Spatial requirements for 15-(R)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid synthesis within the cyclooxygenase active site of murine COX-2 - Why acetylated COX-1 does not synthesize 15-(R)-HETE

The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspi rin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site . Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeico satetraenoic acid (18-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HET E), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any p roducts. Because the COX isoforms are structurally similar and share approx imately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated CO X-1 to make 11- and 15-HETE. Residues Val-434, Arg-513, and Val-523 were pr edicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed ed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 t o their COX-I equivalents resulted in abrogation of 11- and 15-HETE product ion after aspirin treatment, confirming the hypothesis that these residues are the major isoform selectivity determinants regulating HETE production. The ability of aspirin-treated R513H mCOX-2 to make 15-HETE, although in re duced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificit y determinant regulating HETE production. Further experiments were undertak en to ascertain whether the steric bulk imparted by the acetyl moiety on Se r-530 prevented the omega-end of arachidonic acid from binding within the t op channel cavity in mCOX-2. Site directed mutagenesis was performed to cha nge Val-228, which resides at the junction of the main cyclooxygenase chann el and the top channel, and Gly-533, which is in the top channel, Both V228 F and G533A produced wild type-like product profiles, but, upon acetylation , neither was able to make HETE products. This suggests that arachidonic ac id orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.