GLYCOLYSIS: FUNCTIONS, ENZYMES, PHASES, PRODUCTS, IMPORTANCE - BIOLOGY - 2025

The glycolysis or glycolysis is the major route of glucose catabolism, whose ultimate aim is to generate energy in the form of ATP and reducing power in the form of NADH, from this carbohydrate.

This route, fully elucidated in the 1930s by Gustav Embden and Otto Meyerhof while studying the consumption of glucose in skeletal muscle cells, consists of the complete oxidation of this monosaccharide and, by itself, represents an anaerobic pathway for the obtaining energy.

Molecular structure of ATP, one of the glycolytic products (Summary of the glycolytic pathway (Source: Tekks at English Wikipedia / CC BY-SA (https://creativecommons.org/licenses/by-sa/3.0) via Wikimedia Commons) via Wikimedia Commons)

It is one of the main metabolic routes, since it occurs, with its differences, in all living organisms that exist, unicellular or multicellular, prokaryotic or eukaryotic, and it is thought to be a chain of reactions that is evolutionarily highly conserved in nature.

In fact, there are some organisms and cell types that depend exclusively on this route to survive.

In the first instance, glycolysis consists of the oxidation of glucose, of 6 carbon atoms, to pyruvate, which has three carbon atoms; with the concomitant production of ATP and NADH, useful for cells from the metabolic and synthetic point of view.

In cells capable of further processing the products obtained from glucose catabolism, glycolysis ends with the production of carbon dioxide and water through the Krebs cycle and the electron transport chain (aerobic glycolysis).

Ten enzymatic reactions take place in the course of the glycolytic pathway, and although the regulation of these reactions may be somewhat different from species to species, the regulatory mechanisms are also fairly conserved.

Functions of glycolysis

From a metabolic point of view, glucose is one of the most important carbohydrates for all living things.

It is a stable and very soluble molecule, which is why it can be transported relatively easily throughout the entire body of an animal or a plant, from where it is stored and / or obtained to where it is needed as cellular fuel.

Structure of glucose (Source: Oliva93 / CC BY-SA (https://creativecommons.org/licenses/by-sa/3.0) via Wikimedia Commons)

The chemical energy contained in glucose is exploited by living cells through glycolysis, which consists of a series of highly controlled steps by which the energy released from the oxidation of this carbohydrate can be “captured” into more usable forms of energy., hence its importance.

Through this route not only energy (ATP) and reducing power (NADH) are obtained, but it also provides a series of metabolic intermediaries that are part of other routes, also important from an anabolic (biosynthetic) and general cellular functioning. Here's a list:

- Glucose 6-phosphate for the Pentose Phosphate Pathway (PPP)

- Pyruvate for lactic fermentation

- Pyruvate for the synthesis of amino acids (alanine, mainly)

- Pyruvate for the tricarboxylic acid cycle

- Fructose 6-phosphate, glucose 6-phosphate and dihydroxyacetone phosphate, which function as “building blocks” in other routes such as the synthesis of glycogen, fatty acids, triglycerides, nucleotides, amino acids, etc.

Energy production

The amount of ATP produced by the glycolytic pathway, when the cell that produces it cannot live under aerobic conditions, is sufficient to supply the energy needs of a cell when it is coupled to different types of fermentation processes.

However, when it comes to aerobic cells, glycolysis also serves as an emergency source of energy and serves as a "preparatory step" prior to the oxidative phosphorylation reactions that characterize cells with aerobic metabolism.

Enzymes involved in glycolysis

Glycolysis is only possible thanks to the participation of the 10 enzymes that catalyze the reactions that characterize this pathway. Many of these enzymes are allosteric and change shape or conformation when they exert their catalytic functions.

There are enzymes that break and form covalent bonds between their substrates and there are others that require specific cofactors to carry out their functions, mainly metal ions.

Structurally speaking, all glycolytic enzymes have a center consisting essentially of parallel β sheets surrounded by α helices and arranged in more than one domain. Furthermore, these enzymes are characterized in that their active sites are usually at the binding sites between domains.

It is also important to note that the main regulation of the pathway passes through the control (hormonal or metabolites) of enzymes such as hexokinase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase.

