# Glycolysis

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Glycolysis is a biochemical pathway by which a molecule of glucose (Glc) is oxidized to two molecules of pyruvic acid (Pyr).

The word glycolysis comes from Greek glyk- (meaning sweet) and lysis (meaning dissolving). It is the initial process of many pathways of carbohydrate catabolism, and serves two principal functions: generation of high-energy molecules (ATP and NADH), and production of a variety of six- or three-carbon intermediate metabolites, which may be removed at various steps in the process for other intracellular purposes (such as nucleotide biosynthesis).

Glycolysis is one of the most universal metabolic processes known, and occurs (with variations) in many types of cells in nearly all types of organisms. which suggests that it is a very ancient metabolic pathway; it may have originated with the first prokaryotes, 3.5 billion years ago or more. Glycolysis ending in pyruvate, lactate or ethanol, produces less energy per glucose molecule than complete aerobic oxidation, and so flux through the pathway is greater in anaerobic conditions. Glycolysis is the main energy source in prokaryotes or eukaryotic cells (i.e. mature erythrocytes) devoid of mitochondria or eukaryotic organisms (i.e. yeast) where local anaerobiosis is compatible with life.

The most common and well-known type of glycolysis is the Embden-Meyerhof pathway, initially elucidated by Gustav Embden and Otto Meyerhof. The term can be taken to include alternative pathways, such as the Entner-Doudoroff Pathway. However, glycolysis will be used here as a synonym for the Embden-Meyerhof pathway.

## Overview

The overall reaction of glycolysis is:

${\displaystyle Glc+2NAD^{+}+2ADP+2P_{i}\to 2NADH+2Pyr+2ATP+2H_{2}O+2H^{+}}$

So, for simple fermentations, the metabolism of one molecule of glucose to two molecules of pyruvate has a net yield of two molecules of ATP. Cells performing respiration use the products of glycolysis in order to synthesize an additional 34 molecules (approximately) of ATP for each glucose molecule oxidized. Unlike most of the molecules of ATP produced via aerobic respiration, those of glycolysis are produced by substrate-level phosphorylation.

In eukaryotes, glycolysis takes place within the cytosol of the cell, while in plant cells, some of the glycolytic reactions are also conserved in the Calvin cycle that functions inside the chloroplast.

## Pathway

### Sequence of reactions

#### Preparatory phase

The first five steps are regarded as a preparatory phase since they actually consume energy as the glucose is converted to two three-carbon sugar phosphates (G3P). The bold abbreviations in the two tables correspond to the nomenclature used in the diagram.

Step Substrate Enzyme Enzyme class Comment
1 glucose Glc hexokinase HK transferase Glucose often comes from the hydrolysis of starch or glycogen. In this step, ATP is used and therefore this reaction has a highly negative change in free energy, and is thus, irreversible.
2 glucose-6-phosphate G6P phosphoglucose isomerase or hexophosphate isomerase PGI isomerase In this reaction, the aldehyde group in glucose is reduced to an alcohol, whereas the adjacent carbon has been oxidized to form a ketone, so that no net change in the redox state of the sugar is observed. While this reaction is not normally favorable, it is driven by a low concentration of F6P, which is constantly consumed during the next step of glycolysis.
3(1) fructose 6-phosphate F6P phosphofructokinase PFK-1 transferase The energy expenditure of another ATP in this step is justified in 2 ways: the glycolytic process (up to this step) is now irreversible, and the formed molecule can now be cleaved into two almost identical molecules.
During fasting PFK1 activity is reduced. This leads to an increase of flux through the gluconeogenesis pathway.
4 fructose 1,6-bisphosphate F1,6BP aldolase ALDO lyase Destablising the molecule in the previous reaction allows the hexose ring to be split by ALDO into two very similar triose sugars, DHAP and GADP.
5 dihydroxyacetone phosphate DHAP triose phosphate isomerase TPI isomerase TPI rapidly interconverts DHAP with glyceraldehyde 3-phosphate (GADP) that proceeds further into glycolysis.
(1) - The third step can also be catalysed by pyrophosphate dependent phosphofructokinase (PFP or PPi-PFK). This enzyme catalyses the same reaction as PFK1 (also known as ATP-PFK), but uses pyrophosphate (PPi) as a phosphate donor instead of ATP. It is a reversible reaction, increasing the flexibility of glycolytic metabolism. This enzyme is not found in animal cells, but is found in most plants, some bacteria, archaea and protists. [1] A more rare ADP-dependent PFK enzyme (ATP-PFK) variant has been indentified in archaean species, such as Pyrococcus furiosus. [2]

