Regulation of mammalian muscle type 6-phosphofructo-1-kinase and its implication for the control of the metabolism


  • Mauro Sola-Penna,

    Corresponding author
    1. Laboratorio de Enzimologia e Controle do Metabolismo (LabECoM) and Laboratório de Oncobiologia Molecular (LabOMol), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
    • LabECoM, Faculdade de Farmácia, UFRJ, Ilha do Fundão, Rio de Janeiro, RJ 21941-590, Brazil. Tel./Fax: +55-21-2560-8438
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    • Tel./Fax: +55-21-2560-8438

  • Daniel Da Silva,

    1. Laboratorio de Enzimologia e Controle do Metabolismo (LabECoM) and Laboratório de Oncobiologia Molecular (LabOMol), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
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  • Wagner S. Coelho,

    1. Laboratorio de Enzimologia e Controle do Metabolismo (LabECoM) and Laboratório de Oncobiologia Molecular (LabOMol), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
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  • Monica M. Marinho-Carvalho,

    1. Laboratorio de Enzimologia e Controle do Metabolismo (LabECoM) and Laboratório de Oncobiologia Molecular (LabOMol), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
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  • Patricia Zancan

    1. Laboratorio de Enzimologia e Controle do Metabolismo (LabECoM) and Laboratório de Oncobiologia Molecular (LabOMol), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
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Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory mechanism that is modulated by a large variety of metabolites, drugs, and intracellular proteins. To date, the mammalian enzyme structure has not yet been resolved. However, it is known that PFK undergoes an intricate oligomerization process, shifting among monomers, dimers, tetramers, and more complex oligomeric structures. The equilibrium between PFK dimers and tetramers is directly correlated with the enzyme regulation, because the dimer exhibits very low catalytic activity, whereas the tetramer is fully active. Several PFK ligands modulate the enzyme, favoring the formation of its dimers or tetramers. The present review integrates recent findings regarding the regulatory aspects of muscle type PFK and discusses their relation to the control of metabolism. © IUBMB IUBMB Life, 62(11): 791–796, 2010.


The glycolytic enzyme 6-phosphofructo-1-kinase (PFK, ATP: D-fructose-6-phosphate-1-phosphotransferase, EC plays a central role in the regulation of glycolysis. This enzyme catalyzes the MgATP-dependent phosphorylation of fructose-6-phosphate (F6P), forming ADP and fructose-1,6-bisphosphate (F1,6BP). This reaction is virtually irreversible under intracellular conditions and is considered to be the pacemaker of the glycolytic flux (1). There has been evidence that the mammalian PFK is derived from the duplication, tandem fusion, and divergence of an ancestral prokaryotic gene. Thus, the bacterial PFK is about half the size of the mammalian enzyme. As a result, mammalian PFK contains additional sites that have evolved into the known allosteric sites for ATP, which exhibits inhibitory properties, and for fructose-2,6-bisphosphate (F2,6BP), which is known as the major physiologic activator of the enzyme (2).

There are three different isoforms of PFK in mammals, named PFK-M, PFK-L, and PFK-P (also called PFK-C) because of their major expression in muscle, liver, and platelets (or cerebrum), respectively. In humans, the isoforms M, L, and P present 85.2, 85.0, and 85.6 kDa, and their genes are assigned to chromosomes 1, 21, and 10, respectively (3). Moreover, it has been shown that PFK-M and PFK-L share 68.6% amino acid sequence identity, PFK-M and PFK-P share 70.3%, and PFL-L and PFL-P share 66.6%. The level of expression of these isoforms is tissue-specific; skeletal muscle is the only tissue that expresses only one isoform (PFK-M), whereas all other tissues express distinct levels of the three isoforms (4). Therefore, PFK-M is considered as the only isoform expressed in all the mammalian cells evaluated. The distinct patterns of PFK isoforms expressed among tissues may contribute to their specific glycolytic rates. As an example, it has recently been shown that the glycolytic efficiency (lactate formed per glucose consumed) of some human mammary tumor cell lines is enhanced in more aggressive cells, which was attributed to a higher expression of PFK-L over the other two isoforms (5).

