Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol.

Kozak KR, Prusakiewicz JJ, Rowlinson SW, Schneider C, Marnett LJ
J Biol Chem. 2001 276 (32): 30072-7

PMID: 11402053 · DOI:10.1074/jbc.M104467200

The endocannabinoid, 2-arachidonylglycerol (2-AG), is an endogenous ligand for the central (CB1) and peripheral (CB2) cannabinoid receptors and has been shown to be efficiently and selectively oxygenated by cyclooxygenase (COX)-2. We have investigated 2-AG/COX-2 interactions through site-directed mutagenesis. An evaluation of more than 20 site-directed mutants of murine COX-2 has allowed for the development of a model of 2-AG binding within the COX-2 active site. Most strikingly, these studies have identified Arg-513 as a critical determinant in the ability of COX-2 to efficiently generate prostaglandin H(2) glycerol ester, explaining, in part, the observed isoform selectivity for this substrate. Mutational analysis of Leu-531, an amino acid located directly across from Arg-513 in the COX-2 active site, suggests that 2-AG is shifted in the active site away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Despite this difference, aspirin-treated COX-2 oxygenates 2-AG to afford 15-hydroxyeicosatetraenoic acid glycerol ester in a reaction analogous to the C-15 oxygenation of arachidonic acid observed with acetylated COX-2. Finally, the differences in substrate binding do not alter the stereospecificity of the cyclooxygenase reaction; 2-AG-derived and arachidonic acid-derived products share identical stereochemistry.

MeSH Terms (34)

Amino Acids Amino Acid Sequence Animals Arachidonic Acid Arachidonic Acids Arginine Binding Sites Cannabinoid Receptor Modulators Cannabinoids Cyclooxygenase 1 Cyclooxygenase 2 DNA Mutational Analysis Endocannabinoids Esters Glycerides Glycerol Hydroxyeicosatetraenoic Acids Isoenzymes Leucine Mass Spectrometry Membrane Proteins Mice Models, Chemical Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutation Oxygen Prostaglandin-Endoperoxide Synthases Prostaglandin H2 Prostaglandins H Protein Binding Protein Isoforms Time Factors

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