Interaction of D-fructose and fructose 1-phosphate with yeast phosphofructokinase and its influence on glycolytic oscillations.

Fermentation of D-fructose- and D-glucose induced glycolytic oscillations of different period lengths in Saccharomyces carlsbergensis. Recent studies suggested, that D-fructose or one of its metabolites interacted with phosphofructokinase (ATP:D-fructo-6-phosphate 1-phosphofructokinase, EC 2.7.1.11), the core of the glycolytic 'oscillator'. In order to explore the kinetics of interaction, the influence of D-fructose and fructose 1-phosphate on purified yeast phosphofructokinase was studied. D-fructose concentrations up to 0.3 mM stimulated the enzyme, while a further increase led to competitive inhibition. The Hill coefficient for fructose 6-phosphate decreased from 2.8 to 1.0. Fructose 1-phosphate acted in a similar way, up to 1 mM activation and inhibition competitive to fructose 6-phosphate at higher concentration (2.0--3.5 mM) with the same effect on the Hill coefficient. The inhibition patterns obtained with D-fructose or fructose 1-phosphate suggest a sequential random reaction mechanism of yeast phosphofructokinase with fructose 6-phosphate and MgATP2-. The mode of interaction of phosphofructokinase with D-fructose and fructose 1-phosphate is discussed. The influence of both effectors resulted in altered enzyme kinetics, which may cause the different period lengths of glycolytic oscillations.     

Properties of phsophofructokinase from the mucosa of rat jejunum and the relation to the lack of Pasteur effect

1. The properties of phosphofructokinase after its slight purification from the mucosa of rat jejunum were studied. 2. The enzyme is inhibited by almost 100% by an excess of ATP (1.6mm), with 0.2mm-fructose 6-phosphate. AMP, ADP, P(i) and NH(4) (+) at 0.2, 0.76, 1.0 and 2mm respectively do not individually prevent the inhibition of phosphofructokinase activity by 1.6mm-ATP with 0.2mm-fructose 6-phosphate to any great extent, but all of them together completely prevent the inhibition of phosphofructokinase by ATP. 3. One of the effects of high concentrations of ATP on the enzyme was to increase enormously the apparent K(m) value for the other substrate fructose 6-phosphate, and this increase is largely counteracted by the presence of AMP, ADP, P(i) and NH(4) (+). At low concentrations of ATP the above effectors individually decrease the concentration of fructose 6-phosphate required for half-maximum velocity and when present together they decrease it further, in a more than additive way. 4. When fructose 6-phosphate is present at a saturating concentration (5mm), 0.3mm-NH(4) (+) increases the maximum velocity of the reaction 3.3-fold; with 0.5mm-fructose 6-phosphate, 4.5mm-NH(4) (+) is required for maximum effect. The other effectors do not change the maximum reaction velocity. 5. The results presented here suggest that NH(4) (+), AMP, ADP and P(i) synergistically decrease the inhibition of phosphofructokinase activity at high concentrations of ATP by decreasing the concentration of fructose 6-phosphate required for half-maximum velocity. Such synergism among the effectors and an observed, low ;energy charge' [(ATP+(1/2)ADP)/(AMP+ADP+ATP)] in conjunction with the possibility of a relatively high NH(4) (+) and fructose 6-phosphate concentration in this tissue, may keep the mucosal phosphofructokinase active and uninhibited by ATP under aerobic conditions, thus explaining the high rate of aerobic glycolysis and the lack of Pasteur effect in this tissue.     

