Phosphofructokinase from the epithelial cells of rat small intestine. Comparison of regulatory properties with those of skeletal muscle, liver and brain phosphofructokinase

The regulatory properties of phosphofructokinase from rat mucosa, liver, brain and muscle were investigated. Mucosal phosphofructokinase displayed cooperativity with respect to fructose 6-phosphate at pH 7.0 and so did the muscle, brain and liver isoenzymes. All these four isoenzymes were inhibited by ATP, the mucosal isoenzyme being the least inhibited. They were also inhibited by citrate and creatine phosphate. AMP, ADP, glucose 1,6-diphosphate, fructose 2,6-bisphosphate and inorganic phosphate were all strong activators for the mucosal, brain, liver and muscle phosphofructokinase, but the mucosal isoenzyme was found to be more activated than the others, accounting for the higher rates of glycolysis observed in mucosa. The results suggest that mucosal phosphofructokinase is unique and different from all the other isoenzymes.     

Some regulatory properties of pea leaf carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase of pea shoots (Pisum sativum L.) was purified 101-fold. Its stability was greatly increased by the addition of substrates and activators. The enzyme was strongly inhibited by micromolar amounts of UMP (Ki less than 2 mum). UDP, UTP, TMP, and ADP were also inhibitory. AMP caused either slight activation (under certain conditions) or was inhibitory. Uridine nucleotides were competitive inhibitors, as was AMP, while ADP was a noncompetitive inhibitor. Enzyme activity was increased manyfold by the activator ornithine. Ornithine acted by increasing the affinity for Mg.ATP by a factor of 8 or more. Other activators were IMP, GMP, ITP, and GTP, IMP, like ornithine, increased the Michaelis constant for Mg.ATP. The activators ornithine, GMP, and IMP (but not GTP and ITP) completely reversed inhibition caused by pyrimidine nucleotides while increasing the inhibition caused by ADP and AMP.     

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.     

The regulation of pea-seed phosphofructokinase by phosphoenolpyruvate

1. Pea-seed phosphofructokinase was purified 27-fold by a combination of fractionation with ethanol and ammonium sulphate. Under the conditions of assay, the enzyme was strongly inhibited by phosphoenolpyruvate. This inhibition was reversed by increasing the concentration of fructose 6-phosphate or magnesium chloride, or by lowering the ATP concentration. 2. Citrate, ADP and AMP inhibited phosphofructokinase and increased the sensitivity to phosphoenolpyruvate inhibition. Sulphate and inorganic phosphate stimulated the enzyme activity and decreased the sensitivity to phosphoenolpyruvate. 3. In the presence of inorganic phosphate and low concentrations of ATP, inhibition by phosphoenolpyruvate ceased and phosphoenolpyruvate became stimulatory. 4. The possible significance of these results in the control of plant carbohydrate metabolism is discussed.     

Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. The aerobic/anaerobic transition: role of arginine phosphate in enzyme control.

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 mumol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 +/- 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6-phosphate, ATP, and Mg2+) at either pH 7.9 OR PH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8, the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate, NAD(P)H,Ca2+, fructose 1,6-bisphosphate, 3-phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K +, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP(0.2- 1.0mM) and to a lesser extent by high concentrations of Pi (20 mM) and adenosine 3' :5'-monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki (ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8, whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values. The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulator of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia, the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.     

Spin-labelled phosphofructokinase. A simple and direct approach to the study of allosteric equilibria under near-physiological conditions.

Rabbit muscle phosphofructokinase, spin-labelled at its most reactive thiol group, has an electron spin resonance spectrum which is very sensitive to the binding of substrates and allosteric effectors. The spectral changes have been interpreted in terms of a concerted allosteric transition between two conformational states with non-exclusive binding of effectors. On this basis MgATP, fructose 6-phosphate plus ATP, and NH+4ions behave as potent positive effectors, inorganic phosphate, sulphate, AMP, fructose 6-phosphate and fructose 1,6-bisphosphate are less potent activators, and free ATP and H+ions are negative effectors, in agreement with the kinetic behaviour, but citrate behaves anomalously. In addition, the allosteric equilibrium can be displaced towards the inhibited state by selectively modifying two further thiol groups. Strong positive cooperativity occurs under suitable conditions with ATP, metal-ATP and fructose 6-phosphate. Biphasic changes of conformation, attributed to binding at the catalytic and inhibitory sites, have been observed in titrations with ATP. The differentiation of the two ATP binding sites arises in the presence of fructose 6-phosphate because of a distinct concerted effect on conformation between the two substrates at the active site. A similar effect occurs between ATP and citrate. Other heterotropic effects are more consistent with simple models; phosphates favour the binding, and reduce the cooperativity, of fructose 6-phosphate and metal-ATP, whereas excess ATP and H+ ions antagonise the binding and increase the cooperativity of fructose 6-phosphate. The observations are related to existing kinetic and binding studies where possible. Anomalous features of the behaviour suggest that the model should be regarded only as a first approximation.     

A conformational transition involved in antagonistic substrate binding to the allosteric phosphofructokinase from Escherichia coli.

