Molecular and kinetic determinants of cyclooxygenase-2 selective substrate metabolism and inhibition

Cyclooxygenases (COXs) and Lipoxygenases (LOXs) oxygenate arachidonic acid to provide an array of potent mediators, which include prostglandins (PGs), thromboxanes, and hydroperoxyeicosatetraenoic acids (HpETE). Endogenous lipoamino acids have been shown to regulate several physiological and pathological processes. We examined the ability of COXs and LOXs to metabolize N-arachidonylglycine and other lipoamino acids. COX-2, 12-LOX and 15-LOX enzymes efficiently oxygenated lipoamino acids. These results suggest that COX-2 and multiple LOXs may regulate lipoamino acid levels during the synthesis of novel oxygenation products.; Along with N-arachidonylglycine, The endocannabinoids 2-arachidonylglycerol (2-AG) and arachidonylethanolamide, are selective substrates for COX-2. The molecular determinants of COX-2 that conferred selective oxygenase activity were found to reside in an active site region, termed the side-pocket. Furthermore, this region was highly conserved among COX-2 genes. These results suggest that the capacity to metabolize lipoamino acids and endocannabinoids may represent an important evolutionary impetus for two separate COX isoforms.; For over 30 years the time-dependent mechanism of COX inhibition by indomethacin has been investigated. We characterized the interactions between the 2-methyl group of indomethacin and a small hydrophobic pocket in the active site of COX-2. When this pocket was blocked by mutagenesis, indomethacin became a reversible inhibitor. Removing the indomethacin 2-methyl group also resulted in reversible inhibition and a near loss of time-dependence. The contacts between the 2-methyl and this hydrophobic pocket are critical for the inhibitory mechanism and efficacy of indomethacin.; We evaluated the potency of weak inhibitors of COX arachidonic acid oxygenation, against COX-2 metabolism of 2-AG. We observed an increase of nearly 4 orders of magnitude in potency. These fast-reversible inhibitors appeared to become tight time-dependent inhibitors with 2-AG. Our observations were consistent even in cellular assays. We revealed the kinetic component of this discrepancy to be the relative magnitudes of the dissociation rate constants between the substrates and inhibitors. More importantly, these data provided insights into the pharmacology of endocannabinoid-derived PG generation.