Target size analysis of prostaglandin endoperoxide synthase

Ruf, Hans Heinrich; Schuhn, Dietmar; Dietz, Reiner; Nastainczyk, Wolfgang; Nielsen, Mogens

doi:10.1111/j.1432-1033.1992.tb16730.x

Cited by 5 publications

(2 citation statements)

References 28 publications

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“…However, this half-of-sites behavior was not supported by subsequent crystallographic data showing fatty acid or substrate analog bound to both monomers in the PGHS-1 and -2 dimers [5-9]. Similarly, radiation inactivation results indicated that the minimal functioning unit of PGHS-1 is the monomer [114]. Thus it was surprising when a PGHS-2 heterodimer comprising one wildtype monomer and one monomer with a G533A mutation known to block cyclooxygenase catalysis displayed the same cyclooxygenase specific activity as the wildtype homodimer [115].…”

Section: Half-of-sites Reactivitymentioning

confidence: 99%

Prostaglandin H synthase: Resolved and unresolved mechanistic issues

Tsai

Kulmacz

2010

Archives of Biochemistry and Biophysics

146

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The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the Habstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.Prostaglandin H synthase (PGHS) is a key enzyme in prostanoid biosynthesis. Mammalian systems have two distinct PGHS isozymes sharing ~60% sequence identity. The constitutive isozyme, PGHS-1, is thought to function usually as a housekeeping enzyme, whereas the inducible isozyme, PGHS-2, is associated with cytokine and mitogen dependent processes, such as inflammation and cell proliferation [1,2]. Both PGHS isozymes catalyze the same two reactions: dioxygenation of arachidonic acid (AA) to yield prostaglandin G 2 (PGG 2 ), containing both a 9-11 endoperoxide and a 15-peroxide group; and a peroxidase reaction, which converts PGG 2 to prostaglandin H 2 (PGH 2 ) where the 15-peroxide is reduced to an alcohol © 2009 Elsevier Inc. All rights reserved. *CORRESPONDING AUTHOR: FAX: 713 500-6810; Ah-Lim.Tsai@uth.tmc.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this e...

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Section: Half-of-sites Reactivitymentioning

confidence: 99%

Prostaglandin H synthase: Resolved and unresolved mechanistic issues

Tsai

Kulmacz

2010

Archives of Biochemistry and Biophysics

146

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“…Its cyclooxygenase activity oxygenates arachidonic acid to the hydroperoxy endoperoxide PGG2, and its peroxidase activity reduces PGG2 to the alcohol PGH2 in the presence of a reducing substrate (eq 1) (Hamberg et al, 1974;Nugteren & Hazelhof, 1973). The holoenzyme is a homodimer of 70-kDa subunits containing one heme per subunit (Van Der Ouderaa et al, 1977;Ruf etal., 1984);each monomer appears to catalyze both activities (Ruf et al, 1992). PGH synthase is inactivated in vitro and in vivo by a variety of agents known as nonsteroidal antiinflammatory drugs (NSAIDs).…”

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confidence: 99%

Inactivation of prostaglandin endoperoxide synthase by acylating derivatives of indomethacin

Wells

Marnett²

1993

Biochemistry

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Derivatives of the potent antiinflammatory agent and cyclooxygenase inhibitor indomethacin were synthesized in which the carboxylic acid moiety was converted into reactive acylating agents. Indomethacin imidazole (indomethacin-IM) and indomethacin N-hydroxysuccinimide (indomethacin-NHS) inactivated both the cyclooxygenase and peroxidase activities when incubated with the apo form of purified prostaglandin endoperoxide synthase (PGH synthase) at a stoichiometry of 1:1. Treatment of the inactivated enzyme with hydroxylamine at neutral pH led to recovery of all peroxidase and about 50% of the cyclooxygenase activity. Hydroxylamine did not regenerate the cyclooxygenase activity of indomethacin-inactivated protein. Reconstitution of the apoprotein with heme protected against inactivation by indomethacin-NHS. Visible spectroscopy established that indomethacin-NHS-inactivated apoenzyme had a reduced capacity to bind heme. Indomethacin-NHS also substantially protected the apoenzyme from cleavage at the trypsin-sensitive Arg277 site. Incubation of [2-14C]indomethacin-NHS with PGH synthase led to incorporation of radioactivity into the protein, but no adduct was detected by reversed-phase HPLC, suggesting it was unstable to the chromatographic conditions. Incubation of indomethacin-NHS with apoprotein followed by HPLC analysis led to the formation of greater amounts of the hydrolysis product indomethacin than did similar treatment of holoprotein. The results suggest that indomethacin-IM and indomethacin-NHS covalently and selectively label PGH synthase near the heme binding site, leading to loss of both catalytic activities of the enzyme.

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