Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

Doyen, J. Rand; Yucer, Nur; Lichtenberger, Lenard M.; Kulmacz, Richard J.

doi:10.1016/j.prostaglandins.2007.12.001

Cited by 10 publications

(8 citation statements)

References 33 publications

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“…Considering the first hypothesis, the critical micelle concentration (CMC) lies in the range of 6-60 μM for most unsaturated fatty acids, depending on pH and the counter-cation used (31)(32)(33)(34), which is consistent with the inflection point of 20 μM (Fig. 2A).…”

Section: Resultssupporting

confidence: 69%

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Alegria

Meireles

Cussiol

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Organic hydroperoxide resistance (Ohr) enzymes are unique Cysbased, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 10 7 -10 8 M −1 ·s −1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 10 7 M −1 ·s −1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a Δohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the Δohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the Δohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H 2 O 2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wildtype strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.hydroperoxides | thiols | Cys-based peroxidase | pathogenic bacteria | Pseudomonas aeruginosa

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Section: Resultssupporting

confidence: 69%

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Alegria

Meireles

Cussiol

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The K M value for hPGHS-1 is about four-fold higher than that observed for oPGHS-1 [39]. This is probably due to the higher level of detergent used in the present assays, as increasing detergent concentration is known to increase the cyclooxygenase K M value [40]. Mutation of Tyr504 to phenylalanine resulted in a 24% decrease in cyclooxygenase specific activity, a smaller change than seen for the analogous mutation in hPGHS-2 [22], but did not change significantly the K M value for arachidonate (Table 1).…”

Section: Resultsmentioning

confidence: 99%

Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Rogge

Liu

Kulmacz

et al. 2009

Journal of Inorganic Biochemistry

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Prostaglandin H synthase isoforms 1 and -2 (PGHS-1 and -2) react with peroxide to form a radical on Tyr385 that initiates the cyclooxygenase catalysis. The tyrosyl radical EPR signals of PGHS-1 and -2 changes over time and are altered by cyclooxygenase inhibitor binding. We characterized the tyrosyl radical dynamics using wild type human PGHS-1 (hPGHS-1) and its Y504F, Y385F and Y385F/Y504F mutats to determine whether the radical EPR signal changes involve Tyr504 radical formation, Tyr385 radical phenyl ring rotation, or both. Reaction of hPGHS-1 with peroxide produced a wide singlet, whereas its Y504F mutant produced only a wide doublet signal, assigned to the Tyr385 radical. The cyclooxygenase specific activity and K M for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K M for peroxide were increased. The Y385F and Y385F/Y504F mutants retained only a small fraction of the peroxidase activity; the former had a much-reduced yield of peroxide-induced radical and the latter essentially none. After binding of indomethacin, a cyclooxygenase inhibitor, hPGHS-1 produced a narrow singlet but the Y504F mutant did not show much radical. These results indicate that peroxideinduced radicals form on Tyr385 and Tyr504 of hPGHS-1, with radical primarily on Tyr504 in the wild type protein; indomethacin binding prevented radical formation on Tyr385 but still on Tyr504. Thus, hPGHS-1 and -2 have different distributions of peroxide-derived radical between Tyr385 and Tyr504. Y504F mutants in both hPGHS-1 and -2 significantly decreased the cyclooxygenase activation efficiency, indicating that formation of the Tyr504 radical is functionally important for both isoforms.

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“…However, in artificial lipid membrane systems, such as liposome vesicles, fatty acids and NSAIDs become sequestered in the large vesicles, subsequently limiting their movement into the enzyme active site and subsequent availability for catalysis and inhibition. As a consequence, the measured kinetic parameters become altered, making data interpretation challenging (18). In contrast to liposome vesicles, nanodisc incorporation confines COX-2 to a much smaller patch of lipid membrane such that substrates and inhibitors that enter the lipid bilayer are immediately localized in close proximity to the entrance of the cyclooxygenase channel.…”

Section: Discussionmentioning

confidence: 99%

“…Rand Doyen and colleagues characterized the kinetics of COX-1 and COX-2 in the presence of phosphatidylcholine molecules of varying acyl chain lengths and physical state (18). These studies revealed that monomeric, micelle, and bilayer forms of phospholipids exhibit an inhibitory effect on cyclooxygenase catalysis, predominantly due to sequestration of substrate (18). MirAfzali and colleagues successfully incorporated COX-1 and COX-2 into large unilamellar vesicles produced from a mixture of DOPC:DOPS that had been doped with oleic acid (19).…”

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confidence: 99%

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs

Orlando

McDougle

Lucido

et al. 2014

Archives of Biochemistry and Biophysics

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Cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. The enzymes associate with one leaflet of the membrane bilayer. We utilized nanodisc technology to investigate the function of human (hu) COX-2 and murine (mu) COX-2 in a lipid bilayer environment. huCOX-2 and muCOX-2 were incorporated into nanodiscs composed of POPC, POPS, DOPC, or DOPS phospholipids. Size-exclusion chromatography and negative stain electron microscopy confirm that a single COX-2 homodimer is incorporated into the nanodisc scaffold. Nanodisc-reconstituted COX-2 exhibited similar kinetic profiles for the oxygenation of AA, eicosapentaenoic acid, and 1-arachidonoyl glycerol compared to those derived using detergent solubilized enzyme. Moreover, changing the phospholipid composition of the nanodisc did not alter the ability of COX-2 to oxygenate AA or to be inhibited by various nonselective NSAIDs or celecoxib. The cyclooxygenase activity of nanodisc-reconstituted COX-2 was reduced by aspirin acetylation and potentiated by the nonsubstrate fatty acid palmitic acid to the same extent as detergent solubilized enzyme, independent of phospholipid composition. The stabilization and maintenance of activity afforded by the incorporation of the enzyme into nanodiscs generates a native-like lipid bilayer environment to pursue studies of COX utilizing solution-based techniques that are otherwise not tractable in the presence of detergents.

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Phospholipid actions on PGHS-1 and -2 cyclooxygenase kinetics

Cited by 10 publications

References 33 publications

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Cyclooxygenase-2 catalysis and inhibition in lipid bilayer nanodiscs

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