MgATP and fructose 6-phosphate interactions with phosphofructokinase from Escherichia coli.

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)

[1]  H. Buc,et al.  [11] Phosphofructokinases from Escherichia coli , 1982 .

[2]  P R Evans,et al.  Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP. , 1991, Biochemistry.

[3]  D. Blangy.  Propriétés allostériques de la phosphofructokinase d'E. coli. Étude de la fixation des ligands par dialyse à l'équilibre , 1971 .

[4]  W. Cleland,et al.  Statistical analysis of enzyme kinetic data. , 2006, Methods in enzymology.

[5]  Linked-function origins of cooperativity in a symmetrical dimer. , 1988, Biophysical chemistry.

[6]  J P Changeux,et al.  On the nature of allosteric transitions: implications of non-exclusive ligand binding. , 1966, Journal of molecular biology.

[7]  G. Weber.  Ligand binding and internal equilibria in proteins. , 1972, Biochemistry.

[8]  J. Monod,et al.  Genetic regulatory mechanisms in the synthesis of proteins. , 1961, Journal of molecular biology.

[9]  W. Cleland.  [20] Substrate inhibition , 1979 .

[10]  G. Weber,et al.  Energetics of ligand binding to proteins. , 1975, Advances in protein chemistry.

[11]  J. Wyman,et al.  LINKED FUNCTIONS AND RECIPROCAL EFFECTS IN HEMOGLOBIN: A SECOND LOOK. , 1964, Advances in protein chemistry.

[12]  Gregory D. Reinhart.  The determination of thermodynamic allosteric parameters of an enzyme undergoing steady-state turnover. , 1983, Archives of biochemistry and biophysics.

[13]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[14]  H. Buc,et al.  Kinetics of the allosteric interactions of phosphofructokinase from Escherichia coli. , 1968, Journal of molecular biology.

[15]  D Deville-Bonne,et al.  Substrate antagonism in the kinetic mechanism of E. coli phosphofructokinase‐1 , 1991, FEBS letters.

[16]  C. Kundrot,et al.  Designing an allosterically locked phosphofructokinase. , 1991, Biochemistry.

[17]  F. Bourgain,et al.  pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli. , 1991, Biochemistry.

[18]  W. Cleland,et al.  The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. 1963. , 1989, Biochimica et biophysica acta.