Tetramer-dimer conversion of phosphofructokinase from Thermus thermophilus induced by its allosteric effectors.
Phosphofructokinase (PFKase) was purified from an extreme thermophile. Thermus thermophilus. Allosteric natures of T. thermophilus PFKase is similar to those of Bacillus stearothermophilus PFKase, that is, hyperbolic plots of the activity versus concentration of fructose 6-phosphate (F6P) were changed into a sigmoidal shape by the addition of phosphoenolpyruvate (PEP), while further addition of ADP caused it to revert to a hyperbolic shape. The native T. thermophilus PFKase has an Mr of 148,000 consisting of four 36,500 subunits. However, it exists as a two-subunit form of Mr 74,000 in the presence of PEP. The two-subunit form was catalytically inactive. The four-subunit enzyme was regenerated by addition of either F6P or Mg.ADP, or by removal of PEP from the solution. This reversible dissociation was observed within a wide range of pH (6.5 to 8.4) and temperature (4 degrees C to 65 degrees C). Thus, unlike PFKase from other sources, the allosteric kinetics of T. thermophilus PFKase can be explained well, at least qualitatively, by the dynamic equilibrium between the active four-subunit form and inactive two-subunit form that is modulated by PEP, F6P and Mg.ADP. Parallel suppression of the PEP-induced conversion in molecular form and kinetics by high concentrations of sulfate and phosphate supports the above explanation. Also, the observation that the degree of PEP inhibition was dependent on the protein concentration of the PFKase in the assay solution is consistent with the presence of this equilibrium.
Xu
J
J
Department of Life Science, Faculty of Science, Tokyo Institute of Technology, Yokohama, Japan.
Oshima
T
T
Yoshida
M
M
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D005636:Fructosephosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D011487:Protein Conformation; D013431:Sulfates / Q000494:pharmacology; D013696:Temperature; D013824:Thermus / Q000187:drug effects / Q000201:enzymology*
D005636:Fructosephosphates; D013431:Sulfates; D000244:Adenosine Diphosphate; C027618:fructose-6-phosphate; D010728:Phosphoenolpyruvate; D010732:Phosphofructokinase-1; D008274:Magnesium