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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 Vmax(app) of 5-LOXwith the AA substrate. The data are fit to eq 4 (Scheme 1) with a Km of 1.9 μM, an α of 3, and a Ki of 8, where β was determined to be 5.4 ±0.08.
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fig5: Effect of ATP on the Vmax(app) of 5-LOXwith the AA substrate. The data are fit to eq 4 (Scheme 1) with a Km of 1.9 μM, an α of 3, and a Ki of 8, where β was determined to be 5.4 ±0.08.

Mentions: A plot of Km(app) versus ATP concentration,with AA as the substrate (Figure 4), yieldedan α of 3.0 ± 0.4 and a Ki of8 ± 6 μM, when fit with eq 2 (Table 2). The values of α and Ki were then utilized in eq 3 and fitto the Vmax/Km data, which yielded a β of 5.5 ± 0.5. The value of βwas also determined from the Vmax data(fit with eq 4 and the values of α and Ki given above), which yielded a β of 5.4± 0.08 (Figure 5) and matched well withthe β value from the Vmax/Km plot. These values indicate mixed hyperbolicallostery [α > 1 (K-type inhibition), and β > 1(V-typeactivation)],51 with the majority of thekinetic change being seen in the value of Vmax. This larger Vmax change is best observedby considering the Vmax/Km value, which is greater than 1 (β/α = 1.8± 0.2) and indicates Vmax/Km allosteric activation. These hyperbolic datasuggest the formation of a catalytically active ternary complex (A·E·S)between 5-LOX and ATP and are consistent with the previous findingof an allosteric site in 5-LOX.31,52 The data are also consistentwith what was observed at saturating ATP concentrations (listed inTable 1).


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 Vmax(app) of 5-LOXwith the AA substrate. The data are fit to eq 4 (Scheme 1) with a Km of 1.9 μM, an α of 3, and a Ki of 8, where β was determined to be 5.4 ±0.08.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Effect of ATP on the Vmax(app) of 5-LOXwith the AA substrate. The data are fit to eq 4 (Scheme 1) with a Km of 1.9 μM, an α of 3, and a Ki of 8, where β was determined to be 5.4 ±0.08.
Mentions: A plot of Km(app) versus ATP concentration,with AA as the substrate (Figure 4), yieldedan α of 3.0 ± 0.4 and a Ki of8 ± 6 μM, when fit with eq 2 (Table 2). The values of α and Ki were then utilized in eq 3 and fitto the Vmax/Km data, which yielded a β of 5.5 ± 0.5. The value of βwas also determined from the Vmax data(fit with eq 4 and the values of α and Ki given above), which yielded a β of 5.4± 0.08 (Figure 5) and matched well withthe β value from the Vmax/Km plot. These values indicate mixed hyperbolicallostery [α > 1 (K-type inhibition), and β > 1(V-typeactivation)],51 with the majority of thekinetic change being seen in the value of Vmax. This larger Vmax change is best observedby considering the Vmax/Km value, which is greater than 1 (β/α = 1.8± 0.2) and indicates Vmax/Km allosteric activation. These hyperbolic datasuggest the formation of a catalytically active ternary complex (A·E·S)between 5-LOX and ATP and are consistent with the previous findingof an allosteric site in 5-LOX.31,52 The data are also consistentwith what was observed at saturating ATP concentrations (listed inTable 1).

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