title: Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide.
abstract: The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.
authors.affiliation: Department of Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Kozak
authors.forename: Jeffery J
authors.initials: JJ
authors.lastname: Prusakiewicz
authors.forename: Scott W
authors.initials: SW
authors.lastname: Rowlinson
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Prudhomme
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Marnett
mesh_terms: D000107:Acetylation; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D063385:Cannabinoid Receptor Modulators; D002186:Cannabinoids / Q000378:metabolism*; D002384:Catalysis; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D063388:Endocannabinoids; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins; D008715:Methionine / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism*; D053284:Polyunsaturated Alkamides; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics; D012756:Sheep; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D002186:Cannabinoids; D063388:Endocannabinoids; D005231:Fatty Acids, Unsaturated; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D008565:Membrane Proteins; D053284:Polyunsaturated Alkamides; D014443:Tyrosine; D012694:Serine; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D008715:Methionine; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse; D010100:Oxygen; C078814:anandamide