Specific suppression of heterotropic interactions in phosphofructokinase by the mutation of leucine 178 into tryptophan.
The leucine residue at position 178 in the allosteric phosphofructokinase from Escherichia coli has been changed into a tryptophan residue by oligonucleotide-directed mutagenesis. The modified enzyme has been purified to homogeneity, and its enzymatic properties show that this single mutation suppresses the heterotropic interactions without affecting the homotropic ones. The mutant has the same saturation curve by fructose 6-phosphate as the wild type, showing that its active site binds this substrate with the same affinity and cooperativity. The regulatory site of the mutant enzyme can bind the effectors, the activator GDP, or the inhibitor phosphoenolpyruvate, as measured by protection against irreversible thermal denaturation. However, the binding of either effector does no longer influence the activity. This specific suppression of the coupling between the regulatory and active sites is not predicted by the concerted model which postulates that the same structural transition between two states R and T is responsible for both homotropic and heterotropic interactions. Leu-178 belongs to neither the active nor the regulatory site but appears as an important residue in the conformational change(s) involved in the regulation by allosteric effectors.
Serre
M C
MC
Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Teschner
W
W
Garel
J R
JR
D000494:Allosteric Regulation; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D007930:Leucine; D009154:Mutation; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000502:physiology*; D011489:Protein Denaturation; D013329:Structure-Activity Relationship; D014364:Tryptophan
D006153:Guanosine Diphosphate; D010728:Phosphoenolpyruvate; D014364:Tryptophan; D010732:Phosphofructokinase-1; D007930:Leucine