COMPARISON OF STRUCTURAL STABILITIES OF PROSTAGLANDIN-H SYNTHASE-1 AND SYNTHASE-2

There are two known isoforms of prostaglandin H synthase (PGHS), a key enzyme in the conversion of arachidonic acid to bioactive prostanoids , The ''constitutive'' isoform, PGHS-1, is thought to have housekeepin g functions, and the ''inducible'' isoform, PGHS-2, has been implicate d in cellular responses to cytokines. The two isoforms have high seque nce conservation in the cyclooxygenase active site and quite similar c rystallographic structures, but differ markedly in their interactions with many cyclooxygenase substrates and inhibitors. We have evaluated the stability of the overall folding, and of the active sites of ovine PGHS-1 and human PGHS-2 using denaturation with guanidinium hydrochlo ride (GdmHCl), Changes in hydrodynamic and cross-linking properties in dicated a dimer --> monomer transition for both isoforms between 0.5 a nd 2 M GdmHCl; the monomers unfolded at higher GdmHCl levels. Changes in overall secondary and tertiary structure, measured by tryptophan fl uorescence and circular dichroism, occurred in two phases for each iso form, with the transition between the phases at 0.2-0.5 M GdmHCl. Disr uption of active site functions (cyclooxygenase, peroxidase, and cyclo oxygenase inhibitor binding activities) began at GdmHCl levels below 0 .2 M, The structural and functional changes were completely reversible up to about 2 M GdmHCl, they were more pronounced at lower protein le vels, and they required lower GdmHCl levels for PGHS-2 than for PGHS-1 , The results are consistent with a four-state denaturation process fo r both isoforms: native dimers --> inactive dimers --> compact monomer s --> unfolded monomers, The first two steps are reversible for both i soforms; PGHS-2 undergoes the first and last steps more readily than P GHS-1. Thus, the structural stability of PGHS-2, both in the active si te regions and in the subunits overall, is distinctly less than that o f PGHS-1, These differences in structural stability may contribute to the isoforms' active site ligand selectivity.