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
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.
- 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.
ADP + ADP ⇄ ATP + AMP
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.