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| ELECTRON-SPIN-RESONANCE INVESTIGATION OF TYROSYL RADICALS OF PROSTAGLANDIN-H SYNTHASE - RELATION TO ENZYME CATALYSIS |
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| Author(s): LASSMANN G, ODENWALLER R, CURTIS JF, DEGRAY JA, MASON RP, MARNETT LJ, ELING TE |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 266 Issue: 30 Pages: 20045-20055 Published: OCT 25 1991 |
| Times Cited: 90 References: 59 |
| Abstract: We have examined, by low temperature ESR, the protein-derived radicals formed by reaction of purified ram seminal vesicle prostaglandin H synthase (PHS). Upon addition of arachidonic acid or 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) to PHS reconstituted with Fe(III)-protoporphyrin IX (Fe-PHS) at -12-degrees-C, an ESR spectrum was observed at -196-degrees-C containing a doublet that rapidly converted into a singlet. These protein-derived radicals were identified as tyrosyl radicals. The addition of a peroxidase substrate, phenol, completely abolished the appearance of the doublet and suppressed the formation of the singlet but did not inhibit eicosanoid formation. Incubation of arachidonic acid with PHS reconstituted with Mn(III)-protoporphyrin IX (Mn-PHS) produced only a broad singlet that exhibited different power saturation behavior than the tyrosyl radicals and decayed more rapidly. This broad singlet does not appear to be a tyrosyl radical. No ESR signals were observed on incubation of PPHP with Mn-PHS, which has cyclooxygenase but not peroxidase activity. Eicosanoid synthesis occurred very rapidly after addition of arachidonic acid and was complete within 1 min. In contrast, the protein-derived radicals appeared at a slower rate and after the addition of the substrate reached maximal levels between 1 and 2 min for Fe-PHS and 4-6 min for Mn-PHS. These results suggest that the observed protein-derived radicals are not catalytically competent intermediates in cyclooxygenase catalysis by either Fe-PHS or Mn-PHS. The peroxidase activity appears to play a major role in the formation of the tyrosyl radicals with Fe-PHS. |
| Document Type: Article |
| Language: English |
Addresses:
1. NIEHS, MOLEC BIOPHYS LAB, RES TRIANGLE PK, NC 27709 USA
2. VANDERBILT UNIV, MED CTR, SCH MED, CTR MOLEC TOXICOL, DEPT BIOCHEM, AB HANCOCK JR MEM LAB CANC RES, NASHVILLE, TN 37232 USA
3. VANDERBILT UNIV, MED CTR, SCH MED, DEPT CHEM, NASHVILLE, TN 37232 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: GM039 |
| ISSN: 0021-9258 |
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| | | | | | | | | Record from Web of Science® | | | | | | | | | |