title: MgATP and fructose 6-phosphate interactions with phosphofructokinase from Escherichia coli.
abstract: A thermodynamic linked-function analysis is presented of the interactions of MgATP and fructose 6-phosphate (Fru-6-P) with phosphofructokinase (PFK) from Escherichia coli in the absence of allosteric effectors. MgATP and Fru-6-P are shown to bind in random fashion by product inhibition of the back-reaction as well as by the kinetically competent binding of each ligand individually as monitored by the consequent changes in the intrinsic fluorescence of E. coli PFK. When Fru-6-P is saturating, the dissociation of MgATP is sufficiently slow that it cannot achieve a binding equilibrium in the steady state, causing the observed Km (49 microM) to significantly exceed the Kd (1.7 microM) deduced from a thermodynamic linkage analysis. The following features distinguish the interactions of MgATP and Fru-6-P with E. coli PFK: MgATP and Fru-6-P antagonize each other's binding to the enzyme in a saturable manner with an overall apparent coupling free energy equal to +2.5 kcal/mol at 25 degrees C; MgATP induces positive cooperativity in the Fru-6-P binding profile, with the Hill coefficient calculated from the Fru-6-P binding curves reaching a maximum of 3.6 when MgATP is saturating; and MgATP exhibits substrate inhibition at low concentrations of Fru-6-P. Simulations based upon the rate equation pertaining to a two-active-site, two-substrate dimer indicate that these features can all result from two independent couplings: an antagonistic MgATP-Fru-6-P coupling extending at least in part between active sites and a MgATP-induced Fru-6-P-Fru-6-P coupling.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
authors.forename: J L
authors.initials: JL
authors.lastname: Johnson
authors.forename: G D
authors.initials: GD
authors.lastname: Reinhart
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates / Q000378:metabolism*; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D005636:Fructosephosphates; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1