Main points of regulation of the glycolytic pathway (Source: Gregor 0492 / CC BY-SA (https://creativecommons.org/licenses/by-sa/3.0) via Wikimedia Commons)

1- Hexokinase (HK)

The first reaction of glycolysis (glucose phosphorylation) is catalyzed by hexokinase (HK), whose mechanism of action appears to consist of a substrate “induced tightening” that promotes the “lock” of the enzyme around ATP and of glucose (its substrates) once it has bound to them.

Depending on the organism that is considered, there may be one or more isoenzymes, whose molecular weight ranges between 50 (around 500 amino acids) and 100 kDa, since they seem to group together in the form of dimers, whose formation is favored by the presence of glucose, magnesium ions and ATP.

Hexokinase has a tertiary structure composed of open alpha and beta sheets, although there are many structural differences in these enzymes.

2- Phosphoglucose isomerase (PGI)

Glucose phosphorylated by hexokinase is isomerized to fructose 6-phosphate via phosphoglucose isomerase (PGI), also known as glucose 6-phosphate isomerase. The enzyme, then, does not remove or add atoms, but rearranges them at the structural level.

This is an active enzyme in its dimeric form (the monomer weighs more or less 66 kDa) and is involved not only in glycolysis, but also in gluconeogenesis, in the synthesis of carbohydrates in plants, etc.

3- Phosphofructokinase (PFK)

Fructose 6-phosphate is a substrate for the phosphofructokinase enzyme, which is capable of re-phosphorylating this molecule using ATP as a phosphoryl group donor, producing fructose 1,6-bisphosphate.

This enzyme exists in bacteria and mammals as a homotetrameric enzyme (made up of four identical subunits of 33 kDa each for bacteria and 85 kDa each in mammals) and in yeasts it is an octamer (made up of larger subunits, between 112 and 118 kDa).

It is an allosteric enzyme, which means that it is positively or negatively regulated by some of its products (ADP) and by other molecules such as ATP and citrate.

4- Aldolasse

Also known as fructose 1,6-bisphosphate aldolase, aldolase catalyzes the catalytic breakdown of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate and the reverse reaction, that is, the union of both sugars for the formation of fructose 1,6-bisphosphate.

In other words, this enzyme cuts the fructose 1,6-bisphosphate right in half, releasing two phosphorylated 3-carbon compounds. Aldolase is also composed of 4 identical subunits, each with its own active site.

The existence of two classes (I and II) of this enzyme has been determined, which are differentiated by the mechanism of the reaction they catalyze and because some (the first) occur in bacteria and "lower" eukaryotes, and the others (the second) are in bacteria, protists and metazoans.

The "higher" eukaryotic aldolase consists of a homotetramer of 40 kDa molecular weight subunits, each consisting of a barrel made up of 8 β / α sheets.

5- Triose phosphate isomerase (TIM)

The two phosphorylated trioses can be interconverted with each other thanks to the action of triose-phosphate isomerase, which allows both sugars to be used throughout glycolysis, ensuring full use of each glucose molecule that enters the pathway.

This enzyme has been described as the "perfect" enzyme, as it catalyzes the described reaction about a trillion times faster than it would occur without your participation. Its active site is in the center of a beta-barrel structure, characteristic of many glycolytic enzymes.

It is a dimeric protein, made up of two identical subunits of approximately 27 kDa, both with a globular structure.

6- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

Glyceraldehyde 3-phosphate produced by the action of aldolase and triose phosphate isomerase serves as a substrate for GAPDH, which is a homotetrameric enzyme (34-38 kDa each subunit) that cooperatively binds to a molecule of NAD + in each of its 4 active sites, as well as 2 phosphate or sulfate ions.

In this step of the pathway, the enzyme allows the phosphorylation of one of its substrates using inorganic phosphate as a phosphoryl group donor, with the concomitant reduction of two NAD + molecules and the production of 1,3-bisphosphoglycerate.

7- Phosphoglycerate kinase (PGK)

Phosphoglycerate kinase is responsible for transferring one of the phosphate groups of 1,3-bisphosphoglycerate to an ADP molecule by phosphorylation at the substrate level. This enzyme uses a mechanism similar to that used by hexokinase, since it closes on contact on its substrates, protecting them from interfering water molecules.

