Ligand-induced conformational transitions in Escherichia coli phosphofructokinase 2: evidence for an allosteric site for MgATP2-.

The binding of ligands to phosphofructokinase 2 (Pfk-2) from Escherichia coli induces changes in the fluorescence emission properties of its single tryptophan residue, Trp88, suggesting that upon binding the protein undergoes a conformational change. This fluorescence probe was used to determine the presence of an allosteric site for MgATP2- in the enzyme. Fructose 6-phosphate (fructose-6-P), the first substrate that binds to the enzyme with an ordered bi-bi mechanism, increases the fluorescence up to 30%. The saturation curve for this compound is hyperbolic with a Kd of 6 microM. The titration of Pfk-2 with MgATP2- causes a quenching of fluorescence of about 30% of its initial value, with a blue shift of 7 nm in the emission maximum. The response is cooperative with a Kd of 80 microM and a Hill coefficient of 2. The interaction of MgATP2- cannot take place at the active site in the absence of fructose-6-P, due to the ordered kinetic mechanism. Addition of compounds that bind to the catalytic site of Pfk-2, such as ATP4- or Mg-AMP-PNP, did not produce significant changes in the fluorescence spectrum of Trp88. However, in the absence of Mg2+, the addition of ATP4- to the enzyme-fructose-6-P complex shows a hyperbolic increase of fluorescence of 8%. Acrylamide steady-state quenching experiments for different enzyme-ligand complexes of Pfk-2, indicate that the tryptophan in the enzyme-MgATP2- complex is exposed to a much smaller extent to the solvent than in the free enzyme or in the enzyme-fructose-6-P complex. The effect of the binding of fructose-6-P or MgATP2- on the polarization fluorescence of the emission of Trp88 in Pfk-2 indicates changes in the local mobility of the Trp88 in both enzyme complexes. The average lifetime for Trp88 in Pfk-2 was found to be unusually large, approximately 7.7 ns, and did not vary significantly with the ligation state of the enzyme, which demonstrates that the quenching or enhancement of fluorescence induced by the ligands is mainly due to the complex formation with Pfk-2. These results demonstrate the presence of an allosteric site for MgATP2- in Pfk-2 from E. coli, responsible for the inhibition of the enzyme activity by this ligand.

A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli.

Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.

A new method of assessing rates of the futile cycle during glycolytic and gluconeogenic metabolism.

A method for assessing rates of the futile cycle is presented, and it is illustrated in vitro. Glycolytic- and gluconeogenic-type futile cycles are simulated for the reactions catalyzed by phosphofructokinase (EC 2.7.1.11) and fructose-1,6-bisphosphatase (EC 3.1.3.11) in assays systems in which the cycle rates in either direction can be varied and determined. While either system is sustaining a net flux of carbons in a given direction, the flux of radioactively labeled carbons in the opposite direction is determined. Different cycle rates are obtained by varying phosphofructokinase activity while keeping fructose-1,6-bisphosphatase activity constant in the "gluconeogenic" simulation and varying fructose-1,6-bisphosphatase while keeping phosphofructokinase activity constant in the "glycolytic" simulation. A direct, linear relationship was found between the cycle rate and the radioactive labeling of fructose 1,6-bisphosphate from [U-14C]glucose 6-phosphate during net gluconeogenic carbon flux. Also, a direct, linear relationship was found between cycle rate and radioactive labeling of fructose-6-P from [U-14C]fructose-1,6-bisP during net glycolytic carbon flux. The applicability, advantages, and problems of the method are discussed.

Glucose metabolism in Escherichia coli and the effect of increased amount of aldolase.

We present a comparative study of Escherichia coli with normal and increased amounts of fructose-1,6-bisphosphate aldolase. Most experiments employed a resting cell system involving a high cell density (so as to obtain the soluble pool by direct extraction) and anaerobic incubation in the presence of chloramphenicol. Glucose use is linear with time with a rate ca. half of that in growth, fermentation is almost quantitative, and metabolite concentrations reach a quasi steady state. Increased amount of aldolase had little effect on glucose flux; fructose-1,6-P2 concentration decreased by ca. one-third, and the extent of equilibration of its two halves, measured by a dismutation procedure on samples taken during metabolism of [6-14C]glucose, increased from 0.33 [(cpm in C1-3)/(cpm in C1-6)] to 0.43. Using the simplest model, that increased amount of aldolase does not perturb net flux or later metabolites, together with the steady-state rate equations for aldolase and triose-P isomerase, we show that the results with resting cells fit with the extra enzyme being fully active, and do not necessitate special assumptions concerning a glycolytic complex, metabolite compartmentation, or secondary mechanisms assuring high metabolite concentration. However, the fit does require that the measured Vmax values substantially underestimate the actual ones. Calculation also shows that the forms of the predicted curves--and hence the fit with experimental data--of fructose-1,6-P2 concentration and labeling as a function of the amount of aldolase are highly dependent on glyceraldehyde-3-P concentration but independent of the kinetic parameters of aldolase.