Effect of substrates and effectors on the reversible inactivation of pig spleen phosphofructokinase by adenosine triphosphate

1. To investigate the mechanism of the reversible inactivation of pig spleen phosphofructokinase by ATP, the effect of order of addition of reactants (substrates, effectors and enzyme solution) was studied by preincubating the enzyme before assay with various combinations of its substrates and effectors. 2. Preincubation of the enzyme with MgATP or ATP at pH7.0 before addition of fructose 6-phosphate caused a rapid and much greater inhibition of activity than that observed when the reaction (carried out at identical substrate concentrations) was initiated with enzyme. 3. The rapid inhibition caused by preincubation with ATP, together with the sigmoidal response to fructose 6-phosphate and activation by AMP, were all blocked by prior photo-oxidation of the enzyme with Methylene Blue, which selectively destroys the inhibitory binding site for ATP [Ahlfors & Mansour (1969) J. Biol. Chem.244, 1247-1251]. 4. Fructose 6-phosphate, but not Mg(2+), protected phosphofructokinase from inhibition during preincubation with ATP in a manner that was sigmoidally dependent on the fructose 6-phosphate concentration. 5. Mg(2+), by protecting the enzyme from the inhibitory effect of preincubation at low pH (7.0) and by preventing its activation during preincubation with fructose 6-phosphate, demonstrated both a weak activating effect in the absence of the other substrates and a stronger inhibitory effect in the presence of fructose 6-phosphate. 6. Positive effectors (K(+), NH(4) (+), AMP and aspartate) protected the enzyme from inhibition during preincubation with MgATP in proportion to their potency as activators, but citrate potentiated the ATP inhibition. P(i) significantly slowed the inactivation process without itself acting as a positive effector. 7. The non-linear dependence of the initial rate of the unmodified enzyme on protein concentration (associated with increased positive homotropic co-operativity to fructose 6-phosphate) was intensified by preincubation with ATP and abolished by photo-oxidation. 8. The results are interpreted in terms of an association-dissociation model which postulates that protonation, at low pH, of a photo-oxidation-sensitive inhibitory site for ATP allows more rapid dissociation of an active tetramer to an inactive dimeric species.     

Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated.

The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically modify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby locking the enzyme into a less active T-state. This enzyme form has a maximum velocity that is 10% that of the native enzyme in the direction of fructose 6-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S0.5 (F6P) = 19 mM and nH = 2.2) at pH 6.8 and a hyperbolic saturation curve for MgATP with a Km identical to that for the native enzyme. The allosteric effectors, fructose 2,6-bisphosphate and AMP, do not affect the S0.5 for F6P but produce a slight (1.5- and 2-fold, respectively) V-type activation with Ka values (effector concentration required for half-maximal activation) of 0.40 and 0.24 mM, respectively. Their activating effects are additive and not synergistic. The kinetic mechanism for the modified enzyme is steady-state-ordered with MgATP as the first substrate and MgADP as the last product to be released from the enzyme surface. The decrease in V and V/K values for the reactants likely results from a decrease in the equilibrium constant for the isomerization of the E:MgATP binary complex, thus favoring an unisomerized form. The V and V/KF6P are pH dependent with similar pK values of about 7 on the acid side and 9.8 on the basic side. The microenvironment of the active site appears to be affected minimally as evidenced by the similarity of the pK values for the groups involved in the binding site for F6P in the modified and native enzymes.     

pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli.

The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors. The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, [Fru-6P]0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active. The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites. Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase. The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate. The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphate dependency of phosphofructokinase 2

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half-maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6-phosphate, without affecting Km for ATP. Citrate and P-enolpyruvate inhibited the enzyme non-competitively with fructose 6-phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic-AMP-dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6-bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6-phosphate and a half-maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6-bisphosphatase by fructose 6-phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.     

Interaction of calmodulin with muscle phosphofructokinase. Interplay with metabolic effectors of the enzyme under physiological conditions

