Urea-induced inactivation, dissociation, and unfolding of the allosteric phosphofructokinase from Escherichia coli.

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.