Once translated, PFK monomers rapidly associate forming dimers. In fact, PFK monomers are too unstable and easily unfold (6, 7); thus, the dimerization of monomers is essential for the maintenance of the enzyme tertiary structure. PFK dimers display minimal catalytic activity, but may associate to form PFK tetramers that are fully active (8, 9). These PFK tetramers are actually considered dimers of PFK dimers and can also associate into more complex structures without compromising their catalytic activity (10). The equilibrium among the distinct PFK oligomeric structures is affected by many physical and chemical conditions (8) such as the enzyme concentration (a higher concentration of the enzyme leads to greater complexity of the oligomer), pH, temperature (an acidic medium and higher temperatures favor the dissociation of oligomers), and allosteric ligands, as will be discussed in the next section.


PFK is tightly regulated and responds to diverse molecules and signals by changing its catalytic activity and behavior. This enzyme is one of the few examples in which inhibition by the substrate occurs. PFK is strongly inhibited by ATP, one of its substrates, showing two distinct sites for this molecule: the catalytic site, presenting a binding constant of ∼0.15 mM, and the inhibitory allosteric site, presenting a binding constant of ∼2.5 mM (11–13). The intracellular range of ATP concentration varies from 2 to 5 mM, depending on the tissue and the metabolic state. Hence, ATP constantly inhibits PFK at different levels, depending on the metabolic state. However, some metabolites, such as ADP, AMP, cAMP, and F2,6BP, counteract the inhibitory effects of ATP on PFK (13–15). On the other hand, other metabolites (e.g., citrate and lactate) potentiate the inhibitory effects of ATP on PFK (11, 13). Other strong activators of PFK are F1,6BP and glucose-1,6-bisphosphate (16). Therefore, the intracellular levels of all these metabolites are critical for the catalytic activity of PFK and normally are responsible for the low levels of ATP inhibition observed under cellular conditions.

Recently, it has been shown that these allosteric modulators of PFK exert stimulatory or inhibitory properties, interfering with the equilibrium between dimers and tetramers of the enzyme. Therefore, it seems plausible that ATP, citrate, and lactate inhibit PFK by stabilizing its dimeric conformation, whereas ADP, AMP, cAMP, and F2,6BP favor the formation of tetramers (7, 11, 13, 17). This characterization of the mechanism of PFK regulation simplifies much of the puzzle concerning the large number of molecules modulating the enzyme. A single mechanism driving the activity of the enzyme toward an active or inactive conformation explains the counterbalanced effects of PFK inhibitors and activators. Thus, it is not difficult to understand that F2,6BP reverses the inhibitory effects of ATP.

It is important to note that the metabolites that regulate the PFK activity that, in turn, modulates the enzyme oligomeric conformation are markers of cell metabolic status. High ATP levels clearly indicate that the glycolysis rate should be reduced, avoiding unnecessary ATP synthesis, which is achieved through inhibition of PFK. Cytosolic citrate is also indicative of a high glycolytic rate because it leaks from the mitochondria after a high input of pyruvate into the organelle, normally due to an elevated rate of oxidative glycolysis. The process of fermentative glycolysis is similar, in that lactate is produced and the accumulation of this metabolite leads to the direct inhibition of PFK. On the other hand, the ADP and AMP that are elevated due to high ATP utilization by the cell counteract the inhibitory effects of the metabolites described previously, signaling the cellular need for ATP synthesis.


PFK is phosphorylated by diverse protein kinases (PKAs) at serine, threonine, or tyrosine residues (18–24). Very little is known regarding the sites in which these phosphorylations occur. Moreover, phosphorylation does not promote great changes in the enzyme activity. However, it modifies the pattern of activation by F6P, decreasing the cooperativity index for this substrate and making the enzyme less susceptible to inhibition by ATP. These effects are due to the stabilization of its tetrameric conformation by phosphorylation (18). Thus, PFK phosphorylation interferes with the regulation of the enzyme due to the modulators that affect the oligomerization of the enzyme. For example, on phosphorylation of muscle type PFK by cAMP-dependent PKA, lactate does not inhibit PFK (11). This effect can be particularly interesting, as PFK is phosphorylated by PKA under epinephrine stimulation in the skeletal muscle (23). Therefore, under certain situations such as during physical activity, the augmented epinephrine in circulation may attenuate the inhibition of PFK by the increased lactate produced due to elevated anaerobic glycolysis.

