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Regulation of Glycolysis
(1) the production of ATP, generated by the degradation of glucose, and
(2) the provision of building blocks for synthetic reactions, such as the formation of fatty acids.
Flux through a metabolic pathway can be regulated in several ways:
1. Availability of substrate
2. Concentration of enzymes responsible for rate-limiting steps (Induction and Repression)
3. Allosteric regulation of enzymes
4. Covalent modification of enzymes (e.g. phosphorylation)
Generally, enzymes that catalyze essentially irreversible steps in metabolic pathways are potential sites for regulatory control. Although most of the reactions of glycolysis are reversible, three are markedly exergonic and must therefore be considered physiologically irreversible.
The enzymes responsible for catalyzing these three steps, hexokinase (or glucokinase) for step 1, phosphofructo kinase-1 for step 3, and pyruvate kinase for step 10, are the primary steps for enzyme regulation.
Availability of substrate (in this case, glucose), is another general point for regulation.
The concentration of these three enzymes in the cell is regulated by hormones that affect their rates of transcription (Induction/repression). Insulin up regulates (induces) the transcription of Glucokinase, phosphofructo kinase-1, and pyruvate kinase, while glucagon down regulates(represses) their transcription. These effects take place over a period of hours to days, and generally reflect whether a person is well-fed or starving.
1) Regulation at the level of Hexokinase and Glucokinase
- The Hexokinase enzyme is allosterically inhibited by the product, glucose-6-phosphate.
- Glucokinase is highly specific for D-glucose, has a much higher Km for glucose (approximately 10.0 mM ), and is not product-inhibited. With such a high Km for glucose, Glucokinase becomes important metabolically only when liver glucose levels are high.The low glucose affinity of Glucokinase in the liver gives the brain and muscles first call on glucose when its supply is limited, whereas it ensures that glucose will not be wasted when it is abundant.
- Glucokinase is an inducible enzyme—the amount present in the liver is controlled by insulin.
2) Regulation of Phospho fructokinase-1
Phospho fructokinase is the “valve” controlling the rate of glycolysis.
a) Role of ATP
- ATP is an allosteric inhibitor of this enzyme.
- In the presence of high ATP concentrations, the Km for fructose-6-phosphate (its substrate) is increased, glycolysis thus “turns off.”
- ATP elicits this effect by binding to a specific regulatory site that is distinct from the catalytic site.
- AMP reverses the inhibitory action of ATP, and so the activity of the enzyme increases when the ATP/AMP ratio is lowered.
- In other words, glycolysis is stimulated as the energy charge falls.
b) Role of Citrate
- Glycolysis also furnishes carbon skeletons for biosyntheses, and so a signal indicating whether building blocks are abundant or scarce should also regulate phosphofructokinase-1.
- Phosphofructokinase-1 is inhibited by citrate, an early intermediate in the citric acid cycle.
- A high level of citrate means that biosynthetic precursors are abundant and additional glucose should not be degraded for this purpose.
- Citrate inhibits phosphofructokinase 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.
c) Role of Fr 2,6 bisphosphate
- Phosphofructokinase-1 is also regulated by D-fructose-2,6-bisphosphate, a potent allosteric activator that increases the affinity of phosphofructokinase for the substrate fructose-6-phosphate.
- Stimulation of phosphofructokinase-1 is also achieved by decreasing the inhibitory effects of ATP.
- Fructose-2,6-bisphosphate increases the net flow of glucose through glycolysis by stimulating phosphofructokinase-1 and, by inhibiting fructose-1,6-bisphosphatase, the enzyme that catalyzes this reaction in the opposite direction.
- Fructose 2,6 bisphosphate is produced from fructose-6-P by Phosphofructokinase-2 enzyme (an isomer of PFK-1)- see the details below-
d) Effect of pH
- A fall in pH also inhibits Phosphofructokinase-1 activity.
- The inhibition of phosphofructokinase-1 by H+ prevents excessive formation of lactic acid and a precipitous drop in blood pH (acidosis).
