Spatial Requirements for 15-(R)-Hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic Acid Synthesis within the Cyclooxygenase Active Site of Murine COX-2

Abstract

The two isoforms of cyclooxygenase, COX-1 and COX-2, are acetylated by aspirin at Ser-530 and Ser-516, respectively, in the cyclooxygenase active site. Acetylated COX-2 is essentially a lipoxygenase, making 15-(R)-hydroxyeicosatetraenoic acid (15-HETE) and 11-(R)-hydroxyeicosatetraenoic acid (11-HETE), whereas acetylated COX-1 is unable to oxidize arachidonic acid to any products. Because the COX isoforms are structurally similar and share approximately 60% amino acid identity, we postulated that differences within the cyclooxygenase active sites must account for the inability of acetylated COX-1 to make 11-and 15-HETE. Residues Val-434, Arg-513, and Val-523 were predicted by comparison of the COX-1 and -2 crystal structures to account for spatial and flexibility differences observed between the COX isoforms. Site-directed mutagenesis of Val-434, Arg-513, and Val-523 in mouse COX-2 to their COX-1 equivalents resulted in abrogation of 11-and 15-HETE production 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 reduced amounts, indicates that this residue is not an alternate binding site for the carboxylate of arachidonate and that it is not the only specificity determinant regulating HETE production. Further experiments were undertaken to ascertain whether the steric bulk imparted by the acetyl moiety on Ser-530 prevented the -end of arachidonic acid from binding within the top channel cavity in mCOX-2. Site-directed mutagenesis was performed to change Val-228, which resides at the junction of the main cyclooxygenase channel and the top channel, and Gly-533, which is in the top channel. Both V228F and G533A produced wild type-like product profiles, but, upon acetylation, neither was able to make HETE products. This suggests that arachidonic acid orientates in a L-shaped binding configuration in the production of both prostaglandin and HETE products.

Identification of Two Cyclooxygenase Active Site Residues, Leucine 384 and Glycine 526, That Control Carbon Ring Cyclization in Prostaglandin Biosynthesis

“…This is in line with previous suggestions on the mechanisms of 15R versus 15S stereocontrol in COX-2. To explain the inversion of oxygenation stereochemistry by the acetylated Ser-530 in the COX-2 active site giving 15R-HETE as product, it was proposed that the increase in size of the residue triggers a realignment of the arachidonic acid -chain (8,47,48). It seems reasonable to propose a similar twisting over of the side chain as the basis for the 15R stereochemistry of diepoxyalcohol product 1.…”

Cyclooxygenase-2-mediated DNA Damage

“…The change in oxygenation stereospecificity induced by aspirin treatment is proposed to arise because acetylated serine-516 in the human COX-2 (serine-530 in the mouse enzyme) forces a realignment of the -chain of AA. This unusual binding conformation appears to be responsible for oxygenation in the R configuration (43,(47)(48)(49).…”

Structural and Functional Basis of Cyclooxygenase Inhibition

“…51,55 This differential inhibition of the COX enzymes by aspirin is due to the larger volume of the COX-2 active site produced by the Val-523 substitution at the mouth of the side pocket. 56 Mutation of Val-523, Arg-513, and Val-434 in COX-2 to their COX-1 equivalents (Ile-523, His-513, Ile-434) results in the inhibition of 15-and 11(R)-hydroxyeicosatetranoic acid production following treatment with aspirin. 56 The crystal structure of COX-1 reacted with 2-bromoacetoxybenzoic acid (an aspirin analogue) demonstrates the incorporation of the bromoacetyl group at Ser-530 ( Figure 8).…”

“…56 Mutation of Val-523, Arg-513, and Val-434 in COX-2 to their COX-1 equivalents (Ile-523, His-513, Ile-434) results in the inhibition of 15-and 11(R)-hydroxyeicosatetranoic acid production following treatment with aspirin. 56 The crystal structure of COX-1 reacted with 2-bromoacetoxybenzoic acid (an aspirin analogue) demonstrates the incorporation of the bromoacetyl group at Ser-530 ( Figure 8). 57 Two rotamers of the bromoacetyl group exist that block the active site channel to different extents; this may help to explain the dissimilar outcomes of acetylation of COX-1 and COX-2.…”