Structural Characterization of Arachidonyl Radicals Formed by Aspirin-treated Prostaglandin H Synthase-2

Tsai, Alan C.; Palmer, Graham; Wu, Gang; Peng, Sheng; Okeley, Nicole M.; Donk, Wilfred A. van der; Kulmacz, Richard J.

doi:10.1074/jbc.m206961200

Cited by 24 publications

(34 citation statements)

References 54 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S3). The second additional type of EPR spectrum observed was similar to that attributed previously to an allyl AA radical [40]. Further analysis by HFEPR (see later text) indicated that the latter spectrum was actually a novel tyrosyl radical signal that will be referred to as NS1c.…”

Section: Resultssupporting

confidence: 75%

“…5 shows the D-band spectrum of PGHS-1 equilibrated anaerobically with AA first and then reacted with EtOOH. X-band spectrum of this sample was dominated by the species of narrow EPR linewidth (data not shown) attributed to an “allyl” radical in earlier studies [40]. Surprisingly, the HFEPR spectrum of the PGHS-1/AA/EtOOH reaction mixture (Fig.…”

Section: Resultsmentioning

confidence: 54%

See 1 more Smart Citation

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Tsai

Rogge

et al. 2011

Journal of Inorganic Biochemistry

Self Cite

View full text Add to dashboard Cite

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O 2 . The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.

show abstract

Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 54%

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Tsai

Rogge

et al. 2011

Journal of Inorganic Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…BCA protein reagent was from Pierce. Polyacrylic acid (sodium salt) 5100 was from Hampton Research Corp. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]AA (1.85 GBq/mmol) was from American Radiolabeled Chemicals. Hexane, isopropyl alcohol, and acetic acid were HPLC grade from Thermo Fisher Scientific, Inc.…”

Section: Methodsmentioning

confidence: 99%

“…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 .…”

mentioning

confidence: 99%

Human Cyclooxygenase-2 Is a Sequence Homodimer That Functions as a Conformational Heterodimer

Dong

Vecchio

Sharma

et al. 2011

Journal of Biological Chemistry

100

231

View full text Add to dashboard Cite

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...

show abstract

“…The initial step in the cyclooxygenase reaction is abstraction of the 13-pro-S-hydrogen atom from AA to yield an allylic arachidonyl radical (13)(14)(15)(16). This hydrogen abstraction involves a protein tyrosyl radical centered on Tyr 385 (17,18).…”

Section: Prostaglandin-endoperoxide H Synthase (Pghs)mentioning

confidence: 99%

Histidine 386 and Its Role in Cyclooxygenase and Peroxidase Catalysis by Prostaglandin-endoperoxide H Synthases

Seibold

Ball

Hsi

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Prostaglandin-endoperoxide H synthases (PGHSs) have a cyclooxygenase that forms prostaglandin (PG) G 2 from arachidonic acid (AA) plus oxygen and a peroxidase that reduces the PGG 2 to PGH 2 . The peroxidase activates the cyclooxygenase. This involves an initial oxidation of the peroxidase heme group by hydroperoxide, followed by oxidation of Tyr 385 to a tyrosyl radical within the cyclooxygenase site. His 386 of PGHS-1 is not formally part of either active site, but lies in an extended helix between Tyr 385 , which protrudes into the cyclooxygenase site, and His 388 , the proximal ligand of the peroxidase heme. When His 386 was substituted with alanine in PGHS-1, the mutant retained <2.5% of the native peroxidase activity, but >20% of the native cyclooxygenase activity. However, peroxidase activity could be restored (10 -30%) by treating H386A PGHS-1 with cyclooxygenase inhibitors or AA, but not with linoleic acid; in contrast, mere occupancy of the cyclooxygenase site of native PGHS-1 had no effect on peroxidase activity. Heme titrations indicated that H386A PGHS-1 binds heme less tightly than does native PGHS- and restores native-like structure to the extended helix. Being less bulky than AA, linoleic acid is apparently unable to reopen this constriction.

show abstract

Structural Characterization of Arachidonyl Radicals Formed by Aspirin-treated Prostaglandin H Synthase-2

Cited by 24 publications

References 54 publications

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Human Cyclooxygenase-2 Is a Sequence Homodimer That Functions as a Conformational Heterodimer

Histidine 386 and Its Role in Cyclooxygenase and Peroxidase Catalysis by Prostaglandin-endoperoxide H Synthases

Contact Info

Product

Resources

About