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ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.

Smyrniotis CJ, Barbour SR, Xia Z, Hixon MS, Holman TR - Biochemistry (2014)

Bottom Line: 5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid.This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation.Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.

ABSTRACT
5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

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Effect of 200 μM ATP on the efficiency of epoxideformation.[LTA4]/[5(S)-HpETE] turnover ratio from10 μM AA substrate as measured by HPLC and UV–vis spectrophotometry.[LTA4] was measured at 280 nm and [5(S)-HpETE] at 234 nm, and both were normalized to their respectiveextinction coefficients. The absolute kinetic activity of wild-type5-LOX ammonium sulfate preparations was ≈60 μmol min–1 mg–1.
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fig3: Effect of 200 μM ATP on the efficiency of epoxideformation.[LTA4]/[5(S)-HpETE] turnover ratio from10 μM AA substrate as measured by HPLC and UV–vis spectrophotometry.[LTA4] was measured at 280 nm and [5(S)-HpETE] at 234 nm, and both were normalized to their respectiveextinction coefficients. The absolute kinetic activity of wild-type5-LOX ammonium sulfate preparations was ≈60 μmol min–1 mg–1.

Mentions: As described above, ATP activates bothAA hydroperoxidation and 5(S)-HpETE epoxidation.However, it is unclear if the epoxidation reaction affects the rateof hydroperoxidation, because of the consumption of the hydroperoxidationproduct, 5(S)-HpETE, and/or production of the 5,6-epoxide,LTA4. To investigate this possibility, a diode-array UV–visspectrophotometer was used to observe the formation of both 5(S)-HpETE (234 nm) and LTA4 (280 nm) simultaneouslyfrom 0 to 20% product formation and determine if the slight increasein the 5(S)-HpETE concentration could affect therate of hydroperoxide formation. The LTA4/5(S)-HpETE ratio of products (the efficiency of epoxide formation) wascalculated throughout the reaction and observed to remain constantup to 20% product formation [LTA4/5(S)-HpETEratio of 0.161 ± 0.004 (Figure 3)]. Withthe addition of 200 μM ATP, this value increased to a constantvalue of 0.247 ± 0.005 (Figure 3) andmatched the value determined for rat PMNL 5-LOX.40 In addition, 5(S)-HETE and 5,12-DiHETEconcentrations were quantified via HPLC and comparable product ratioswere observed [0.19 ± 0.02 (no ATP) and 0.25 ± 0.02 (200μM ATP)], confirming the accuracy of the dual-wavelength assay(Figure 3). These data indicate that the consumptionof 5(S)-HpETE, and the subsequent production of the5,6-epoxide, does not affect the relative rates of hydroperoxidationand epoxidation.


ATP allosterically activates the human 5-lipoxygenase molecular mechanism of arachidonic acid and 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid.

Smyrniotis CJ, Barbour SR, Xia Z, Hixon MS, Holman TR - Biochemistry (2014)

Effect of 200 μM ATP on the efficiency of epoxideformation.[LTA4]/[5(S)-HpETE] turnover ratio from10 μM AA substrate as measured by HPLC and UV–vis spectrophotometry.[LTA4] was measured at 280 nm and [5(S)-HpETE] at 234 nm, and both were normalized to their respectiveextinction coefficients. The absolute kinetic activity of wild-type5-LOX ammonium sulfate preparations was ≈60 μmol min–1 mg–1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215895&req=5

fig3: Effect of 200 μM ATP on the efficiency of epoxideformation.[LTA4]/[5(S)-HpETE] turnover ratio from10 μM AA substrate as measured by HPLC and UV–vis spectrophotometry.[LTA4] was measured at 280 nm and [5(S)-HpETE] at 234 nm, and both were normalized to their respectiveextinction coefficients. The absolute kinetic activity of wild-type5-LOX ammonium sulfate preparations was ≈60 μmol min–1 mg–1.
Mentions: As described above, ATP activates bothAA hydroperoxidation and 5(S)-HpETE epoxidation.However, it is unclear if the epoxidation reaction affects the rateof hydroperoxidation, because of the consumption of the hydroperoxidationproduct, 5(S)-HpETE, and/or production of the 5,6-epoxide,LTA4. To investigate this possibility, a diode-array UV–visspectrophotometer was used to observe the formation of both 5(S)-HpETE (234 nm) and LTA4 (280 nm) simultaneouslyfrom 0 to 20% product formation and determine if the slight increasein the 5(S)-HpETE concentration could affect therate of hydroperoxide formation. The LTA4/5(S)-HpETE ratio of products (the efficiency of epoxide formation) wascalculated throughout the reaction and observed to remain constantup to 20% product formation [LTA4/5(S)-HpETEratio of 0.161 ± 0.004 (Figure 3)]. Withthe addition of 200 μM ATP, this value increased to a constantvalue of 0.247 ± 0.005 (Figure 3) andmatched the value determined for rat PMNL 5-LOX.40 In addition, 5(S)-HETE and 5,12-DiHETEconcentrations were quantified via HPLC and comparable product ratioswere observed [0.19 ± 0.02 (no ATP) and 0.25 ± 0.02 (200μM ATP)], confirming the accuracy of the dual-wavelength assay(Figure 3). These data indicate that the consumptionof 5(S)-HpETE, and the subsequent production of the5,6-epoxide, does not affect the relative rates of hydroperoxidationand epoxidation.

Bottom Line: 5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid.This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation.Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.

ABSTRACT
5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

Show MeSH
Related in: MedlinePlus