Dietary fish oil containing 3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the 6 fatty acid arachidonic acid (AA) and the 3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil

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...

Pain associated with inflammation involves prostaglandins synthesized from arachidonic acid (AA) through cyclooxygenase-2 (COX-2) pathways while thromboxane A 2 formed by platelets from AA via cyclooxygenase-1 (COX-1) mediates thrombosis. COX-1 and COX-2 are both targets of nonselective nonsteroidal antiinflammatory drugs (nsNSAIDs) including aspirin whereas COX-2 activity is preferentially blocked by COX-2 inhibitors called coxibs. COXs are homodimers composed of identical subunits, but we have shown that only one subunit is active at a time during catalysis; moreover, many nsNSAIDS bind to a single subunit of a COX dimer to inhibit the COX activity of the entire dimer. Here, we report the surprising observation that celecoxib and other coxibs bind tightly to a subunit of COX-1. Although celecoxib binding to one monomer of COX-1 does not affect the normal catalytic processing of AA by the second, partner subunit, celecoxib does interfere with the inhibition of COX-1 by aspirin in vitro. X-ray crystallographic results obtained with a celecoxib/COX-1 complex show how celecoxib can bind to one of the two available COX sites of the COX-1 dimer. Finally, we find that administration of celecoxib to dogs interferes with the ability of a low dose of aspirin to inhibit AA-induced ex vivo platelet aggregation. COX-2 inhibitors such as celecoxib are widely used for pain relief. Because coxibs exhibit cardiovascular side effects, they are often prescribed in combination with low-dose aspirin to prevent thrombosis. Our studies predict that the cardioprotective effect of low-dose aspirin on COX-1 may be blunted when taken with coxibs.arachidonic acid | adrenic acid | nonsteroidal antiinflammatory drugs | platelet | prostaglandin

Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 (also called cyclooxygenases (COXs)-1 and -2) catalyze the committed step in prostaglandin biosynthesis. Both isoforms are targets of nonsteroidal anti-inflammatory drugs (NSAIDs). PGHSs are homodimers that exhibit half-of-sites COX activity; moreover, some NSAIDs cause enzyme inhibition by binding only one monomer. To learn more about the cross-talk that must be occurring between the monomers comprising each PGHS-1 dimer, we analyzed structures of PGHS-1 crystallized under five different conditions including in the absence of any tightly binding ligand and in the presence of non-specific NSAIDs and of a COX-2 inhibitor. When crystallized with sub-stoichiometric amounts of an NSAID, both monomers are often fully occupied with inhibitor; thus, the enzyme prefers to crystallize in a fully occupied form. In comparing the five structures, we only observe changes in the positions of residues 123-129 and residues 510-515. In cases where one monomer is fully occupied with an NSAID and the partner monomer is incompletely occupied, an alternate conformation of the loop involving residues 123-129 is seen in the partially occupied monomer. We propose, based on this observation and previous cross-linking studies, that cross-talk between monomers involves this mobile 123-129 loop, which is located at the dimer interface. In ovine PGHS-1 crystallized in the absence of an NSAID, there is an alternative route for substrate entry into the COX site different than the well-known route through the membrane binding domain. KeywordsCOX-1; Prostaglandin Endoperoxide H Synthase; NSAIDs; Aspirin; Nimesulide; Flurbiprofen; Diclofenac; Heterodimer; Crystallography; Merohedral twinning Prostaglandin endoperoxide H synthases (PGHSs) + -1 and -2, also known as cyclooxygenases (COXs)-1 and -2, are ER resident proteins that are bound to a single face of the ER membrane bilayer. PGHSs-1 and -2 are responsible for catalyzing the conversion of arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ) in the committed step of prostaglandin (PG) biosynthesis ( 1-5 ). PGH 2 is then converted to biologically active PGs including PGD 2 , PGE 2 , PGF 2α or PGI 2 (prostacyclin) or thromboxane A 2 (TxA 2 ) ( 6 ). Both PGHS isoforms are targets of non-selective nonsteroidal anti-inflammatory drugs (nsNSAIDs), such as aspirin and ibuprofen, while PGHS-2 can also be blocked selectively by diarylheterocyclic "COX-2 specific" inhibitors called coxibs ( 7 ).Both PGHS-1 and -2 are homodimers composed of ~72 kDa subunits that are tightly bonded to one another via an interface spanning 2500 Å 2 ( 8 ) PGHS crystal structures have provided limited information about the nature of the differences between monomers of the biological dimer. Recently, we determined that ovPGHS-1 crystals frequently display merohedral twinning ( 15,(17)(18)(19) ) and that solving a structure in space group P6 5 , which contains the biological dimer in the asymmetric unit, can unmask asymmetry between the partner monomers. Here, we ...

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...