Heart phosphofructokinase: allosteric kinetics with fructose 6-sulfate.
The allosteric regulation of heart phosphofructokinase was studied at pH 6.9 with an alternative substrate, fructose 6-sulfate. The alternative substrate allowed kinetic studies to be carried out at high enzyme concentrations (0.1 mg/ml) where the effect of allosteric ligands on enzyme physical structure has been studied. A Km for ATP binding (8-10 muM) in the presence of saturating AMP concentrations was found which agreed well with the value obtained at pH 8.2, ATP inhibitory effects closely followed saturation of its substrate site. Hill plots for ATP inhibition gave an interaction coefficient of 3.5 indicating cooperatively between at least four enzyme subunits. Neither AMP nor fructose 6-sulfate affected the cooperativity between the ATP inhibitory sites but only increased the inhibitory threshold. As the ATP concentration was increased from suboptimal to inhibitory levels, interaction coefficients for AMP and fructose 6-sulfate changed from 1 to 2. Increasing citrate concentration resulted in an increase in the interaction coefficient for fructose 6-sulfate to a value of 1.9. Citrate inhibition was synergistic with ATP inhibition with an interaction coefficient of 2. The data indicate that  allosteric kinetics of the enzyme can be shown at high enzyme concentrations with the alternative substrate. ATP inhibition appears to involve interaction between at least four subunits, while citrate, AMP, and fructose 6-sulfate interact minimally with two subunits.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D005632:Fructose / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D010732:Phosphofructokinase-1 / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012756:Sheep; D013464:Sulfuric Acids / Q000494:pharmacology*
D013464:Sulfuric Acids; D005632:Fructose; D000249:Adenosine Monophosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1