Enolase

Enolase is an enzyme, more particularly, an active metalloenzyme. This enzyme belongs to the family of lyases, the hydro-lyases, breaking the carbon-oxygen bonds, and is present in all tissues and organisms involved in glycolysis or fermentation. The optimal pH of this enzyme is 6.5 in humans.Its main function is to intervene in the 9th step of glycolysis (the penultimate step in this metabolic pathway), a step in which occurs the dehydration of 2-phosphoglycerate (2-PG) in phosphoenolpyruvate (PEP), a product that will be used in the next and final step for the production of energy (ATP).
Enolase has three different isoforms: the ENO1 or alpha-enolase (in muscle tissue); ENO2 or gamma-enolase or neuro specific enolase (in neurons); ENO3 or beta-enolase (in skeletal muscle cells). Enolase has a molecular weight of about 100000 Daltons (depending on the isoform). In humans, the α-enolase has two antiparallel subunits, which have two domains that establish hydrophobic interactions. The subunits interact via salt bridges, involving arginine and glutamate.
The specific enolase to neurons is released in a wide variety of diseases, such as multiple sclerosis or stroke, or myocardial infarction.
In several medical experiments, it was employed enolase concentrations in samples in an attempt to diagnose certain conditions and its severity. Several studies demonstrated that different levels of enolase may also be associated with tumor growth or with the occurrence of myocardial infarction or stroke, so it was inferred that the levels of enolase serve as an indicative of the prognostic evaluation of victims of cardiac arrest.
Enolase inhibitors have been utilized in health care for the treatment and prevention various diseases, such as anti-trypanosome drugs and more recently as anticancer agents. Enolase can be inhibited by fluoride ion (F-). The fluoride forms a complex with magnesium and phosphate, which binds to the active center of the enzyme rather than the substrate 2-PG, preventing the conversion of 2-PG into PEP, decreasing the production of PEP and, consequently, ATP.
Intake of fluoride-containing water inhibits the catalytic activity of enolase of bacteria present in oral cavity (highly dependent on glycolysis due to the anaerobic environment), interrupting glycolysis and, thus, bacterial fermentation (decreased acid production), preventing the formation of dental caries.

Text written by:
Inês Carvalho
Junjie Lin
Maria Alves
Susana Pinto
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Regulation of pyruvate oxidation (part 2)

The pyruvate dehydrogenase complex activity is regulated mainly by two distinct mechanisms – allosteric mechanisms and reversible covalent modification.

In fact, there are some allosteric modulators of the complex, which in this particular case belong to the class of negative modulators, ie, inhibitors of its catalytic activity:

- Acetyl-CoA – it is the product of the reaction, thus, it makes sense that the molecule of acetyl-CoA has an inhibitory effect on its own synthesis

- NADH – one of the products of the reaction is NADH, so the reasoning is equivalent to that carried over to the acetyl-CoA molecule. Furthermore, as already mentioned in other posts of this blog, NADH may be involved in ATP synthesis (in cellular respiration), so, its presence indicates a potential for a high energy state into the cell. Accordingly, and as the oxidation of pyruvate to acetyl-CoA is part of catabolism, whose main objective is to obtain energy, it makes sense that NADH inhibits catabolism and, in particular, this reaction.

Regarding the reversible covalent modification, this enzyme complex is inhibited by phosphorylation and activated by dephosphorylation. This process is mediated by two different enzymes… The one that phosphorylates is called pyruvate dehydrogenase kinase, whereas the one that dephosphorylates it is the pyruvate dehydrogenase phosphatase.

Factors that activate the kinase, leading to the phosphorylation of pyruvate dehydrogenase complex (ie, inhibitors of its catalytic activity):

- Acetyl-CoA and NADH – in addition to its direct effects on the pyruvate dehydrogenase complex, as allosteric inhibitors, these two molecules also trigger the phosphorylation of the complex, promoting its inhibition, which means that they can act, therefore, through two distinct mechanisms

Factors that inhibit the kinase, favoring the balance towards the dephosphorylated form of pyruvate dehydrogenase complex (ie, activating its catalytic activity):

- NAD⁺ – for this molecule it can be done the reverse rationale made for NADH. That is, the presence of NAD⁺ indicates an energy deficit on the cell, so it is needed to activate the catabolism to counteract this deficit.

