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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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Related in: MedlinePlus

Effect of ATP on the Km(app) of 5-LOXwith the 5(S)-HpETE substrate. The data are fit toeq 2 (Scheme 1), witha Km of 14 μM. α and Ki were determined to be 1.6 ± 0.08 and12 ± 5 μM, respectively.
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fig6: Effect of ATP on the Km(app) of 5-LOXwith the 5(S)-HpETE substrate. The data are fit toeq 2 (Scheme 1), witha Km of 14 μM. α and Ki were determined to be 1.6 ± 0.08 and12 ± 5 μM, respectively.

Mentions: Similar ATP titration experimentswere also performed with 5(S)-HpETE as the substrate,which showed an α valueof 1.56 ± 0.08 (K-type inhibition) and a Ki value of 12 ± 5 μM (Table 2), from the Km(app) plot (Figure 6). The α value was half the α valuefor AA activation, indicative of a weaker ATP allosteric effect on Km with 5(S)-HpETE as the substrate.However, the Ki value was comparable tothat of AA activation, indicating similar ATP binding affinities andmost likely the same ATP binding site for both hydroperoxidation andepoxidation. The β value was determined to be 5.1 ± 0.1(Figure 7), which is V-type activation, similarto that seen for AA. The β/α ratio is nearly 2-fold greaterfor 5(S)-HpETE (3.3 ± 0.2) than for AA (1.8± 0.2), indicating that ATP activates the rate of substrate capturefor 5(S)-HpETE more so than AA. These results agreewith our kinetic results at saturating ATP concentrations (Table 1), suggesting that the allosteric effect causedby ATP binding has largely similar consequences for both the hydroperoxidationof AA and the epoxidation of 5(S)-HpETE, but withsubtle differences in magnitude.


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 ATP on the Km(app) of 5-LOXwith the 5(S)-HpETE substrate. The data are fit toeq 2 (Scheme 1), witha Km of 14 μM. α and Ki were determined to be 1.6 ± 0.08 and12 ± 5 μM, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Effect of ATP on the Km(app) of 5-LOXwith the 5(S)-HpETE substrate. The data are fit toeq 2 (Scheme 1), witha Km of 14 μM. α and Ki were determined to be 1.6 ± 0.08 and12 ± 5 μM, respectively.
Mentions: Similar ATP titration experimentswere also performed with 5(S)-HpETE as the substrate,which showed an α valueof 1.56 ± 0.08 (K-type inhibition) and a Ki value of 12 ± 5 μM (Table 2), from the Km(app) plot (Figure 6). The α value was half the α valuefor AA activation, indicative of a weaker ATP allosteric effect on Km with 5(S)-HpETE as the substrate.However, the Ki value was comparable tothat of AA activation, indicating similar ATP binding affinities andmost likely the same ATP binding site for both hydroperoxidation andepoxidation. The β value was determined to be 5.1 ± 0.1(Figure 7), which is V-type activation, similarto that seen for AA. The β/α ratio is nearly 2-fold greaterfor 5(S)-HpETE (3.3 ± 0.2) than for AA (1.8± 0.2), indicating that ATP activates the rate of substrate capturefor 5(S)-HpETE more so than AA. These results agreewith our kinetic results at saturating ATP concentrations (Table 1), suggesting that the allosteric effect causedby ATP binding has largely similar consequences for both the hydroperoxidationof AA and the epoxidation of 5(S)-HpETE, but withsubtle differences in magnitude.

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