REGULATION OF GLYCOLYSIS REGULATION OF PFK1

REGULATION OF GLYCOLYSIS REGULATION OF PFK1

11.3.      Regulation of PFK1

PFK-1 has a homotetrameric enzyme composed of four identical subunits. Like other allosteric proteins (hemoglobin) and enzymes (ATCase) the binding of allosteric effectors and substrates is communicated to each of the active sites. PFK exist in two state of conformations, called the T and R states. These two conformational states are in equilibrium:

PFK-1 is allosterically inhibited by high levels of ATP and citrate.

AMP is an allosteric activator of PFK-1. AMP reverses the inhibition due to high concentrations of AMP. AMP binds preferentially to the R state of PFK.

AMP in the cell is formed by the enzyme adenylate kinase. Small decrease in the concentration of ATP can drastically increase the level of AMP. The steady state concentration of ATP in the cell is 10 times greater than the concentration of ADP, and 50 times the concentration of AMP. As a result of the activity of adenylate kinase, a 10% decrease in the concentration of ATP results in 400 % increase in the concentration of AMP.

ADP is another allosteric effector of PFK-1. The activity of PFK-1 is dependent on the ATP, ADP and AMP concentrations which are all functions of the cellular energy status.

ATP feedback inhibition

The function of the glycolytic pathway is to generate ATP. ATP is both a substrate and an allosteric inactivator. The enzyme has two binding sites for ATP. One is the substrate binding site and the other one is an inhibitory site. The PFK-1 substrate binding site binds ATP equally well in both the T and R states. The inhibitory ATP binding site only binds ATP when the enzyme is in the T conformation. The other substrate fructose-6-phosphate binds only to the R state.

High concentrations of ATP shift the equilibrium towards the T conformation which decreases the affinity of the enzyme for F-6-P. Fructose-1,6-bisphosphatase is another important site of gluconeogenesis regulation. high concentrations of citrate activates Fructose-1,6-bisphosphatase and high concentrations of AMP  inhibit it.

AMP enhances the inhibition of Fructose-2,6-BP. Allosteric effectors of fructose-1, 6-bisphosphatase and  Phosphofructokinase are common. These effectors reciprocally regulate both enzymes.

This provides a way for gluconeogenesis and glycolysis to be coordinated such that when one pathway is active the other pathway is inactive. If both pathways were active at the same time, the net result would be the net hydrolysis of 2ATPs and 2GTPs per reaction cycle. Both pathways are highly exergonic so there is no thermodynamic barrier to such cycling. The cycle shown above, cycling fructose-6-phosphate and fructose-1,6-bisphosphate is called as a substrate cycle.  The net result of the substrate cycling is the net hydrolysis of ATP.

Fructose-2,6-bisphosphate is an allosteric activator of PFK-1. β-Dfructose 2-6-bisphosphate binds to the R-state of the enzyme and increases the affinity of the enzyme for fructose-6-phosphate.

Fructose-2,6-bisphosphate is synthesized by phosphofructokinase-2 (PFK-2). During hyperglycemic condition the insulin hormones increases the activity of PFK-2. PFK-2 produce Fructose-2,6-bisphosphate. which work as allosteric activator of PFK-1and activates glycolysis. β-D-fructose 2-6-bisphosphate also decreases the inhibitory effects of ATP.

PFK-2 is a bifunctional enzyme that behaves as a phosphatase and kinase. Insulin hormones activates the kinase activity of PFK-2 and glucagon activates the phosphatase activity of PFK-2.  When phosphorylated in response to the hormone glucagon (released into blood in reponse to low blood sugar).

It functions as a  kinase when dephosphorylated in response to the hormone insulin (high blood sugar). Fructose-2,6-bisphosphate, produced via hormone-induced covalent modification of PFK-2, is an indicator of high levels of available glucose and allosterically activates PFK-1. Accumulated fructose-1,6-bisphosphate activates pyruvate kinase, providing a feed-forward mechanism of control (i.e., fructose-1,6-bisphosphate is an allosteric activator).

PFK-2 is, in turn, activated by a dephosphorylation reaction catalyzed by phosphoprotein phosphatase (PPP), an enzyme activated by insulin. Insulin hormone activates phosphoprotein phosphatase enzyme which dephosphorylates the PFK-2. dephosphorylated form of  PFK-2 work as kinase.

Glucagon, released by pancreatic a-cells when blood glucose is low, activates the phosphatase function of PFK-2, thereby reducing the level of fructose-2,6-bisphosphate in the cell.  Glucagon work through GPCR pathways and its effect are mediated by cyclic AMP. cAMP binds to and activates protein kinase A (PKA). PKA then initiates a signal cascade of phosphorylation/dephosphorylation reactions. PKA phosphorylates PFK-2. Phosphorylated PFK-2 work as phosphatase and convert fructose-2,6-bisphosphate into fructose-6-phosphate.

Muscle and adipose tissue has insulin-sensitive glucose transporters. Insulin promotes the translocation of glucose transporters to the cell plasma membrane. Insulin works through RTK pathways and the signal cascade activates a transcription factor SREBP1c, a sterol regulatory element binding protein. This increase the synthesis of glucokinase and pyruvate kinase.

Citrate binds preferentially to the T state of PFK-1. Thus high concentrations of citrate inactivate the enzyme.

11.4.      AMPK: A METABOLIC MASTER SWITCH

AMP-activated protein kinase (AMPK) is an enzyme that plays a central role in energy metabolism. AMP is activated when the cell has high AMP:ATP ratio. AMPK is involved in lipid and glucose metabolism. Once activated phosphorylates target proteins (enzymes and transcription factors). AMPK switches off anabolic pathways (e.g., protein and lipid synthesis) and switches on catabolic pathways (e.g., glycolysis and fatty acid oxidation). AMPK promotes glycolysis in cardiac and skeletal muscle during exercise. It increase the number of glucose transporters to the plasma membrane. In cardiac cells, AMPK stimulates glycolysis by activating PFK-2.

11.5.      Pyruvate kinase

Pyruvate kinase is the third regulated enzyme of glycolysis. Pyruvate kinase  is regulated by various allosteric effectors like AMP, ATP and Acetyl –CoA. A high AMP concentration (an indicator of low energy production) activates pyruvate kinase. In contrast, pyruvate kinase is inhibited by a high ATP concentration (an indicator that the cell’s energy requirements are being met). Acetyl-CoA, which accumulates when ATP is in rich supply, inhibits pyruvate kinase.

F-1,6-BP activates the  Pyruvate kinase  in the liver, a second example of feed forward stimulation. Alanine (a biosynthetic product of pyruvate) is an allosteric inhibitor of pyruvate kinase. Phosphorylation of pyruvate kinase is regulated by blood glucose level, just like PFK. High glucagon (low blood sugar) causes phosphorylation, which in this case renders the enzyme inactive.

11.6.      Hemolytic Anemia

Pyruvate kinase deficiency causes hemolytic anemia, since red blood cells depend entirely on glycolysis for ATP synthesis. Pyruvate kinase deficiency causes accumulation of 1,3 BPG and PEP. The concentration  of pyruvate and lactate are lower than normal in this disorder.

Acetyl CoA is an allosteric effector of both glycolysis and gluconeogenesis. Acetyl-CoA inhibits pyruvate kinase and reciprocally activates pyruvate carboxylase. Acetyl CoA also inactivates pyruvate dehydrogenase providing a regulatory link between glycolysis and the citric acid cycle. High concentrations of acetyl-CoA are indicative of high energy supplies. When the energy supply is high, metabolites are directed towards storage in the form of glycogen.


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