Reaction and Free Radical Kinetics of Prostaglandin H Synthase with Manganese Protoporphyrin IX as the Prosthetic Group

Kulmacz, Richard J.; Palmer, Graham; Wei, Chunhong; Tsai, Ah‐Lim

doi:10.1021/bi00184a011

Cited by 39 publications

(60 citation statements)

References 39 publications

(84 reference statements)

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“…Some published evidence has suggested the formation of an enzymatic tyrosyl radical during the catalysis of Mn-PHS-1 (29), but that suggestion has not been supported with unequivocal ESR data (30,31). To test whether tyrosyl radicals are formed in the Mn-enzyme and to determine whether iminoxyl radical formation is a more general phenomenon upon exposure of tyrosyl radicals to NO ⅐ , the experiments were repeated with Mn-PHS-2.…”

Section: Resultsmentioning

confidence: 98%

“…Reconstitution of PHS-2 with manganese-heme rather than iron-heme results in a protein that retains cyclooxygenase activity but has greatly decreased peroxidase activity (29). Some published evidence has suggested the formation of an enzymatic tyrosyl radical during the catalysis of Mn-PHS-1 (29), but that suggestion has not been supported with unequivocal ESR data (30,31).…”

Section: Resultsmentioning

confidence: 99%

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Nitric Oxide Trapping of the Tyrosyl Radical of Prostaglandin H Synthase-2 Leads to Tyrosine Iminoxyl Radical and Nitrotyrosine Formation

Gunther¹,

Hsi²,

Curtis³

et al. 1997

Journal of Biological Chemistry

162

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The determination of protein nitrotyrosine content has become a frequently used technique for the detection of oxidative tissue damage. Protein nitration has been suggested to be a final product of the production of highly reactive nitrogen oxide intermediates (e.g. peroxynitrite) formed in reactions between nitric oxide (NO ⅐ ) and oxygen-derived species such as superoxide. The enzyme prostaglandin H synthase-2 (PHS-2) forms one or more tyrosyl radicals during its enzymatic catalysis of prostaglandin formation. In the presence of the NO ⅐ -generator diethylamine nonoate, the electron spin resonance spectrum of the PHS-2-derived tyrosyl radical is replaced by the spectrum of another free radical containing a nitrogen atom. The magnitude of the nitrogen hyperfine coupling constant in the latter species unambiguously identifies it as an iminoxyl radical, which is likely formed by the oxidation of nitrosotyrosine, a stable product of the addition of NO ⅐ to tyrosyl radical. Addition of superoxide dismutase did not alter the spectra, indicating that peroxynitrite was not involved. Western blot analysis of PHS-2 after exposure to the NO ⅐ -generator revealed nitrotyrosine formation. The results provide a mechanism for nitric oxide-dependent tyrosine nitration that does not require formation of more highly reactive nitrogen oxide intermediates such as peroxynitrite or nitrogen dioxide.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Nitric Oxide Trapping of the Tyrosyl Radical of Prostaglandin H Synthase-2 Leads to Tyrosine Iminoxyl Radical and Nitrotyrosine Formation

Gunther¹,

Hsi²,

Curtis³

et al. 1997

Journal of Biological Chemistry

162

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“…Guaiacol (o-methoxyphenol), α-aminolevulinic acid, Lubrol PX and lactose were purchased from Sigma. Sheep apo-PGHS-1 and holoenzymes replenished with hemin or mangano protoporphyrin IX were prepared as described previously [12].…”

Section: Methodsmentioning

confidence: 99%

“…15-HETE and 15-KETE were identified using the authentic compounds produced by the reaction of 15-HPETE with Fe-or Mn-PGHS, respectively [12,16]. Fe-or Mn-PGHS (0.5 μM) was reacted with [1-14 C]-15-HPETE (100 μM) in the presence of guaiacol (980 μM) for 1 min.…”