Phosphorylation of PFK results from stimulation by hormones such as epinephrine, insulin, or serotonin in the skeletal muscle (19, 23–27). These hormones all have the ability to activate glycolysis by stimulating glucose consumption in the skeletal muscles (19, 23, 24). There is a direct correlation between the phosphorylation of PFK and the activation of glycolysis triggered by these hormones, because the blockage of the former event impedes the latter (19, 24). However, PFK phosphorylation sites are not all the same, because epinephrine induces phosphorylation on serine residues, whereas insulin and serotonin promote phosphorylation on PFK tyrosine residues (19, 28). Interestingly, insulin may actually promote additional phosphorylations on serine residues of PFK, because in type 1 diabetes mellitus, there is a decrease in phosphorylated PFK serine residues (24). Regardless of which residue is targeted, phosphorylation seems to augment PFK activity in tissue extracts and glycolysis. The divergence between the effects of phosphorylation on the PFK activity evaluated using purified enzyme or tissue extracts is due to the intracellular redistribution of the enzyme on phosphorylation that occurs in the latter.


Muscle type PFK reversibly associates with distinct cellular components such as actin filaments (f-actin), microtubules, and the integral membrane anion transporter, band 3 (16, 29–33). The association with f-actin stabilizes the tetrameric conformation of PFK and, thus, stimulates the enzyme activity, whereas the association with microtubules or band 3 inhibits the enzyme due to stabilization of the dimers (16, 30, 34). The association/dissociation process is dynamic and regulated, playing a central role in the control of PFK activity and of the entire glycolytic flux. As an example, the level of oxygen controls the PFK association/dissociation with band 3 in erythrocytes because of the competition between PFK and deoxyhemoglobin that also binds to band 3 at the same site that PFK binds (34). Therefore, at low levels of O2, deoxyhemoglobin displaces PFK from band 3, activating the enzyme and glycolysis. On the other hand, at high O2 levels, the oxyhemoglobin formed dissociates from band 3 and, thus, PFK binds to band 3, resulting in the inhibition of the enzyme and the shift of glucose metabolism to the pentose phosphate pathway, which is critical in supplying NADPH to attenuate the highly oxidative environment (34).

Phosphorylation of PFK increases its affinity for f-actin and, thus, regulates the fraction of the enzymes associated with this cellular ultrastructure. Therefore, stimulation of skeletal muscle with epinephrine, insulin, or serotonin augments the f-actin-bound PFK fraction (19, 23, 25, 35). This association is described as part of the mechanism by which these hormones activate PFK and the entire glycolytic flux (19, 23–25, 36). In fact, this association is also described as being responsible for the increased PFK activity and glycolytic flux in cancer cells (5, 14, 37–42). Cancer cells exhibit an increased fraction of f-actin-associated PFK when compared with normal cells, which is confirmed by evaluating human breast cancer tissues (37). This pattern is directly correlated with the incidence of metastasis in human subjects (37). The association of PFK with f-actin is also shown to increase the glycolytic efficiency, that is, the rate of lactate produced per glucose consumed (5, 38). In other words, PFK association with f-actin is partially responsible for the so-called “Warburg effect,” a characteristic of tumors that accounts for ability to proliferate (43). Association of PFK to f-actin can prevent PFK inhibition by lactate (11). This fact is particularly important for tumor proliferation because these cells produce large amounts of lactate (5, 43), which would inhibit PFK if it were not associated with the cytoskeleton.


PFK is a calmodulin (CaM) binding protein, presenting two CaM-binding sites per protomer, each with distinct binding affinities (44). The high-affinity binding site presents a Kd of ∼3 nM, whereas the low-affinity binding sites present a Kd of ∼1 μM (44). CaM binding to PFK is dependent on Ca2+ concentrations and responds to the intracellular range of Ca2+ fluctuations (44, 45). Therefore, PFK binds to CaM in response to the elevation of the intracellular Ca2+ concentrations. The effects of CaM on PFK vary according to the number of CaM bound per PFK protomer. When both CaM sites are occupied, PFK is strongly inhibited, and this has been attributed to the stabilization of the dimeric conformation of the enzyme by CaM (44). Recent studies have demonstrated that the exclusive binding of CaM to the high-affinity site on PFK is responsible for the dissociation of the tetramers of the enzyme into dimers. However, these PFK dimers with one molecule of CaM bound per monomer are not inhibited and exhibit catalytic activity similar to the tetramers (13, 45). As the intracellular levels of CaM are not compatible with the affinity of the second CaM-binding site, it may be that CaM can only bind to its high-affinity site on PFK (45).