#### Pay-off phase

The second half of glycolysis is known as the pay-off phase, characterised by a net gain of the energy-rich molecules ATP and NADH. Since glucose leads to two triose sugars in the preparatory phase, each reaction in the pay-off phase occurs twice per glucose molecule. This yields 2 NADH molecules and 4 ATP molecules, leading to a net gain of 2 NADH molecules and 2 ATP molecules from the gylcolytic pathway per glucose.

Step Substrate Enzyme Enzyme class Comment
6 glyceraldehyde 3-phosphate GADP glyceraldehyde 3-phosphate dehydrogenase GAP oxidoreductase Triose sugars are dehydrogenated and inorganic phosphate is added to them. The released electrons are used to reduce NAD+ into NADH + H+.
7 1,3-bisphosphoglycerate 1,3BPG phosphoglycerate kinase PGK transferase A reaction that converts ADP to ATP by an enzymatic transfer of a phosphate to ADP; is an example of substrate-level phosphorylation.
8 3-phosphoglycerate 3PG phosphoglyceromutase PGAM mutase Notice that this enzyme is a mutase and not an isomerase. While an isomerase changes the oxidation state of the carbons being reacted, a mutase does not.
9 2-phosphoglycerate 2PG enolase ENO lyase
10 phosphoenolpyruvate PEP pyruvate kinase PK transferase Another example of substrate-level phosphorylation that converts ADP, the discharged form of ATP, into a charged ATP molecule, forming pyruvate (Pyr).

### Entry of sugars

The first step in glycolysis is phosphorylation of Glc by a family of enzymes called HKs to form G6P. In the liver, an isozyme of hexokinase called GCK is used, which differs primarily in regulatory properties. This reaction consumes 1 ATP, but the energy is well-spent - it keeps [Glc]i low as to allow continuous entry of Glc through its plasma membrane transporters; prevents Glc leakage out - the cell lacks such transporters for G6P; activates Glc preparing it for the next metabolic changes.

G6P is then rearranged into F6P by GPI. Fru can also enter the glycolytic pathway via phosphorylation at this point.

### Control of flux

The flux through the glycolytic pathway must be adjusted in response to conditions both inside and outside the cell. The rate is regulated to meet two major cellular needs: (1) the production of ATP, and (2) the provision of building blocks for biosynthetic reactions. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are effectively irreversible. In metabolic pathways, such enzymes are potential sites of control, and all these three enzymes serve this purpose in glycolysis.

There are several different ways to regulate the activity of an enzyme. An immediate form of control is feedback via allosteric effectors or by covalent modification. A slower form of control is transcriptional regulation that controls the amounts of these important enzymes.

#### Hexokinase

Hexokinase is inhibited by glucose-6-phosphate (G6P), the product it forms through the ATP driven phosphorylation. This is necessary to prevent an accumulation of G6P in the cell when flux through the glycolytic pathway is low. Glucose will enter the cell but since the hexokinase is not active it can readily diffuse back to the blood through the glucose transporter in the plasma membrane. If hexokinase remained active during low glycolytic flux the G6P would accumulate and the extra solute would cause the cells to enlarge due to osmosis.

In liver cells, the extra G6P is stored as glycogen. In these cells hexokinase is not expressed, instead glucokinase catalyses the phosphorylation of glucose to G6P. This enzyme is not inhibited by high levels of G6P and glucose can still be converted to G6P and then be stored as glycogen. This is important when blood glucose levels are high. During hypoglycemia the glycogen can be converted back to G6P and then converted to glucose by a liver specific enzyme glucose 6-phosphatase. This reverse reaction is an important role of liver cells to maintain blood sugars levels during fasting. This is critical for neuron function since they can only use glucose as an energy source.

#### Phosphofructokinase-1

Phosphofructokinase is an important control point in the glycolytic pathway since it is immediately downstream of the entry points for hexose sugars.

