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)     

Persistent binding of MgADP to the E187A mutant of Escherichia coli phosphofructokinase in the absence of allosteric effects

MgADP binding to the allosteric site enhances the affinity of Escherichia coli phosphofructokinase (PFK) for fructose 6-phosphate (Fru-6-P). X-ray crystallographic data indicate that MgADP interacts with the conserved glutamate at position 187 within the allosteric site through an octahedrally coordinated Mg(2+) ion [Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994]. Lau and Fersht reported that substituting an alanine for this glutamate within the allosteric site of PFK (i.e., mutant E187A) causes MgADP to lose its allosteric effect upon Fru-6-P binding [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. However, these authors later reported that MgADP inhibits Fru-6-P binding in the E187A mutant. The inhibition presumably occurs by preferential binding to the inactive (T) state complex of the Monod-Wyman-Changeux two-state model [Lau, F. T.-K., and Fersht, A. R. (1989) Biochemistry 28, 6841-6847]. The present study provides an alternative explanation of the role of MgADP in the E187A mutant. Using enzyme kinetics, steady-state fluorescence emission, and anisotropy, we performed a systematic linkage analysis of the three-ligand interaction between MgADP, Fru-6-P, and MgATP. We found that MgADP at low concentrations did not enhance or inhibit substrate binding. Anisotropy shows that MgADP binding at the allosteric site occurred even when MgADP produced no allosteric effect. However, as in the wild-type enzyme, the binding of MgADP to the active site in the mutant competitively inhibited MgATP binding and noncompetitively inhibited Fru-6-P binding. These results clarified the mechanism of a three-ligand interaction and offered a nontraditional perspective on allosteric mechanism.     

Equilibrium binding studies of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus

A tryptophan-shifted mutant of phosphofructokinase (PFK) from Bacillus stearothermophilus has been constructed. This mutant, which is functionally similar to wild-type, provides the opportunity to examine the allosteric properties of PFK under equilibrium conditions. The unique fluorescence properties of the tryptophan-shifted mutant enzyme, W179F/F230W, have been utilized to deduce the thermodynamics of ligand binding and the allosteric perturbations in the absence of catalytic turnover. Specifically, phospho(enol)pyruvate (PEP) and MgADP binding to the mutant PFK can be directly observed using tryptophan fluorescence, and dissociation constants for these ligands have been measured to be equal to 2.71 +/- 0.04 and 90.4 +/- 3.5 microM, respectively. In addition, the homotropic couplings for the allosteric ligands have been assessed for the first time. PEP binds cooperatively with a Hill number of 2.9 +/- 0.3, while MgADP binding is not cooperative. The equilibrium couplings between these ligands and the substrate fructose 6-phosphate (Fru-6-P) have also been determined and follow the same trends with temperature observed under steady-state kinetic assay conditions using wild-type PFK, indicating that the presence of bound MgATP has little influence on the allosteric interactions. Like wild-type PFK, the coupling free energies for the mutant result from largely compensating enthalpy and entropy components at 25 degrees C. Furthermore, the sign of each coupling free energy, which signifies the nature of the allosteric effect, is opposite that of the enthalpy contribution and is therefore due to the larger absolute value of the associated entropy change. This characteristic stands in direct contrast to the thermodynamic basis of the allosteric response in the homologous PFK from E. coli in which the sign of the coupling free energy is established by the sign of the coupling enthalpy.     

Mechanism of substrate inhibition in Escherichia coli phosphofructokinase

Phosphofructokinase from Escherichia coli (EcPFK) is a homotetramer with four active sites, which bind the substrates fructose-6-phosphate (Fru-6-P) and MgATP. In the presence of low concentrations of Fru-6-P, MgATP displays substrate inhibition. Previous proposals to explain this substrate inhibition have included both kinetic and allosteric mechanisms. We have isolated hybrid tetramers containing one wild type subunit and three mutated subunits (1:3). The mutated subunits contain mutations that decrease affinity for Fru-6-P (R243E) or MgATP (F76A/R77D/R82A) allowing us to systematically simplify the possible allosteric interactions between the two substrates. In the absence of a rate equation to explain the allosteric effects in a tetramer, the data have been compared to simulated data for an allosteric dimer. Since the apparent substrate inhibition caused by MgATP binding is not seen in hybrid tetramers with only a single native MgATP binding site, the proposed kinetic mechanism is not able to explain this phenomenon. The data presented are consistent with an allosteric antagonism between MgATP in one active site and Fru-6-P in a second active site.     

Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior

Arg252 of fructose-6-phosphate 1-kinase (PFK) from Bacillus stearothermophilus has been proposed to be involved in the binding of the substrate Fru-6-P. We demonstrate here that mutation of this residue to alanine converts the enzyme to a form with characteristics similar to those of its allosterically tight form. The mutant enzyme exhibits a high affinity for its inhibitor phosphoenolpyruvate (a 68-fold difference compared to wild type) and a dramatically decreased Fru-6-P affinity (1500-fold increase in Km). It is more sensitive to inhibition by high ATP concentrations than the wild type, and this inhibition is relieved by ADP, GDP, or higher Fru-6-P concentrations. In contrast, mutation of Arg252 to lysine increases the affinity of the enzyme for P-enolpyruvate by only 2-fold and increases its Km for Fru-6-P by only 50-fold. Sigmoidal kinetics with respect to Fru-6-P in the presence of P-enolpyruvate were observed with Hill numbers of 2.2, 2.4, and 1.7 for wild-type B. stearothermophilus PFK and the Arg252 to lysine and to alanine mutations, respectively. Unlike fructose-6-phosphate 1-kinase from Escherichia coli, in the absence of P-enolpyruvate, B. stearothermophilus PFK exhibits a hyperbolic profile with respect to Fru-6-P concentration. B. stearothermophilus PFK is sensitive to inhibition by high ATP concentrations and competitively inhibited by GDP or ADP. Our data indicate that Arg252 of B. stearothermophilus PFK plays a major role in both Fru-6-P binding and allosteric interaction between the subunits. However, this residue does not seem to participate directly in the catalytic process.     

Role of Ser530, Arg292, and His662 in the allosteric behavior of rabbit muscle phosphofructokinase.

Fructose-2,6-bisphosphate (Fru-2,6-P(2)) is a potent allosteric activator of the ATP-dependent phosphofructokinase (PFK) in eukaryotes. Based on the sequence homology between rabbit muscle PFK and two bacterial PFKs and the crystal structures of the latter, Ser(530), Arg(292) and His(662) of the rabbit enzyme are implicated as binding sites for Fru-2,6-P(2). We report here the effects of three mutations, S530D, R292A, and H662A on the activation of rabbit muscle PFK by Fru-2,6-P(2). At pH 7.0 and the inhibitory concentrations of ATP, the native enzyme gives a classic sigmoidal response to changes in Fru-6-P concentration in the absence of Fru-2,6-P(2) and a nearly hyperbolic response in the presence of the activator. Under the same conditions, no activation was seen for S530D. On the other hand, H662A can be activated but requires a 10-fold or higher concentration of Fru-2,6-P(2). Limited activation was observed for mutant R292A. A model illustrating the sites for recognition of Fru-2,6-P(2) in rabbit muscle PFK as well as the mechanism of allosteric activation is proposed.     

Identification of substrate contact residues important for the allosteric regulation of phosphofructokinase from Eschericia coli

The side chains of Escherichia coli phosphofructokinase (EcPFK) that interact with bound substrate, fructose 6-phosphate (Fru-6-P), are examined for their potential roles in allosteric regulation. Mutations that severely decrease Fru-6-P affinity and/or k(cat)/K(m) were created at each contact residue, with the exception of the catalytic base, D127. Even though Fru-6-P affinity was greatly decreased for R162E, M169A, E222A/H223A, and R243E, the mutated proteins retained the ability to be activated by MgADP and inhibited by phosphoenolpyruvate (PEP). R252E did not show an allosteric response to either MgADP or PEP. The H249E mutation retained MgADP activation but did not respond to PEP. R72E, T125A, and R171E maintained allosteric inhibition by PEP. Both R72E and T125A displayed a MgADP-dependent decrease in k(cat) but no MgADP-dependent K-type effects. R171E maintained MgADP-dependent K-type activation but also displayed a MgADP-dependent decrease in k(cat). Localization of mutations that alter MgADP activation near the transferred phosphate group indicates the importance of the 1-methoxy region of Fru-6-P in allosteric regulation by MgADP. A region near the 6'-phosphate may be similarly important for PEP inhibition. R252 is uniquely positioned between the 1'- and 6'-phosphates of bound Fru-1,6-BP, and the mutation at this position may alter both allosterically responsive regions. The differential functions of specific regions in the Fru-6-P contact residues support different mechanisms for allosteric activation and inhibition. In addition, the lack of correlation between mutations that decrease Fru-6-P affinity and those that abolish allosteric communications supports the independence of affinity and allosteric coupling.     