The formation of active PFK dimers bound to CaM are of great regulatory relevance for the enzyme because this conformation is not affected by the allosteric modulators that inhibit PFK and stabilize its dimers (13). Therefore, once intracellular Ca2+ increases, PFK binds to CaM, which counteracts the inhibitory effects of ATP on the enzyme (13). This process is similar to the effects described for F2,6BP, which is considered to be the most relevant activator of PFK (15). Thus, CaM should be considered at least as relevant as F2,6BP with respect to the activation of PFK and glycolysis. Furthermore, CaM counteracts the inhibitory effects of ATP, citrate, lactate, and the combination of these metabolites more efficiently than F2,6BP (13). This higher efficiency is explained by the fact that F2,6BP counteracts the aforementioned inhibitory effects by shifting the oligomeric equilibrium of PFK in a direction opposite to that of the inhibitors. On the other hand, CaM binds to the dimers formed in the presence of the inhibitors, turning them into active forms (13). These effects are illustrated in Fig. 1, which also summarizes other structural and functional consequences of the PFK effectors. The relevance of the stimulatory effects of CaM on PFK is also related to hormonal signals such as insulin. It has been shown that insulin triggers a transient intracellular Ca2+ fluctuation because of the aperture of voltage-dependent calcium channels (26). Moreover, CaM-antagonists are able to attenuate the activation of PFK and glycolysis by insulin (36), supporting the claim that CaM participates in these insulin effects.

Figure 1.

Schematic model of PFK activity and oligomeric structure modulation. PFK monomer illustration is a schematic model and does not represent the actual structure of the enzyme.


PFK is a potential target for the control of cell growth because of the close relationship between its enzymatic activity and the rate of glycolysis as well as the relevance of glycolysis for tumorigenesis. Two main inhibitory strategies have been adopted: (a) the direct inhibition of the enzyme and (b) interference with the enzyme's ability to associate with the cytoskeleton (11, 12, 14, 19, 24, 38, 39, 46–49). Actually, these two strategies are interrelated because the pharmaceuticals that directly inhibit PFK act by dissociating the enzyme tetramers into dimers, which also affects the ability of PFK to associate with different components of the cytoskeleton. Clotrimazole and acetylsalicylic acid are examples of this category of pharmaceuticals. They directly inhibit PFK because of the dissociation of the enzyme tetramers (12, 14, 39). Moreover, these drugs also promote the detachment of the enzyme from the cytoskeleton (14, 38, 40, 41). As a consequence, these drugs counteract the Warburg effect on tumor cells, that is, the increased glycolytic rate, even in the presence of oxygen, decreases their proliferation and viability (14, 38, 40, 41). Other drugs such as vinblastine, paclitaxel, lidocaine, and bupivacaine mainly affect the association of the enzyme with the cytoskeleton (46, 48, 49). Paclitaxel, because of its stabilizing properties on microtubules, decreases PFK activity and enhance the fraction of the enzyme bound to this ultrastructure (46, 49). On the other hand, lidocaine and bupivacaine detach PFK from f-actin, decreasing this fraction of the enzyme and, consequently, its activity and the entire glycolysis process (48). Regardless of whether they promote attachment to microtubules or the detachment from f-actin, all of these drugs inhibit PFK activity, glycolysis, and tumor cell viability (11, 12, 14, 19, 24, 38, 39, 46–49). Less is known whether these mechanisms are involved on the clinical usage of these drugs. However, it is a possible explanation for some of their effects.


Muscle type PFK is a multiregulated enzyme and its activity drives the rate of glycolysis. Therefore, PFK plays a central role in metabolism and in various other cell functions, such as cell growth and viability. Despite the fact that several regulatory properties of this enzyme are well described in biochemistry textbooks, some of its physiologically important characteristics have been neglected. Among them, the inhibition of the enzyme by lactate and its activation by CaM in response to Ca2+ fluctuations should be highlighted. Lactate is the final product of anaerobic glycolysis and of aerobic glycolysis, that is, the Warburg effect (important for tumor growth), and its effect on the inhibition of PFK is an important mechanism of feedback inhibition in glycolysis. The effects of lactate on PFK can be potentiated or counteracted by other inhibitors or activators of the enzyme, indicating the complexity of PFK regulation. Nevertheless, CaM appears to be a potent PFK activator that can override the inhibitory effects of several physiologically relevant negative modulators of the enzyme, such as ATP, citrate, and lactate. Moreover, CaM activates PFK in a calcium-dependent fashion, contributing to the glycolytic stimulation by several hormones, such as insulin and serotonin. Figure 1 summarizes the major regulatory aspects discussed here. In conclusion, it should be emphasized that PFK might be a prominent intervention target in cancer treatment because inhibiting its activity reduces tumor cell viability.