EFFECTOR-INDUCED DISSOCIATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE DISCRIMINATED BY UREA SOLVATION

The dissociation of glyceraldehyde-3-phosphate dehydrogenase (GAPD) fr om 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 a nd selective chemical modification at TRP-193. The results are in agre ement with a consecutive two-step model of dissociation of the tetrame r and the dimer (C-T = 0.42 M urea < C-D* = 1.39 M urea). The binding of effector(s) destabilizes the oligomeric structures (Delta G(T) cha nges from -1.00 to -0.54 kcal/mol; Delta G(D) 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 delta Delta G approximate to 1 kcal/mol, owing to TRP-193 ... TY R-39 ... TYR-92 cluster destruction. By using lobster GAPD atomic coor dinates (PDB file 1GPD) and pig muscle GAPD amino-acid sequence, a ten tative molecular model was constructed and the subunit contacts in ter ms of the Lee-Richard static accessibilities were described. A detaile d analysis of the dissociation as a transfer of the buried residues fr om the molecular interface to the urea solutions was performed. (C) Mu nksgaard 1995.