Prostaglandin endoperoxide H synthases 1 and 2, also known as cyclooxygenases (COXs) 1 and 2, convert arachidonic acid (AA) to prostaglandin endoperoxide H 2 . Prostaglandin endoperoxide H synthases are targets of nonspecific nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors called coxibs. PGHS-2 is a sequence homodimer. Each monomer has a peroxidase and a COX active site. We find that human PGHS-2 functions as a conformational heterodimer having a catalytic monomer (E cat ) and an allosteric monomer (E allo ). Heme binds tightly only to the peroxidase site of E cat , whereas substrates, as well as certain inhibitors (e.g. celecoxib), bind the COX site of E cat . E cat is regulated by E allo in a manner dependent on what ligand is bound to E allo . Substrate and nonsubstrate fatty acids (FAs) and some COX inhibitors (e.g. naproxen) preferentially bind to the COX site of E allo . AA can bind to E cat and E allo , but the affinity of AA for E allo is 25 times that for E cat . Palmitic acid, an efficacious stimulator of human PGHS-2, binds only E allo in palmitic acid/murine PGHS-2 co-crystals. Nonsubstrate FAs can potentiate or attenuate actions of COX inhibitors depending on the FA and whether the inhibitor binds E cat or E allo . Our studies suggest that the concentration and composition of the free FA pool in the environment in which PGHS-2 functions in cells, the FA tone, is a key factor regulating PGHS-2 activity and its responses to COX inhibitors. We suggest that differences in FA tone occurring with different diets will likely affect both baseline prostanoid synthesis and responses to COX inhibitors.Prostaglandin endoperoxide H synthases (PGHSs), 2 also known generically as cyclooxygenases (COXs), convert arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ) in the committed step of prostanoid biosynthesis (1-6). PGHS-1 and PGHS-2 are products of different genes. In general, PGHS-1 is constitutively expressed, whereas PGHS-2 expression is inducible (4, 5, 7). The enzymes are embedded in the luminal monolayer of the endoplasmic reticulum and inner membrane of the nuclear envelope (8 -11).PGHSs catalyze two different reactions, a COX reaction and a peroxidase (POX) reaction. COX catalysis begins with abstraction of the 13-pro-S-hydrogen from AA by the enzyme in the rate-determining step to generate an arachidonyl radical (3,6,12,13). Two molecules of O 2 are then sequentially added to the arachidonyl chain with concomitant rearrangements to form PGG 2 . PGG 2 can be reduced to PGH 2 by the POX activity. The purified isoforms are about equally efficient in catalyzing the conversion of AA to PGH 2 (1-6).PGHSs are homodimers composed of tightly associated monomers with identical sequences (14). Each monomer comprising a PGHS homodimer has a physically distinct COX and POX active site. Dissociation of the dimers into monomers only occurs upon denaturation (14). Kulmacz and Lands (15,16) provided the first evidence that the monomers of PGHS homodimers differ. They found that maximal COX activity of...

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

In an effort to understand the reaction mechanism of a B2 metallo-β-lactamase, steady-state and presteady state kinetic and rapid-freeze quench EPR studies were conducted on ImiS and its reaction with imipenem and meropenem. pH Dependence studies revealed no inflection points in the pH range of 5.0 -8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Sitedirected mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s −1 and a slower rate of fluorescence regain of 98 s −1 . Stopped-flow UV-Vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s −1 . These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid-freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 milliseconds that is assigned to a reaction intermediate that has a 5-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 milliseconds. Taken together, these results allow for a reaction mechanism to be offered for the B2 metallo-β-lactamases and demonstrates that the mononuclear Zn(II)-and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.Bacterial resistance to antibiotics is a growing clinical concern (1,2). Zn(II)-containing β-lactamases (metallo-β-lactamases, MβL's) contain 1-2 moles of Zn(II) per mole of enzyme, hydrolyze all known cephalosporins, carbapenems and penicillins, are not inhibited by clavulanic acid and other classical β-lactamase inhibitors, and have no known clinically-useful inhibitor towards them (3,4). Previous studies have shown that there is significant structural and mechanistic diversity among the MβL's, leading to the grouping of the enzymes into three distinct subclasses: B1, B2, and B3 (5,6). Sequence identity ranges from 25-40% between members in one subclass and from 10-20% between members in different subclasses. Subclass B1 enzymes have been found in strains of Bacillus, Bacteroides, Pseudomonas, Serratia, and Chryseobacterium, and subclass B3 enzymes have been found in strains of Stenotrophomonas, Legionella, Fluoribacter, Janthinobacterium and Caulobacter (3,4). Enzymes from the B1 and B3 subclasses have broad substrate profiles and require two Zn(II) † This work was supported by the National Institutes of Health (GM40052 to MWC; AI056231 to BB, and EB001980 to the Medical College of Wisconsin).*To whom correspondence should be addressed: M. W. Crowder, e-mail: crowdemw@muohio.edu, phone: (513) 529-7274, fax: (513) 529-5715. NIH Public Access Author ManuscriptBiochemistry. A...

