Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid 2-arachidonylglycerol.
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.
Kozak
K R
KR
Departments of Biochemistry and Chemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Prusakiewicz
J J
JJ
Rowlinson
S W
SW
Schneider
C
C
Marnett
L J
LJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D016718:Arachidonic Acid / Q000737:chemistry; D001095:Arachidonic Acids; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D063385:Cannabinoid Receptor Modulators; D002186:Cannabinoids / Q000378:metabolism*; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D004252:DNA Mutational Analysis; D063388:Endocannabinoids; D004952:Esters / Q000737:chemistry; D005989:Glycerides / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007930:Leucine / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D044262:Prostaglandin H2; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011463:Prostaglandins H / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D013997:Time Factors
D000596:Amino Acids; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D002186:Cannabinoids; D063388:Endocannabinoids; D004952:Esters; D005989:Glycerides; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D008565:Membrane Proteins; D011463:Prostaglandins H; D020033:Protein Isoforms; D016718:Arachidonic Acid; D044262:Prostaglandin H2; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C094503:glyceryl 2-arachidonate; D001120:Arginine; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse; D007930:Leucine; D005990:Glycerol; D010100:Oxygen