Regulation of rabbit muscle phosphofructokinase by phosphorylation.
Muscle phosphofructokinase is one of the glycolytic enzymes whose partitioning between the particulate and soluble fractions in skeletal muscle is linked to the biological activity of the muscle. The formation of the enzyme-actin complex is apparently regulated by phosphorylation of the enzyme. In order to understand the role of phosphorylation on the regulatory mechanism of phosphofructokinase, the self-association of the phosphorylated and dephosphorylated forms of phosphofructokinase was studied by investigating the sedimentation velocity at pH 7.0 and 23 degrees C in different solvent constituents. The results show that both the phosphorylated and dephosphorylated forms of the enzyme exhibit the same mechanism of assembly. The effects of allosteric effectors are dependent on the phosphorylation state of the enzyme. The presence of 0.2 mM fructose-6-phosphate, one of the two substrates, leads to a significant enhancement in the formation of octomers without altering the equilibrium constant for tetramerization for either phosphorylated or dephosphorylated enzyme. The presence of 10 mM citrate, an allosteric inhibitor, leads to the formation of a significant amount of dimer, an inactive form of the enzyme. Citrate decreases the propensities of the dephosphorylated and phosphorylated forms of the enzyme to tetramerize 3000 times and 100 times, respectively. Based on the mode of subunit assembly, bimodal sedimentation velocity profiles can be obtained by simulation. Furthermore, simulation showed that the seemingly very different profiles reported in the literature can be accounted for by various combinations of equilibrium constants. In summary, this study showed that the propensity of subunit assembly is affected differentially by specific metabolites and the phosphorylation state of phosphofructokinase.
Cai
G Z
GZ
E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104, USA.
Callaci
T P
TP
Luther
M A
MA
Lee
J C
JC
D000818:Animals; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D013816:Thermodynamics
D010732:Phosphofructokinase-1