Reversible ligand-induced dissociation of a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus

The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.5) for the substrate, fructose-6-phosphate (Fru-6-P), as compared to the wild-type enzyme. The dissociation constant for the inhibitor, phospho(enol)pyruvate (PEP), increases 2-fold, and the coupling parameter, Q(ay), decreases 2-fold. This suggests that while the mutant displays a slightly decreased affinity for PEP, PEP is still an effective inhibitor once bound. The new position of the tryptophan in W179Y/Y164W is approximately 6 A from the Fru-6-P portion of the active site. A 25% decrease in fluorescence intensity is observed upon Fru-6-P binding, and an 80% decrease in fluorescence intensity is observed with PEP binding. In addition, the intrinsic fluorescence polarization increases from 0.327 +/- 0.001 to 0.353 +/- 0.001 upon Fru-6-P binding, but decreases to 0.290 +/- 0.001 when PEP binds. Most notably, the presence of PEP induces dissociation of the tetramer. Dissociation of the tetramer into dimers occurs along the active site interface and can be monitored by the loss in activity or the loss in tryptophan fluorescence that is observed when the enzyme is titrated with PEP. Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

Steady-state fluorescence of Escherichia coli phosphofructokinase reveals a regulatory role for ATP.

We have investigated the effects of ligands and effectors on the intrinsic fluorescence of Escherichia coli phosphofructokinase (PFK). We have found that the substrate fructose 6-phosphate (Fru6P) or the allosteric activator ADP can quench the fluorescence up to 35%. The response is hyperbolic with Ks[Fru6P] of 20 microM and Ks[ADP] of 13 microM. The allosteric inhibitor phosphoenolpyruvate (PEP) converts the hyperbolic response with respect to Fru6P to a sigmoidal response. AMP-PNP, a nonhydrolyzable analogue of ATP, also inhibits the Fru6P fluorescence response. PFK mutant KA213, which is insensitive to effectors, has a decreased fluorescence response with respect to ADP, and PEP does not convert the Fru6P response to sigmoidicity. However, its fluorescence response with respect to Fru6P is decreased by ATP or AMP-PNP. Taken together, these results suggest that, in the absence of effectors or ligands, E. coli PFK exists in a state with high affinity for Fru6P ("R" state). This state can be altered to a low affinity ("T" state) by PEP binding to the allosteric site or by ATP binding to the enzyme.     

A new transient activator of phosphofructokinase during initiation of rapid glycolysis in brain

The tissue contents of previously known allosteric effectors of brain phosphofructokinase (EC 2.7.1.11) (PFK) and the kinetic behavior of isolated PFK were investigated during the initiation of rapid glycolytic flux in freeze-blown rat brain. Comparing 0- with 5-s brains revealed that there was a 4-fold drop in total tissue content of Fru-6-P and a 5.6-fold increase in Fru-1,6-P2 consistent with activation of PFK. Additionally, analysis of brain content showed a 15-fold increase in AMP, a 3-fold decrease in ATP, a 3-fold decrease in Pi, and a 1.6-fold increase in NH4+. There was no change in Fru-2,6-P2, H+, citrate, or Glc-1,6-P2 or the kinetic profiles of isolated PFK for ATP inhibition or Fru-2,6-P2 activation. We concluded that the observed change in PFK activity could be accounted for only partially by changes in the concentrations of adenine nucleotides and other known effectors. High performance liquid chromatography fractions of extracts obtained from 5-s brains showed the activator with a mobility identical to ribose 1,5-P2 and gave 2 nmol/g (wet weight) at 0 s, 10 nmol/g at 5 s, and 2 nmol/g at 20 s. Assay of PFK in the presence of effectors determined to be in tissue at 5 s showed that addition of 10 nmol/ml ribose 1,5-P2 gave a 4-fold activation of PFK. Based on the rapidity of its formation, its potency of activation, and its similarity in chemical properties to authentic ribose 1,5-P2, we conclude that ribose 1,5-P2 served as the initial activator of PFK in brain.     

