearance is largely predominant, whereas at low
[O2] (<5µM),Q becomes the major product,
as a result of the very low P appearance rate.
View this table:
[in this window]
[in a new window]
|
Table III
Estimated values of the kinetic parameters for the model shown in
Scheme 1
Values determined by other authors are indicated. The numbers in
parentheses indicate the reference in which the value is cited. Each
parameter has been estimated according to the method described under
"Experimental Procedures," except k5 and
k7 for which the following values have been used:
5.4 × 108 (28) and 107 (27), respectively. The
KmS value is based on the estimated value of
KM for linoleic acid (see "Experimental Procedures").
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 8.
Comparison of experimental data and
simulations carried out using the estimated values of the kinetic
parameters shown in Table III. Vertical lines represent
standard deviations. A, evolution of the initial rate of
oxygen consumption as a function of linoleate concentration for
experimental data ( ) and model simulation (solid line).
[soybean lipoxygenase-1] = 9.3 nM;
[O2]initial = 230 µM.
B, experimental time-dependent variations in
oxygen ( ) or HPOD ( ) concentration, and corresponding model
simulation (solid line). [soybean lipoxygenase-1] = 7.7 nM, [O2]initial = 245.8 µM, [LA]initial = 43.6 µM,
[HPOD]initial = 16.7 µM. C,
experimental time-dependent variations in oxygen
concentration ( ) and respective model simulation (solid
line). [soybean lipoxygenase-1] = 7.98 nM,
[O2]initial = 236.9 µM,
[LA]initial = 22.4 µM,
[HPOD]initial = 126.4 µM. Note that as
P and Q (cf. Scheme 1) or 13-HPOD and 9-HPOD cannot be distinguished experimentally when measuring enzyme kinetics, [HPOD] here refers to the total amount of hydroperoxides produced, i.e. [P] + [Q] or [13-HPOD] + [9-HPOD].
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
Simulation of the initial rates
d[P]/dt (dotted line) and
d[Q]/dt (solid line) as a function of
[O2] with [LA] = 50 µM (A) or
300 µM (B). [soybean lipoxygenase-1] = 9.3 nM.
|
|
The 9-HPOD percentage relative to ([13-HPOD] + [9-HPOD]) can
be simulated with Equations 3 and 4, i.e. on the basis of
the rates for P and Q formation and their
hypothesized regio-isomeric composition (13:9 ratio = 95:5 and
50:50 for P and Q, respectively) with
|
(Eq. 20)
|
When calculated according to Equation 19 (Fig.
10), the 9-HPOD percentage tends to be
about 6-7% with high (>200 µM) oxygen concentrations,
whereas the 13-HPOD:9-HPOD ratio tends to be 50:50 at low oxygen
concentrations (
55:45 at [O2] = 5 µM).
The results and simulations presented here are therefore in good
agreement with the observed data and the hypothesis presented
above.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 10.
Simulation of % 9-HPOD as a function of
[O2] with [LA] = 50 µM (a) or
300 µM (b). % 9-HPOD is estimated on
the basis of the initial rates d[P]/dt and
d[Q]/dt as described under "Results." [soybean
lipoxygenase-1] = 9.3 nM.
|
|
 |
DISCUSSION |
The results presented here show that regiospecificity of soybean
lipoxygenase-1 strongly depends upon oxygen concentration. Furthermore,
this altered specificity can be expressed during a reaction with
"classical" conditions, i.e. with a buffer equilibrated with air before the reaction initiation.
These data suggest that when the lipoxygenase reaction is carried out
under oxygen concentrations limiting relative to substrate, dioxygenation specificity varies with the reaction course. During the
first phase (OC), the initial oxygen concentration is high, and the
13-HPOD:9-HPOD ratio is 95:5. During the second phase (SP), the oxygen
concentration is low, and the remaining substrate (not transformed
during the first phase) is dioxygenated with a 13-HPOD:9-HPOD
ratio
50:50.
To account for these experimental findings, we have expanded upon the
model presented by Schilstra et al. (1), which is based on
that proposed by Ludwig et al. (7). In this model, the
ES· complex can dissociate into E and S·. The occurrence
of such a dissociation is highly probable, since we have shown that the
enzyme releases substrate-based free radical species under low oxygen
concentrations. Our contribution is to specify that the released
pentadienyl radical S· can associate with O2,
leading to formation of a hydroperoxide in a nonspecific manner. The
model thus obtained presents two product appearance pathways with
different regio-isomeric composition: 13:9 ratio = 95:5 or
50:50. The equations describing the kinetic scheme have been derived
and simulations clearly show that the ratio between these two competing
pathways, and thus the overall regiospecificity, is dependent upon
oxygen concentration.
