Oxygen Concentration Determines Regiospecificity in Soybean Lipoxygenase-1 Reaction Via a Branched Kinetic Scheme*

Hugues BerryDagger , Hélène DébatDagger , and Véronique Larreta Garde§

From the Dagger  Laboratory of Enzyme Technology, UPRES A 6022 CNRS, University of Compiègne, B.P. 20.529, 60205 Compiègne, France and § Errmece, Department of Life Sciences, University of Cergy-Pontoise, 2, avenue Adolphe Chauvin, 95302 Cergy-Pontoise Cedex, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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. beta -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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).


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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; SObardot 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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. beta -Carotene (Sigma) was prepared as described in Ref. 21 and stored at -20 °C as a 0.23 mM stock solution, assuming epsilon 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 epsilon  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.

beta -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 beta -carotene solution circulating as a closed circuit into a spectrophotometer with a Gilson Minipuls 2 pump at maximal flow. Absorbance variations caused by beta -carotene cooxidation or dilution were thus recorded continuously at 460 nm without contact between enzyme and beta -carotene.

Absorbance variations caused by dilution by the filtrate of the beta -carotene solution were calculated as Adilution = (A0 × V0)/(V0 + (X × t)) with A0, initial absorbance; V0, initial beta -carotene volume (ml); and X, filtrate flow into the beta -carotene tank (ml/min). Variations caused by beta -carotene cooxidation were calculated as Delta Abeta -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 [O2approx  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).


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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.

                              
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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, square ). Moreover, the specificity varies with substrate concentration; the 9-HPOD percentage increases with increasing linoleic acid concentrations (Fig. 2).


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Fig. 2.   Regiospecificity of the dioxygenation reaction at the end of the pseudo-stationary phase (SP) without bubbling (open circle ) or with continuous bubbling of the buffer throughout the reaction with either air (bullet ) or oxygen (square ). 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).


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Fig. 3.   Regiospecificity of the dioxygenation reaction at the end of the oxygen consumption phase OC (square ) or at the end of the stationary phase SP with N2 flushing the reactor headspace at the beginning of SP (black-square). [LA]initial = 300 µM; [O2]initial = 230 µM.

                              
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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 approx  55:45). This ratio is similar to that observed in the auto-oxidation reaction (24).


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Fig. 4.   Regiospecificity of the dioxygenation reaction initiated at low initial oxygen concentration (initial N2 bubbling) in buffer (square ) or 2 M sorbitol containing buffer (open circle ). [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
<FR><NU>(%9−<UP>HPOD</UP>)<SUB><UP>SP</UP></SUB></NU><DE>100</DE></FR>= (Eq. 1a)
<FENCE><FR><NU>([<UP>S</UP>]<SUB>i</SUB>×((%9−<UP>HPOD</UP>)<SUB><UP>DP</UP></SUB>/100))−([<UP>O</UP><SUB>2</SUB>]<SUB>i</SUB>×0.05)</NU><DE>[<UP>S</UP>]<SUB>i</SUB>−[<UP>O</UP><SUB>2</SUB>]<SUB>i</SUB></DE></FR></FENCE>
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 approx  50:50.


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Fig. 5.   Observed specificity at low initial oxygen concentration (<5 µM) (black-square) or calculated specificity during the reaction phase SP of a reaction initiated at [O2] = 230 µM (square ). 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.


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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 beta -carotene bleaching to detect the presence of a free radical dissociated from the enzyme. Soybean lipoxygenase-1 is known to catalyze cooxidation (bleaching) of beta -carotene in the presence of linoleic acid (21) resulting in a decrease in beta -carotene absorbance at 460 nm. This bleaching is caused by soybean lipoxygenase-1-produced free radicals derived from substrate. To separate the enzyme from beta -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 beta -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 beta -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).


