Higher oxidation states of prostaglandin H synthase

Karthein, R.; Dietz, Reiner; Nastainczyk, Wolfgang; Ruf, Hans Heinrich

doi:10.1111/j.1432-1033.1988.tb13792.x

Cited by 273 publications

(220 citation statements)

References 35 publications

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“…Our currcnt isotope substitution experiments have ruled out the possibility that this enzyme contains a tyrosyl radical as known for ribonucleotide reductase [13] or prostaglandin H synthase [14]. EPR spectroscopic data indicate that the unpaired clcctron is carbon-centered.…”

Section: Discussionmentioning

confidence: 95%

The free radical of pyruvate formate‐lyase

Unkrig

Neugebauer

Knappe

1989

European Journal of Biochemistry

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The first-derivative EPR spectrum of the active form of Escherichiu coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of I spin per active site.The formate analogue hypophosphite has been characterized as a specific k,,, inhibitor of pyruvate formatelyase which destroys the enzyme radical. Protein-linked 1 -hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178,. EPR spectroscopy of this system has now identified the corresponding a-phosphoryl radical as a reaction intermediate [g = 2.0032; u(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0°C. This finding proves that the enzyme radical is a hydrogen-atomtransferring coenzymic element.Pyruvate formate-lyase is the key enzyme of the anaerobic glucose degradation route in Escherichia coli cells [l]. It shows unique mechanisms of enzyme regulation and catalysis. Transcription of the structural gene (~f l )is highly controlled by anaerobic induction [2]. At the post-translational level, pyruvate formate-lyase is interconverted by specific converter enzymes between a fully inactive form (Ei) and an active form (E,) that contains a persistent organic free radical [3]. Pyruvate is nonoxidatively processed to acetyl-CoA by a two-step reaction pattern with an intermediary acetyl-enzyme:(2) Addressing structure/function relationships, we have recently mapped the covalent catalytic SH-group as the Cys-419 residue of the polypeptide chain [4]. These studies also showed that the acetyl-enzyme intermediate will react with the formate analogue hypophosphite to produce l-hydroxyethylphosphonate, linked as a thioester to Cys-418. The C-P bond-forming process, which resembles pyruvate synthesis from formate (reversal of Eqn l), has now been investigated by EPR spectroscopy. We have identified a substrate-based radical intermediate, thus demonstrating for the first time a functional role of the free radical of pyruvate formate-lyase. Our findings substantiate the notion that the reversible C-C bond cleavage/synthesis in the first half-reaction of the catalytic cycle occurs by a radical mechanism.We also report in this paper the EPR and ultraviolet spectroscopic characterization of the enzyme radical, its quantification, and several isotope substitution experiments for approachi...

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Section: Discussionmentioning

confidence: 95%

The free radical of pyruvate formate‐lyase

Unkrig

Neugebauer

Knappe

1989

European Journal of Biochemistry

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“…In addition to the unstable tyrosyl radical of prostaglandin H synthase (41), stable tyrosyl radicals in ribonucleotide reductase and photosystem II (42,43) have been detected. Furthermore, tyrosyl radicals have been detected in oxidatively damaged hemoproteins such as myoglobin (44), hemoglobin (45), and cytochrome c (46).…”

Section: Discussionmentioning

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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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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“…This initial event increases the overall oxidative state of the enzyme by generating radical species in the protein [11,12] through intramolecular electron transfer. Generation of the Tyr385 radical in the cyclooxygenase catalytic pocket [13] leads to abstraction of an hydrogen from arachidonic acid, which initiates the oxygenation that leads to formation of prostaglandin G 2 (PGG 2 ). We previously found that hydroperoxide substrates that drive the peroxidase activity antagonize the inhibition of PGHSs by sodium salicylate [14].…”

Section: Introductionmentioning

confidence: 99%

Acetylation of prostaglandin H2 synthases by aspirin is inhibited by redox cycling of the peroxidase

Bala

Chin

Logan

et al. 2008

Biochemical Pharmacology

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Aspirin exerts its unique pharmacological effects by irreversibly acetylating a serine residue in the cyclooxygenase site of prostaglandin-H 2 -synthases (PGHSs). Despite the irreversibility of the inhibition, the potency of aspirin varies remarkably between cell types, suggesting that molecular determinants could contribute to cellular selectivity. Using purified enzymes, we found no evidence that aspirin is selective for either of the two PGHS isoforms, and we showed that hydroperoxide substrates of the PGHS peroxidase inhibited the rate of acetylation of PGHS-1 by 68%. Using PGHS-1 reconstituted with cobalt protoporphyrin, a heme devoid of peroxidase activity, we demonstrated that reversal by hydroperoxides of the aspirin-mediated acetylation depends upon the catalytic activity of the PGHS peroxidase. We demonstrated that inhibition of PGHS-2 by aspirin in cells in culture is reversed by 12-hydroperoxyeicosatetraenoic acid dose-dependently (ED 50 = 0.58 ± 0.15 μM) and that in cells with high levels of hydroperoxy-fatty acids (RAW264.7) the efficacy of aspirin is markedly decreased as compared to cells with low levels of hydroperoxides (A549; IC 50 s = 256 ± 22 μM and 11.0 ± 0.9 μM, respectively). Together, these findings indicate that acetylation of the PGHSs by aspirin is regulated by the catalytic activity of the peroxidase which yields a higher oxidative state of the enzyme.

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Higher oxidation states of prostaglandin H synthase

Cited by 273 publications

References 35 publications

The free radical of pyruvate formate‐lyase

The free radical of pyruvate formate‐lyase

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

Acetylation of prostaglandin H2 synthases by aspirin is inhibited by redox cycling of the peroxidase

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