Comparison of Structural Stabilities of Prostaglandin H Synthase-1 and -2

Xiao, Guang; Chen, Wei; Kulmacz, Richard J.

doi:10.1074/jbc.273.12.6801

Cited by 46 publications

(32 citation statements)

References 53 publications

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“…PGHSs undergo a substrate turnover-dependent, reactionbased suicide inactivation (15) Previous studies of PGHS dimers have indicated that the enzymes exhibit half of sites catalytic activity (6,7) and that the monomers are tightly associated (21). Thus, PGHSs must be conformational heterodimers during catalysis and inhibition and cross-talk must occur between partner monomers.…”

Section: Effects Of Fas On Cox Activities Of Pghss-mentioning

confidence: 99%

Cyclooxygenase Allosterism, Fatty Acid-mediated Cross-talk between Monomers of Cyclooxygenase Homodimers

Yuan

Sidhu

Kuklev

et al. 2009

Journal of Biological Chemistry

116

213

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Prostaglandin endoperoxide H synthases (PGHSs) 1 and 2, also known as cyclooxygenases (COXs), catalyze the oxygenation of arachidonic acid (AA) in the committed step in prostaglandin (PG) biosynthesis. PGHSs are homodimers that display half of sites COX activity with AA; thus, PGHSs function as conformational heterodimers. Here we show that, during catalysis, fatty acids (FAs) are bound at both COX sites of a PGHS-2 dimer. Initially, an FA binds with high affinity to one COX site of an unoccupied homodimer. This monomer becomes an allosteric monomer, and it causes the partner monomer to become the catalytic monomer that oxygenates AA. A variety of FAs can bind with high affinity to the COX site of the monomer that becomes the allosteric monomer. Importantly, the efficiency of AA oxygenation is determined by the nature of the FA bound to the allosteric monomer. When tested with low concentrations of saturated and monounsaturated FAs (e.g. oleic acid), the rates of AA oxygenation are typically 1.5-2 times higher with PGHS-2 than with PGHS-1. These different kinetic behaviors of PGHSs may account for the ability of PGHS-2 but not PGHS-1 to efficiently oxygenate AA in intact cells when AA is a small fraction of the FA pool such as during "late phase" PG synthesis. Prostaglandin endoperoxide H synthases (PGHSs)2 also known generically as cyclooxygenases (COXs) convert arachidonic acid (AA), two O 2 molecules, and two electrons from one or more unknown reductants to prostaglandin H 2 (PGH 2 ) (1-5). The conversion involves two steps. First, the cyclooxygenase activity of PGHSs catalyzes the introduction of two O 2 molecules into the AA backbone forming prostaglandin G 2 . Prostaglandin G 2 can be reduced to PGH 2 by the peroxidase (POX) activity of the enzyme. There is a functional interplay between the COX and POX sites in that the heme group at the POX site needs to be oxidized by a peroxide to oxidize Tyr-385 in the active COX site to a tyrosyl radical. The Tyr-385 radical abstracts the 13-proS hydrogen from AA to initiate the COX reaction in the rate-determining step.PGHSs are homodimers with monomer molecular masses of ϳ72 kDa (1). Previous studies have indicated that there is crosstalk between the COX activities of the partner monomers and that the COX function exhibits half of sites activity with AA (6). That is, only one monomer of a dimer catalyzes AA oxygenation at any given time. Furthermore, the binding of inhibitors of the 2-phenylpropionic acid class such as flurbiprofen (FBP) or ibuprofen to one COX site is sufficient to inhibit all COX activity of a dimer (6, 7). The existence of half of sites activity establishes that PGHSs are conformational heterodimers when the rate-determining step in catalysis occurs.Despite exhibiting half of sites activity, there has been little evidence for cooperativity between the two COX sites during substrate turnover, and no physiological importance has been attached to the half of sites activity. However, we recently found in testing PGHS-2 with AA and another COX substr...

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Section: Effects Of Fas On Cox Activities Of Pghss-mentioning

confidence: 99%

Cyclooxygenase Allosterism, Fatty Acid-mediated Cross-talk between Monomers of Cyclooxygenase Homodimers

Yuan

Sidhu

Kuklev

et al. 2009

Journal of Biological Chemistry

116

213

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“…27 Sheep COX-1 was one of the first membrane proteins to be crystallized and to have its structure solved, 28 and there are now many COX crystal structures available, including several with bound inhibitors. The asymmetric unit of the ovine COX-1/flurbiprofen crystals was shown to contain two identical monomers exhibiting extensive contacts in a large subunit interface.…”

Section: Cyclooxygenase Enzymes: Structure and Mechanismsmentioning

confidence: 99%

Structural and Functional Basis of Cyclooxygenase Inhibition

2007

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“…(24,25). Disruption of the interface occurs only under denaturing conditions that also perturb the major folding domains of the proteins and eliminate catalytic activity (6). To detect possible interactions between PGHS monomers, we investigated the relationship between the monomers in two different huPGHS-2 heterodimers, native͞G533A huPGHS-2 and native͞R120Q huPGHS-2.…”

Section: Physical Properties Of the Native͞r120q Hupghs-2 Heterodimermentioning

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.

113

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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Comparison of Structural Stabilities of Prostaglandin H Synthase-1 and -2

Cited by 46 publications

References 53 publications

Cyclooxygenase Allosterism, Fatty Acid-mediated Cross-talk between Monomers of Cyclooxygenase Homodimers

Cyclooxygenase Allosterism, Fatty Acid-mediated Cross-talk between Monomers of Cyclooxygenase Homodimers

Structural and Functional Basis of Cyclooxygenase Inhibition

Partnering between monomers of cyclooxygenase-2 homodimers

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