The binding of fructose 6-phosphate, ATP or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate), ADP, and phosphoenolpyruvate to Escherichia coli phosphofructokinase has been studied by changes in the protein fluorescence and/or equilibrium dialysis. The results lead to the following conclusions: (1) tetrameric phosphofructokinase can bind four ATP but only two fructose-6-phosphate, and this binding occurs without cooperativity; (2) only two conformational states, T and R, with respectively a high and a low fluorescence, seem accessible to phosphofructokinase, which exists as a mixture of one-third R and two-third T states in the absence of ligand; (3) the substrate fructose 6-phosphate and the allosteric activator ADP bind preferentially to the low-fluorescence R state, while the other substrate, ATP [or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate)], and the allosteric inhibitor phosphoenolpyruvate bind to the high-fluorescence T state; (4) the binding of a given ligand is cooperative, with a Hill coefficient of 2, only when this binding is accompanied by a complete shift from one state to the other; for instance, the binding of the ATP analogue adenylyl 5'-(beta,gamma-methylenediphosphonate) to the T state is cooperative only in the presence of fructose 6-phosphate which favors the R state. This behavior is qualitatively consistent with a concerted transition, but quite different from that described earlier for phosphofructokinase from steady-state activity measurements (Blangy et al., 1968). This discrepancy suggests that the allosteric properties of phosphofructokinase are due in part to ligand binding and in part to the kinetics of the enzymatic reaction.     

Heart phosphofructokinase: allosteric kinetics with fructose 6-sulfate.

The allosteric regulation of heart phosphofructokinase was studied at pH 6.9 with an alternative substrate, fructose 6-sulfate. The alternative substrate allowed kinetic studies to be carried out at high enzyme concentrations (0.1 mg/ml) where the effect of allosteric ligands on enzyme physical structure has been studied. A Km for ATP binding (8-10 muM) in the presence of saturating AMP concentrations was found which agreed well with the value obtained at pH 8.2, ATP inhibitory effects closely followed saturation of its substrate site. Hill plots for ATP inhibition gave an interaction coefficient of 3.5 indicating cooperatively between at least four enzyme subunits. Neither AMP nor fructose 6-sulfate affected the cooperativity between the ATP inhibitory sites but only increased the inhibitory threshold. As the ATP concentration was increased from suboptimal to inhibitory levels, interaction coefficients for AMP and fructose 6-sulfate changed from 1 to 2. Increasing citrate concentration resulted in an increase in the interaction coefficient for fructose 6-sulfate to a value of 1.9. Citrate inhibition was synergistic with ATP inhibition with an interaction coefficient of 2. The data indicate that allosteric kinetics of the enzyme can be shown at high enzyme concentrations with the alternative substrate. ATP inhibition appears to involve interaction between at least four subunits, while citrate, AMP, and fructose 6-sulfate interact minimally with two subunits.     

Evolution of the allosteric ligand sites of mammalian phosphofructo-1-kinase

Mammalian phosphofructokinase (PFK) has evolved by a process of tandem gene duplication and fusion to yield a protein that is more than double the size of prokaryotic PFKs. On the basis of complete conservation of active site residues in the N-terminal half of the eukaryotic enzyme with those of the bacterial PFKs, one assumes that the active site of the eukaryotic PFK is located in the N-terminal half. Again using sequence comparisons, the four allosteric ligand sites of mammalian PFK have been thought to arise from the duplicated catalytic and regulatory sites of the ancestral PFK. Previous site-directed mutagenesis studies [Li et al. (1999) Biochemistry 38, 16407-16412; Chang and Kemp (2002) Biochem. Biophys. Res. Commun. 290, 670-675] have identified the origins of the citrate and fructose 2,6-bisphosphate sites. Here, site-directed mutagenesis of two arginine residues (Arg-433 and Arg-429) of mouse phosphofructokinase is used to identify the ATP inhibitory site, and, by inference, the AMP/ADP site. Mutation of the residues to alanine reduced ATP inhibition in the case of Arg-429 and eliminated ATP inhibition in the instance of Arg-433. The Arg-433 mutant could be inhibited by citrate, and that inhibition could be reversed by fructose 2,6-bisphosphate and cyclic AMP, a high-affinity ligand for the AMP/ADP binding site. It is concluded that the two inhibitors, ATP and citrate, of mammalian PFK interact with sites that have evolved from the duplicated phosphoenolpyruvate/ADP allosteric site of the ancestral PFK. The two sites for activators, fructose 2,6-bisphosphate and AMP or ADP, have evolved from the catalytic site of the ancestral precursor.     

Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei.

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.     

Decreased phosphofructokinase activity in skeletal muscle of diabetic rats

The activities of phosphofructokinase, aldolase and pyruvate kinase were diminished in extracts from skeletal muscle of streptozotocin diabetic rats, whereas the activities of glucose phosphate isomerase and phosphoglucomutase were not changed. Treatment of diabetic rats with insulin restored the activity of phosphofructokinase to normal. A kinetic study of the partially purified enzyme from normal and diabetic rats showed identical Michaelis constants for ATP and equal sensitivity to inhibition by excess of this substrate. Extracts of quick frozen muscle from diabetic rats had higher levels of citrate (an inhibitor of phosphofructokinase) and lower levels of D-fructose-1,6-bisphosphate and D-glucose-1,6-bisphosphate (activators of this enzyme). The levels of D-fructose-6-phosphate, D-glucose-6-phosphate, ATP, ADP and AMP were the same for the two groups. Our data suggest that the in vivo decrease of phosphofructokinase activity in skeletal muscle of diabetic rats is due to a decrease in the level of the enzymatically active protein as well as to an unfavorable change in the level of several of its allosteric modulators.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

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.     