Section: Product Analysis By Hplc and Electron-spray Ionization Mass mentioning

confidence: 99%

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Yeh¹,

Tsai²,

Wang³

2007

Archives of Biochemistry and Biophysics

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Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are atypical cytochrome P450s. They do not require NADPH or dioxygen for isomerization of prostaglandin H 2 (PGH 2 ) to produce prostacyclin (PGI 2 ) and thromboxane A 2 (TXA 2 ). PGI 2 and TXA 2 have opposing actions on platelet aggregation and blood vessel tone. In this report, we use a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), to explore the active site characteristics of PGIS and TXAS. The two enzymes transformed 15-HPETE not only into 8,, like many microsomal P450s, but also to 15-ketoeicosatetraenoic acid (15-KETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). 13-OH-14,15-EET and 15-KETE result from homolytic cleavage of the O-O bond, whereas 15-HETE results from heterolytic cleavage, a common peroxidase pathway. About 80% of 15-HPETE was homolytically cleaved by PGIS and 60% was homolytically cleaved by TXAS. The V max of homolytic cleavage is 3.5-fold faster than heterolytic cleavage for PGIS-catalyzed reactions (1100 min −1 vs. 320 min −1 ) and 1.4-fold faster for TXAS (170 min −1 vs. 120 min −1 ). Similar K M values for homolytic and heterolytic cleavages were found for PGIS (∼60 μM 15-HPETE) and TXAS (∼80 μM 15-HPETE), making PGIS a more efficient catalyst for the 15-HPETE reaction.Keywords prostacyclin synthase; thromboxane synthase; cytochrome P450; peroxide bond cleavage; homolytic; heterolytic; hydroperoxides; epoxyalcohol Prostacyclin synthase (PGIS) and thromboxane synthase (TXAS) are members of the cytochrome P450 superfamily, which uses heme as the prosthetic group and has a cysteine thiolate proximal ligand [1,2]. PGIS and TXAS convert prostaglandin H 2 (PGH 2 ) to prostacyclin (PGI 2 ) and thromboxane A 2 (TXA 2 ), respectively. PGI 2 inhibits platelet activation and aggregation and induces vasodilation, whereas TXA 2 stimulates platelet secretion, aggregation and vasoconstriction [3]. Both PGI 2 and TXA 2 are rapidly hydrolyzed To whom correspondence should be addressed: Lee-Ho Wang Division of Hematology Department of Internal Medicine 6431 Fannin Houston, TX 77030 Tel: 713-500-6794 Fax: 713-500-6810 e-mail: lee-ho.wang@uth.tmc.edu 1 The abbreviations used are: PGIS, prostacyclin synthase; PGI 2 , prostacyclin; PGHS, prostaglandin H synthase; TXAS, thromboxane synthase; TXA 2 , thromboxane A 2 ; 8,11-eicosatrienoic acid; ESI/MS, electron-spray ionization/ mass spectrometry; EPR, electron paramagnetic resonance; SVD, singular value decomposition.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 early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Hecker and Ullrich proposed a caged radical mechanism for TXAS ...

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“…This is presumably due to slower Intermediate I formation (15)(16)(17) and fits well with the long lag phase observed for cyclooxygenase catalysis by Mn-PGHS-1 (30). Although the MnPPIX redox changes appeared to be somewhat faster than formation of tyrosyl radical with both 2 and 5 equiv of 15-HPETE, the difference may be due to the wavelength used for our kinetic measurement, 420 nm, which tracks not only the conversion from Intermediate I to Intermediate II but also the preceding step of Intermediate I formation, which is quite rapid (11).…”

Section: Formation and Decay Of Tyr385 Radical And Mn 4+ =O During Thmentioning

confidence: 99%

Oxyferryl Heme and Not Tyrosyl Radical Is the Likely Culprit in Prostaglandin H Synthase-1 Peroxidase Inactivation

Rogge

Wang

et al. 2006

Biochemistry

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Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G 2 . Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.Prostagladin H synthases (PGHSs) catalyze the first two steps in biosynthesis of prostaglandins, the cyclooxygenase reaction that converts arachidonic acid (AA) 1 to prostaglandin (PG)G 2 and the peroxidase reaction that reduces PGG 2 to PGH 2 (1). The cyclooxygenase reaction is dependent upon formation of the Tyr385 radical during the peroxidase reaction (2). A simplified mechanistic model is shown in Figure 1. According to † This work was supported by U.S. Public Health Service Grants GM44911 (to A.-L.T.), GM52170 (to R.J.K.), and GM55807 (to G.P.) and Postdoctoral Fellowship DK61929 (to C.E.R.), and Welch Foundation Grant C636 (to G.P. Reaction with peroxide drives the redox events that lead to strong feedback amplification of cyclooxygenase catalysis, and it also drives the irreversible self-inactivation of both peroxidase and cyclooxygenase activities that imposes an upper limit on prostanoid synthesis (7,8).Cyclooxygenase inactivation induced by peroxide occurs on the same time scale as peroxidase inactivation, although the former seems more sensitive to the peroxide structure (9,1...

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Reaction and Free Radical Kinetics of Prostaglandin H Synthase with Manganese Protoporphyrin IX as the Prosthetic Group

Cited by 39 publications

References 39 publications

Nitric Oxide Trapping of the Tyrosyl Radical of Prostaglandin H Synthase-2 Leads to Tyrosine Iminoxyl Radical and Nitrotyrosine Formation

Nitric Oxide Trapping of the Tyrosyl Radical of Prostaglandin H Synthase-2 Leads to Tyrosine Iminoxyl Radical and Nitrotyrosine Formation

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

Oxyferryl Heme and Not Tyrosyl Radical Is the Likely Culprit in Prostaglandin H Synthase-1 Peroxidase Inactivation

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