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Glycolysis is the metabolic pathway that converts glucose (C6H12O6) into pyruvate and, in most organisms, occurs in the liquid part of cells (the cytosol). The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH).[1] Glycolysis is a sequence of ten reactions catalyzed by enzymes.
The wide occurrence of glycolysis in other species indicates that it is an ancient metabolic pathway.[2] Indeed, the reactions that make up glycolysis and its parallel pathway, the pentose phosphate pathway, can occur in the oxygen-free conditions of the Archean oceans, also in the absence of enzymes, catalyzed by metal ions, meaning this is a plausible prebiotic pathway for abiogenesis.[3]
The most common type of glycolysis is the Embden–Meyerhof–Parnas (EMP) pathway, which was discovered by Gustav Embden, Otto Meyerhof, and Jakub Karol Parnas. Glycolysis also refers to other pathways, such as the Entner–Doudoroff pathway and various heterofermentative and homofermentative pathways. However, the discussion here will be limited to the Embden–Meyerhof–Parnas pathway.[4]
The glycolysis pathway can be separated into two phases:[5]
- Investment phase – wherein ATP is consumed
- Yield phase – wherein more ATP is produced than originally consumed
Overview
The overall reaction of glycolysis is:
The use of symbols in this equation makes it appear unbalanced with respect to oxygen atoms, hydrogen atoms, and charges. Atom balance is maintained by the two phosphate (Pi) groups:[6]
- Each exists in the form of a hydrogen phosphate anion ([HPO42−), dissociating to contribute 2H+ overall
- Each liberates an oxygen atom when it binds to an adenosine diphosphate (ADP) molecule, contributing 2 O overall
Charges are balanced by the difference between ADP and ATP. In the cellular environment, all three hydroxyl groups of ADP dissociate into −O− and H+, giving ADP3−, and this ion tends to exist in an ionic bond with Mg2+, giving ADPMg−. ATP behaves identically except that it has four hydroxyl groups, giving ATPMg2−. When these differences along with the true charges on the two phosphate groups are considered together, the net charges of −4 on each side are balanced.
For simple fermentations, the metabolism of one molecule of glucose to two molecules of pyruvate has a net yield of two molecules of ATP. Most cells will then carry out further reactions to "repay" the used NAD+ and produce a final product of ethanol or lactic acid. Many bacteria use inorganic compounds as hydrogen acceptors to regenerate the NAD+.
Cells performing aerobic respiration synthesize much more ATP, but not as part of glycolysis. These further aerobic reactions use pyruvate, and NADH + H+ from glycolysis. Eukaryotic aerobic respiration produces approximately 34 additional molecules of ATP for each glucose molecule, however most of these are produced by a mechanism vastly different from the substrate-level phosphorylation in glycolysis.
The lower-energy production, per glucose, of anaerobic respiration relative to aerobic respiration, results in greater flux through the pathway under hypoxic (low-oxygen) conditions, unless alternative sources of anaerobically oxidizable substrates, such as fatty acids, are found.
| Metabolism of common monosaccharides, including glycolysis, gluconeogenesis, glycogenesis and glycogenolysis |
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History
The pathway of glycolysis as it is known today took almost 100 years to fully elucidate.[7] The combined results of many smaller experiments were required in order to understand the intricacies of the entire pathway.