- ADP – the reasoning is equivalent to the one mentioned above, as to say that the cell is accumulating ADP means that it is spending ATP. Thus, it will need to produce again ATP

- Pyruvate – pyruvate is the substrate of the reaction, and its presence will activate the pyruvate dehydrogenase complex by inhibiting the phosphorylation process (and thus its inhibition) of the pyruvate dehydrogenase complex

- Coenzyme A (CoA) – this cofactor plays a co-substrate role, so that its presence will affect catalytic activity of the complex in a similar manner to that described for pyruvate

Factors that activate the phosphatase, leading to dephosphorylation of pyruvate dehydrogenase complex (ie, activating its catalytic activity):

- Ca²⁺ - calcium ion is an important modulator of the metabolism. In this particular case, this ion acts (in the muscle) at the level of pyruvate dehydrogenase phosphatase, activating it (by promoting its dephosphorylation). Put simply, the calcium ion is an indicator of muscle contraction, so it makes perfect sense that in the context of working muscles, the catabolism becomes active, so that there is ATP available for the process

Regulation of pyruvate oxidation (part 1)

The pyruvate dehydrogenase complex is mainly regulated by two distinct mechanisms: allosteric regulation and reversible covalent modification. Both can act (and act indeed!) at the same time, and there are molecules (activators and inhibitors) involved in both processes simultaneously.

Activators of the pyruvate dehydrogenase complex

- AMP and ADP – AMP and ADP are two molecules that are obtained when ATP is used as a source of chemical energy (ATP can be cleaved either to ADP or AMP). Therefore, both molecules indicate a low energy state, so it makes sense that they function as activators of processes that allow for energy, the catabolic processes. Since the oxidation of pyruvate is part of the catabolism, this process is activated by AMP and ADP.
- CoA – this is one of the cofactors of the enzyme that appears included in the products (pyruvate is both decarboxylated, oxidized and combined with CoA). Thus, as it is one of the molecules that will react with the substrate, its presence activates the enzyme.
- NAD+ – like the molecule of CoA, NAD+ is also used in the reaction, where it appears in the products (in the form of NADH). Furthermore, since the NADH can be used to promote ATP synthesis (in the cellular respiration), where it is oxidized to NAD+, the latter is an indicator of a low energy state in the cell. For all of this, it makes sense that this molecule is an activator of pyruvate oxidation.
- Ca2+ (muscle) – the calcium ion is an important mediator of various cellular responses. One of them is muscle contraction. Therefore, as this ion is an indicator of muscle contraction, which is an ATP-consuming process, it is advantageous for muscle cell that it can be simultaneously used as an activator of catabolism and, in particular of the oxidation of pyruvate. Thus, with the same messenger, muscle performs contraction and activates catabolism.
- Pyruvate – pyruvate is the substrate of the pyruvate dehydrogenase complex, so it makes sense that it can act as an activator.
- Dephosphorylation – complex pyruvate dehydrogenase is active in its dephosphorylated form.

Inhibitors of pyruvate dehydrogenase complex

- ATP – the main goal of catabolism is to produce energy, mainly in the form of ATP. If the cell has already ATP, or NADH (which, as mentioned above, can lead to the production of ATP), catabolism is inhibited.
- Acetyl-CoA – this is the product of the reaction, so it is natural that it has an inhibitory role in the process.
- Long chain fatty acids - some fatty acids, particularly those with long chains, can act as inhibitors of this reaction.
- Phosphorylation - pyruvate dehydrogenase complex is inactivated by reversible phosphorylation.
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Oxidation of pyruvate

Under aerobic conditions, most eukaryotic cells and various bacteria oxidize the pyruvate, produced in glycolysis, to CO2 and H2O, instead of reducing it to lactate or ethanol.
 Production of acetyl-CoA from pyruvateThe molecules of acetyl-CoA are the form in which the Krebs cycle accepts most of its fuel. Pyruvate is oxidized to acetyl-CoA and CO2 by an enzyme complex (3 enzymes) called pyruvate dehydrogenase complex. This complex is located in the mitochondria (eukaryotes) or cytosol (prokaryotes).
 The oxidation of pyruvate to acetyl-CoA is an example of an irreversible oxidative decarboxylation. The irreversibility of the reaction was demonstrated by proving that when radiolabelled CO2 was used, pyruvate  with radioactive carbon was not be obtained.
In addition to acetyl-CoA and CO2, this reaction produces a molecule of NADH from NAD+.The pyruvate dehydrogenase complex requires the action of five cofactors: thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), coenzyme A (CoA), nicotinamide adenine dinucleotide (NAD +) and lipoate. 4 necessary vitamins in human nutrition are vital components of this system: thiamine (for TPP), riboflavin (for FAD), niacin (to NAD) and pantothenate (for CoA).

