Association and activation of fructose 1,6-bisphosphase during unfolding and refolding: spectroscopic and enzymatic studies

Fructose 1,6-biphosphase is a well-characterized oligomer enzyme, and many effectors allosterically control its activity. In this report, we compared the activity, allosteric properties, and conformational changes in its denaturant-induced unfolding processes. In addition, a trpytophan residue has been introduced into the interface between the C1 and C2 subunits to investigate conformational changes during unfolding. Results show that the denaturation curves of WT FruP₂ase detected by various methods do not agree, and the dissociation occurs first with a monomeric form existing around 0.4 M GdmCl as shown by gel filtration. The dissociation of all mutants is accompanied by changes in fluorescence intensity. The results suggest that the unfolding of FruP₂ase is a complicated, multiphase process. The activation of FruP₂ase by GdmCl at low concentrations can be interpreted as a consequence of the effect of monovalent cation. In the refolding experiments, it is found that Mg²⁺ is not only essential for enzyme activity, but also can assist the enzyme in refolding and association by preventing the formation of aggregates.