High levels of ATP inhibit the PFK enzyme by lowering its affinity for F6P. ATP causes this control by binding to a specific regulatory site that is distinct from the catalytic site. This is a good example of allosteric control. AMP can reverse the inhibitory effect of ATP. A consequence is that PFK is tightly controlled by the ratio of ATP/AMP in the cell. This makes sense since these molecules are direct indicators of the energy charge in the cell.

Since glycolysis is also a source of carbon skeletons for biosynthesis, a negative feedback control to glycolysis from the carbon skeleton pool is useful. Citrate is an example of a metabolite that regulates phosphofructokinase by enhancing the inhibitory effect of ATP. Citrate is an early intermediate in the citric acid cycle, and a high level means that biosynthetic precursors are abundant.

Low pH also inhibits phosphofructokinase activity and prevents the excessive rise of lactic acid during anaerobic conditions that could otherwise cause a drop in blood pH (acidosis), (a potentially life threatening condition).

Fructose 2,6-bisphosphate (F2,6BP) is a potent activator of phosphofructokinase (PFK-1) that is synthesised when F6P is phosphorylated by a second phosphofructokinase (PFK2). This second enzyme is inactive when cAMP is high, and links the regulation of glycolysis to hormone activity in the body. Both glucagon and adrenalin cause high levels of cAMP in the liver. The result is lower levels of liver fructose 2,6-bisphosphate such that gluconeogenesis (glycolysis in reverse) is favored. This is consistent with the role of the liver in such situations since the response of the liver to these hormones is to releases glucose to the blood.

### Energy pay-off

Each molecule of GADP is then oxidized by a molecule of NAD+ in the presence of GAP, forming 1,3-bisphosphoglycerate. In the next step, PGK generates a molecule of ATP while forming 3-phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have been synthesized. This step, one of the two substrate-level phosphorylation steps, requires ADP; thus, when the cell has plenty of ATP (and little ADP) this reaction does not occur. Because ATP decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway. PGAM then forms 2-phosphoglycerate; ENO then forms phosphoenolpyruvate; and another substrate-level phosphorylation then forms a molecule of Pyr and a molecule of ATP by means of the enzyme PK. This serves as an additional regulatory step.

After the formation of F1,6bP, many of the reactions are energetically unfavorable. The only reactions that are favorable are the 2 substrate-level phosphorylation steps that result in the formation of ATP. These two reactions pull the glycolytic pathway to completion.

## Follow-up

The ultimate fate of pyruvate and NADH produced in glycolysis depends upon the organism and the conditions, most notably the presence or absence of oxygen and other external electron acceptors.

In aerobic organisms, pyruvate typically enters the mitochondria where it is fully oxidized to carbon dioxide and water by pyruvate decarboxylase and the set of enzymes of the citric acid cycle (also known as the TCA or Krebs cycle). The products of pyruvate are sequentially dehydrogenated as they pass through the cycle conserving the hydrogen equivalents via the reduction of NAD+ to NADH. NADH is ultimately oxidized by an electron transport chain using oxygen as final electron acceptor to produce a large amount of ATP via the action of the ATP synthase complex, a process known as oxidative phosphorylation. A small amount of ATP is also produced by substrate-level phosphorylation during the TCA cycle.

Although human metabolism is primarily aerobic, under hypoxic (or partially anaerobic) conditions, for example in overworked muscles that are starved of oxygen or in infarcted heart muscle cells, pyruvate is converted to the waste product lactate. This and similar reactions are known as fermentation, and they are a solution to maintaining the metabolic flux through glycolysis in response to an anaerobic or severely hypoxic environment.

Although fermentation does not produce much energy, it is critical for an anaerobic or hypoxic cell, since it regenerates NAD+ that is required for glycolysis to proceed. This is important for normal cellular function, as glycolysis is the only source of ATP in anaerobic or severely hypoxic conditions.

There are several types of fermentation wherein pyruvate and NADH are anaerobically metabolized to yield any of a variety of products with an organic molecule acting as the final hydrogen acceptor. For example, the bacteria involved in making yogurt simply reduce pyruvate to lactic acid, whereas yeast produces ethanol and carbon dioxide. Anaerobic bacteria are capable of using a wide variety of compounds, other than oxygen, as terminal electron acceptors in respiration: nitrogenous compounds (such as nitrates and nitrites), sulphur compounds (such as sulphates, sulphites, sulphur dioxide, and elemental sulphur), carbon dioxide, iron compounds, manganese compounds, cobalt compounds, and uranium compounds.

