Mechanism of Free Radical Oxygenation of Polyunsaturated Fatty Acids by Cyclooxygenases

Rouzer, Carol A.; Marnett, Lawrence J.

doi:10.1021/cr000068x

Cited by 219 publications

(190 citation statements)

References 248 publications

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“…The oxidation of resveratrol occurred at the peroxidase active site and had an obligatory requirement for peroxide substrate. Although characterization of a mechanism-based inactivator for COX is complicated by the fact that the enzymes undergo self-inactivation (12,13), it was found that in the presence of peroxide and resveratrol there was significantly more COX-1 inactivation than could be accounted for by self-inactivation alone (Fig. 4A).…”

Section: Mechanism-based Inactivation Of Cox-1 By Resveratrol-mentioning

confidence: 99%

“…Because stable covalent modification of COX-1 was not observed, inactivation must result from a "hit-and-run" mechanism in which the unstabilized m-hydroquinone radical generates a protein radical that goes on to inactivate the enzyme. Such protein radicals are believed to be responsible for the normal phenomenon of peroxidase self-inactivation (12,13). In this mechanism, the oxidized m-hydroquinone product leaves the enzyme (Scheme 2B).…”

Section: Mechanism-based Inactivation Of Cox-1 By Resveratrol-mentioning

confidence: 99%

“…Their potential cardioprotective role is discussed. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]AA (51 mCi/mmol), Sephadex G-25, and Tween 20 were purchased from Sigma. PGF 2␣ , PGE 2 , and PGD 2 were purchased from Biomol Research Laboratories.…”

mentioning

confidence: 99%

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Resveratrol is a Peroxidase-mediated Inactivator of COX-1 but Not COX-2

Szewczuk

Forti

Stivala

et al. 2004

Journal of Biological Chemistry

212

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Resveratrol (3,4 ,5-trihydroxy-trans-stilbene) is a phytoalexin found in grapes that has anti-inflammatory, cardiovascular protective, and cancer chemopreventive properties. It has been shown to target prostaglandin H 2 synthase (COX)-1 and COX-2, which catalyze the first committed step in the synthesis of prostaglandins via sequential cyclooxygenase and peroxidase reactions. Resveratrol discriminates between both COX isoforms. It is a potent inhibitor of both catalytic activities of COX-1, the desired drug target for the prevention of cardiovascular disease, but only a weak inhibitor of the peroxidase activity of COX-2, the isoform target for nonsteroidal anti-inflammatory drugs. We have investigated the unique inhibitory properties of resveratrol. We find that it is a potent peroxidase-mediated mechanism-based inactivator of COX-1 only (k inact ‫؍‬ 0.069 ؎ 0.004 s ؊1 , K i(inact) ‫؍‬ 1.52 ؎ 0.15 M), with a calculated partition ratio of 22. Inactivation of COX-1 was time-and concentration-dependent, it had an absolute requirement for a peroxide substrate, and it was accompanied by a concomitant oxidation of resveratrol. Resveratrolinactivated COX-1 was devoid of both the cyclooxygenase and peroxidase activities, neither of which could be restored upon gel-filtration chromatography. Inactivation of COX-1 by [ 3 H]resveratrol was not accompanied by stable covalent modification as evident by both SDS-PAGE and reverse phase-high performance liquid chromatography analysis. Structure activity relationships on methoxy-resveratrol analogs showed that the m-hydroquinone moiety was essential for irreversible inactivation of COX-1. We propose that resveratrol inactivates COX-1 by a "hit-and-run" mechanism, and offers a basis for the design of selective COX-1 inactivators that work through a mechanism-based event at the peroxidase active site.

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Section: Mechanism-based Inactivation Of Cox-1 By Resveratrol-mentioning

confidence: 99%

Section: Mechanism-based Inactivation Of Cox-1 By Resveratrol-mentioning

confidence: 99%

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Resveratrol is a Peroxidase-mediated Inactivator of COX-1 but Not COX-2

Szewczuk

Forti

Stivala

et al. 2004

Journal of Biological Chemistry

212

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“…aspirin ͉ flurbiprofen ͉ heme ͉ ibuprofen ͉ prostaglandin P rostaglandin endoperoxide H synthases (PGHSs) 1 and 2 catalyze the committed step in prostaglandin biosynthesis (1)(2)(3). They are both targets of nonsteroidal antiinflammatory drugs, and PGHS-2 is the target of cyclooxygenase (COX) 2 inhibitors (4,5).…”

mentioning

confidence: 99%

“…PGHS-1 and PGHS-2 function only as homodimers although each subunit has both a POX and a COX active site (1)(2)(3)6). It is not clear whether each monomer functions independently or whether cross-talk occurs between monomers comprising a dimer.…”