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Fig. 7.   Absorbance variations during beta -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 (bullet ) or air (open circle ) bubbling. [LA]initial = 1 mM; [beta -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.
<FR><NU>d[<UP>S</UP>]</NU><DE>dt</DE></FR>=<UP>−</UP>f<SUB>2</SUB>[X<SUB>2</SUB>]−<FENCE>k<SUB>6</SUB>[<UP>S</UP>]<RAD><RCD><FR><NU>k<SUB>4</SUB></NU><DE>k<SUB>7</SUB>(k<SUB>3</SUB>[<UP>O</UP><SUB>2</SUB>]+k<SUB>4</SUB>)</DE></FR> f<SUB>2</SUB>[X<SUB>2</SUB>]</RCD></RAD></FENCE> (Eq. 2)
<FR><NU>d[<UP>O</UP><SUB>2</SUB>]</NU><DE>dt</DE></FR>=<FR><NU>d[<UP>S</UP>]</NU><DE>dt</DE></FR> (Eq. 3)
<FR><NU>d[P]</NU><DE>dt</DE></FR>=<UP>−</UP>f<SUB>1P</SUB>[X<SUB>1</SUB>]+<FENCE><FR><NU>k<SUB>3</SUB>[<UP>O</UP><SUB>2</SUB>]</NU><DE>k<SUB>3</SUB>[<UP>O</UP><SUB>2</SUB>]+k<SUB>4</SUB></DE></FR> f<SUB>2</SUB>[X<SUB>2</SUB>]</FENCE> (Eq. 4)
<FR><NU>d[Q]</NU><DE>dt</DE></FR>=<UP>−</UP>f<SUB>1Q</SUB>[X<SUB>1</SUB>]+<FENCE>k<SUB>6</SUB>[<UP>S</UP>]<RAD><RCD><FR><NU>k<SUB>4</SUB></NU><DE>k<SUB>7</SUB>(k<SUB>3</SUB>[<UP>O</UP><SUB>2</SUB>]+k<SUB>4</SUB>)</DE></FR> f<SUB>2</SUB>[X<SUB>2</SUB>]</RCD></RAD></FENCE> (Eq. 5)
where
f<SUB>1P</SUB>=<FR><NU>k<SUB>1</SUB>[P]</NU><DE>K<SUB>mP</SUB>(1+([S]/K<SUB>iS</SUB>))+[P]+[Q]</DE></FR> (Eq. 6)
f<SUB>1Q</SUB>=<FR><NU>k<SUB>1</SUB>[Q]</NU><DE>K<SUB>mP</SUB>(1+([S]/K<SUB>iS</SUB>))+[P]+[Q]</DE></FR> (Eq. 7)
f<SUB>2</SUB>=<FR><NU>k<SUB>2</SUB>[S]</NU><DE>K<SUB>mS</SUB>(1+(P/K<SUB>iP</SUB>))+[S]</DE></FR> (Eq. 8)
[X<SUB>1</SUB>]=[E]+[ES]+[EP]+[EQ] (Eq. 9)
[X<SUB>2</SUB>]=[E∗]+[E∗S]+[E∗P] (Eq. 10)
With the steady state approximation, i.e.
<FR><NU>d[X<SUB>1</SUB>]</NU><DE>dt</DE></FR>=<FR><NU>d[X<SUB>2</SUB>]</NU><DE>dt</DE></FR>=<FR><NU>d[ES<SUP>⋅</SUP>]</NU><DE>dt</DE></FR>=0 (Eq. 11)
the system (1-9) simplifies to
<FR><NU>d[S]</NU><DE>dt</DE></FR>=<FR><NU>d[<UP>O</UP><SUB>2</SUB>]</NU><DE>dt</DE></FR>=<UP>−</UP>(&ngr;<SUP>E</SUP><SUB><UP>O</UP></SUB>+&ngr;<SUP>SE</SUP><SUB><UP>O</UP></SUB>+&ngr;<SUB>hP</SUB>+&ngr;<SUB>hQ</SUB>) (Eq. 12)
<FR><NU>d[P]</NU><DE>dt</DE></FR>=&ngr;<SUP>E</SUP><SUB><UP>O</UP></SUB>−&ngr;<SUB>hP</SUB> (Eq. 13)
<FR><NU>d[Q]</NU><DE>dt</DE></FR>=&ngr;<SUP>SE</SUP><SUB><UP>O</UP></SUB>−&ngr;<SUB>hQ</SUB> (Eq. 14)
where
&ngr;<SUP>E</SUP><SUB><UP>O</UP></SUB>=<FR><NU>[X<SUB>0</SUB>]</NU><DE>D</DE></FR>(k<SUB>1</SUB>k<SUB>2</SUB>k<SUB>3</SUB>[S][<UP>O</UP><SUB>2</SUB>]([P]+[Q])) (Eq. 15)
&ngr;<SUP>SE</SUP><SUB><UP>O</UP></SUB>=k<SUB>6</SUB>[<UP>S</UP>]<RAD><RCD><FR><NU>[X<SUB>0</SUB>]</NU><DE>D</DE></FR> <FR><NU>k<SUB>1</SUB>k<SUB>2</SUB>k<SUB>4</SUB>[S]([P]+[Q])</NU><DE>k<SUB>7</SUB></DE></FR></RCD></RAD> (Eq. 16)
&ngr;<SUB>hP</SUB>=<FR><NU>[X<SUB>0</SUB>]</NU><DE>D</DE></FR>(k<SUB>1</SUB>k<SUB>2</SUB>k<SUB>4</SUB>[P][S]) (Eq. 17)
&ngr;<SUB>hQ</SUB>=<FR><NU>[X<SUB>0</SUB>]</NU><DE>D</DE></FR>(k<SUB>1</SUB>k<SUB>2</SUB>k<SUB>4</SUB>[Q][S]) (Eq. 18)
and
       [X<SUB>0</SUB>]=[X<SUB>1</SUB>]+[X<SUB>2</SUB>]+[ES<SUP>·</SUP>]
        D=<FENCE>k<SUB>2</SUB>k<SUB>4</SUB>[S]<FENCE>K<SUB>mP</SUB><FENCE>1+<FR><NU>[S]</NU><DE>K<SUB>iS</SUB></DE></FR></FENCE>+[P]+[Q]</FENCE></FENCE>
         +<FENCE>k<SUB>1</SUB>(k<SUB>3</SUB>[<UP>O</UP><SUB>2</SUB>]+k<SUB>4</SUB>)([P]+[Q])<FENCE>K<SUB>mS</SUB><FENCE>1+<FR><NU>[P]</NU><DE>K<SUB>iP</SUB></DE></FR></FENCE>+[S]</FENCE></FENCE>
         +{k<SUB>1</SUB>k<SUB>2</SUB>[S]([P]+[Q])} (Eq. 19)
Equations 14-17 show four different types of rates; two "hydroperoxidase" rates (nu hP and nu hQ) corresponding to the rate at which P andQ, respectively, are consumed by the enzyme activation, and two dioxygenation rates (nu OE and nu OSE) corresponding, respectively, to the rate at which the fully enzymatic and semienzymatic reactions occur. Thus nu OE represents the rate concerning the appearance of P, and nu 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.