## Intermediates for other pathways

This article concentrates on the catabolic role of glycolysis with regard to converting potential chemical energy to usable chemical energy during the oxidation of glucose to pyruvate. Many of the metabolites in the glycolytic pathway are also used by anabolic pathways, and, as a consequence, flux through the pathway is critical to maintain a supply of carbon skeletons for biosynthesis.

From an energy perspective, NADH is either recycled to NAD+ during anaerobic conditions, to maintain the flux through the glycolytic pathway, or used during aerobic conditions to produce more ATP by oxidative phosphorylation. From an anabolic metabolism perspective, the NADH has a role to drive synthetic reactions, doing so by directly or indirectly reducing the pool of NADP+ in the cell to NADPH, which is another important reducing agent for biosynthetic pathways in a cell.

## High aerobic glycolysis

During anaerobic conditions, glycolysis is the cellular mechanism to obtain ATP, by fermentation. However, in mammalian cells, glycolysis is coupled with aerobic respiration. In the presence of oxygen, mitochondria take up pyruvate, the end-product of glycolysis, and further oxidize it into CO2 and water. As a result, the flux through the glycolytic pathway is lower during aerobic conditions since the full oxidation of one molecule of pyruvate (equivalent to one-half molecule of glucose) can lead to 18 times more ATP. Malignant rapidly-growing tumor cells, however, have glycolytic rates that are up to 200 times higher than that of their normal tissues of origin, despite the ample availability of oxygen. A classical explanation holds that the local depletion of oxygen within the tumor is the cause of the high glycolytic rate in tumor cells. Nevertheless, there is also strong experimental evidence that attributes these high aerobic glycolytic rates to an overexpressed form of mitochondrially-bound hexokinase [3] responsible for driving the high glycolytic activity when oxygen is not necessarily depleted. This phenomenon was first described in 1930 by Otto Warburg, and hence it is referred to as the Warburg Effect. This has a current important medical application, as aerobic glycolysis by malignant tumors is utilized clinically to diagnose and monitor treatment responses of cancers by imaging uptake of 2-18F-2-deoxyglucose (a radioactive modified hexokinase substrate) with positron emission tomography (PET) [4], [5].

## Alternative nomenclature

Some of the metabolites in glycolysis have alternative names and nomenclature. In part, this is because some of them are common to other pathways, such as the Calvin cycle.

This article Alternative names Alternative nomenclature
1 glucose Glc dextrose
2 glucose-6-phosphate G6P
3 fructose 6-phosphate F6P
4 fructose 1,6-bisphosphate F1,6BP fructose 1,6-diphosphate FBP, FDP, F1,6DP
5 dihydroxyacetone phosphate DHAP
6 glyceraldehyde 3-phosphate GADP 3-phosphoglyceraldehyde PGAL, G3P, GALP,GAP
7 1,3-bisphosphoglycerate 1,3BPG glycerate 1,3-bisphosphate,
glycerate 1,3-diphosphate,
1,3-diphosphoglycerate
PGAP, BPG, DPG
8 3-phosphoglycerate 3PG glycerate 3-phosphate PGA, GP
9 2-phosphoglycerate 2PG glycerate 2-phosphate
10 phosphoenolpyruvate PEP
11 pyruvate Pyr

## Recent advances

A new study reveals that, in response to oxidative stress, organisms can redirect their metabolic flux from glycolysis to the pentose phosphate pathway, the pathway that provides the reducing power for the main cellular redox systems. This ability is conserved between yeast and animals, showing its importance in the adaptation to oxidative stress [6].

## References

1. Reeves, R. E.; South D. J., Blytt H. J. and Warren L. G. (1974). "Pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase. A new enzyme with the glycolytic function 6-phosphate 1-phosphotransferase.". J Biol Chem 249: 7737–7741. PMID 4372217.
2. Selig, M.; Xavier K. B., Santos H. and Schönheit P. (1997). "Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium Thermotoga.". Arch Microbiol 167: 217-232. PMID 9075622.
3. PET Scan: PET Scan Info Reveals .... Retrieved on December 5, 2005.
4. 4320139 549..559. Retrieved on December 5, 2005.