Phosphofructokinase 1 (PFK 1) regulation

Phosphofructokinase 1 (PFK 1) regulation

  • Phosphofructokinase is the most important control element in the mammalian glycolytic pathway. This enzyme is the pacemaker of glycolytic pathway.
  • Phosphofructokinase (PFK) is the enzyme that controls the third step of glycolysis (rate limiting step), the conversion of fructose-6-phosphate (F6P) into fructose-1, 6-biphosphate (F1, 6BP). It works by transferring a phosphate group from ATP to F6P.
  • This step is subject to extensive regulation since it is not only highly exergonic under physiological conditions, but also because it is a committed step – the first irreversible reaction unique to the glycolytic pathway.
  • Glucose 6 phosphate is not solely a glycolytic intermediate. Before this enzyme’s reaction, glucose-6-phosphate can be oxidized via pentose phosphate pathway to form NADPH, or be converted to glucose-1-phosphate for glycogenesis.

PFK 1 is an allosteric enzyme made up of 4 subunits. In addition to substrate binding sites, PFK 1 has several binding sites for allosteric effectors.

PFK 1 exists in 2 states: T-state (Enzymatically inactive) and R-state (Enzymatically active)

(F6P binds with a high affinity to the R state but not the T state enzyme)

ATP, citrate, and hydrogen ions, Fatty acids are allosteric inhibitors of the enzyme

How the allosteric inhibitors act?

  • The allosteric inhibitor binds to an enzyme at a regulatory site. As result the shape of the active site is altered so that the enzyme can no longer bind to its substrate. Hence they are negative allosteric modulators.
  • Allosteric inhibitors facilitate the formation of the T state, thereby inhibiting enzyme activity.

How the allosteric activators act?

 Allosteric activators bind to the allosteric site/regulatory site of the enzyme to facilitate the formation of the R state by inducing structural changes in the enzyme. By doing so they enhance the enzyme activity. They are positive allosteric modulators.

AMP, Pi and fructose 2, 6-bisphosphate are allosteric activators of the enzyme

Figure 1:Summary of the regulators affecting PFK-1 activity

Role of ATP in PFK 1 regulation

  • PFK1 is allosterically inhibited by high levels of ATP. ATP lowers the affinity of the enzyme for fructose 6-phosphate.
  • The activity of the enzyme increases when the cellular ATP/AMP ratio is lowered (Reason: AMP reverse the inhibitory action of ATP)
  • A high concentration of ATP converts the hyperbolic binding curve of fructose 6-phosphate into a sigmoidal one. ATP elicits this effect by binding to a specific regulatory site that is distinct from the catalytic site.


Figure 2:Allosteric regulation of muscle PFK-1 by ATP, shown by a substrate-activity curve.
  • At low concentrations of ATP, the Km for fructose 6-phosphate is relatively low, enabling the enzyme to function at a high rate at relatively low concentrations of fructose 6-phosphate.
  • When the concentration of ATP is high, Km for fructose 6-phosphate is greatly increased, as indicated by the sigmoid relationship between substrate concentration and enzyme activity.

The activity of PFK-1 increases when the energy charge of the cell is low (when there is a need for ATP) whereas it decreases when the energy charge of the cell is high (when ATP concentration in the cell is high). How?

Answer: PFK1 has two sites with different affinities for ATP which is both a substrate and an inhibitor for the enzyme.

  • When the nucleotide is produced faster than it is consumed, its cellular concentration is high.
  • Under such condition ATP, binding to its allosteric site, inhibits PFK-1 by reducing the affinity of the enzyme for fructose 6-phosphate.
  • When ATP consumption exceeds its production, ADP and AMP concentrations rises (in particular that of AMP) due to the reaction catalyzed by adenylate kinase that form ATP from ADP.


The activity of phosphofructokinase 1 depends on the cellular energy status:

  • When ATP is plentiful, enzyme activity decreases. If there is already plenty of ATP in the cell, glycolysis does not need to make more.
  • When AMP levels increase and ATP levels fall, enzyme activity increases.

Role of citrate in PFK 1 regulation

  • Glycolysis also furnishes carbon skeletons for biosyntheses and so a signal indicating whether building blocks are abundant or scarce should also regulates PFK 1.
  • PFK 1 is inhibited by citrate (a key intermediate in the aerobic oxidation of pyruvate, fatty acids, and amino acids)
  • High levels of citrate means that biosynthetic precursors are abundant and additional glucose should not be degraded for this purpose.
  • Citrate inhibits PFK 1 by enhancing the inhibitory effect of ATP.
  • Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated.

Effect of pH in PFK1 regulation

  • A fall in pH inhibits PFK 1 activity.
  • The inhibition of PFK 1 by H+ prevents excessive formation of lactic acids and precipitous drop in blood pH.

Role of AMP in PFK 1 regulation

  • PFK1 is allosterically activated by a high concentration of AMP.
  • When consumption of ATP is more than production, AMP concentration raises and it act allosterically to relieve enzyme inhibition by ATP.

Role of Fructose 2, 6 bisphosphate in PFK1 regulation

  • Fructose 2, 6-bisphosphate (Fru-2,6-P2) is the most potent stimulator (allosteric activator) of PFK1
  • Fructose 2, 6- bisphosphate activates phosphofructokinase by increasing its affinity for fructose 6-phosphate and diminishing the inhibitory effect of ATP.

The concentration of Fructose-2, 6 bisphosphate in cells is controlled through regulation of the synthesis and breakdown by PFK-2/FBPase-2.

The primary regulators of this are the hormones insulin, glucagon, and epinephrine which affect the enzyme through phosphorylation/dephosphorylation reactions.

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