Document Detail

Identification of two cyclooxygenase active site residues, Leucine 384 and Glycine 526, that control carbon ring cyclization in prostaglandin biosynthesis.
MedLine Citation:
PMID:  14594816     Owner:  NLM     Status:  MEDLINE    
The cyclooxygenase (COX) reaction of prostaglandin (PG) biosynthesis begins with the highly specific oxygenation of arachidonic acid in the 11R configuration and ends with a 15S oxygenation to form PGG2. To obtain new insights into the mechanisms of stereocontrol of oxygenation, we mutated active site residues of human COX-2 that have potential contacts with C-11 of the reacting substrate. Although the 11R oxygenation was not perturbed, changing Leu-384 (into Phe, Trp), Trp-387 (Phe, Tyr), Phe-518 (Ile, Trp, Tyr), and Gly-526 (Ala, Ser, Thr, Val) impaired or abrogated PGG2 synthesis, and typically 11R-HETE was the main product formed. The Gly-526 and Leu-384 mutants formed, in addition, three novel products identified by LC-MS, NMR, and circular dichroism as 8,9-11,12-diepoxy-13R-(or 15R)-hydro(pero)xy derivatives of arachidonic acid. Mechanistically, we propose these arise from a free radical intermediate in which a C-8 carbon radical displaces the 9,11-endoperoxide O-O bond to yield an 8,9-11,12-diepoxide that is finally oxygenated stereospecifically in the 13R or 15R configuration. Formation of these novel products signals an arrest in the normal course of prostaglandin synthesis just prior to closing of the 5-membered carbon ring, and points to a crucial role for Leu-384 and Gly-526 in the correct positioning of the reacting fatty acid intermediate. Some of the Gly-526 and Leu-384 mutants catalyzed both formation of PGG2 (with the normal 15S configuration) and the 13R- or 15R-oxygenated diepoxides. This result suggests that oxygenation specificity can be determined by the orientation of the reacting fatty acid radical and is not a predetermined outcome based solely on the structure of the cyclooxygenase active site.
Claus Schneider; William E Boeglin; Alan R Brash
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Publication Detail:
Type:  In Vitro; Journal Article; Research Support, U.S. Gov't, P.H.S.     Date:  2003-10-31
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  279     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2004 Feb 
Date Detail:
Created Date:  2004-02-02     Completed Date:  2004-04-01     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4404-14     Citation Subset:  IM    
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
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MeSH Terms
Amino Acid Substitution
Arachidonic Acids / biosynthesis,  chemistry
Catalytic Domain / genetics
Chromatography, High Pressure Liquid
Cyclooxygenase 2
Free Radicals / chemistry
Glycine / chemistry
Hela Cells
Isoenzymes / chemistry*,  genetics,  metabolism*
Leucine / chemistry
Membrane Proteins
Models, Molecular
Molecular Structure
Mutagenesis, Site-Directed
Nuclear Magnetic Resonance, Biomolecular
Prostaglandin-Endoperoxide Synthases / chemistry*,  genetics,  metabolism*
Prostaglandins G / biosynthesis*,  chemistry
Recombinant Proteins / chemistry,  genetics,  metabolism
Grant Support
Reg. No./Substance:
0/Arachidonic Acids; 0/Free Radicals; 0/Isoenzymes; 0/Membrane Proteins; 0/Prostaglandins G; 0/Recombinant Proteins; 51982-36-6/prostaglandin G2; 56-40-6/Glycine; 61-90-5/Leucine; 73347-42-9/11-hydroperoxyeicosa-5,8,12,14-tetraenoic acid; EC 2; EC protein, human; EC Synthases

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