Fatty Acid Binding to the Allosteric Subunit of Cyclooxygenase-2 Relieves a Tonic Inhibition of the Catalytic Subunit.

Prostaglandin endoperoxide H synthase-2 (PGHS-2), also called cyclooxygenase-2 (COX-2), converts arachidonic acid to PGH2 PGHS-2 is a conformational heterodimer composed of allosteric (Eallo) and catalytic (Ecat) subunits. Fatty acids (FAs) bind to Arg-120 of Eallo increasing to different degrees, depending on the FA, the Vmax of its Ecat partner. We report here that movement of helical residues 120-122 and loop residues 123-129 of Eallo underlies the allosteric effects of FAs and allosteric COX-2 inhibitors, including naproxen and flurbiprofen. An S121P substitution in both PGHS-2 monomers yields a variant (S121P/S121P PGHS-2) that has 1.7-1.8 times the Vmax of native PGHS-2 and is relatively insensitive to activation by FAs or inhibition by allosteric inhibitors. The S121P substitution in Eallo is primarily responsible for these effects. In X-ray crystal structures, the Cα atoms of helical residues 119-122 of S121P/S121P PGHS-2 are displaced from their normal positions. Additionally, the S121P/S121P PGHS-2 variants in which Pro-127 and Ser-541 are replaced by cysteines spontaneously forms Cys-127 to Cys-541 cross-links between monomers. This is unlike the corresponding native PGHS-2 variant and suggests that S121P substitutions also unhinge the loop involving residues 123-129. We conclude the following: (a) the region involving residues 120-129 of unoccupied Eallo tonically inhibits Ecat; (b) binding of an activating FA (e.g. arachidonic, palmitic, or oleic acid) to Eallo or an S121P substitution in Eallo repositions this region to increase Ecat activity; and (c) allosteric COX inhibitors act by preventing FA binding to Eallo and additionally by relocating Eallo residues to inhibit Ecat.