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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Video about pyruvate

Here it is another musical video, this time about pyruvate.

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

Video on the oxidation of pyruvate and Krebs cycle

Here is a video about what happens to pyruvate in the mitochondria, where it is used as an energy source. It is a very interesting video, with enough detail at the level of biochemical reactions.

Fates of pyruvate (main aspects)

Lactic fermentation

- Main goal: to regenerate NAD⁺ through the reduction of pyruvate

- Subcellular localization: cytosol

- Conditions required to occur: anaerobic and/or absence of mitochondria

- Number of biochemical reactions: 1

- Energetic output (per pyruvate molecule): -1 NADH

- Final product (per pyruvate molecule): 1 lactate

Pyruvate oxidation

- Type of metabolic pathway: catabolic, linear

- Main goal: energy production from pyruvate oxidation

- Subcellular localization: mitochondrial matrix

- Conditions required to occur: aerobic

- Number of biochemical reactions: 1

- Energetic output (per pyruvate molecule): +1NADH

- Final product (per pyruvate molecule): 1 acetyl-CoA