Effector‐induced dissociation of glyceraldehyde‐3‐phosphate dehydrogenase discriminated by urea solvation

Abstract

The dissociation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) from pig muscle in water solutions (0.1 M phosphate, pH 7) at increased urea concentrations was studied by means of frontal-gel chromatography, intrinsic (TRP) fluorescence, differential absorption spectroscopy and selective chemical modification at TRP-193. The results are in agreement with a consecutive two-step model of dissociation of the tetramer and the dimer ( c t = 0.42 M urea< C b = 1.39 M urea). The binding of effector(s) destabilizes the oligomeric structures (AGT changes from -1.00 to -0.54 kcal/mol; AGD from -2.30 to -1.22 kcal/mol). The introduction of the bulky Koshland-reagent group to TRP-193 at the subunit-subunit interface leads to a decrease of the stability with 6AG x 1 kcal/mol, owing to TRP-193.. .TYR-39.. .TYR-92 cluster destruction. By using lobster GAPD atomic coordinates (PDB file IGPD) and pig muscle GAPD amino-acid sequence, a tentative molecular model was constructed and the subunit contacts in terms of the Lee-Richard static accessibilities were described. A detailed analysis of the dissociation as a transfer of the buried residues from the molecular interface to the urea solutions was performed. 0 Munksgaard 1995.