An in vitro model showing different rates of substrate cycle for phosphofructokinases of Escherichia coli with different kinetic properties.

An in vitro assay model is introduced for the coupled assay of phosphofructokinase (PFK) and fructose-bisphosphatase. The model is applied to the study of three PFK of Escherichia coli: two isoenzymes, phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2), and a mutant form of phosphofructokinase-2 (PFK-2*). Results show that for a variety of conditions the PFK-1/fructose-bisphosphatase pair gives the lowest and the PFK-2*/fructose-bisphosphatase pair the highest rates of substrate cycle, with the PFK-2/fructose-bisphosphatase pair in an intermediate position. The effects of variables such as maximum activity ratios and MgATP concentration were explored. The possible role of MgATP in decreasing the futile cycle of the PFK-2/fructose-bisphosphatase pair is described. The results are discussed in terms of possible metabolic consequences of PFK-2* and of predictions of the model to be tested in vivo.

Determination of the molecular weight of proteins by electrophoresis in slab gels with a transverse pore gradient of crosslinked polyacrylamide in the absence of denaturing agents.

The molecular weight of proteins under nondenaturing conditions can be determined through polyacrylamide electrophoresis by comparing their relative mobilities at different gel concentrations with the relative mobilities of standard proteins under the same conditions (J. L. Hedrick and A. J. Smith (1968) Arch. Biochem. Biophys. 126, 155). This work describes a procedure that eliminates the need for several gels of different acrylamide concentrations with the use of a slab gel with a transverse pore gradient of crosslinked polyacrylamide.

Influence of ligands on the aggregation of the normal and mutant forms of phosphofructokinase 2 of Escherichia coli.

The aggregation states of Escherichia coli phosphofructokinase 2 (Pfk-2) and of a mutant enzyme (Pfk-2*) altered in the inhibitory allosteric site for MgATP were measured in the presence and in the absence of substrates and products of the reaction. When sucrose gradient ultracentrifugation experiments were performed in the absence of added ligands, both enzymes sedimented as dimers. Likewise, at low concentrations of both substrates (0.1 mM) the aggregation state of Pfk-2 and Pfk-2* corresponded to a dimer. However, in the presence of 1 mM MgATP alone, Pfk-2 sedimented as a tetramer, whereas Pfk-2* sedimented as a dimer. At a low fructose 6-phosphate concentration (0.1 mM) and an inhibitory concentration of MgATP (4 mM), Pfk-2 sedimented as a tetramer. However, at the same MgATP concentration but at a higher fructose-6-P concentration (1 mM), a condition under which Pfk-2 is not inhibited by the Mg-nucleotide complex, the enzyme sedimented as a dimer. Pfk-2* is not inhibited under these conditions and sedimented as a dimer in each case. Thus, the effectiveness of MgATP in promoting the aggregation of Pfk-2 and Pfk-2* parallels the inhibitability of the enzymes by the nucleotide complex. However, ATP4-, a potent inhibitor of Pfk-2 and Pfk-2* that binds to the catalytic site of the enzymes, had no effect upon their aggregation states. Possibly Pfk-2* is not able to form a tetramer because of an alteration in the regulatory site for the Mg-nucleotide complex.

Phosphate modification of fructose-1,6-bisphosphate aldolase in Escherichia coli.

When E. coli carrying multicopy plasmids for fructose-1,6-P2 aldolase or phosphoglycerate kinase was grown in the presence of 32Pi, there was label at the position of cognate high level polypeptide after SDS-PAGE. As tested for aldolase, the label was resistant to acetone, RNase, and hot TCA treatments, and was also observed by immunoprecipitation, which was competed for by purified aldolase. Incorporation of label also occurred in the presence of chloramphenicol. Immunoprecipitation revealed apparent aldolase labeling in the wild type strain as well.

Evolution and the structural domains of proteins.

Domains are regarded as the basic units of globular proteins and are associated to protein function, folding, and evolution. The occurrence of similar domains in different proteins has led to the proposal of divergent evolution from a common ancestral gene that has been duplicated and fused with a variety of different genes, which suggests that large proteins have been constructed using a modular mechanism in which domains are the building blocks. The appearance of proteins with new or different functions could then arise by exon recombination since these protein coding units frequently correspond to protein structural domains. Some aspects of the evolution of phosphofructokinase are assessed from this standpoint.

AMP-insensitive fructose bisphosphatase in Escherichia coli and its consequences.

Inhibition of fructose bisphosphatase (EC 3.1.3.11) by AMP is thought to be an important control mechanism, and, for the Escherichia coli enzyme, is the only control known. We here report on a mutant E. coli fructose bisphosphatase almost insensitive to this inhibition. The purified enzyme is normal in other respects. A strain with this enzyme instead of the wild-type enzyme grows normally in a variety of media. A strain with a very high level of the wild-type enzyme also grows normally. A strain with the very high level of mutant enzyme, however, does show growth abnormalities, but they are not clearly associated with the AMP insensitivity.

