Allosteric activation increases the maximum velocity of E. coli phosphofructokinase.
Several mutations that cause a decrease of 25 to 65% of the catalytic activity, were introduced at different positions in the phosphofructokinase from Escherichia coli, and the influence of the allosteric activator GDP on these mutants was measured. In the case of the wild-type enzyme, GDP converts the highly cooperative saturation towards fructose-6-phosphate into a hyperbolic saturation with almost no change in the maximum velocity. The mutants Glu148 --> Leu, Leu178 --> Val and Leu178 --> Trp are still cooperative for fructose-6-phosphate, and their cooperativity is also abolished or markedly decreased by GDP. In addition, GDP acts on these mutants as an activator of maximum velocity, and increases their catalytic rate constants by 35 to 65% depending on the mutation. The Leu178 --> Val mutant is even as active as the wild-type enzyme in the presence of GDP. The Thr125 --> Ser mutation decreases the maximum velocity by 60% and also suppresses the cooperativity towards fructose-6-phosphate. Accordingly, the only effect of GDP on the Thr125 --> Ser mutant is on its maximum velocity and not on its affinity for fructose-6-phosphate. However, the maximum velocity of this mutant is not increased by GDP but reduced by 33%. These results show that GDP affects the maximum velocity of these mutants and suggest that the activation by GDP of the wild-type enzyme measured by steady-state kinetics could be partially due to an increase of the maximum velocity, and not exclusively to an increase of the affinity for fructose-6-phosphate.
Auzat
I
I
Laboratoire d'Enzymologie et de Biochimie Structurales, Gif-sur-Yvette, France.
Le Bras
G
G
Garel
J R
JR
D000494:Allosteric Regulation; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*
D006153:Guanosine Diphosphate; D010732:Phosphofructokinase-1