The first steps in understanding glycolysis began in the nineteenth century with the wine industry. For economic reasons, the French wine industry sought to investigate why wine sometimes turned distasteful, instead of fermenting into alcohol. French scientist Louis Pasteur researched this issue during the 1850s, and the results of his experiments began the long road to elucidating the pathway of glycolysis.[8] His experiments showed that fermentation occurs by the action of living microorganisms, yeasts, and that yeast's glucose consumption decreased under aerobic conditions of fermentation, in comparison to anaerobic conditions (the Pasteur effect).[9]
Insight into the component steps of glycolysis were provided by the non-cellular fermentation experiments of Eduard Buchner during the 1890s.[10][11] Buchner demonstrated that the conversion of glucose to ethanol was possible using a non-living extract of yeast, due to the action of enzymes in the extract.[12]: 135–148 This experiment not only revolutionized biochemistry, but also allowed later scientists to analyze this pathway in a more controlled laboratory setting. In a series of experiments (1905-1911), scientists Arthur Harden and William Young discovered more pieces of glycolysis.[13] They discovered the regulatory effects of ATP on glucose consumption during alcohol fermentation. They also shed light on the role of one compound as a glycolysis intermediate: fructose 1,6-bisphosphate.[12]: 151–158
The elucidation of fructose 1,6-bisphosphate was accomplished by measuring CO2 levels when yeast juice was incubated with glucose. CO2 production increased rapidly then slowed down. Harden and Young noted that this process would restart if an inorganic phosphate (Pi) was added to the mixture. Harden and Young deduced that this process produced organic phosphate esters, and further experiments allowed them to extract fructose diphosphate (F-1,6-DP).
Arthur Harden and William Young along with Nick Sheppard determined, in a second experiment, that a heat-sensitive high-molecular-weight subcellular fraction (the enzymes) and a heat-insensitive low-molecular-weight cytoplasm fraction (ADP, ATP and NAD+ and other cofactors) are required together for fermentation to proceed. This experiment begun by observing that dialyzed (purified) yeast juice could not ferment or even create a sugar phosphate. This mixture was rescued with the addition of undialyzed yeast extract that had been boiled. Boiling the yeast extract renders all proteins inactive (as it denatures them). The ability of boiled extract plus dialyzed juice to complete fermentation suggests that the cofactors were non-protein in character.[13]
In the 1920s Otto Meyerhof was able to link together some of the many individual pieces of glycolysis discovered by Buchner, Harden, and Young. Meyerhof and his team were able to extract different glycolytic enzymes from muscle tissue, and combine them to artificially create the pathway from glycogen to lactic acid.[14][15]
In one paper, Meyerhof and scientist Renate Junowicz-Kockolaty investigated the reaction that splits fructose 1,6-diphosphate into the two triose phosphates. Previous work proposed that the split occurred via 1,3-diphosphoglyceraldehyde plus an oxidizing enzyme and cozymase. Meyerhoff and Junowicz found that the equilibrium constant for the isomerase and aldoses reaction were not affected by inorganic phosphates or any other cozymase or oxidizing enzymes. They further removed diphosphoglyceraldehyde as a possible intermediate in glycolysis.[15]
With all of these pieces available by the 1930s, Gustav Embden proposed a detailed, step-by-step outline of that pathway we now know as glycolysis.[16] The biggest difficulties in determining the intricacies of the pathway were due to the very short lifetime and low steady-state concentrations of the intermediates of the fast glycolytic reactions. By the 1940s, Meyerhof, Embden and many other biochemists had finally completed the puzzle of glycolysis.[15] The understanding of the isolated pathway has been expanded in the subsequent decades, to include further details of its regulation and integration with other metabolic pathways.
Sequence of reactions
Summary of reactions
Preparatory phase
The first five steps of Glycolysis are regarded as the preparatory (or investment) phase, since they consume energy to convert the glucose into two three-carbon sugar phosphates[5] (G3P).
| d-Glucose (Glc) | Hexokinase glucokinase (HK) a transferase |
α-d-Glucose-6-phosphate (G6P) | |
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| ATP | H+ + ADP | ||
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Once glucose enters the cell, the first step is phosphorylation of glucose by a family of enzymes called hexokinases to form glucose 6-phosphate (G6P). This reaction consumes ATP, but it acts to keep the glucose concentration inside the cell low, promoting continuous transport of blood glucose into the cell through the plasma membrane transporters. In addition, phosphorylation blocks the glucose from leaking out – the cell lacks transporters for G6P, and free diffusion out of the cell is prevented due to the charged nature of G6P. Glucose may alternatively be formed from the phosphorolysis or hydrolysis of intracellular starch or glycogen.
In animals, an isozyme of hexokinase called glucokinase is also used in the liver, which has a much lower affinity for glucose (Km in the vicinity of normal glycemia), and differs in regulatory properties. The different substrate affinity and alternate regulation of this enzyme are a reflection of the role of the liver in maintaining blood sugar levels.
