Question

Essay Question: Describe the allosteric regulation of phosphfructokinase during glycolysis. Please be very descriptive.

Answer 1

The enzyme Phosphofructokinase-1(PFK-1) catalyzes the transfer of the phosphoryl group from ATP to fructose-6-Phosphate and yeild Fructose-1-6-bis phosphate. This reaction is irreversible under normal cellular conditions and is the first 'committed' step in glycolysis. The reaction goes as follows-

  

( The reaction occurs in presense of Mg²⁺ ion).

Now, Glucose-6-Phosphate which is an isomer of Fructose-phosphate, can flow either into glycolysis or some other pathways such as pentose phosphate pathway or glycogenesis. PFK-1 catalyzes the reaction that commits the glucose into glycolytic pathway. PFK is a complex tetrameric enzyme. in case of mammals it is composed of three different subunits( M, L,P) in different combinations in different tissue types. For example, in muscles PFK-1 is composed of four M subunits becoming a homotetramer whereas in erythrocytes it may exists in M2L2. ML3, M3L combinations alongwith M4 and L4 homotetramers.

In addition to the substrate-binding site this complex enzyme has several allosteric regulatory sites where allosteric activators or inhibitors can bind.

Different allosteric effectors of PFK-1 and their regulation mechanisms-

1.ATP, ADP/AMP

ATP is not only a substrate for PFK-1 but also an end product of glycolysis. So at presence of high cellular ATP level activity of PFK-1 is decreased. High level of ATP signals that ATP is being produced in the cells than it is being consumed. ATP binds to one allosteric site of PFK-1 and lowers its affinity towards another substrate fructose-6-phosphate.

When ADP and AMP concentration gets high in the cell, it signals that the consumption of ATP has outpaced the formation of the same. They act allosterically to relieve this inhibition of PFK-1by ATP. Thus higher enzyme activity is produced when ADP and AMP concentration is high and ATP is low.

2. Citrates-

Citrate which is an important intermediate substance of several pathways such as aerobic oxidation of pyruvate, amino acids and fatty acids, is an allosteric regulator of PFK-1. High concentration of citrate in the cell increases the inhibitory effects of ATP. As a result glucose cannot commit to the glycolytic pathway and flows into other metabolic pathways.

In this case citrate serves as an intercellular signal the tissue is meeting its needs by energy-yielding pathways such as beta oxidation of fatty acids.

To elaborate further on the subject of allosteric regulation of the enzyme we have to acknowledge another pathway- Gluconeogenesis that takes place simultaneously in the cells by which glucose is synthesized and transported to the tissues where the glycogen storage has been exhausted and no glucose is available. the reactions in gluconeogenesis takes place in an opposite fashion with that of glycolysis.

The corresponding step to glycolytic reaction written above is the convertion of Fructose-1-6-bisphosphate to fructose-6-phosphate by an enzyme named FBPase-1. PFK-1 and FBPase-1 are regulated in a coordinated reciprocal manner. When sufficient concentration of citrate and acetyl-CoA or a high proportion of cell's adenylates are present in the form of ATP, gluconeogenesis is favored. When AMP level increases it promotes glycolysis by stimulating PFK-1..

3.The hormonal regulation of glycolysis and gluconeogenesis is mediated by Fructose 2,6- bisphosphate, an allosteric effector for the enzymes PFK-1 and FBPase-1. When fructose-2,6-bisphosphate binds to the allosteric site on PFK-1. it increases the affinity for its substrate fructose-6-phosphate and reduces its affinity for the allosteric inhibitors like citrate and ATP. The cellular concentration of Fructose 2,6-bisphosphate is set by the relative rates of its formation and breakdown. It is formed from Fructose 6 -Phosphate by Phosphofructokinase-2 (PFK-2) and broken down by Fructose 2-6- bisphosphatase (FBPase-2).

PFK is also inhibited by glucagon which increases theadenyl cyclase activity in liver thus concentration of cAMP increases. cAMP then activates cAMP-dependent protein kinase which phosphorylates bifunctional protein PFK-2/FBPase-2 enhancing FBPase -2 activity and decreases PFK-2 activity. Thus glucagon inhibits glycolysis and stimulates gluconeogenesis.

Serotonin(5-HT) increases PFK activity by binding it which causes tyrosine residue of PFK to be phosphorylated via phospholypase C.