The values of most of the estimated kinetic parameters are highly
correlated with previous studies based on the basic kinetic scheme
proposed by Schilstra et al. (1), where parameters were estimated with a mathematical treatment similar to that used here (1,
29, 31). One exception nevertheless is the value of the parameter
k2, which describes the enzyme-catalyzed
substrate deprotonation step. This parameter is two orders of magnitude higher than that determined in other studies (29, 31). Furthermore, the
bimolecular rate constant k6, which expresses
the propagation of the semienzymatic pathway (leading to Q)
has been estimated in the present study at 23.5 × 103
M
1 s
1. For the auto-oxidation
reaction, the parameter describing the propagation of the free radical,
nonenzymatically catalyzed reaction has been reported to be
approximately 62 M
1 s
1 (30),
which is 400 times lower than the value determined here. As a result,
the k2 value also increases to account for the
rate of Q production. Nevertheless, during auto-oxidation,
the reaction is only initiated by linoleate deprotonation, and
maintained by the propagation steps. In our model, the semienzymatic
way is not only initiated by the k2 step, but
this step also maintains the reaction and provides S· radicals
throughout the reaction. The reaction is therefore different from a
classical free radical reaction.
Modification of soybean lipoxygenase-1 regiospecificity at low oxygen
concentrations has already been reported. In conditions where
O2 concentration is limiting relative to substrate,
regiospecificity variations have been demonstrated with rabbit
reticulocyte lipoxygenase (7) and presented as a support for the
validity of the kinetic model. Our data are consistent with these
observations, but the amplitude of the effect is much larger with
soybean lipoxygenase-1; at low oxygen concentrations, the observed
13-HPOD:9-HPOD ratio is approximately 50:50.
Nevertheless, this work is the first to attempt to explain this
phenomenon and to incorporate its mechanism into the catalytic scheme
of soybean lipoxygenase-1. Moreover, this new completed scheme implies
two important characteristics of lipoxygenase catalysis.
First, it implies that, during lipoxygenase catalysis, the lipid
substrate is deprotonated before molecular oxygen enters the
reaction. Interestingly, this new feature has recently been demonstrated (25).
Furthermore, this model involves a branched mechanism that gives rise
to two products that are not distinguishable experimentally when
measuring enzyme kinetics. In a recent study (32), it has been
considered that such a mechanism would seem unlikely, because the
branch in the mechanism would be isotopically insensitive. The overall
kinetic isotope effect would result from both pathways (insensitive and
sensitive) and thus be lowered. This would be in contrast to the
extremely large isotope effect observed during soybean lipoxygenase-1
catalysis (32).
In the model presented here (Scheme 1), the steps described by
k2 and k3 have been shown
to be isotopically sensitive (32), but the step described by
k4 (i.e. the proposed branch) is not isotopically sensitive as it does not involve proton exchange at the
enzyme catalytic site. Since the ratio between the steps described by
k3 (leading to P) and
k4 (leading to Q) is oxygen dependent
in our scheme (as shown in Fig. 9), the contribution of the insensitive
pathway to the overall isotope effect should be very low at high oxygen
concentrations, leading to a strong kinetic isotope effect under these
conditions.
With decreasing oxygen concentrations, the ratio between the sensitive
and insensitive pathway is modified in favor of the insensitive one so
that the latter becomes predominant when [O2] < 5 µM. This model therefore predicts an increase in the
kinetic isotope effect with increasing oxygen concentration, which has actually been observed (25, 32). The branched model presented here can
therefore explain the magnitude of the observed isotope effect.
We thank Dr S. Mottelet (Division
Mathematiques Appliquées, Université de Compiègne,
France) for precious help concerning the Matlab® program
for parameter estimations, and R. Sousa Yeh, professional scientific
translator (Paris, France), for critically reviewing the language of
the manuscript. We also are grateful to Prof. M. Le Meste (ENS-BANA,
Dijon, France) for the use of the EPR spectrometer and helpful advice
on spectra interpretation.