A phosphofructokinase mutant of Escherichia coli altered in its allosteric properties impairs gluconeogenic growth.

The regulation of metabolic fluxes is accomplished by modulation of key enzyme catalyzed reactions. This modulation takes place partially through the control of the catalytic activity of enzymes labelled as regulatory enzymes. The kinetic behavior of many regulatory enzymes can be explained in terms of multiple binding sites for effector molecules. Of these, the ones that play their control over catalysis by binding at an allosteric site have been considered of much importance. Nevertheless, proof that the effects observed in vitro, are in fact responsible for the physiological regulation in vivo, is scarce. In this regard, mutant enzymes altered in their allosteric properties might be useful. This will be illustrated with an enzyme considered crucial for the regulation of carbohydrate metabolism, namely phosphofructokinase. We present here the comparison of some of the kinetic and structural properties of wild type phosphofructokinase-2 of E. coli and of a mutant form which impairs gluconeogenic growth, an indication of the significance of the in vivo regulation. The main differences between the enzymes are their kinetic reaction mechanism, inhibitability by ATP, and aggregation states in the presence of substrates and effectors. So far these differences support only speculations as to the mechanism of the gluconeogenic impairment observed in strains that contain the mutant enzyme, a few of which are offered.

Effect of ATP on phosphofructokinase-2 from Escherichia coli. A mutant enzyme altered in the allosteric site for MgATP.

The activity of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2* was measured over a wide range of Mg2+ and ATP concentrations. MgATP2- inhibited only the Pfk-2 enzyme, with a degree of cooperativity of 1.5. This inhibition was relieved upon increasing the fructose-6-P concentration or by lowering the pH of the reaction mixture. Other nucleotides used as phosphate donors instead of ATP did not inhibit. MgATP2- was the true substrate for both enzymes and their Km values for this compound were not affected by an increase of the free Mg2+ concentration. However, free Mg2+ partially relieved the MgATP2- inhibition of Pfk-2 under conditions where the ATP4- concentration was negligible, without changes in the degree of cooperativity. ATP4- acted as a strong competitive inhibitor of both Pfk-2 and Pfk-2* with respect to MgATP2- with Ki values of 10 and 8 microM, respectively. ADP, AMP, and cAMP did not prevent the MgATP2- inhibition of Pfk-2. These results suggest the presence of an allosteric site for MgATP2- in Pfk-2 responsible for the MgATP2- inhibition, which is altered in Pfk-2* as a consequence of the structural mutation.

Kinetic mechanism of phosphofructokinase-2 from Escherichia coli. A mutant enzyme with a different mechanism.

The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.

Fructose bisphosphatase from Escherichia coli. Purification and characterization.

Escherichia coli fructose-1,6-bisphosphatase has been purified for the first time, using a clone containing an approximately 50-fold increased amount of the enzyme. The procedure includes chromatography in phosphocellulose followed by substrate elution and gel filtration. The enzyme has a subunit molecular weight of approximately 40,000 and in nondenaturing conditions is present in several aggregated forms in which the tetramer seems to predominate at low enzyme concentrations. Fructose bisphosphatase activity is specific for fructose 1,6-bisphosphate (Km of approximately 5 microM), shows inhibition by substrate above 0.05 mM, requires Mg2+ for catalysis, and has a maximum of activity around pH 7.5. The enzyme is susceptible to strong inhibition by AMP (50% inhibition around 15 microM). Phosphoenolpyruvate is a moderate inhibitor but was able to block the inhibition by AMP and may play an important role in the regulation of fructose bisphosphatase activity in vivo. Fructose 2,6-bisphosphate did not affect the rate of reaction.

An alteration in phosphofructokinase 2 of Escherichia coli which impairs gluconeogenic growth and improves growth on sugars.

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

Protein dye affinity chromatography using immobilized tetraiodofluorescein.

The chromatographic behavior of a heterogeneous protein mixture and of a series of homogeneous proteins on the immobilized dye tetraiodofluorescein has been observed and analyzed. Less than 6%, of the millimolar concentration of dye immobilized to a porous agarose matrix is accessible to protein. The affinity of a protein for immobilized dye is dramatically increased by insertion of apolar spacer atoms between the dye and the matrix. Dye columns constructed with a 9-atom spacer can be used to advantage for the retention and competitive elution of proteins not found previously amenable to dye chromatography.

Purification and characterization of dog liver glucokinase.

Glucokinase from dog liver has been purified to homogeneity by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation, gel filtration chromatography, and affinity chromatography on glucosamine-Sepharose. The purified enzyme was characterized with respect to stability, molecular weight, amino acid composition, SH groups, and physicochemical and kinetic properties. A molecular weight of 49,000 and 47,000 was estimated by sodium dodecylsulfate gel electrophoresis and gel filtration in non-denaturing conditions, respectively, indicating a monomeric structure for the enzyme. Glucokinase exhibits a sigmoidal saturation function for glucose with a Hill coefficient of 1.5 and a half-saturation value of 4mM at pH 7.5.