The Productive Conformation of Arachidonic Acid Bound to Prostaglandin Synthase

Malkowski, Michael G.; Ginell, Stephan L.; Smith, William L.; Garavito, R. Michael

doi:10.1126/science.289.5486.1933

Cited by 271 publications

(381 citation statements)

References 33 publications

(15 reference statements)

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“…However, observation of the crystal structures of PGHS-1 and PGHS-2 dimers suggests that both subunits are identical and both bind substrates and inhibitors (14,15,17,24,25). We performed differential scanning calorimetr y (DSC) on the native huPGHS-2 homodimer, the R120Q huPGHS-2 homodimer, and the native͞R120Q huPGHS-2 heterodimer in the presence and absence of FBP, a time-dependent COX inhibitor, and Sibuprofen (S-IBP), a simple competitive inhibitor, to determine whether we could obtain physical evidence for a nonuniformity of subunits (Fig.…”

Section: Physical Properties Of the Native͞r120q Hupghs-2 Heterodimermentioning

confidence: 99%

“…G533A mutants of PGHSs oxygenate fatty acids with low efficiencies (11-15). Arg-120 is found near the mouth of the COX active site of PGHSs and is involved in binding the carboxyl group of fatty acid substrates (11,16,17). An R120Q huPGHS-2 mutant can oxygenate AA but not eicosapentaenoic acid (EPA) (18); furthermore, FBP is Ϸ1,000 times less potent as an inhibitor of R120Q huPGHS-2 than native huPGHS-2.…”

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confidence: 99%

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Partnering between monomers of cyclooxygenase-2 homodimers

Yuan

Rieke

Rimon

et al. 2006

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

113

142

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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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Section: Physical Properties Of the Native͞r120q Hupghs-2 Heterodimermentioning

confidence: 99%

mentioning

confidence: 99%

Partnering between monomers of cyclooxygenase-2 homodimers

Yuan

Rieke

Rimon

et al. 2006

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

113

142

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“…[37][38][39][40] Interestingly, a crystal structure of mCOX-2 is available in which a mixture of structures was observed containing substrate (AA) and product (PGH 2 ) bound in the cyclooxygenase active site. 32 Although the product species is ambiguous (it could be PGG 2 or PGH 2 ), the carboxylate of the molecule is positioned near Arg-120 and Tyr-355 and the ω-end is bound in the top channel, similar to arachidonic acid. 41 This conformation of product, in which the PGG 2 /PGH 2 species hydrogen-bonds with the constriction site residues and bends in an L-shaped fashion at Tyr-385, suggests that arachidonic acid was positioned properly for catalysis.…”

Section: Cyclooxygenase Enzymes: Structure and Mechanismsmentioning

confidence: 99%

“…The aliphatic backbone projects up into the top of the cyclooxygenase active site from the hydrophobic channel and then makes a sharp bend in the vicinity of Tyr-385 ( Figure 5). 32,33 In these structures, the vast majority of contacts between the substrate and various protein residues in the active site involve van der Waals interactions. The ω-end of arachidonic acid binds in a narrow channel at the top of the active site and is surrounded by six aromatic amino acids.…”

Section: Cyclooxygenase Enzymes: Structure and Mechanismsmentioning

confidence: 99%

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Structural and Functional Basis of Cyclooxygenase Inhibition

2007

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Prostaglandin EndoperoxideH₂Synthases‐1 and ‐2

Garavito

2004

Handbook of Metalloproteins

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The prostaglandin endoperoxide H 2 synthase (PGHS) isozymes 1 and 2 are membrane bound, heme‐dependent enzymes that catalyze the committed step in the conversion of polyunsaturated fatty acids to prostanoids and thromboxanes. The PGHS isozymes, which are also known as cyclooxygenases, produce prostaglandin H 2 in two sequential enzymatic steps – a bis‐oxygenase (cyclooxygenase) reaction generates prostaglandin G 2 from arachidonic acid and a hydroperoxidase reaction creates the final product, prostaglandin H 2 . The PGHS isozymes are also the primary targets of nonsteroidal anti‐inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, and recent pharmacological research efforts have led to the development of isoform selective drugs like Celebrex® and Vioxx®. In this chapter, we discuss the biochemistry, enzymology, and the structural biology of the PGHS isozymes and their relevance to prostanoid physiology and NSAID pharmacology.

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The Productive Conformation of Arachidonic Acid Bound to Prostaglandin Synthase

Cited by 271 publications

References 33 publications

Partnering between monomers of cyclooxygenase-2 homodimers

Partnering between monomers of cyclooxygenase-2 homodimers

Structural and Functional Basis of Cyclooxygenase Inhibition

Prostaglandin EndoperoxideH₂Synthases‐1 and ‐2

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The Productive Conformation of Arachidonic Acid Bound to Prostaglandin Synthase

Cited by 271 publications

References 33 publications

Partnering between monomers of cyclooxygenase-2 homodimers

Partnering between monomers of cyclooxygenase-2 homodimers

Structural and Functional Basis of Cyclooxygenase Inhibition

Prostaglandin EndoperoxideH2Synthases‐1 and ‐2

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Prostaglandin EndoperoxideH₂Synthases‐1 and ‐2