Monday, July 18, 2011

Regulation of glycolysis

Glycolysis provides different cell molecules important for cellular proper functioning. The main donor of chemical energy in most of our processes is ATP. However, it also provides precursors for many other processes such as synthesis of amino acids, for example. Thus, it is essential to have a strict regulation of glycolysis, so that the cell can respond to different needs of ATP or other metabolites.

During his studies on fermentation of glucose by yeast, Louis Pasteur discovered that the rate and amount of glucose consumed was higher in anaerobic than in aerobic conditions! At first glance this may seem strange, because aerobic metabolism is normally associated with something more advantageous for the cell. In fact, the biochemical explanation is simple: under anaerobic conditions one molecule of glucose generates 2 molecules of ATP, but under aerobic conditions generates 30 or 32 molecules of ATP. Simplifying, if one thinks that in five minutes the yeast will need to get 30 ATP, this means that under aerobic conditions it will only need to spend a molecule of glucose in that time, while under anaerobic conditions, as the process is less profitable in the energy point of view, it is necessary to spend 15 molecules of glucose. That is, the flow of glucose through the glycolytic pathway is regulated depending on cell ATP levels (as well as adequate supplies of glycolytic intermediates to biosynthetic roles).

As I have mentioned in previous posts, glycolysis has 10 reactions, and there are three regulatory points (irreversible reactions). The enzymes that catalyze them are the hexokinase (reaction 1), PFK-1 (reaction 3) and pyruvate kinase (reaction 10). As these reactions are the limiting steps of glycolysis, changes in speed of action of their enzymes will alter the overall speed of the glycolytic pathway.

Of the three regulatory enzymes, the main one is PFK-1. This may seem strange, because indeed the most logical situation was that the main regulatory enzyme was the first ... Again, there is a very simple explanation for this. What is happening is that hexokinase is an enzyme also common to other metabolic processes (synthesis of glycogen and pentose phosphate pathway). In other words, despite being a regulatory enzyme, it is not unique to glycolysis. Thus, the main point of regulation of glycolysis has to be the second, ie, the reaction catalyzed by PFK-1.

One thing I usually tell to my students is that in this part of metabolic regulation is preferable to understand why certain molecules activate and inhibit some pathways, thus avoiding to memorize endless lists of modulators... Of course it is not always possible to make a direct argument for understand why some molecules act as activators and others as inhibitors, but whenever possible I will develop this idea... Let's move on to a list of the main modulators of glycolysis.
Activators of hexokinase:
- Fructose-1-phosphate (liver) – It competes with fructose-6-phosphate to the regulatory protein of glucocinase, canceling its inhibitory effect.
- Inorganic phosphate (Pi) – It is a player in the glycolytic process (involved in reaction 6) so it makes sense that if it has a regulatory role, is a stimulating one.
Inhibitors of hexokinase:
- Glucose-6-phosphate (muscle) - It makes sense that functions as an inhibitor because it is the reaction product. If we have too much product, we will not need to continue to produce more ...
- Fructose-6-phosphate (liver) – It is the product of the following reaction (reaction 2), but can be interpreted the same way as the molecule before. That is, if we are to accumulate the intermediate formed from the reaction product, there is no point in continuing to make more product. This inhibition occurs through a protein called regulator protein of glucocinase.

