On the role of molecular oxygen in lipoxygenase activation: comparison and contrast of epidermal lipoxygenase-3 with soybean lipoxygenase-1 - PubMed (original) (raw)
Comparative Study
. 2010 Dec 17;285(51):39876-87.
doi: 10.1074/jbc.M110.180794. Epub 2010 Oct 5.
Affiliations
- PMID: 20923767
- PMCID: PMC3000969
- DOI: 10.1074/jbc.M110.180794
Comparative Study
On the role of molecular oxygen in lipoxygenase activation: comparison and contrast of epidermal lipoxygenase-3 with soybean lipoxygenase-1
Yuxiang Zheng et al. J Biol Chem. 2010.
Abstract
The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O(2) concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O(2) in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O(2) with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O(2) within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity.
Figures
SCHEME 1
FIGURE 1.
Increased O2 concentration or the A451G mutation shortened the lag phase in the reaction of eLOX3 with 9_E_,11_Z_,14_Z_-20:3ω6. Note that this synthetic fatty acid contains a conjugated diene with strong 235 nm absorbance, whereas the oxygenation product has a conjugated triene absorbing at 270 nm (15); consequently, reaction is associated with an absorbance decrease at 235 nm. A, reaction progress curves monitored at 235 nm. Reaction was conducted in sodium phosphate buffer, pH 7.5, at room temperature and was started by addition of enzyme. Wild-type eLOX3 concentration is 0.10 μ
m
. A451G eLOX3 concentration is 0.13 μ
m
. 9_E_,11_Z_,14_Z_-20:3 ω6 concentration is 18–20 μ
m
. B, statistical bar representation of the lag phase duration of the reactions in A. The lag phase duration is defined as the time axis intercept of a straight line through the portion of the reaction progress curve where the rate is maximal (10). The data are presented as the mean ± S.D. For each of the reactions of wild-type eLOX3 in air- and O2-saturated buffer, n = 4. For the reaction of A451G eLOX3 in air-saturated buffer, n = 3. p < 0.01 for every pair of reactions.
FIGURE 2.
Increased O2 concentration or the A451G mutation decreased the rate of the reaction of eLOX3 with 13_S_-HPODE. A, reaction progress curves monitored at 235 nm. Reaction was conducted in sodium phosphate buffer, pH 7.5, at room temperature and was started by addition of enzyme. Wild-type eLOX3 concentration is 0.25 μ
m
. A451G eLOX3 concentration is 0.32 μ
m
. Note that the oxygenation rate of wild-type eLOX3 at 0.25 μ
m
is similar to the oxygenation rate of A451G eLOX3 at 0.32 μ
m
(Fig. 1). 13_S_-HPODE concentration is 52–55 μ
m
. B, statistical bar representation of the initial rate of the reactions in A. The data are presented as the mean ± S.D. For each of the reactions of wild-type eLOX3 in air- and O2-saturated buffer, n = 4. For the reaction of wild-type eLOX3 under anaerobic conditions, n = 3. For the reaction of A451G eLOX3 in air-saturated buffer, n = 3. p < 0.01 for every pair of reactions.
FIGURE 3.
RP- and SP-HPLC analysis of the products from the reactions with 13_S_-HPODE. Analysis of wild-type eLOX3 (A–C and F–H), A451G eLOX3 (D and I), and soybean LOX-1 following reaction with 13_S_-HPODE (E and J) is shown. For wild-type eLOX3, O2 concentration was varied in the incubation, from 0 (A and F) to 240 μ
m
(B and G) to 1.2 m
m
(C and H). For A451G eLOX3 and soybean LOX-1, NDGA was included in the incubation. RP-HPLC analysis used a Waters Symmetry C18 column (0.46 × 25 cm), a flow rate of 1 ml/min, and a solvent system of methanol/water/acetic acid (80:20:0.01, by volume). SP-HPLC analysis used a Beckman Silica Ultrasphere column (0.46 × 25 cm), a flow rate of 1 ml/min and a solvent system of hexane/isopropyl alcohol/acetic acid (100:2:0.02). *, control experiments indicated that this peak was not an enzymatic product.
FIGURE 4.
GC-MS analysis of products I and II. Mass spectra of the TMS ether methyl ester derivative of the TPP-reduced major products (product I and II in Fig. 3) from the reaction of soybean LOX-1 or A451G eLOX3 with 13_S_-HPODE in the presence of NDGA are shown. A, product I; B, product II.
SCHEME 2
FIGURE 5.