This enzyme, like others that use two or more substrates, have a binding site for ADP and another for sugar phosphate.

Unlike the other enzymes described, this protein is a 44 kDa monomer with a bilobal structure, composed of two domains of the same size connected by a narrow “cleft”.

8- Phosphoglycerate mutase

The 3-phosphoglycerate undergoes a change from the phosphate group towards carbon 2, in the middle of the molecule, which represents a strategic instability site that facilitates the subsequent transfer of the group to an ATP molecule in the last reaction of the pathway.

This rearrangement is catalyzed by the enzyme phosphoglycerate mutase, a dimeric enzyme for humans and tetrameric for yeast, with a subunit size close to 27 kDa.

9- Enolase

Enolase catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate, a necessary step for the generation of ATP in the next reaction.

It is a dimeric enzyme composed of two identical 45 kDa subunits. It depends on magnesium ions for its stability and for the conformational change necessary to bind to its substrate. It is one of the enzymes most abundantly expressed in the cytosol of many organisms and performs functions in addition to glycolytic ones.

10- Pyruvate kinase

The second substrate-level phosphorylation that occurs in glycolysis is catalyzed by pyruvate kinase, which is responsible for the transfer of the phosphoryl group from phosphoenolpyruvate to ADP and for the production of pyruvate.

This enzyme is more complex than any of the other glycolytic enzymes and in mammals it is a homotetrameric enzyme (57 kDa / subunit). There are, in vertebrates, at least 4 isoenzymes: L (in liver), R (in erythrocytes), M1 (in muscle and brain) and M2 (fetal tissue and adult tissues).

Phases of glycolysis (step by step)

The glycolytic pathway consists of ten sequential steps and begins with one molecule of glucose. During the process, the glucose molecule is “activated” or “prepared” with the addition of two phosphates, inverting two ATP molecules.

Subsequently, it is "cut" into two fragments and finally it is chemically modified a couple of times, synthesizing four ATP molecules along the way, so that the net gain in the route corresponds to two ATP molecules.

From the above, it can be inferred that the route is divided into an energy "investment" phase, fundamental for the complete oxidation of the glucose molecule, and another energy "gain" phase, where the energy used initially is replaced and two are gained. net ATP molecules.

- Energy investment phase

1- The first step of the glycolytic pathway consists of the phosphorylation of glucose mediated by hexokinase (HK), for which the enzyme uses one molecule of ATP for each molecule of glucose that is phosphorylated. It is an irreversible reaction and depends on the presence of magnesium ions (Mg2 +):

Glucose + ATP → Glucose 6-phosphate + ADP

2- The glucose 6-phosphate thus produced is isomerized to fructose 6-phosphate thanks to the action of the enzyme phosphoglucose isomerase (PGI). This is a reversible reaction and does not involve additional energy expenditure:

Glucose 6-phosphate → Fructose 6-phosphate

3- Subsequently, another energy inversion step involves the phosphorylation of fructose 6-phosphate to form fructose 1,6-bisphosphate. This reaction is catalyzed by the enzyme phosphofructokinase-1 (PFK-1). Like the first step in the pathway, the phosphate group donor molecule is ATP and it is also an irreversible reaction.

Fructose 6-phosphate + ATP → Fructose 1,6-bisphosphate + ADP

4- In this step of glycolysis, the catalytic breakdown of fructose 1,6-bisphosphate into dihydroxyacetone phosphate (DHAP), a ketosis, and glyceraldehyde 3-phosphate (GAP), an aldose, occurs. This aldol condensation is catalyzed by the enzyme aldolase and is a reversible process.

Fructose 1,6-bisphosphate → Dihydroxyacetone phosphate + glyceraldehyde 3-phosphate

5- The last reaction of the energy inversion phase consists of the interconversion of the triose phosphate DHAP and GAP catalyzed by the enzyme triose-phosphate isomerase (TIM), a fact that does not require additional energy intake and is also a reversible process.

Dihydroxyacetone phosphate ↔ Glyceraldehyde 3-phosphate

- Energy gain phase

6- Glyceraldehyde 3-phosphate is used "downstream" in the glycolytic pathway as a substrate for an oxidation reaction and another for phosphorylation, catalyzed by the same enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

The enzyme catalyzes the oxidation of the molecule's C1 carbon to a carboxylic acid and its phosphorylation in the same position, producing 1,3-bisphosphoglycerate. In the course of the reaction, 2 molecules of NAD + are reduced for each molecule of glucose and 2 molecules of inorganic phosphate are used.