The enzymes that compose the pyruvate dehydrogenase complex are pyruvate dehydrogenase, and dihydrolipoyl transacetilase dihydrolipoyl dehydrogenase. Each of these enzymes is present in multiple copies.
 Animals deprived of thiamine are usually unable to oxidize pyruvate. This has implications especially in the brain that usually gets all its energy from glucose oxidation, a process that necessarily involves the oxidation of pyruvate. Beriberi is a vitamin deficiency caused by thiamine deficiency. It is characterized by a loss of neuronal function. This disease is more common in populations that eat predominantly white rice (polished), because it is in rice husks that most of its thiamine is found.

Mutations in genes that encode subunits of this enzyme complex, as well as a diet deficient in thiamine can have serious consequences.










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

Alcoholic fermentation

The first reaction requires the presence of Mg2 + and the second reaction regenerates NAD+ from NADH (one molecule per molecule of pyruvate). As I mentioned in the post of lactic fermentation, this is the aim of fermentation, the regeneration of NAD+ so that glycolysis can continue to occur.
The pyruvate decarboxylase enzyme is normally present in yeast used in the manufacture of beverages and bread. The gasification of champagne and other alcoholic beverages, as well as the bubbles present in the bread crumbs are originated by the decarboxylation of pyruvate.

The alcoholic fermentation does not occur in our bodies. Bear in mind that when I say this, I am not saying that we can not metabolize ethanol, as this is an independent process of fermentation. What I am saying is that it is impossible for our body to convert glucose into ethanol.

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

Lactic fermentation

This reaction is clearly favored in the forward direction. In situations of hypoxia (intense muscular effort, for example), or absence of mitochondria, the cell is unable to regenerate NAD+ from NADH through the respiratory chain. It does so using the conversion of pyruvate to lactate, which consumes NADH and releases NAD+ release. It should be noted that the NAD+ is essential for glycolysis to continue to occur, thereby to obtain energy through the catabolism of sugars. Each molecule of pyruvate converted to lactate regenerates a molecule of NAD+. The lactate formed is sent through the bloodstream to the liver where it is converted to glucose in gluconeogenesis. The question that arises is: "So if you can recycle lactate, converting it back into glucose, why is the liver that has to do this and not the muscle, since it is the muscle that produces lactate? If so, the muscle could use directly the product of fermentation to restore the levels of metabolic fuel." In fact, at a first glance it may make sense to think in this way. However, the synthesis of glucose through gluconeogenesis is very expensive, in terms of energy, so that after an intense physical effort, it did not make sense that the muscle has to spend additional energy to synthesize glucose. Thus, the recovery of an intense effort includes not only the restoration of ATP levels in muscle but also an extra consumption of oxygen in the liver, necessary for the synthesis of ATP to be used in gluconeogenesis from lactate. In other words, after muscular efforts, is the liver that has to use lactate, allowing a faster and more efficient muscle recovery. This process is called the Cori cycle.

Dring anaerobic work the concentration of lactate in the muscle fibers can increase about 30 times and it is a commonplace to say that it is this accumulation of lactate ion which causes fatigue. However, the experimental evidence shows that although the concentration of lactate is directly related to the degree of fatigue it does not interfere with the the muscle contractile activity. Fatigue, muscle pain and cramping experienced after an intense physical effort are the result of an acidification caused by lactic acid in muscle (the pH can drop from 7 to 6.5 !!!). The pKa of lactic acid is about 4, which causes that at the cell pH (≈ 7) or plasma (≈ 7.4) occurs the dissociation of lactic acid to lactate + H+. This accumulation of H+ will interfere with the contractile capacity of muscle fibers and will also invade the synaptic cleft. Thus, the inability of the neuromuscular junction in relaying the nerve impulses to muscle fibers is due probably to a lower release of the chemical transmitter acetylcholine by nerve endings, due to acidification of the interstitial fluid and alteration of protein structures (acetylcholine receptors) by the action of H+. This system provides energy for physical activities that result in fatigue after about 60-120 seconds. It is therefore the primary metabolic process associated with activities such as running up to 400-800 m, swimming events of 100-200m, and also provides energy for high intensity moments in football, basketball, volleyball, tennis, among others. The common denominator of these activities is the support of high-intensity efforts lasting 1-2 minutes. Even the best trained athletes are unable to sprint for more than a minute. A highly competitive athlete needs about 30 minutes to recover from a 100m sprint. Some lactobacilli and streptococcus ferment lactose to lactic acid in milk. The ionization of lactic acid lowers the pH and causes denaturation of the casein (main milk protein) and other milk proteins. When this denaturation is controlled, and occurs in the right conditions, you get the yogurt or cheese.

 

In short, the fermentation is not used to get energy under anaerobic conditions (this misconception is very common ...). It serves to regenerate NAD+ so that glycolysis can continue to occur in the absence of O2, as glycolysis is the process that will produce ATP!