mentioning

confidence: 99%

Partnering between monomers of cyclooxygenase-2 homodimers

Yuan

Rieke

Rimon

et al. 2006

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

115

143

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Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2 convert arachidonic acid to prostaglandin H2 in the committed step of prostanoid biosynthesis. These enzymes are pharmacological targets of nonsteroidal antiinflammatory drugs and cyclooxygenase (COX) 2 inhibitors. Although PGHSs function as homodimers and each monomer has its own COX and peroxidase active sites, the question of whether there is cross-talk between monomers has remained unresolved. Here we describe two heterodimers in which a native subunit of human PGHS-2 has been coupled to a subunit having a defect within the COX active site at some distance from the dimer interface. Native͞G533A PGHS-2, a heterodimer with a COX-inactive subunit, had the same specific COX activity as the native homodimer. Native͞R120Q PGHS-2, a heterodimer in which both subunits can oxygenate arachidonic acid but in which the R120Q subunit cannot bind the COX inhibitor flurbiprofen, was inhibited by flurbiprofen to about the same extent as native PGHS-2. These results imply that native PGHS-2 exhibits half-ofsites reactivity. Isothermal titration calorimetry established that only one monomer of the native PGHS-2 homodimer binds flurbiprofen tightly. In short, binding of ligand to the COX site of one monomer alters its companion monomer so that it is unable to bind substrate or inhibitor. We conclude that PGHS monomers comprising a dimer, although identical in the resting enzyme, differ from one another during catalysis. The nonfunctioning subunit may provide structural support enabling its partner monomer to catalyze the COX reaction. This subunit complementarity may prove to be characteristic of other dimeric enzymes having tightly associated monomers.aspirin ͉ flurbiprofen ͉ heme ͉ ibuprofen ͉ prostaglandin P rostaglandin endoperoxide H synthases (PGHSs) 1 and 2 catalyze the committed step in prostaglandin biosynthesis (1-3). They are both targets of nonsteroidal antiinflammatory drugs, and PGHS-2 is the target of cyclooxygenase (COX) 2 inhibitors (4, 5). The enzymes have two catalytic activities: (i) a COX that converts arachidonic acid (AA) to a prostaglandin endoperoxide, prostaglandin G 2 (PGG 2 ), and (ii) a peroxidase (POX) that reduces PGG 2 to PGH 2 . Oxidation of the heme group at the POX active site by a peroxide is required to activate the COX by generating a tyrosyl radical at Tyr-385 at the COX active site (1-3). The Tyr-385 radical is involved in abstracting the hydrogen atom from the fatty acid substrate in the initial and rate-determining step in COX catalysis.PGHS-1 and PGHS-2 function only as homodimers although each subunit has both a POX and a COX active site (1-3, 6). It is not clear whether each monomer functions independently or whether cross-talk occurs between monomers comprising a dimer. Titrations of PGHS-1 with time-dependent COX inhibitors such as flurbiprofen (FBP) and indomethacin indicate that complete inhibition requires only one inhibitor molecule to be bound per dimer (7). Other studies with PGHS-1 using low concentrations of PGHS-2-spe...

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Co‐Oxidation by Cyclooxygenases

Szewczuk

Penning

2009

CP Toxicology

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Cyclooxygenases (COXs; prostaglandin H(2) synthases) catalyze the bis-dioxygenation of arachidonic acid (AA) to generate prostaglandin (PG) G(2) followed by the peroxidative cleavage of PGG(2) to yield PGH(2), the precursor to all of the vasoactive PGs. These enzymes utilize a Fe-protoporhyrin IX (heme) co-factor to catalyze peroxide bond cleavage, which puts the Fe at a higher oxidation state (Fe(3+) → Fe(5+)). The heme Fe requires two electrons (e(-)) to return to its resting state (Fe(3+)) for the next round of catalysis. Peroxide bond cleavage thus occurs via compound I and compound II, observed for horseradish peroxidase. To return to Fe(3+), electrons come from "co-reductants" and their subsequent oxidation by the enzyme is known as "co-oxidation". The protocols in this unit are aimed at characterizing this side reaction of COXs.

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Mechanism of Free Radical Oxygenation of Polyunsaturated Fatty Acids by Cyclooxygenases

Cited by 219 publications

References 248 publications

Resveratrol is a Peroxidase-mediated Inactivator of COX-1 but Not COX-2

Resveratrol is a Peroxidase-mediated Inactivator of COX-1 but Not COX-2

Partnering between monomers of cyclooxygenase-2 homodimers

Co‐Oxidation by Cyclooxygenases

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