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC₅₀) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.     

The control of rat-heart phosphofructokinase by citrate and other regulators

1. The effects of ATP, inorganic phosphate and citrate on the relationship between fructose 6-phosphate concentration and initial velocity of reaction has been investigated with a partially purified preparation of rat-heart phosphofructokinase. 2. At low concentrations of ATP (<80mum) rate curves for fructose 6-phosphate approximated to Michaelis-Menten kinetics. At higher ATP concentrations rate curves were sigmoid, the K(m) for fructose 6-phosphate increased and the reaction appeared to be first-order with respect to fructose 6-phosphate at concentrations above its K(m) and of a higher order at concentrations below its K(m). Inorganic phosphate lowered the K(m) for fructose 6-phosphate and the concentration at which the apparent kinetic order decreased. 3. At 40mum-ATP, citrate was an activator at low concentration (<100mum) and an inhibitor at higher concentrations. At 0.5mm-ATP, citrate was inhibitory at all concentrations tested. 4. A new method for phosphofructokinase assay using [U-(14)C]fructose 6-phosphate is described which allows measurements to be made of the velocity of the forward reaction at known concentrations of the products of the reaction. With this method confirmatory evidence has been obtained that concentrations of ATP, AMP, phosphate and citrate may regulate phosphofructokinase in the perfused rat heart.     

The purine nucleotide cycle. Control of phosphofructokinase and glycolytic oscillations in muscle extracts.

Linked oscillations of the glycolytic pathway and the purine nucleotide cycle were studied in particle-free extracts of rat skeletal muscle. Under the conditions used, an accumulation of about 1 muM fructose diphosphate can trigger a sudden increase in phosphofructokinase activity. The activation by fructose diphosphate depends on the presence of AMP. When the AMP concentration drops, phosphofructokinase becomes inhibited, even though the fructose disphosphate concentration remains high. It is concluded that the oscillatory behavior can be of advantage for maintaining a high average [ATP]/[ADP] ratio.     

The purification, characterization and regulatory properties of liver phosphofructokinase in the Arabian one-humped camel (Camelus dromedarius)

1. Phosphofructokinase from camel liver was purified to homogeneity more than 3600-fold, and the yield of the preparation was 46%. 2.The sodium dodecyl sulphate-treated purified enzyme migrated as a single band in 10% polyacrylamide gel. 3. The enzyme is a tetramer, with a monomer Mr 90,000. 4. The regulatory properties of the purified enzyme from camel liver were studied at pH 7.0. 5. The enzyme displayed cooperativity with respect to fructose 6-phosphate and was inhibited by high concentrations of ATP. 6. The enzyme was also inhibited by citrate, phosphocreatine and 2,3-bisphosphoglycerate. 7. On the other hand, ADP, AMP, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate were all found to be strong activators for camel liver phosphofructokinase.     

The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition.

1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a 'prior-isomerization' pathway; at higher concentrations a limited contribution from a 'substrate-guided' pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.     

Binding and regulatory properties of phosphofructokinase from swine kidney

Summary The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P₂ reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P₂ the reaction showed a sigmoidal dependence on fructose 6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K_0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate.The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The K_D for fructose 6-P was 13 µM and in the presence of 0.5 mM ATP it increased to 27 µM. The addition of 0.3 mM citrate also increased the K_D for fructose 6-P to about 40 µM. AMP, 10 µM, decreased the K_D to 5 µM and the addition of fructose 2,6-phosphate decreased the K_D for fructose 6-P to 0.9 µM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The K_D of the site with the highest affinity for ATP was 4 µM, and it increased to 15 µM in the presence of fructose 2,6-bisphosphate. The addition of 50 µM fructose 1,6-bisphosphate increased the K_D for ATP to 5.9 µM. AMP increased the K_D to 5.9 µM whereas 0.3 mM citrate decreased the K_D for ATP to about 2 µM. The K_D for AMP, was 2.0 µM; the K_D for cyclic AMP was 1.0 µM; the K_D for ADP was 0.9 µM; the K_D for fructose 1,6-bisphosphate was 0.5 µM; the K_D for citrate was 0.4 µM and the K_D for fructose 2,6-bisphosphate was about 0.1 µM. A maximum of about 4 moles of AMP, ADP and cyclic AMP and fructose 2,6-bisphosphate were bound per mole of enzyme. Taken collectively, these and previous studies (9) indicate that fructose 2,6-phosphate is a very effective activator of swine kidney phosphofructokinase. This effector binds to the enzyme with a very high affinity, and significantly decreases the binding of ATP at the inhibitory site on the enzyme.     

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.