Regulation of Glycolysis.

Regulation of Glycolysis

In this answer to the question I will look at how the pathway of glycolysis is regulated in the cells, and how the enzymes involved respond to changes in conditions both within the cell and in the surrounding fluids.

Before I talk about the regulation of this pathway however, I will briefly describe what glycolysis is. For a more detailed discussion of this, see the notes.

Glycolysis is the first step in the respiration of glucose. It can occur under aerobic or anaerobic conditions but yields different products in each of these. It occurs in the cytoplasm of cells as it does not require any specific organelles such as mitochondria. The overall reaction scheme is shown here:

In the reaction, ATP is produced (net gain of 2 ATP molecules for 1 glucose molecule). This is one of the reasons why the pathway must be regulated. The body’s demand for energy changes depending on external conditions such as temperature and how much exercise the organism is doing. The cell needs to have a way to increase the rate of glycolysis when ATP is in demand and decrease it again as the demand diminishes. This is important in order to avoid wasting the energy we take in as food. ATP is not a long term energy store, so if we do not require immediate energy, it is more beneficial to store the glucose as something else such as glycogen rather than metabolise it immediately.

The pyruvate created in the reaction can also be used to synthesise biological molecules, hence the abundance of these molecules in the cell also has an effect on the rate of glycolysis.

In most metabolic pathways, the reactions are controlled by enzymes that catalyse non-reversible reactions. In glycolysis, these are Hexokinase (HK), Phosphofructokinase (PFK) and Pyruvate kinase (PK). Indeed, each of these does play a role in control, however it is PFK that has the biggest role, and it is this that I shall concentrate on the most.

This is a preview of the whole essay

The reason PFK is the main regulatory step in the pathway, is because it is the first committed step, that is, the first step where an irreversible reaction gives rise to a unique intermediate that is not used in other pathways. For example, HK also catalyses a non-reversible reaction, but the product of this reaction (Glucose-6-Phosphate) is also used to make glycogen and NADPH. The product of the PFK reaction, Fructose-1,6-bisphosphate, on the other hand is an intermediate used only in glycolysis.

The rate of enzyme controlled reactions can be controlled in two ways. Either the enzyme can be inhibited in some way. This is a short term effect that can alter the rate almost within a matter of seconds. Alternatively, the rate of translation of the enzyme can be altered. This will change the concentration of the enzyme, hence altering the rate of reaction. Since this involves many stages: changing rate of transcription followed by translation, it is a more long term effect that can take several hours to have any effect. For the rest of this question, I will concentrate of the former method I described.

PFK is inhibited allosterically by ATP. When an ATP molecule binds to one of the 4 allosteric sites, the graph of rate against substrate concentration changes from a hyperbolic shape to a sigmoidal one:

AMP reverses this process, increasing the efficiency of the enzyme. There are two main reasons why AMP is chosen to do this rather than the more obvious choice of ADP:

1) under extreme conditions, the following reaction occurs:

2ADP → AMP + ATP

2) as the concentration of AMP in the cell is always lower than that of ATP, it can be used as a sensitive control mechanism. This is because a small % change in [ATP] will be magnified into a larger % change of [AMP].

A drop in pH will also inhibit PFK as this can prevent a build up of lactate which could eventually lead to a drop in blood pH and acidosis. When the [H+] starts to build up, the rate of the PFK reaction therefore decreases, halting the glycolytic pathway and preventing the pyruvate from building up.

PFK is also inhibited by citrate. This works by increasing the inhibition power of ATP. A build up of citrate in the cell normally means that biosynthetic precursors are abundant because citrate is one of the first compounds in the citric acid cycle, and therefore no more pyruvate molecules are needed for structural purposes.

An important chemical in the regulation of glycolysis is Fructose-2,6-bisphosphate (F-2,6-BP). This molecule activates PFK allosterically by diminishing the effect of ATP in a similar way to H+, and also by increasing the affinity the enzyme has for the substrate (Fructose-6-phosphate).

The levels of F-2,6-BP are controlled by a single bi-functional protein with three separate domains. One of these is a regulatory region, one is the PFK2 enzyme and the third is the FBPase2 enzyme. This comes in M and L isozymes, the M being more prominent in muscles and the L form in the liver. In the liver, this protein also helps to regulate glucose levels.

When the blood glucose level is low, glucagons is released into the blood. This causes a cyclic AMP cascade event which in turn causes phosphorylation of a serine residue by protein kinase A. This activates FBPase2 which lowers the concentration of F-2,6-BP by hydrolysing one of the phosphate groups. This stops glucose from being used in glycolysis (by deactivating PFK) and hence leaves glucose in the blood in times of stress.

On the other hand, when levels of glucose are high, this residue is not phosphorylated so PFK2 is activated so F-6-P is phosphorylated into F-2,6-BP, increasing the rate of glucose metabolism by glycolysis which helps to lower the levels of glucose in the blood.

Other regulatory enzymes are involved in glycolysis as I mentioned earlier. Hexokinase is inhibited by G-6-P which builds up when PFK in inhibited. This prevents glucose being converted into unusable G-6-P and leaves it in the blood for other cells to use. However, in the liver, an isozyme of hexokinase exists (Glucokinase). This has 50 times less affinity for glucose than HK and is NOT inhibited by G-6-P. This allows the liver to build up G-6-P which it then uses for glycogen production in times of glucose surplus. Also, because glucokinase has such a smaller affinity for glucose than HK, when blood glucose levels are scarce, the liver cells do not take up glucose, instead they leave what little glucose there is for brain and other cells.

Pyruvate kinase is the final enzyme involved in the glycolytic pathway and is the other enzyme that is involved in the control of the pathway. It also comes in L and M forms in a similar way to the FBPase2 enzyme. This enzyme controls the out-flow of pyruvate because it is the last enzyme involved. ATP inhibits PK allosterically (as well as inhibiting PFK). This stops excess pyruvate being formed, instead allows the intermediates to build up to an extent, although even this is prevented by the HK regulation described earlier. Fructose-1,6-bisphosphate (the product of the previous irreversible step) activates PK to prepare it for the influx of intermediates it would then be about to receive after the activation of PFK.

Alanine can be formed in one step from pyruvate. Alanine also inhibits PK, signalling an abundance of building blocks – as opposed to the ATP which signals an abundance of energy charge.

When glucose levels are low, only the L form is phosphorylated by AMP cascade events, inhibiting it allowing muscle and brain cells to use the glucose first

In summary, glycolysis is a finely tuned pathway of reactions that contains essentially three control points. The main events that affect the rate of glycolysis are: