The coupling between the peroxidase and cyclooxygenase activities of prostaglandin H synthase (PGHS) has been proposed to be mediated by a critical tyrosyl radical through a branched chain mechanism (Dietz, R., Nastainczyk, W., and Ruf, H. H. (1988) Eur. J. Biochem. 171, 321-328). In this study, we have examined the ability of PGHS isoform-1 (PGHS-1) tyrosyl radicals to react with arachidonate. Anaerobic addition of arachidonate following formation of the peroxide-induced wide doublet or wide singlet tyrosyl radical led to disappearance of the tyrosyl radicals and emergence of a new EPR signal, which is distinct from known PGHS-1 tyrosyl radicals. The new radical was clearly derived from arachidonate because its EPR line shape changed when 5,6,8,9,11,12,14,15-octadeuterated arachidonate was used. Subsequent addition of oxygen to samples containing the fatty acyl radical resulted in regeneration of tyrosyl radical EPR. In contrast, the peroxide-generated tyrosyl radical in indomethacin-treated PGHS-1 (a narrow singlet) failed to react with arachidonate, consistent with the cyclooxygenase inhibition by indomethacin. These results indicate that the peroxide-generated wide doublet and wide singlet tyrosyl radicals serve as immediate oxidants of arachidonate bound at the cyclooxygenase active site to form a carbon-centered fatty acyl radical, which reacts with oxygen to form a hydroperoxide. These observations represent the first direct evidence of chemical coupling between the peroxidase reaction and arachidonate oxygenation in PGHS-1 and support the proposed role for a tyrosyl radical in cyclooxygenase catalysis.
Two isoforms of prostaglandin H synthase have been described: isoform-1 (PGHS-1), which is ascribed a role in basal or housekeeping prostaglandin synthesis; and isoform-2 (PGHS-2), which has been found to be strongly inducible in many tissues and has been associated with inflammatory processes. Recent observations have indicated that cyclooxygenase catalysis by the two isoforms can be differentially regulated when both are present simultaneously (Reddy, S. T., and Herschman, H. R. To compare the levels of hydroperoxide required for cyclooxygenase initiation in the two PGHS isoforms, we have examined the ability of a hydroperoxide scavenger, glutathione peroxidase, to suppress the cyclooxygenase activity of purified preparations of human PGHS-2, ovine PGHS-2, and ovine PGHS-1. Half-maximal prostaglandin synthetic activity was found to require a much lower hydroperoxide level with human PGHS-2 (2.3 nM) and ovine PGHS-2 (2.2 nM) than with ovine PGHS-1 (21 nM). Similar results were obtained when cyclooxygenase activity was monitored by chromatographic analyses of radiolabeled arachidonate metabolites or with oxygen electrode measurements. Mixing four parts of ovine PGHS-1 with one part of human PGHS-2 did not markedly change the sensitivity of the overall cyclooxygenase activity to inhibition by glutathione peroxidase, indicating that the PGHS-1 activity was not easily initiated by PGHS-2 activity in the same vessel. Effective catalysis by PGHS-2 can thus proceed at hydroperoxide levels too low to sustain appreciable catalysis by PGHS-1. This difference in catalytic characteristics provides a biochemical mechanism for differential control of prostaglandin synthesis by the two PGHS isoforms, even when both are present in the same intracellular compartment.
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...