Why is phosphofructokinase rather than hexokinase the pacemaker of glycolysis? The reason becomes evident on noting that glucose 6-phosphate is not solely a glycolytic intermediate. Glucose 6-phosphate can also be converted into glycogen or it can be oxidized by the pentose phosphate pathway to form NADPH or it can be used in the Uronic acid pathway depending upon the cellular requirement.
The first irreversible reaction unique to the glycolytic pathway, the committed step, is the phosphorylation of fructose 6- phosphate to fructose 1,6-bisphosphate. Thus, it is highly appropriate for phosphofructokinase-1 to be the primary control site in glycolysis. In general, the enzyme catalyzing the committed step in a metabolic sequence is the most important control element in the pathway.
3) Regulation of pyruvate Kinase
a) Allosteric modification
Pyruvate kinase possesses allosteric sites for numerous effectors. It is activated by AMP and fructose-1,6-bisphosphate and inhibited by ATP, acetyl-Co A, and alanine (Figure-1).
b) Covalent modification
Liver pyruvate kinase is regulated by covalent modification. Hormones such as glucagon activate a c-AMP-dependent protein kinase, which transfers a phosphoryl group from ATP to the enzyme (Figure-1). The phosphorylated form of pyruvate kinase is more strongly inhibited by ATP and alanine and has a higher Km for phosphoenol pyruvate ( PEP), so that, in the presence of physiological levels of PEP, the enzyme is inactive. Then PEP is used as a substrate for glucose synthesis in the gluconeogenesis pathway. This hormone-triggered phosphorylation, prevents the liver from consuming glucose when it is more urgently needed by brain and muscles.
In the well fed state, under the influence of Insulin the dephosphorylated form predominates, the enzyme remains active and the glucose utilization is promoted so as to maintain the blood glucose concentration within the physiological range.
Figure –1- Regulation of pyruvate kinase by allosteric effectors and by covalent modification. The enzyme becomes active in the low energy state (excess of AMP), while it becomes inactive in the energy rich state(high ATP concentration ). Alanine is produced from pyruvate by transamination, its high concentration also signifies excess pyruvate/ product of glycolysis, hence the pathway is switched off in conditions of excess alanine. Similarly excess Acetyl co A produced from pyruvate also inhibits this enzyme by allosteric modification.In the presence of high glucose concentration the enzyme remains in the dephosphorylated or active form whereas in conditions of starvation/low glucose concentration, the enzyme remains in the phosphorylated or inactive form to inhibit glycolysis.
4) Effect of hypoxia
Hypoxia stimulates glycolysis, by stimulating the peripheral uptake of glucose by increasing the number of glucose transporters and also by increasing the activities of glycolytic enzymes.
5) Role of hormones
Insulin promotes glycolysis by
- Inducing the regulatory enzymes (Glucokinase, PFK-1 and Pyruvate kinase)
- Covalently modifying pyruvate kinase enzyme (maintaining it in the dephosphorylated form)
Glucagon inhibits glycolysis by
- Repressing the key regulatory enzymes
- Covalently modifying pyruvate kinase enzyme (maintaining it in the phosphorylated form)
Role of fructose 2,6 bisphosphate
Fr 2, 6 bisphosphate is an important regulator of glycolysis. It is a positive modifier of Phosphofructokinase -1 enzyme.
Two enzymes regulate its concentration by its synthesis (phosphorylating fructose 6-phosphate) and degradation (dephosphorylating fructose 2,6- bisphosphate).
Synthesis of fructose-2,6-bisphosphate
Fructose 2,6-bisphosphate is formed in a reaction catalyzed by phosphofructokinase- 2 (PFK2), a different enzyme from phosphofructokinase-1.
Degradation of Fructose 2,6-bisphosphate
Fructose 2,6-bisphosphate is hydrolyzed to fructose 6-phosphate by a specific phosphatase, fructose2,6 bisphosphatase 2 (FBPase2).