Influence of pH on the regulatory kinetics of rat liver phosphofructokinase: a thermodynamic linked-function analysis

The relationship between pH and the MgATP inhibition of rat liver phosphofructokinase has been quantiatively evaluated by utilization of a thermodynamic linked-function approach. This approach obviates the need to presuppose discrete inhibited and active states of the enzyme. The behavior of the apparent Michaelis constant for fructose 6-phosphate (Fru-6-P) over a 100-fold concentration range of MgATP conforms to the behavior predicted by the linked-function theory in that, a high concentrations of MgATP, saturation of the inhibitory effect is achieved, a result not predicted by a mutually exclusive two-state model. This behavior is described by the relationship Ka = Ka0[(Kix0 + [X])]/(Kix0 + Q[X])], where Ka is the apparent Michaelis constant for Fru-6-P, Ka0 is the Michaelis constant for Fru-6-P in the absence of MgATP, Kix0 is the dissociation constant of MgATP in the absence of Fru-6-P, and Q is the coupling term that quantitatively describes the finite degree of antagonism between MgATP and Fru-6-P. The free energy of interaction between MgATP and Fru-6-P, obtained from Q, is 1.9 kcal/mol at 25 degrees C. Ka0 and Kix0 are 0.17 and 0.3 mM, respectively. The influence of pH on these three parameters was then systematically investigated, and only Ka0 increased substantially with decreasing pH. Consequently, it is concluded that decreasing the pH does not increase the apparent Ka for Fru-6-P by augmenting the binding or inhibition by MgATP to a significant extent but rather by directly affecting the intrinsic affinity of the enzyme for Fru-6-P. The pK for this effect is 8.1.     

Phosphoenolpyruvate carboxylase of Escherichia coli. Specificity of some compounds as activators at the site for fructose 1,6-bisphosphate, one of the allosteric effectors

An investigation was performed to elucidate some unusual phenomena which had been observed with phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli. (i) Fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP--the allosteric activators--were competitive with each other in the activation. (ii) Some analogs of PEP such as DL-2-phospholactate and 2-phosphoglycolate, which behaved as inhibitors in the presence of the activator (acetyl-CoA or dioxane), activated the enzyme to some extent in the absence of the activator. (iii) Ammonium sulfate deprived the enzyme of sensitivity to Fru-1,6-P2 or GTP but had no effect on the sensitivity to other effectors. It was found that the activation by the analogs was lost upon desensitization of the enzyme to Fru-1,6-P2 by reaction with 2,4,6-trinitrobenzene sulfonate. The activation by the analogs was not observed in the presence of 200 mM ammonium sulfate. In the presence of lower concentrations (0.1 mM) of PEP, ammonium sulfate activated the enzyme at concentrations less than 700 mM but had an inhibitory effect on the desensitized enzyme. These findings suggest that the unusual phenomena described above are a result of binding of the phosphate esters and sulfate ions with the Fru-1,6-P2 site of the enzyme or the active site depending on the reaction conditions.     

Phosphoenolpyruvate carboxylase of Escherichia coli. The role of lysyl residues in the catalytic and regulatory functions.

Phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of E. coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased. The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP.     

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

Influence of fructose 2,6-bisphosphate and MgATP on rat liver phosphofructokinase at pH 7: evidence for a complex interdependence.

The relationship between fructose 2,6-bisphosphate (Fru-2,6-BP) activation and MgATP inhibition of rat liver phosphofructokinase has been comprehensively evaluated at pH 7. When either ligand is varied at a fixed concentration of the other, its influence on the concentration of fructose 6-phosphate (Fru-6-P) required to produce half-maximal velocity, Ka, is usually well described by the same simple, single-modifier linkage expression that described the actions of these ligands at pH 9. However, the effects of both ligands together cannot be described by the same overall linkage relationship that described their actions at pH 9. Specifically, despite an overall antagonistic relationship between the binding of MgATP and that of Fru-2,6-BP, very low concentrations of Fru-2,6-BP appear to facilitate the binding of MgATP to an appreciable degree. Also, MgATP at high concentration appears to inhibit the binding of Fru-2,6-BP to a significantly greater extent than its actions at lower concentration would predict. These additional features of MgATP-Fru-2,6-BP interaction have been incorporated into an overall linkage expression describing the actions of both MgATP and Fru-2,6-BP on Ka for Fru-6-P. The best fit parameters predict the data to within an average standard error of +/- 21%.     

Purification and characterization of phosphofructokinase in bovine parotid gland.