Effect of NDGA on the rates of reaction with 13_S_-HPODE. NDGA (20 μ
m
) stimulated the reaction of soybean LOX-1 (0.06 μ
m
A), A451G eLOX3 (0.32 μ
m
, B), or wild-type eLOX3 (0.25 μ
m
, C) with 13_S_-HPODE (55 μ
m
). The incubations were performed in sodium phosphate buffer, pH 7.5, and initiated by addition of enzyme. NDGA was added at the indicated point. For the reaction of A451G eLOX3 or soybean LOX-1, a second equal aliquot of enzyme was added at the indicated point.
FIGURE 6.
RP-HPLC analysis of the products from the reactions with 15_S_-HPETE. Products from reactions of 15_S_-HPETE with A451G eLOX3 (A), soybean LOX-1 (B), and wild-type eLOX3 (C) are shown. RP-HPLC analysis used a Waters Symmetry C18 column (0.46 × 25 cm), a flow rate of 1 ml/min, and a solvent system of methanol/water/acetic acid (80:20:0.01, by volume).
FIGURE 7.
Progress curves of the anaerobic reactions of A451G eLOX3 (0.23 μm) with 13_S_-HPODE (90 μm). Reactions were conducted in the absence or presence of fatty acids (arachidonic acid or methyl arachidonate, 70 μ
m
) in sodium phosphate buffer, pH 7.5, started by addition of enzyme, and monitored at 280 nm. The anaerobic conditions are described under “Experimental Procedures.”
FIGURE 8.
RP-HPLC analysis of the monomeric products from the anaerobic reaction of A451G eLOX3 with 13_S_-HPODE and arachidonic acid. RP-HPLC analysis used a Waters Symmetry C18 column (0.46 × 25 cm), a flow rate of 1 ml/min, and a solvent system of methanol/water/acetic acid (80:20:0.01, by volume).
FIGURE 9.
RP-HPLC-UV and LC-ESI-MS analysis of the dimer products from the anaerobic reaction of A451G eLOX3 with 13_S_-HPODE and arachidonic acid. RP-HPLC analysis used a Phenomenex C18 column (2.6 μm, 0.3 × 10 cm), a flow rate of 0.3 ml/min, and a solvent system of methanol/water (90:10 by volume) containing 10 m
m
ammonium acetate. MS analysis detected negative ions in the range from m/z 500 to 650. Under these conditions, arachidonic acid eluted at 3.0 min (data not shown).
FIGURE 10.
Mass spectra of 15-HETE formed in the reaction of A451G eLOX3 with 13_S_-HPODE and arachidonic acid. A, reaction with [16O]13_S_-HPODE in H216O. B, reaction with [18O]13_S_-HPODE in H216O. C, reaction with [16O]13_S_-HPODE in 83% H218O. The mass spectra were obtained in LC-ESI-MS analysis using a Phenomenex C18 column (2.6 μm, 0.3 × 10 cm), a flow rate of 0.3 ml/min, and a solvent system of methanol/water (80:20, by volume), containing 10 m
m
ammonium acetate. MS analysis detected negative ions in the range from m/z 200 to 700.
FIGURE 11.
RP-HPLC analysis of the products from anaerobic reactions with 15_S_-HPETE. A, A451G eLOX3. B, soybean LOX-1. C, wild-type eLOX3. RP-HPLC analysis used a Waters Symmetry C18 column (0.46 × 25 cm), a flow rate of 1 ml/min, and a solvent system of methanol/water/acetic acid (80:20:0.01, by volume).
FIGURE 12.
Activation of LOX from ferrous to the active ferric species by reaction with fatty acid hydroperoxide (LOOH). The initial fatty acid alkoxyl radical intermediate rearranges to an enzyme-associated epoxyallylic radical intermediate, Fe(III)-OH[L(O)•]. This enzyme-intermediate complex can cycle back to the free ferrous LOX giving epoxyalcohol product via the hydroperoxide isomerase “oxygen rebound” pathway or dissociate, resulting in the free ferric LOX available for dioxygenase catalysis. Dissociation is facilitated by reaction with O2, giving an epoxyallylic peroxy radical L(O)-OO• that cannot react with Fe(III)-OH, will thus leave the enzyme, and will be reduced in the solution to the epoxyallylic hydroperoxide products we identified (products I and II in Fig. 3). Spontaneous dissociation, almost absent with eLOX3, but more prominent with soybean LOX-1 or A451G eLOX3, under anaerobic conditions can also lead to enzyme oxidation, with the dissociated epoxyallylic radical undergoing nonenzymatic transformations in the solution to various products, including fatty acid dimers.
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