Activators of PFK-1:
- Fructose-2,6-bisphosphate (liver) – It is the most significant allosteric regulator of PFK-1, reducing its affinity for the inhibitors ATP and citrate. It is produced in response to insulin and degraded in response to glucagon.
- Fructose-6-phosphate – It is the substrate, so it makes sense that if we have much substrate the enzyme is activated.
- ADP and AMP – They are produced when ATP is spent, thus indicating a low energy state. Therefore, it makes perfect sense that they can activate glycolysis, so that the cell can replenish their normal energy values. They activate the enzyme because they relieve the inhibition caused by ATP.
Inhibitors of PFK-1:
- Glucagon (liver) - This hormone is produced in a state of hypoglycemia and aims to raise the concentration of glucose in the blood. So it makes perfect sense that it inhibits glycolysis, because this process consumes glucose, which will further accentuate the reduced blood glucose concentration. As mentioned earlier, the glucagon decreases the levels of fructose-2,6-bisphosphate
- ATP - The main objective of glycolysis is to produce energy (ATP). So if the cell already has ATP, it makes sense that glycolysis is inhibited, thus preventing an unnecessary waste of a precious metabolic fuel as glucose! ATP inhibits PFK-1 because it decreases the affinity of the enzyme for its substrate, fructose-6-phosphate.
- Citrate – It stresses the inhibitory effect of ATP. This molecule is the first intermediate of the following step of aerobic catabolism, the Krebs cycle. So if we are accumulating Krebs cycle intermediates, it is useless to continue to perform glycolysis.
- Phosphoenolpyruvate – It is an intermediate of glycolysis that is formed in the penultimate reaction. If there is an accumulation of this intermediate, the reactions above have to be inhibited in order to prevent a further accumulation of the molecule.
- H+ - This enzyme is particularly sensitive to changes in pH, functioning as a "switch" that turns off, for example, when we make an exaggerated lactic fermentation (produces H+), preventing an even greater acidification.

Activators of pyruvate kinase:
- ADP - The reason is the same as mentioned above for the PFK-1, ie, is an indicator of an energy deficit, so it will lead to an activation of glycolysis.
- Fructose 1,6-bisphosphate - an intermediate of glycolysis that is formed in a reaction prior to the one catalyzed by pyruvate kinase. So if we are accumulating an intermediate produced in an earlier stage, we have to activate this enzyme in order to counteract this accumulation (as when a dam is accumulating too much water, and to restore normal values
​​is necessary to open the gate ...).
- Dephosphorylation (liver) - Induced, for example, by insulin, which makes sense, given that insulin is produced in a situation of excess blood sugar (hyperglycemia) and will activate the process (one of them is glycolysis!) that consume glucose in order to lower the blood glucose concentration.
Inhibitors of pyruvate kinase:
- ATP – It is a carrier of chemical energy and one of the end products of glycolysis, so there is no need to continue the breakdown of glucose. It decreases the affinity of the enzyme for phosphoenolpyruvate.
- Acetyl-CoA – It is the molecule in which the product of this reaction (pyruvate) is converted in the case of aerobic catabolism. Therefore, if acetyl-CoA accumulates, it makes no sense to continue to synthesize pyruvate, so the enzyme is inhibited.
- Long-chain fatty acids.
- Phosphorylation (liver) - Induced, for example, by the action of glucagon, which, as mentioned earlier, will have as main function to raise blood glucose levels. To this end, it inhibits, for example, glycolysis.
- NADH - as we shall see in more detail when I speak of cellular respiration, NADH has potential to create molecules of ATP, which signals a high energy state of the cell. In this situation, it is not necessary to resort to glycolysis for more energy.
- Alanine - This amino acid (one of the 20 standard amino acids) can lead to pyruvate (the reaction product of pyruvate kinase!), by removal of its amino group. So if there is a molecule that can directly lead to pyruvate, we do not need to spend more glucose.

In short, we can make some generalizations about the regulation of metabolic pathways, which will be useful to understand the regulation of other processes.
First, energy molecules such as ATP, or potential energy, such as NADH are, in general, inhibitors of catabolism. This is very easy to understand if we think that the main objective of the catabolism is to produce energy. If the cell already has this ability it does not need to degrade more nutrients to produce energy! The oppposite reasoning applies to ADP, AMP and NAD+, because any one of these molecules indicates an energy deficit in the cell (remember that when we spend ATP we obtain ADP or AMP, and when we spend NADH we obtain NAD+...) so it will be necessary to restore energy levels, and this activates the catabolism.
Second, the reaction product or intermediates formed from this (products of reactions following the reaction we are considering) are inhibitors. On the other hand, the substrate, or intermediaries which will originate the substrate (formed in reactions prior to the reaction that we are considering) are activators.

Main bibliographic sources:
- Quintas A, Freire AP, Halpern MJ, Bioquímica - Organização Molecular da Vida, Lidel
- Nelson DL, Cox MM, Lehninger - Principles of Biochemistry, WH Freeman Publishers

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