INACTIVATION PRECEDES CHANGES IN ALLOSTERIC PROPERTIES AND CONFORMATION OF D-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AND FRUCTOSE-1,6-BISPBOSPHATASE DURING DENATURATION BY GUANIDINIUM CHLORIDE

It has been shown that inactivation of several enzymes precedes overall conformational changes of the enzyme molecules as a whole during denaturation [Tsou (1993) Science, 262, 380-381]. However, the relation between inactivation, loss of allosteric properties of oligomeric enzymes and unfolding of the enzyme molecule during denaturation remain little explored. These have now been compared for D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6-bisphosphatase (FruP(2)ase) during denaturation by guanidinium chloride (GdmCl). GAPDH is completely inactivated at 0.3 M GdmCl but at this GdmCl concentration it still binds NAD(+) with negative co-operativity. At 0.4 M GdmCl, inactivation of FruP(2)ase reaches completion whereas its allosteric properties, including the heterotropic effect of AMP inhibition and K+ activation with positive co-operativity, are only partially affected. Much higher GdmCl concentrations are required to bring about unfolding of the overall structures of both enzymes.