Coupling Subunit-Specific States to Allosteric Regulation in Homodimeric Cyclooxygenase-2.

The homodimeric cyclooxygenase enzymes (COX-1 and COX-2) oxygenate arachidonic acid (AA) to generate prostaglandins. COX-2 behaves as a conformational heterodimer in solution comprised of allosteric (E_allo) and catalytic (E_cat) subunits that function cooperatively. We previously utilized ¹⁹F-nuclear magnetic resonance spectroscopy (¹⁹F-NMR) to show that the cyclooxygenase active site entrances in a COX-2 homodimer construct exhibited composite tightened and relaxed states that are dependent upon the type of ligand bound. A third state, hypothesized to represent the alteration of a loop comprised of residues 120-129, was also detected in the presence of ligands that allosterically potentiate activity. We report here studies that couple the use of ¹⁹F-NMR with COX-2 heterodimer constructs to characterize states arising in the individual subunits. Glycine and proline substitutions at Ser-121 were introduced to examine how these mutations alter the 120-129 loop. In the presence of AA, the subunits exhibited asymmetry, with tightened and relaxed states observed in E_allo and E_cat, respectively. Allosteric ligand binding resulted in a shift to equivalent symmetrical states, with tightened states observed in the presence of the allosteric inhibitor flurbiprofen and relaxed states observed in the presence of the allosteric potentiator palmitic acid. The S121P substitution results in a shift to equivalent relaxed states, as well as an alteration of the 120-129 loop in the absence of bound ligand. We put forth a model linking the observed differential states arising from allosteric ligand binding with structural transitions across the dimer interface that govern the regulation of cyclooxygenase activity.