X-RAY CRYSTAL STRUCTURE OF MOUSE PROSTAGLANDIN H₂ SYNTHASE-2. Ravi G. Kurumbail, William C. Stallings, Anna M. Stevens, Jina Pak, Daniel A. Gildehaus, Roderick A. Stegeman, James Gierse, Karen Seibert, Peter C. Isakson. Monsanto, Mail Zone BB4K, 700 Chesterfield Pkwy, St. Louis, Missouri 63198

The integral membrane protein Prostaglandin H₂ Synthase or cyclooxygenase (COX) is the pharmacological target of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) such as aspirin and ibuprofen. COX is a bifunctional enzyme that catalyzes the conversion of arachidonic acid to prostaglandin H₂ through a cyclooxygenase reaction followed by a peroxidase activity. Two isoforms of COX have been identified: COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and organs and is essential for stomach mucosal membrane integrity, normal renal function and hemostasis. By contrast, COX-2 is primarily localized in inflammatory cells and tissues and is induced in response to cytokines, mitogens or hormones. It has been shown that COX-2 is responsible for the elevated levels of prostaglandin production during inflammation. Selective inhibition of COX-2 could lead to the production of superior anti-inflammatory drugs without the side effects associated with common NSAIDs.

We have determined the three-dimensional structure of mouse COX-2 co-crystallized with flurbiprofen at 2.5 Å resolution by molecular replacement methods using the structure of sheep seminal vesicles COX-1. The crystals belong to the space group P2_l2_l2 with two dimers of COX-2 in the asymmetric unit. The crystals were frozen to near liquid nitrogen temperature to facilitate data collection from a single crystal. The amino acid sequence of the two isoforms are 60% identical but COX-2 has an insertion of 18 amino acids near the carboxy terminus. The structure is being refined by X-plor and the current R-factor is 24%. The overall structure of the two COX isoforms are well conserved, particularly near the cyclooxygenase active site. However, the NSAID binding site in COX-2 is considerably enlarged due to subtle changes in the binding pocket. Additional electron density is observed for COX-2 near the carboxy terminus.