Substrates bound to the cyclooxygenase‐2 homodimer exhibit alternate conformations per monomer

COX‐1 and COX‐2 are membrane‐associated heme‐containing homodimers that generate prostaglandin H 2 from arachidonic acid(AA) in the committed step of prostaglandin and thromboxane biogenesis and are the targets for nonsteroidal anti‐inflammatory drugs. Both isoforms behave as heterodimers during catalysis, exhibiting half‐of‐sites reactivity. To elucidate the atomic level interactions that are involved with binding and conformational positioning of substrates for catalysis, we determined the X‐ray crystal structures of AA, eicosapentaenoic acid (EPA), and 1‐arachidonoylglycerol (1‐AG) bound to murine COX‐2 to 2.1–2.4Å resolution. AA, EPA, and 1‐AG bind in different conformations in each monomer constituting the homodimer in their respective structures, such that one monomer exhibits nonproductive binding and the other productive binding of the substrate in the cyclooxygenase active site. Comparison of COX crystal structures revealed the involvement of Leu‐531 in allowing for these novel substrate conformations in COX‐2, but mutagenesis of Leu‐531 did not result in a significant decrease in V max or K m , in contrast to similar studies with COX‐1. We conclude that the mobility of Leu‐531 increases the accessible volume at the opening of the cyclooxygenase active site, and contributes to the observed ability of COX‐2 to oxygenate a broader spectrum of substrates compared to COX‐1. This work was supported by NIH grant R01 GM077176 from the NIGMS and by an Arthritis Investigator Award from the Arthritis Foundation as part of the Segal Osteoarthritis Initiative.