Further kinetic characterization of the non-allosteric phosphofructokinase from Escherichia coli K-12.
The labile non-allosteric form of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to a specific activity of 107 U/mg (2078-fold) from aerobic cultures of Escherichia coli K-12. The enzyme has an isoelectric point (pI) of 5.1, a native molecular weight of 67 000 +/- 3000 and a subunit weight of 34 000 +/- 400. A number of divalent metal ions can substitute for Mg2+ in the enzyme reaction in decreasing order Mn2+ > Mg2+ > Co2+ > Ca2+. In the presence of excess Mg2+, nucleotides do not affect the Km for fructose 6-phosphate with a value of 0.042 mM. The order of efficiency for nucleotides to act as phosphoryl donors is ATP > ITP > GTP > UTP > CTP. This remains unchanged in the presence of excess Mn2+, but V is increased 2.4-fold with ATP. A 2 : 1 ratio of Mn2+/nucleotide 5'-triphosphate produced an equivalent dissociation constant of 1.1 mM for all nucleotides, which was markedly decreased at a high Mn2+ level. The rate of enzyme catalysis was found to be dependent on the concentration of MnATP2-. Mn2+ at non-limiting values does affect the binding of fructose 6-phosphate to the enzyme.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D013379:Substrate Specificity; D014544:Uridine Triphosphate / Q000378:metabolism
D007293:Inosine Triphosphate; D003035:Cobalt; D008345:Manganese; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D008274:Magnesium; D002118:Calcium; D014544:Uridine Triphosphate