Fatty Acid Substrate Specificities of Human Prostaglandin-endoperoxide H Synthase-1 and −2

Laneuville, Odette; Breuer, Debra K.; Xu, Naxing; Huang, Zhiheng; Gage, Douglas A.; Watson, J. Throck; Lagarde, Michel; DeWitt, David L.; Smith, William L.

doi:10.1074/jbc.270.33.19330

Cited by 250 publications

(109 citation statements)

References 60 publications

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“…2). Moreover, the K M values for AA with the native͞native huPGHS-2 and native͞G533A huPGHS-2 were both Ϸ5 M, the same as the value reported previously for native huPGHS-2 (20). Finally, the POX activities of native͞native, G533A͞G533A, and native͞G533A huPGHS-2 were similar to one another.…”

supporting

confidence: 69%

Partnering between monomers of cyclooxygenase-2 homodimers

Yuan

Rieke

Rimon

et al. 2006

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

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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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supporting

confidence: 69%

Partnering between monomers of cyclooxygenase-2 homodimers

Yuan

Rieke

Rimon

et al. 2006

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

Self Cite

115

143

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“…One of the best known and oldest NSAIDs, aspirin, along with the new more COX-2-selective version of aspirin, APHS (7), differ from all other NSAIDs in that they covalently modify the same serine residue (Ser-530) in both COX-1 (6) and COX-2 (7,21,27). Biochemical and structural studies suggest that residues at the mouth of the substrate access channel, specifically Arg-120 and Tyr-355, facilitate binding of aspirin in this region of the protein by ionic and hydrogen-bonding interactions (14,15,17,18,20,22,28). This increases the probability of reaction of the acetyl group with the hydroxyl group of Ser-530.…”

Section: Discussionmentioning

confidence: 99%

Spatial Requirements for 15-(R)-Hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic Acid Synthesis within the Cyclooxygenase Active Site of Murine COX-2

Rowlinson¹,

Crews²,

Goodwin³

et al. 2000

Journal of Biological Chemistry

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The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11-and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11-and 15-HETE production after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the -end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.

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“…Although the mechanism(s) by which these beneficial effects occurs is unknown, a potentially important aspect related to the biological activity of -3 PUFAs is their ability to interfere with the arachidonic acid cascade that generates proinflammatory eicosanoids (2,38). EPA not only can replace arachidonic acid in phospholipid bilayers but is also a competitive inhibitor of COX, reducing the production of 2-series PGs and thromboxane, in addition to the 4-series leukotrienes (11,12). Recent studies have also shown that 3-and 5-series eicosanoids derived from EPA are either less biologically active or inactive compared with the former products and are thus considered to exert effects that are less inflammatory (10,13,39,40).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, EPA reduces the production of 2-series PGs and thromboxane and 4-series leukotrienes from arachidonate. These latter compounds are known to possess potent pro-inflammatory activities (11,12). On the other hand, eicosanoids derived from EPA are either less biologically active or inactive compared with those from arachidonate and are thus considered to exert effects that are less inflammatory (13).…”

mentioning

confidence: 99%

Identification of Novel Autoxidation Products of the ω-3 Fatty Acid Eicosapentaenoic Acid in Vitro and in Vivo

Yin

Brooks

Gao

et al. 2007

Journal of Biological Chemistry

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Increased intake of fish oil rich in the -3 fatty acids eicosapentaenoic acid (EPA, C20:5 -3) and docosahexaenoic acid (DHA, C22:6 -3) reduces the incidence of human disorders such as atherosclerotic cardiovascular disease. However, mechanisms that contribute to the beneficial effects of fish oil consumption are poorly understood. Mounting evidence suggests that oxidation products of EPA and DHA may be responsible, at least in part, for these benefits. Previously, we have defined the free radical-induced oxidation of arachidonic acid in vitro and in vivo and have proposed a unified mechanism for its peroxidation. We hypothesize that the oxidation of EPA can be rationally defined but would be predicted to be significantly more complex than arachidonate because of the fact that EPA contains an addition carbon-carbon double bond. Herein, we present, for the first time, a unified mechanism for the peroxidation of EPA. Novel oxidation products were identified employing state-ofthe-art mass spectrometric techniques including Ag ؉ coordination ionspray and atmospheric pressure chemical ionization mass spectrometry. Predicted compounds detected both in vitro and in vivo included monocylic peroxides, serial cyclic peroxides, bicyclic endoperoxides, and dioxolane-endoperoxides.

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Fatty Acid Substrate Specificities of Human Prostaglandin-endoperoxide H Synthase-1 and −2

Cited by 250 publications

References 60 publications

Partnering between monomers of cyclooxygenase-2 homodimers

Partnering between monomers of cyclooxygenase-2 homodimers

Spatial Requirements for 15-(R)-Hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic Acid Synthesis within the Cyclooxygenase Active Site of Murine COX-2

Identification of Novel Autoxidation Products of the ω-3 Fatty Acid Eicosapentaenoic Acid in Vitro and in Vivo

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