Background: Cyclooxygenase-2 (COX-2), a target of coxibs, aspirin, and related drugs, is a sequence homodimer that functions as a conformational heterodimer. Results: Kinetic and aspirin labeling studies indicate that COX-2 is composed of two equivalent, stable populations of conformational heterodimers. Conclusion: COX-2 is processed and folds into a pre-existent conformational heterodimer. Significance: COX-2 half-site functionality results from COX-2 folding into a stable conformational heterodimer.

Prostaglandin endoperoxide H synthases (PGHS)-1 and -2, also called cyclooxygenases, convert arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ) in the committed step of prostaglandin biosynthesis. Both enzymes are homodimers, but the monomers often behave asymmetrically as conformational heterodimers during catalysis and inhibition. Here we report that aspirin maximally acetylates one monomer of human (hu) PGHS-2. The acetylated monomer of aspirin-treated huPGHS-2 forms 15-hydroperoxyeicosatetraenoic acid from AA, whereas the nonacetylated partner monomer forms mainly PGH 2 but only at 15 to 20% of the rate of native huPGHS-2. These latter conclusions are based on the findings that the nonsteroidal anti-inflammatory drug diclofenac binds a single monomer of native huPGHS-2, having an unmodified Ser530 to inhibit the enzyme, and that diclofenac inhibits PGH 2 but not 15-hydroperoxyeicosatraenoic acid formation by acetylated huPGHS-2. The 18R-and 17R-resolvins putatively involved in resolution of inflammation are reportedly formed via aspirin-acetylated PGHS-2 from eicosapentaenoic acid and docosahexaenoic acid, respectively, so we also characterized the oxygenation of these omega-3 fatty acids by aspirin-treated huPGHS-2. Our in vitro studies suggest that 18R-and 17R-resolvins could be formed only at low rates corresponding to less than 1 and 5%, respectively, of the rates of formation of PGH 2 by native PGHS-2.

In an effort to probe the structure of a group Bb metallo-beta-lactamase, Co(II)-substituted ImiS was prepared and characterized by electronic absorption, NMR, and EPR spectroscopies. ImiS containing 1 equiv of Co(II) (Co(II)(1)-ImiS) was shown to be catalytically active. Electronic absorption studies of Co(II)(1)-ImiS revealed the presence of two distinct features: (1) an intense sulfur to Co(II) ligand to metal charge transfer band and (2) less intense, Co(II) ligand field transitions that suggest 4-coordinate Co(II) in Co(II)(1)-ImiS. (1)H NMR studies of Co(II)(1)-ImiS suggest that one histidine, one aspartic acid, and one cysteine coordinate the metal ion in Co(II)(1)-ImiS. The addition of a second Co(II) to Co(II)(1)-ImiS did not result in any additional solvent-exchangeable NMR resonances, strongly suggesting that the second Co(II) does not bind to a site with histidine ligands. EPR studies reveal that the metal ion in Co(II)(1)-ImiS is 4-coordinate and that the second Co(II) is 5/6 coordinate. Taken together, these data indicate that the catalytic site in ImiS is the consensus Zn(2) site, in which Co(II) (and by extrapolation Zn(II)) is 4-coordinate and bound by Cys221, His263, Asp120, and probably one solvent water molecule. These studies also show that the second, inhibitory metal ion does not bind to the consensus Zn(1) site and that the metal ion binds at a site significantly removed from the active site. These results give the first structural information on metallo-beta-lactamase ImiS and suggest that the second metal binding site in ImiS may be targeted for inhibitors.

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