Regulation of concentration of Fructose 2,6-bisphosphate
The striking finding is that both PFK2 and FBPase2 are present in a single 55–kd polypeptide chain. This bifunctional enzyme contains an N-terminal regulatory domain, followed by a kinase domain and a phosphatase domain (Figure-2). The bifunctional enzyme itself probably arose by the fusion of genes encoding the kinase and phosphatase domains.
Figure –2- Orientation of functional domains of bifunctional enzyme
In the liver, the concentration of fructose 6-phosphate rises when blood-glucose concentration is high, and the abundance of fructose 6-phosphate accelerates the synthesis of Fr 2,6-BP (Figure-3). Hence, an abundance of fructose 6–phosphate leads to a higher concentration of Fr–2,6–BP, which in turn stimulates phosphofructokinase-1. The product of PFK-1 catalyzed reaction Fr 1,6 bisphosphate further stimulates pyruvate kinase enzyme. Such a process is called feed forward stimulation.
The activities of PFK2 and FBPase2 are reciprocally controlled by phosphorylation of a single serine residue. When glucose is scarce, a rise in the blood level of the hormone glucagon triggers a cyclic AMP cascade, leading to the phosphorylation of this bifunctional enzyme by protein kinase A. This covalent modification activates FBPase2 and inhibits PFK2, lowering the level of F-2,6-BP. Thus, glucose metabolism by the liver is curtailed (Figure-3). Conversely, when glucose is abundant, the enzyme loses its attached phosphate group. This covalent modification activates PFK2 and inhibits FBPase2, raising the level of F-2,6-BP and accelerating glycolysis.
Figure- 3- In the bifunctional enzyme, upon phosphorylation by c-AMP mediated cascade, the bisphosphatase enzyme becomes active while PFK-2 becomes inactive, the net concentration of fructose 2,6 bisphosphate decreases, glycolysis is inhibited. Dephosphorylation brings about the opposite effects, PFK-2 activity is increased while bisphosphatase activity is decreased, the net concentration of fructose 2,6 bisphosphate is increased , PFK-1 activity is stimulated and rate of glycolysis is also increased.
Thus, when glucose is abundant as during fed state, glycolysis is stimulated and when glucose is limiting as during fasting or starvation glycolysis is inhibited. These effects are brought about by hormones affecting the concentration of fr 2,6 bisphosphate through action on the bifunctional enzymes fr 2,6 bisphosphatase and PFK-2.
Summary of regulation of glycolysis
|Sr. No.||Features||Hexokinase||Glucokinase||Phosphofructokinase-1||Pyruvate kinase|
|1)||Induction/Repression||Non inducible||Inducible-Induced by insulin, repressed by Glucagon||Inducible-Induced by insulin , repressed by Glucagon||Inducible-Induced by insulin, repressed by Glucagon|
|2)||Effect of substrate||Activity increases in the presence of excess glucose||Active only in the presence of large concentration of glucose due to high Km||Activity increases with the increasing concentration of glucose||Activity increases with the increasing concentration of glucose|
|3)||Feed back inhibition||Inhibited by Glucose-6-P||Not inhibited||Not inhibited unless there is a block at some enzymatic level as in pyruvate kinase deficiency||Inhibited by increasing concentration of pyruvate|
|4)||Allosteric modification||–||–||ATP and Citrate are negative modifier whereas AMP and fructose 2,6 bisphosphate are positive modifiers||ATP, Acetyl coA and Alanine are negative modifiers while AMP and fructose 1,6 bisphosphate are positive modifiers.|
|5)||Covalent modification||–||–||–||Active in the dephosphorylated form while inactive in the Phosphorylated form|
|6)||Role of hormones||Insulin stimulates,Glucagon inhibits||Insulin stimulates,Glucagon inhibits||Insulin stimulates,Glucagon inhibits||Insulin stimulates,Glucagon inhibits|
|7)||Effect of hypoxia||Activity increases||Activity increases||Activity increases||Activity increases|
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