EPR titration of ovine prostaglandin H synthase with hemin

Ruf, Hans Heinrich; Schuhn, Dietmar; Nastainczyk, Wolfgang

doi:10.1016/0014-5793(84)80189-1

Cited by 28 publications

(15 citation statements)

References 13 publications

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“…1 clearly shows the subsequent formation of two intermediates of PGH synthase during its reaction with PGG2. The spectrum of the native enzyme with bands at 410 nm and 630 nm was typical of ferric high-spin heme, which has been proven by EPR [5,61. The first spectral species, termed intermediate I, was formed within the first 20 ms of the reaction were obtained by separate experiments where native enzyme was mixed with buffer and 2-propanol without PPG2.…”

Section: Resultsmentioning

confidence: 79%

Higher oxidation states of prostaglandin H synthase

Dietz

Nastainczyk

Ruf

1988

European Journal of Biochemistry

233

224

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The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 °C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1= 1.4 × 107 M−1 s−1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+· FeIVO). Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2= 65 s−1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome‐c peroxidase, was assigned to a two‐electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+· which was formed by an intramolecular electron transfer from tyrosine to the porphyrin‐π‐cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.

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Section: Resultsmentioning

confidence: 79%

Higher oxidation states of prostaglandin H synthase

Dietz

Nastainczyk

Ruf

1988

European Journal of Biochemistry

233

224

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“…The inactivation by N-acetylimidazole was quite different from the inactivation by other tyrosine-specific reagents, tetranitromethane and pnitrobenzenesulfonyl fluoride. With the latter reagents, the holoenzymc was much more susceptible to inactivation than the apoprotein, and the inactivation of the cyclooxygenase proceeded faster than the inactivation of the peroxidase (Kulniacz and Ren, 1990;Scherer et al, 1991). After reaction with N-acetylimidazole, the apoprotein could not longer bind ferric heme as the prosthctic group, which is required for both activities.…”

Section: Discussionmentioning

confidence: 99%

“…From spectroscopic findings, a special role has been suggested for tyrosyl residues. In the native enzyme, heme is in its high-spin ferric state, exhibiting an EPR spectrum with a rather high rhombicity (Ruf et al, 1984;Karthein et al, 1987;Kulmacz et al, 3987;Kulmacz and Ren, 1990). This rhombicity, together with the absence of a super-hyperfine interaction from an Nc of a potential histidine ligand in the ferrous NO adduct, led to the suggestion of tyrosinate as an axial ligand of the heme iron in native PGH synthase (Karthein et al, 1987).…”

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confidence: 99%

Chemical modification of prostaglandin endoperoxide synthase by N‐acetylimidazol

Karthein

Strieder

Ruf

1992

European Journal of Biochemistry

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Prostaglandin H synthase apoprotein, without its prosthetic heme group, was inactivated by Nacetylimidazole under conditions typical for the O-acetylation of tyrosyl residues. A spontaneous reactivation occurred above pH 7.5 at 22 "C, which indicated spontaneous hydrolysis of acetylated residues. Below pH 7.5, where stable inactivation was observed, reactivation was achieved by reaction with hydroxylamine. Both enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, were inactivated and reactivated simultaneously and to the same extent. In contrast to the apoprotein, the holoenzyme with heme was not inactivated by N-acetylimidazole. The number of acetyl groups, as determined as hydroxamate after the reaction with hydroxylamine at pH 8.2, was 2.5 k 0.4 for the apoprotein and 1 .O If: 0.24 for the holoenzyme. The specific binding of heme as the prosthetic group was no longer observed by EPR (signals at g = 6.7 and 5.3) when hemin was added to the N-acetylimidazole-reacted apoprotein. Treatment of N-acetylimidazole-reacted apoprotein with hydroxykamine restored the specific binding of heme. The N-acetylimidazole-reacted apoprotein supplemented with hemin and reacted with hydroperoxides, neither showed electronic absorption spectra of higher oxidation states nor an EPR doublet signal due to a tyrosyl radical.These results demonstrate that heme protects against the inactivating modification by Nacetylimidazole and that this modification prevents binding of the prosthetic heme group necessary for both enzymic activities. The absence of the prosthetic heme group explains the concomitant loss of cyclooxygenase and peroxidase activities, as well as the absence of higher oxidation states and the tyrosyl radical. We suggest that the acetylation of a residue in the heme pocket, most probably a tyrosine, although a histidine cannot be definitely disproved, exerts the inhibiting effect. This residue could be the axial ligand of the heme or in close contact to the heme. The results also show that the inhibition by N-acetylimidazole does not involve the acetylation of Ser530 which causes the inhibition by acetylsalicylic acid of cyclooxygenase. [The numbering of amino acids in ovine prostaglandin H synthase is according to DeWitt, D.

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“…Both activities are reconstituted following addition of Fe3+-PPIX, whereas reconstitution with Mn3+-PPIX produces an enzyme with high cyclooxygenase but low peroxidase activity (40). There is some disagreement about the stoichiometry of heme binding, but most estimates place it between one and two hemes per homodimer (41,42). The amino acid sequence ofPGH synthase is available from its cDNA sequence (43)(44)(45).…”

Section: Introductionmentioning

confidence: 99%

Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates.

Marnett

1990

Environ Health Perspect

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Prostaglandin-H synthase is unique among enzymes of the plant and animal kingdom in its ability to biosynthesize and metabolize hydroperoxides. Its cyclooxygenase activity oxygenates polyunsaturated fatty acids to hydroperoxy endoperoxides, and its peroxidase activity reduces the hydroperoxy group to hydroxy groups. Higher oxidation states of the peroxidase oxidize reducing substrates to electron-deficient derivatives that react with macromolecular nucleophiles. In the case of aromatic amines, the electrondeficient derivatives are mutagenic to bacterial and mammalian cells. 3-Dicarbonyl compounds and retinoic acid are oxidized to carbon-centered radicals that react with 02 to form peroxyl free radicals. Peroxyl radicals are the most stable oxy radicals and are able to diffuse some distance from the site of their generation. Peroxyl radicals are also formed during lipid peroxidation and in the reaction of polyunsaturated fatty acid hydroperoxides with metal complexes and metalloproteins. Peroxyl radicals epoxidize isolated doubled bonds of compounds such as 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene ; 3,4-dihydroxy-3,4-dihydrobenzo(a)anthracene; and aflatoxin B1. The epoxide products represent the ultimate carcinogenic forms of the respective compounds. Techniques for quantitating the extent of peroxidase dependent or peroxyl radical-dependent metabolism in vivo make use of differences in the structure or stereochemnistry of reactive intermediates formed by peroxidases relative to cytochromes P450. Differences in the relative amounts of hydrolysis products and DNA adducts derived from anti-and syn-dihydrodiolepoxides following application of BP-7,8-diol to mouse skin in vivo indicate peroxyl radicals play a significant role in metabolism of BP-7,8-diol in uninduced animals.

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EPR titration of ovine prostaglandin H synthase with hemin

Cited by 28 publications

References 13 publications

Higher oxidation states of prostaglandin H synthase

Higher oxidation states of prostaglandin H synthase

Chemical modification of prostaglandin endoperoxide synthase by N‐acetylimidazol

Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates.

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