2Glyceraldehyde 3-phosphate + 2NAD + + 2Pi → 2 (1,3-bisphosphoglycerate) + 2NADH + 2H

In aerobic organisms, each NADH produced in this way passes through the electron transport chain to serve as a substrate for the synthesis of 6 ATP molecules by oxidative phosphorylation.

7- This is the first ATP synthesis step in glycolysis and involves the action of phosphoglycerate kinase (PGK) on 1,3-bisphosphoglycerate, transferring a phosphoryl group (substrate-level phosphorylation) from this molecule to a molecule of ADP, yielding 2ATP and 2 molecules of 3-phosphoglycerate (3PG) for each molecule of glucose.

2 (1,3-bisphosphoglycerate) + 2ADP → 2 (3-phosphoglycerate) + 2ATP

8- 3-phosphoglycerate serves as a substrate for the enzyme phosphoglycerate mutase (PGM), which converts it into 2-phosphoglycerate by displacement of the phosphoryl group from carbon 3 to carbon 2 through a two-step reaction that is reversible and dependent on magnesium ions (Mg + 2).

2 (3-phosphoglycerate) → 2 (2-phosphoglycerate)

9- The enolase enzyme dehydrates 2-phosphoglycerate and produces phosphoenolpyruvate (PEP) through a reaction that does not merit the addition of additional energy and that aims to produce a high-energy compound, capable of donating its phosphoryl group in the following reaction.

2 (2-phosphoglycerate) → 2 Phosphoenolpyruvate

10- Phosphoenolpyruvate is a substrate for the enzyme pyruvate kinase (PYK), which is responsible for the transfer of the phosphoryl group in this molecule to an ADP molecule, thus catalyzing another phosphorylation reaction at the substrate level.

In the reaction, 2ATP and 2 pyruvate molecules are produced for each glucose and the presence of potassium and magnesium in ionic form is necessary.

2Phosphoenolpyruvate + 2ADP → 2Pyruvate + 2ATP

The net yield of glycolysis, in this way, consists of 2ATP and 2NAD + for each glucose molecule that enters the pathway.

If it is cells with aerobic metabolism, then the total degradation of a glucose molecule produces between 30 and 32 ATP through the Krebs cycle and the electron transport chain.

Products of glycolysis

The general reaction of glycolysis is as follows:

Glucose + 2NAD + + 2ADP + 2Pi → 2Pyruvate + 2ATP + 2NADH + 2H +

Therefore, if it is analyzed briefly, it could be ensured that the main products of the glycolytic pathway are pyruvate, ATP, NADH and H.

However, the metabolic fate of each reaction intermediary depends, to a large extent, on cellular needs, which is why all intermediates can be considered as reaction products, and can be listed as follows:

- Glucose 6-phosphate

- Fructose 6-phosphate

- Fructose 1,6-bisphosphate

- Dihydroxyacetone phosphate and glyceraldehyde 3-phosphate

- 1,3-bisphosphoglycerate

- 3-phosphoglycerate and 2-phosphoglycerate

- Phosphoenolpyruvate and pyruvate

Importance

Despite the fact that glycolysis, by itself (one could speak of anaerobic glycolysis), produces only about 5% of the ATP that can be extracted from the aerobic catabolism of glucose, this metabolic pathway is essential for several reasons:

- It serves as a “quick” source of energy, especially in situations where an animal has to come out of a state of rest quickly, for which the aerobic oxidation processes would not be fast enough.

- The "white" skeletal muscle fibers in the human body, for example, are fast-twitch fibers and depend on anaerobic glycolysis to function.

- When, for some reason, a cell needs to do without some of its mitochondria (which are the organelles that carry out oxidative phosphorylation of part of the glycolytic products, among other things) the cell becomes more dependent on the energy obtained by glycolytic pathway.

- Many cells depend on glucose as a source of energy via glycolytics, among them the red blood cells, lacking internal organelles, and the cells of the eye (particularly those of the cornea) that do not have a high density of mitochondria.

References

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