Intermediates on the reassociation pathway of phosphofructokinase I from Escherichia coli

Abstract

The folding and association pathway of the allosteric phosphofructokinase from Escherichia coli has been investigated after complete denaturation of the protein in guanidine hydrochloride by spectroscopicla methods, fluorescence and circular dischroism. Three successive processess can be observed during the renaturation of this protein. First, a fast reaction, detected by flurescence, results in the formation of a (partially) structures monomer. Second, two monomers associated into a dimeric species. This step involves the shielding of the unique tryptophan residue, Trp 311, from the aqueous solvent, and it corresponds to the formation of the interface containing the effector binding site. The presence of STPD uring renaturation increases the rate of formation of this dimeric species. The other ligands of enzyme have no effect on this reaction as well as on the whole reactivation. Finally, the enzymatic activity is regained during the third slowest step. This last reaction is due to the association of two dimers into the native tetrameric structure. The presence of fructose 6-phosphate does not increase the rate of reactivation, even though this ligand strongly stabilizes the native enzyme against denaturation by bridging this interface corresponding to the active site. The self-assembly of phsophofructokinase from E. coli from its unfolded and separated chains follows a specific order in the formation of the interactions between subunits and involves a dimeric intermediate with a defined geometry.