1. Phosphofructokinase (PFK) was purified from bovine parotid gland to 750-fold with the specific activity of 67.5 units/mg protein by Cibacron Blue F3GA affinity chromatography, and TSK DEAE-5PW ion-exchange and TSK G4000SW size exclusion chromatographies on HPLC. 2. On gel-filtration, molecular weight of the native PFK was estimated to 400,000. 3. PFK was a heterotetramer composed of three kinds of subunit with molecular weights of 92,000 (C-type), 88,000 (M-type) and 86,000 (L-type), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometrically, relative amounts of C-, M- and L-type subunit were 1:1:2. 4. Under the physiological conditions of fructose 6-phosphate (Fru-6-P) and ATP concentrations and pH, PFK activity was suppressed and hardly detectable. 5. Fru-6-P relieved PFK from the ATP inhibition. 6. Fructose 2,6-bisphosphate (Fru-2,6-P2) and AMP activated PFK with a reduction of S0.5 for Fru-6-P and subunit cooperativity. Fru-2,6-P2 was more effective than AMP.     

Influence of fructose 2,6-bisphosphate on the aggregation properties of rat liver phosphofructokinase

The influence that fructose 2,6-bisphosphate (Fru-2,6-BP) has on the aggregation properties of rat liver phosphofructokinase has been studied by observing the fluorescence polarization of the enzyme covalently bound to the fluorescent probe pyrenebutyric acid. Fru-2,6-BP dramatically slows the dissociation of the high molecular weight aggregate forms of the enzyme when the enzyme is diluted to 3.2 micrograms/ml (4 X 10(-8) M subunits). Furthermore, Fru-2,6-BP is a strong promoter of reassociation to tetramer and larger forms if the enzyme has been previously allowed to dissociate to the dimer in its absence. Unlike many other positive effectors of liver phosphofructokinase, Fru-2,6-BP is also able to overcome the tendency of MgATP to promote tetramer formation and instead stabilize a very high degree of high molecular weight aggregate formation even in the presence of MgATP. The apparent affinity of liver phosphofructokinase for Fru-2,6-BP was measured by its ability to promote reassociation and compared to that for Fru-1,6-BP. The apparent dissociation constant for Fru-2,6-BP under these conditions is 36 microM, about 40-fold lower than the value of 1.4 mM measured for Fru-1,6-BP. Both ligands demonstrate synergism with the substrate Fru-6-P, which can lower the dissociation constant for Fru-2,6-BP 9-fold to 4 microM and that for Fru-1,6-BP 5-fold to 0.28 mM. These data are interpreted to suggest that influencing the aggregation state of rat liver phosphofructokinase may be one way in which Fru-2,6-BP achieves its effects on the enzyme in vivo.     

Evidence for a catalytic Mg2+ ion and effect of phosphate on the activity of Escherichia coli phosphofructokinase-2: regulatory properties of a ribokinase family member

Phosphofructokinase-2 (Pfk-2) from Escherichia coli belongs to the ribokinase family of sugar kinases. One of the signatures observed in amino acid sequences from the ribokinase familiy members is the NXXE motif, which locates at the active site in the ribokinase fold. It has been suggested that the effect of Mg2+ and phosphate ions on enzymatic activity, observed in several adenosine kinases and ribokinases, would be a widespread feature in the ribokinase family, with the conserved amino acid residues in the NXXE motif playing a role in the binding of these ions at the active site [Maj, M. C., et al. (2002) Biochemistry 41, 4059-4069]. In this work we study the effect of Mg2+ and phosphate ions on Pfk-2 activity and the involvement of residue E190 from the NXXE motif in this behavior. The kinetic data are in agreement with the requirement of a Mg2+ ion, besides the one present in the metal-nucleotide complex, for catalysis in the wild-type enzyme. Since the response to free Mg2+ concentration is greatly affected in the E190Q mutant, we conclude that this residue is required for the proper binding of the catalytic Mg2+ ion at the active site. The E190Q mutant presents a 50-fold decrease in the kcat value and a 15-fold increment in the apparent Km for MgATP(2-). Inorganic phosphate, typically considered an activator of adenosine kinases, ribokinases, and phosphofructokinases (nonhomologous to Pfk-2) acted as an inhibitor of wild-type and E190Q mutant Pfk-2. We suggest that phosphate can bind to the allosteric site of Pfk-2, producing an inhibition pattern qualitatively similar to MgATP(2-), which can be reversed to some extent by increasing the concentration of fructose-6-P. Given that the E190Q mutant presents alterations in the inhibition by MgATP(2-) and phosphate, we conclude that the E190 residue has a role not only in catalysis but also in allosteric regulation.