A hydroperoxide-induced tyrosyl radical has been proposed as a key cyclooxygenase intermediate for the "basal" isoform of prostaglandin H synthase (PGHS-1). In the present study with the "inducible" isoform (PGHS-2), hydroperoxide was also found to generate a radical in high yield, a wide singlet at g = 2.0058 (29 G peak to trough). Reaction of PGHS-2 with a tyrosine-modifying reagent, tetranitromethane (TNM), resulted in cyclooxygenase inactivation and a much narrower radical EPR signal (22 G peak to trough). Addition of a cyclooxygenase inhibitor, nimesulide, similarly resulted in a narrow PGHS-2 radical. In PGHS-1, cyclooxygenase inhibition by tyrosine nitration with TNM or by active site ligands leads to generation of a narrow EPR instead of a wide EPR, with both signals originating from authentic tyrosyl radicals, indicating that the hydroperoxide-induced radicals in PGHS-2 are also tyrosyl radicals. Treatment of PGHS-2 with aspirin (acetyl salicylic acid, ASA) was previously shown to result in acetylation of a specific serine residue, cyclooxygenase inhibition, and increased lipoxygenase activity. Acetylation of PGHS-1 by ASA, in contrast, inhibited both lipoxygenase and cyclooxygenase activity. We now have found the ASA-treated PGHS-2 radical to be indistinguishable from that in control PGHS-2. Addition of nimesulide to ASA-treated PGHS-2 inhibited the lipoxygenase and resulted in a narrow radical EPR like that seen in PGHS-2 treated with TNM or nimesulide alone. Retention of PGHS-2 oxygenase activity was thus associated with retention of the native radical, and loss of activity was associated with alteration of the radical. Both native and ASA-treated PGHS-2 produced only the R stereoisomer of 11- and 15-HETE, demonstrating that the lipoxygenase stereochemistry was not changed by ASA. Native and ASA-treated PGHS-2 had lipoxygenase K(m) values considerably higher than that of the control PGHS-2 cyclooxygenase. Taken together, these results suggest that the same PGHS-2 tyrosyl radical serves as the oxidant for both cyclooxygenase and lipoxygenase catalysis and that acetylation of PGHS-2 by ASA favors arachidonate binding in an altered conformation which results in abstraction of the pro-R hydrogen from C13 and formation of 11(R)- and 15(R)-HETE.
A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29 -31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA⅐) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36 -38G, but was also able to convert AA to an AA⅐ with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting -protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carboncentered AA⅐.
Prostaglandin H synthase has both a heme-dependent peroxidase activity and a cyclooxygenase activity. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by an interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical [Stubbe, J. A. (1989) Annu. Rev. Biochem. 58, 257-285]. We have examined the kinetics of radical formation with both ethyl hydroperoxide (EtOOH) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and have analyzed the effects of indomethacin (a selective cyclooxygenase inhibitor) and tetranitromethane (TNM; a selective agent for nitration of tyrosyl residues) on the synthase. At -14 degrees C both EtOOH and 15-HPETE generated within 5 s a free radical species whose electron paramagnetic resonance spectrum was dominated by a doublet centered at g = 2.005 (splitting of approximately 16 G; overall peak-to-trough width of 35 G) that has been attributed to tyrosyl radical. The doublet subsequently gave way to a singlet with a similar peak-to-trough width; the doublet-to-singlet transition was complete in 20-60 s. The intensity of the doublet/singlet combination peaked at 0.6 spins/heme after 120 s with EtOOH and at about 0.3 spins/heme after 20 s with 15-HPETE; the radical intensity declined slowly with EtOOH but more rapidly with 15-HPETE. Reaction of the indomethacin-synthase complex with EtOOH resulted in a narrower (peak-to-trough width of 24 G) singlet free radical signal, with no evidence of an earlier doublet; the intensity of the singlet peaked at 0.45 spins/heme after about 300 s. Reaction of TNM-treated synthase with EtOOH resulted in a singlet almost identical with that seen for the indomethacin-synthase complex. Reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both cyclooxygenase and peroxidase activity, with the former being lost rapidly and completely while the latter was lost slowly and to about 50%. Ibuprofen, a competitive cyclooxygenase inhibitor, slowed the rate of inactivation of the cyclooxygenase by about 20-fold. The rate of inactivation of the cyclooxygenase activity in synthase apoenzyme by TNM was also about 20-fold less than that observed with the holoenzyme. Amino acid analyses revealed that TNM-reacted holoenzyme with less than 10% residual activity contained 1.8 nitrotyrosines/subunit; apoenzyme reacted under the same conditions had greater than 80% of the original activity and contained 0.7 nitrotyrosine/subunit.(ABSTRACT TRUNCATED AT 400 WORDS)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.