The hysteretic calmodulin-induced inactivation of muscle phosphofructokinase and the calmodulin-mediated reactivation are essentially dependent on environmental conditions. The interplay of calmodulin during these reactions and at allosteric conditions with Mg . ATP, fructose 6-phosphate, adenosine 5'-[beta, gamma-imido]triphosphate and with the allosteric effectors AMP, ADP, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate was studied by two techniques. (a) A two-step technique with a preincubation of enzyme, calmodulin and effectors in close to physiological concentrations before dilution into an optimal activity assay. It reveals aggregation and slowly reversible conformation changes. (b) A direct assay of dilute enzyme at allosteric conditions. Dominating in the interplay of calmodulin with metabolic effectors is the competitive-like action of calmodulin on Mg . ATP binding to the regulatory sites of the enzyme. At high enzyme concentrations in the absence of hexose phosphates, i.e. at noncatalytic conditions calmodulin counteracts the stabilization of the highly active tetrameric form caused by Mg . ATP. In the allosteric assay it counteracts the ATP-induced allosteric inhibition. In both cases calmodulin acts synergistic with AMP and ADP. To a minor degree calmodulin also counteracts the stabilization of the tetrameric form caused by fructose 6-phosphate and hexose bisphosphate, now however antagonistically to AMP and ADP. By the demonstrated interactions the enzyme can be slowly and hysteretically shifted between an active tetrameric and an inactive dimeric state under control metabolic conditions and of Ca2+ and calmodulin. Resting conditions will inactivate and high contractile activity reactivate available enzyme.     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

Affinity chromatography of phosphofructokinase.

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N⁶-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N⁶-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg²⁺). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the “dead-end” complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.     

The stimulus-secretion coupling of glucose-induced insulin release. Fasting-induced adaptation of key glycolytic enzymes in isolated islets.

The rate of glucose and fructose 6-phosphate phosphorylation in islet homogenates is reduced by prior fasting of the donor rats. In fed rats, the velocity of glucose phosphorylation at increasing glucose concentrations (0.1 to 100 mM) is compatible with the presence of two enzyme activities. A preferential effect of fasting upon the high Km enzyme activity can be documented either at low ATP concentration which enhances the fractional contribution of the high Km enzyme activity, or in the presence of glucose 6-phosphate, which suppresses the low Km enzyme activity. Islet phosphofructokinase activity was characterized by inhibition by citrate or high ATP concentrations, and relief from ATP inhibition by AMP. Fasting reduces the activity of phosphofructokinase without altering its sensitivity to ATP and AMP. Cyclic AMP fails to overcome the effect of fasting upon phosphofructokinase. The activity of phosphoglucoisomerase is unaffected by fasting. The fasting-induced adaptation of key glycolytic enzymes could account, in part at least, for reduced metabolism of glucose in islets from fasted rats.     

Fructose 6-phosphate prevents the proteolyzed derivative of Escherichia coli phosphofructokinase from dissociation and inactivation

N-terminal sequence analysis shows that the limited proteolysis of Escherichia coli phosphofructokinase results in the removal of the 40-50 C-terminal residues of each chain. When tetrameric, this proteolyzed derivative is still active albeit insensitive to allosteric effectors (Le Bras, G., and Garel, J.-R. (1982) Biochemistry 21, 6656-6660). In the absence of fructose 6-phosphate, the proteolyzed phosphofructokinase spontaneously loses its activity and dissociates into dimeric species. This inactivation/dissociation is slowed down by the binding of fructose 6-phosphate to only part of the sites; it is completely prevented by the saturation of all four fructose 6-phosphate sites. The other substrate ATP does not protect the proteolyzed phosphofructokinase against this inactivation/dissociation. This inactivation/dissociation is not due to denaturation and can be reversed in some conditions by the addition of fructose 6-phosphate. The active tetrameric structure of phosphofructokinase is stable when either the C-terminal segment is not removed or the fructose 6-phosphate sites are occupied.     

Phosphofructokinase from bumblebee flight muscle. Molecular and catalytic properties and role of the enzyme in regulation of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle

