Tetramer-dimer equilibrium of phosphofructokinase and formation of hybrid tetramers.
Moderate concentrations of KSCN inactivate the allosteric phosphofructokinase from Escherichia coli by dissociating the subunit interface that contains the binding site for the substrate fructose-6-phosphate. At a given KSCN concentration, the activity varies with the concentration of protein as expected from a simple equilibrium between active tetramers and inactive dimers. The equilibrium constants for the dissociation of a tetramer into dimers have been determined in 0.4 M KSCN for the wild-type enzyme and the noncooperative mutant T125S, the hypercooperative mutant E148A-R152A, and the inactive mutant D127S. The stability of the tetrameric structure is decreased by the mutations E148A-R152A that are in the interface and increased by the mutation T125S that does not belong to it. There could be an inverse correlation between the cooperativity of the saturation by fructose-6-phosphate (in absence of any effector) and the stability of the interface that contains its binding site. Hybrid tetramers can be formed upon reassociation of a dimer from an active phosphofructokinase (wild-type, T125S, or E148-R152A) with a dimer from the inactive D127S mutant, and their stability and cooperativity toward fructose-6-phosphate have been measured without purifying them. The results indicate that the formation of a hybrid interface involves some flexibility of the two dimers and that the allosteric coupling between distant sites could be related to the plasticity and instability of the interactions across this interface.
Le Bras
G
G
Laboratoire d'Enzymologie du CNRS, Gif-sur-Yvette, France.
Auzat
I
I
Garel
J R
JR
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D046911:Macromolecular Substances; D010732:Phosphofructokinase-1 / Q000737:chemistry* / Q000648:ultrastructure; D017354:Point Mutation; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013861:Thiocyanates
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D013861:Thiocyanates; D010732:Phosphofructokinase-1; C009941:potassium thiocyanate