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

Fates of pyruvate

The fate of pyruvate depends on cell type and metabolic conditions. There are three main destinations for pyruvate:

(1) aerobic organisms and tissues, under aerobic conditions - pyruvate is oxidized, with loss of the carboxylic group, resulting in the acetyl group from acetyl-CoA, which is then oxidized to CO₂ in the Krebs cycle;

(2) Aerobic tissues in conditions of low oxygen (muscle hypoxia, for example), some tissues under aerobic conditions (red cells, for example, because they lack mitochondria), or some anaerobic organisms - pyruvate is reduced to lactate by lactic fermentation . In muscle under conditions of hypoxia, NADH is not reoxidized to NAD⁺ and NAD⁺ is required for glycolysis. The reduction of pyruvate to lactate allows the use of as a donor of electrons to regenerate NAD⁺;

(3) Some tissues of plants, some invertebrates, protists and micro-organisms under anaerobic conditions or hypoxia - pyruvate is converted to ethanol + CO₂ (alcoholic fermentation).

While glycolysis can occur in anaerobic conditions, this fact has a price, because it reduces the amount of ATP formed per molecule of glucose (from 30 or 32 it passes for only 2 ATP!), and, therefore, it is needed more glucose oxidation under these conditions.

What happens to pyruvate is directly related to the amount of NAD⁺ and FAD in the cell. As these amounts are very small, there must be mechanisms to transform the NADH + H⁺ and FADH₂ back into NAD⁺ and FAD, respectively. This is done by transfer of the electrons from NADH + H⁺ and FADH₂ to other molecules, which can occur by fermentation or respiration. The distinction between these is not (contrary to what is generally thought) that one of the processes uses directly the O₂ and the other not! O₂ is only required for oxidative phosphorylation, and not for the oxidation of pyruvate. Unlike aerobic metabolism that depends and are limited by the oxygen supply, the anaerobic glycolysis does not depend on the availability of oxygen and can increase speed up to 1000 times the speed at rest, ie, 2 ATP / glucose can represent many ATP/minute.

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

Utilization of different monosaccharides on glycolysis

There is nothing new in the fact that glycolysis serves to degrade glucose, the most abundant monosaccharide in our diet. But what happens to other monosaccharides we eat? Can we degrade them for energy? If so, can we rely on glycolysis for this?
The answer to both questions is yes. Indeed, glycolysis allows the degradation not only of glucose but also of other monosaccharides such as fructose, galactose and mannose. For that, these monosaccharides have to be converted into glycolytic intermediates.
FructoseFructose is obtained from the diet in foods such as fruits, honey and cereals, for example.

 

In extrahepatic tissues, which lack the enzyme glucocinase, fructose is converted to fructose-6-phosphate by hexokinase, the first enzyme of glycolysis. It should be noted that the hexokinase uses glucose as preferred substrate, but can also phosphorylate other monosaccharides such as fructose, for example. Since fructose-6-phosphate is an intermediate of glycolysis, it can then follow the normal glycolytic pathway.

Alternatively, in liver, fructose has to undergo a different process, because the glucocinase is very specific for glucose, failing therefore to phosphorylate fructose. In this case, fructose is phosphorylated by frutocinase, producing fructose-1-phosphate. Thereafter, the aldolase, which is the 4^th enzyme of glycolysis, acts on fructose-1-phosphate, cleaving it to dihydroxyacetone phosphate and glyceraldehyde. The former molecule is already an intermediate of glycolysis, so it can proceed in that pathway, but not the latter. Therefore, the glyceraldehyde is subsequently phosphorylated by glyceraldehyde kinase, leading to glyceraldehyde-3-phosphate, which is also a glycolytic intermediate.
GalactoseGalactose is found mainly in dairy products because it is a component of lactose.

  

 

Galactose is first phosphorylated to galactose-1-phosphate by galactocinase. Then galactose-1-phosphate will receive an UDP molecule, resulting in UDP-galactose. This reaction is catalyzed by galactose-1-phosphate uridyl transferase. UDP-galactose is then epimerized to UDP-glucose, by the action of UDP-galactose-4-epimerase. The UDP-glucose undergoes an exchange of its UDP portion by a phosphoryl group, producing glucose-1-phosphate, which is then converted to glucose-6-phosphate (glycolytic intermediate) by the action of phosphoglucomutase (this enzyme also participates in the metabolism of glycogen...).

Mannose
Mannose can be found, for example, in beans, peas, or similar vegetables.

Mannose is phosphorylated by hexokinase originating mannose-6-phosphate. The mannose-6-phosphate is converted to fructose-6-phosphate (a glycolytic intermediate) by fosfomanose isomerase.

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

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