Abstract
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 housekeeping functions, and the "inducible" isoform, PGHS-2, has been implicated in cellular responses to cytokines. The two isoforms have high sequence conservation in the cyclooxygenase active site and quite similar crystallographic 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 hydrochloride (GdmHCl). Changes in hydrodynamic and cross-linking properties indicated a dimer --> monomer transition for both isoforms between 0.5 and 2 M GdmHCl; the monomers unfolded at higher GdmHCl levels. Changes in overall secondary and tertiary structure, measured by tryptophan fluorescence and circular dichroism, occurred in two phases for each isoform, with the transition between the phases at 0.2-0.5 M GdmHCl. Disruption of active site functions (cyclooxygenase, peroxidase, and cyclooxygenase 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 levels, and they required lower GdmHCl levels for PGHS-2 than for PGHS-1. The results are consistent with a four-state denaturation process for both isoforms: native dimers --> inactive dimers --> compact monomers --> unfolded monomers. The first two steps are reversible for both isoforms; PGHS-2 undergoes the first and last steps more readily than PGHS-1. Thus, the structural stability of PGHS-2, both in the active site regions and in the subunits overall, is distinctly less than that of PGHS-1. These differences in structural stability may contribute to the isoforms' active site ligand selectivity.
Highlights
Prostaglandin H synthase (PGHS)1 catalyzes two key steps in prostanoid biosynthesis: oxygenation and cyclization of arachidonic to form PGG2 and the reduction of the hydroperoxide of PGG2 to form PGH2
Solvent Exposure of Tryptophan Residues in oPGHS-1 and hPGHS-2—oPGHS-1 has nine, and hPGHS-2 six, tryptophan residues [3, 30], which are potentially useful in fluorimetric monitoring of unfolding in these proteins
1.2 tryptophan residues in oPGHS-1 and 0.5 tryptophan residues in hPGHS-2 were found to be exposed to solvent at 0.05% Tween 20 (Fig. 1)
Summary
Materials—Glycerol, guaiacol, hydrogen peroxide, hemin chloride, D-tryptophan, NAc-Trp-OEt, lysozyme, standards for size exclusion chromatography, 25% glyceraldehyde solution, sodium deoxycholate, and NaBH4 were from Sigma. UV absorbance spectra were recorded for each protein (16.7 M for oPGHS-1 and hPGHS-2 or 20 M for lysozyme) in 0.1 M potassium phosphate, pH 7.2, containing the desired level of Tween 20 and either 0 or 20% (v/v) glycerol. Concentrated solutions of enzyme were diluted into the phosphate buffer containing 0.1% Tween 20 and the desired level of GdmHCl and incubated at room temperature for at least 30 min (unless otherwise noted) before removal of aliquots to assess intrinsic fluorescence, CD, enzymatic activity, inhibitor binding, cross-linking, or hydrodynamic properties. OPGHS-1 was diluted to 0.15 or 1.5 M in 0.1 M potassium phosphate, pH 7.2, containing 0.1% Tween 20 and 0 –3 M GdmHCl (total volume, 250 l) and preincubated at room temperature for 30 – 45 min before addition of 20 l of 25% glutaraldehyde. The intrinsic fluorescence intensities of unbound flurbiprofen and indomethacin were found to be constant over the range of 0 –2 M GdmHCl
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