The binding of arachidonic acid in the cyclooxygenase active site of mouseprostaglandin endoperoxide synthase-2 (COX-2) - A putative L-shaped binding conformation utilizing the top channel region

The chemical mandates for arachidonic acid conversion to prostaglandin G(2) within the cyclooxygenase (COX) active site predict that the substrate wil l orient in a kinked or L-shaped conformation. Molecular modeling of arachi donic acid in sheep COX-1 confirms that this L-shaped conformation is possi ble, with the carboxylate moiety binding to Arg-120 and the omega-end posit ioned above Ser-530 in a region termed the top channel. Mutations of Gly-53 3 to valine or leucine in the top channel of mCOX-2 abolished the conversio n of arachidonic acid to prostaglandin G(2), presumably because of a steric clash between the omega-end of the substrate and the introduced side chain s. A smaller G533A mutant retained partial COX activity. The loss of COX ac tivity with these mutants was not the result of reduced peroxidase activity , because the activity of all mutants was equivalent to the wild-type enzym e and the addition of exogenous peroxide did not restore full COX activity to any of the mutants. However, the Gly-533 mutants were able to oxidize th e carbon 18 fatty acid substrates linolenic acid and stearidonic acid, whic h contain an allylic carbon at the omega-5 position. In contrast, linoleic acid, which is like arachidonic acid in that its most omega-proximal allyli c carbon is at the omega-8 position, was not oxidized by the Gly-533 mutant s, Finally, the ability of Gly-533 mutants to efficiently process omega-5 a llylic substrates suggests that the top channel does not serve as a product exit route indicating that oxygenated substrate diffuses from the cyclooxy genase active site in a membrane proximal direction.