                              
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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").


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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 (open circle ) and model simulation (solid line). [soybean lipoxygenase-1] = 9.3 nM; [O2]initial = 230 µM. B, experimental time-dependent variations in oxygen (open circle ) or HPOD (triangle ) 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 (open circle ) 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].


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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
%9 <UP>HPOD</UP>=<FR><NU>0.5×(d[Q]/dt)+0.05×(d[P]/dt)</NU><DE>(d[Q]/dt)+(d[P]/dt)</DE></FR> (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 (approx 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.


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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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 approx  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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

To whom correspondence should be addressed. Tel.: 33-1-34-25-66-05; Fax: 33-1-34-25-65-20; E-mail: larreta{at}u-cergy.fr.

1 The abbreviations used are: HPOD, hydroperoxyoctadecadienoic acid; 13:9 ratio, ratio between the two regio-isomer products 13-HPOD and 9-HPOD, calculated with 9-HPOD(%) = [9-HPOD] × 100/([13-HPOD] + [9-HPOD]); LA, linoleic acid; OC, oxygen consumption phase; SP, pseudo-stationary phase; DP, headspace oxygen dissolution phase; P, fully enzymatically formed hydroperoxide product; Q, semienzymatically formed hydroperoxide product; S·, pentadienyl radical; SObardot 2, peroxyl radical; k1, k2, k4, monomolecular rate constants; k3, k5, k6, k7, bimolecular rate constants; KmS, KiS, KmP, KiP, equilibrium (dissociation) constants.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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