Phosphofructokinase from the flight muscle of bumblebee was purified to homogeneity and its molecular and catalytic properties are presented. The kinetic behavior studies at pH 8.0 are consistent with random or compulsory-order ternary complex. At pH 7.4 the enzyme displays regulatory behavior with respect to both substrates, cooperativity toward fructose 6-phosphate, and inhibition by high concentration of ATP. Determinations of glycolytic intermediates in the flight muscle of insects exposed to low and normal temperatures showed statistically significant increases in the concentrations of AMP, fructose 2,6-bisphosphate, and glucose 6-phosphate during flight at 25 degrees C or rest at 5 degrees C. Measuring the activity of phosphofructokinase and fructose 1,6-bisphosphatase at 25 and 7.5 degrees C, in the presence of physiological concentrations of substrates and key effectors found in the muscle of bumblebee kept under different environmental temperatures and activity levels, suggests that the temperature dependence of fructose 6-phosphate/fructose 1,6-bisphosphate cycling may be regulated by fluctuation of fructose 2,6-bisphosphate concentration and changes in the affinity of both enzymes for substrates and effectors. Moreover, in the presence of in vivo concentrations of substrates, phosphofructokinase is inactive in the absence of fructose 2,6-bisphosphate.     

An equilibrium binding study of the interaction of fructose 6-phosphate and fructose 1,6-bisphosphate with rabbit muscle phosphofructokinase.

Equilibrium binding studies of the interaction of rabbit muscle phosphofructokinase with fructose 6-phosphate and fructose 1,6-bisphosphate have been carried out at 5 degrees in the presence of 1-10 mM potassium phosphate (pH 7.0 and 8.0), 5 mM citrate (pH 7.0), or 0.22 mm adenylyl imidodiphosphate (pH 7.0 and 8.0). The binding isotherms for both fructose 6-phosphate and fructose 1,6-bisphosphate exhibit negative cooperativity at pH 7.0 and 8.0 in the presence of 1-10 mM potassium phosphate at protein concentrations where the enzyme exists as a mixture of dimers and tetramers (pH 7.0) or as tetramers (pH 8.0) and at pH 7.0 in the presence of 5 mM citrate where the enzyme exists primarily as dimers. The enzyme binds 1 mol of either fructose phosphate/mol of enzyme monomer (molecular weight 80,000). When enzyme aggregation states smaller than the tetramer are present, the saturation of the enzyme with either ligand is paralleled by polymerization of the enzyme to tetramer, by an increase in enzymatic activity and by a quenching of the protein fluorescence. At protein concentrations where aggregates higher than the tetramer predominate, the fructose 1,6-bisphosphate binding isotherms are hyperbolic. These results can be quantitatively analyzed in terms of a model in which the dimer is associated with extreme negative cooperativity in binding the ligands, the tetramer is associated with less negative cooperativity, and aggregates larger than the tetramer are associated with little or no cooperativity in the binding process. Phosphate is a competitive inhibitor of the fructose phosphate sites at both pH 7.0 and 8.0, while citrate inhibits binding in a complex, noncompetitive manner. In the presence of the ATP analog adenylyl imidodiphosphate, the enzyme-fructose 6-phosphate binding isotherm is sigmoidal at pH 7.0, but hyperbolic at pH 8.0. The characteristic sigmoidal initial velocity-fructose 6-phosphate isotherms for phosphofructokinase at pH 7.0, therefore, are due to an heterotropic interaction between ATP and fructose 6-phosphate binding sites which alters the homotropic interactions between fructose 6-phosphate binding sites. Thus the homotropic interactions between fructose 6-phosphate binding sites can give rise to positive, negative, or no cooperativity depending upon the pH, the aggregation state of the protein, and the metabolic effectors present. The available data suggest the regulation of phosphofructokinase involves a complex interplay between protein polymerization and homotropic and heterotropic interactions between ligand binding sites.     

The characteristics of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferases from Sansevieria trifasciata leaves and Phaseolus coccineus stems

Three different molecular forms of pyrophosphate-dependent phosphofructokinase have been isolated: one from Sansevieria trifasciata leaves and two from Phaseolus coccineus stems. The form isolated from S. trifasciata has the molecular weight of about 115,000. The apparent molecular weights for the two forms from mung bean were approximately 220,000 and 450,000. All three forms have the same pH optima, an absolute requirement for Mg2+ ions both in the forward and reverse reaction, but differ in their sensitivity toward fructose 2,6-bisphosphate. Kinetic properties of the partially purified enzymes have been investigated in the presence and absence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase from S. trifasciata exhibited hyperbolic kinetics with all substrates tested. The saturation curves of the enzyme (form A) from mung bean for pyrophosphate, fructose 6-phosphate and fructose 1,6-bisphosphate were sigmoidal in the absence of fructose 2,6-bisphosphate. In the presence of fructose 2,6-bisphosphate these kinetics became hyperbolic.     

