Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP.
The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2+ and ATP concentrations. MgATP2- inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5. This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture. Other nucleotides used as phosphate donors instead of ATP did not inhibit. MgATP2- was the true substrate for both enzymes and their Km values for this compound were not affected by an increase of the free Mg2+ concentration. However, free Mg2+ partially relieved the MgATP2- inhibition of Pfk-2 under conditions where the ATP4- concentration was negligible, without changes in the degree of cooperativity. ATP4- acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2- with Ki values of 10 and 8 microM, respectively. ADP, AMP, and cAMP did not prevent the MgATP2- inhibition of Pfk-2. These results suggest the presence of an allosteric site for MgATP2- in Pfk-2 responsible for the MgATP2- inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.
Guixé
V
V
Babul
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000495:Allosteric Site; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity
D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D008274:Magnesium