Self-association of human erythrocyte phosphofructokinase. Kinetic behaviour in dependence on enzyme concentration and mode of association.

The kinetic behaviour of human erythrocyte phosphofructokinase has been analyzed over a relative wide range of enzyme concentration (0.01 -- 1.7 mug/ml). The kinetic cooperativity which becomes apparent when the enzymic reaction rate is plotted versus the fructose 6-phosphate concentration decreases with increasing enzyme concentration. Simultaneously, a decrease of the half-saturation concentration for fructose 6-phosphate [S]0.5 is observed. Maximum velocity passes through a maximum at increasing enzyme concentrations. Sets of curves representing specific enzymic activity of phosphofructokinase versus enzyme concentration obtained at various fixed concentrations of fructose 6-phosphate and ATP are analyzed. The shapes of these curves are interpreted in terms of an association model of human erythrocyte phosphofructokinase, in which an inactive dimer (Mr 190000) and active multimers of the dimeric form are involved. The conclusion is drawn that the sigmoidal shape of the plots of the enzymic reaction rate versus fructose 6-phosphate concentration is partially caused by a displacement of the equilibrium between different states of association of phosphofructokinase to multimers by this substrate. On the other hand, the inhibition of the enzyme by high concentrations of ATP may be partially caused by a shift of this equilibrium to the state of the inactive dimer.     

Plastid and cytosolic phosphofructokinases from the developing endosperm of ricinus communis: II. Comparison of the kinetic and regulatory properties of the isoenzymes

A steady-state kinetic analysis of plastid phosphofructokinase at pH 8.2 is consistent with the enzyme having a sequential reaction mechanism. Cytosolic phosphofructokinase probably has a similar mechanism. At pH 7.0 plastid phosphofructokinase shows cooperative binding of fructose 6-phosphate and is inhibited by higher concentrations of ATP. In contrast cytosolic phosphofructokinase shows normal kinetics at both pH 8.2 and 7.0 with respect to fructose 6-phosphate and is not inhibited by ATP. In the case of plastid phosphofructokinase the affinity for fructose 6-phosphate increases as the pH is raised from 7 to 8.2 whereas cytosolic phosphofructokinase is affected in an opposite manner. Phosphate is the principal activator of plastid phosphofructokinase since the cooperative kinetics toward fructose 6-phosphate are shifted toward Michaelis-Menten kinetics by 1 mm sodium phosphate and this concentration of phosphate relieves the inhibition by ATP. Both isoenzymes are inhibited by phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate at pH 7.2. Plastid phosphofructokinase is most strongly inhibited by phosphoenol pyruvate with the I_0.5 value varying from 0.08 to 0.5 μm depending on substrate concentrations; phosphate reverses this inhibition. In contrast cytosolic phosphofructokinase is much less inhibited by phosphoenolpyruvate with an I_0.5 approximately 1000-fold higher. Cytosolic phosphofructokinase is powerfully inhibited by 3-phosphoglycerate with an I_0.5 value of 60 μm and this appears to be the principal regulator of this isoenzyme. The two isoenzymes of phosphofructokinase in the endosperm appear, therefore, to be regulated differently. Plastid phosphofructokinase is inhibited by phosphoenolpyruvate and ATP and is activated by phosphate; whereas the cytosolic enzyme is inhibited principally by 3-phosphoglycerate and this inhibition is only partially relieved by phosphate. Some of the differences reported previously for phosphofructokinases from different plant tissues may, therefore, be due to varying ratios of the cytosolic and plastid isoenzymes.     

Chloroplast phosphofructokinase in the green alga, Dunaliella marina: partial purification and kinetic and regulatory properties

The aim of this work was to study the pathway(s) of sugar phosphate metabolism in chloroplasts of the unicellular green alga, Dunaliella marina (Volvocales). Phosphofructokinase, detectable in crude cell extracts, copurifled with intact chloroplasts on sucrose density gradients. In isolated chloroplasts, phosphofructokinase activity displayed latency to the same degree as chloroplast marker enzymes. From the quantitative distribution of enzyme activities in fractionated cells, it is concluded that there is an exclusive localization of phosphofructokinase in chloroplasts. In addition, no separation into multiple forms could be achieved. For the study of regulatory properties, chloroplast phosphofructokinase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose chromatography. The pH optimum of the enzyme activity was 7.0 and was not altered with varying concentrations of substrates or low-molecular-weight effectors. Fructose 6-phosphate showed a sigmoidal saturation curve whose shape was further changed with varying protein concentrations of the preparation. The second substrate, ATP, gave a hyperbolic saturation curve with a Michaelis constant of 60 μm. At a Mg²⁺ concentration of 2.5 mm, ATP concentrations exceeding 1 mm inhibited the enzyme in a positive cooperative manner. The same type of inhibition was observed with other phosphorylated intermediates of carbon metabolism, the most efficient being phosphoenolpyruvate, glycolate 2-phosphate, glycerate 3-phosphate, and glycerate 2-phosphate. Inorganic phosphate was the only activator found for phosphofructokinase. With nonsaturating fructose 6-phosphate concentrations, P_i activated in a positive cooperative fashion, while no activation occurred with saturating fructose 6-phosphate concentrations. In the presence of either an activator or an inhibitor, the sigmoidal shape of the fructose 6-phosphate saturation curve was altered. Most notably, the activator P_i could relieve the inhibitory action of ATP, phosphoenolpyruvate, glycerate 3-phosphate, glycerate 2-phosphate, and glycolate 2-phosphate. Based on these experimental findings, the regulatory properties of D. marina chloroplast phosphofructokinase are discussed with respect to its playing a key role in the regulation of chloroplast starch metabolism during a light/dark transition. All available evidence is compatible with the interpretation that phosphofructokinase is active only in the dark thus channeling starch degradation products into glycolysis.     

Studies on heart phosphofructokinase. Use of fructose 6-sulfate as an alternative substrate to study the mechanism of action and active site specificity.

Fructose 6-sulfate was synthesized by direct sulfurylation of fructose and was isolated by two selective steps: (a) conversion of the 6-sulfuryl ester to fructose 1-phosphate-6-sulfate with phosphofructokinase; (b) conversion of fructose 1-phosphate-6-sulfate to fructose 6-sulfate by fructose-1,6-diphosphatase. Utilizing crystalline sheep heart phosphofructokinase, kinetic studies with the alternative substrate were carried out at pH 8.2 which is optimal for nonallosteric kinetics. The data are consistent with an ordered addition of the two substrates with the first, MgATP, being at thermodynamic equilibrium. The Vmax and Km obtained with fructose 6-sulfate were 0.03- and 100-fold, respectively, that obtained with the natural substrate. The study suggests that the divalent phosphoryl moiety is intimately involved in the active site conformation. Identification of the product of the reaction, fructose 1-phosphate-6-sulfate, was confirmed through studies with aldolase, fructose-1,6-diphosphatase, and by 31P NMR. The utilization of fructose 6-sulfate as a substrate by yeast glucose-6-phosphate isomerase could not be demonstrated.     

Effects of AMP and Cibacron blue F3G-A on the fructose 6-phosphate binding of yeast phosphofructokinase.

The positive effector 5′-AMP of yeast phosphofructokinase does not influence the binding of fructose 6-phosphate to the enzyme. Cibacron blue F3G-A considered an ATP analogue decreases the affinity of the enzyme to fructose 6-phosphate without exerting an effect on the cooperativity of fructose 6-phosphate binding. The peculiarities of the interactions of AMP and Cibacron blue with fructose 6-phosphate binding demonstrate compatibility of the allosteric kinetics with the binding behavior of the enzyme.     

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.