Cofactors: Mg2+
| α-d-Glucose 6-phosphate (G6P) | Phosphoglucoisomerase (PGI) an isomerase |
β-d-Fructose 6-phosphate (F6P) | |
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G6P is then rearranged into fructose 6-phosphate (F6P) by glucose phosphate isomerase. Fructose can also enter the glycolytic pathway by phosphorylation at this point.
The change in structure is an isomerization, in which the G6P has been converted to F6P. The reaction requires an enzyme, phosphoglucose isomerase, to proceed. This reaction is freely reversible under normal cell conditions. However, it is often driven forward because of a low concentration of F6P, which is constantly consumed during the next step of glycolysis. Under conditions of high F6P concentration, this reaction readily runs in reverse. This phenomenon can be explained through Le Chatelier's Principle. Isomerization to a keto sugar is necessary for carbanion stabilization in the fourth reaction step (below).
| β-d-Fructose 6-phosphate (F6P) | Phosphofructokinase (PFK-1) a transferase |
β-d-Fructose 1,6-bisphosphate (F1,6BP) | |
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| ATP | H+ + ADP | ||
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The energy expenditure of another ATP in this step is justified in 2 ways: The glycolytic process (up to this step) becomes irreversible, and the energy supplied destabilizes the molecule. Because the reaction catalyzed by phosphofructokinase 1 (PFK-1) is coupled to the hydrolysis of ATP (an energetically favorable step) it is, in essence, irreversible, and a different pathway must be used to do the reverse conversion during gluconeogenesis. This makes the reaction a key regulatory point (see below).
Furthermore, the second phosphorylation event is necessary to allow the formation of two charged groups (rather than only one) in the subsequent step of glycolysis, ensuring the prevention of free diffusion of substrates out of the cell.
The same reaction can also be catalyzed by pyrophosphate-dependent phosphofructokinase (PFP or PPi-PFK), which is found in most plants, some bacteria, archea, and protists, but not in animals. This enzyme uses pyrophosphate (PPi) as a phosphate donor instead of ATP. It is a reversible reaction, increasing the flexibility of glycolytic metabolism.[17] A rarer ADP-dependent PFK enzyme variant has been identified in archaean species.[18]
Cofactors: Mg2+
| β-d-Fructose 1,6-bisphosphate (F1,6BP) | Fructose-bisphosphate aldolase (ALDO) a lyase |
d-Glyceraldehyde 3-phosphate (GADP) | Dihydroxyacetone phosphate (DHAP) | ||
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+ | 
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Destabilizing the molecule in the previous reaction allows the hexose ring to be split by aldolase into two triose sugars: dihydroxyacetone phosphate (a ketose), and glyceraldehyde 3-phosphate (an aldose). There are two classes of aldolases: class I aldolases, present in animals and plants, and class II aldolases, present in fungi and bacteria; the two classes use different mechanisms in cleaving the ketose ring.
Electrons delocalized in the carbon-carbon bond cleavage associate with the alcohol group. The resulting carbanion is stabilized by the structure of the carbanion itself via resonance charge distribution and by the presence of a charged ion prosthetic group.
| Dihydroxyacetone phosphate (DHAP) | Triosephosphate isomerase (TPI) an isomerase |
d-Glyceraldehyde 3-phosphate (GADP) | |
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Triosephosphate isomerase rapidly interconverts dihydroxyacetone phosphate with glyceraldehyde 3-phosphate (GADP) that proceeds further into glycolysis. This is advantageous, as it directs dihydroxyacetone phosphate down the same pathway as glyceraldehyde 3-phosphate, simplifying regulation.
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.[5] 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 glycolytic pathway per glucose.
| Glyceraldehyde 3-phosphate (GADP) | Glyceraldehyde phosphate dehydrogenase (GAPDH) an oxidoreductase |
d-1,3-Bisphosphoglycerate (1,3BPG) | |
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| NAD+ + Pi | NADH + H+ | ||
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The aldehyde groups of the triose sugars are oxidised, and inorganic phosphate is added to them, forming 1,3-bisphosphoglycerate.
The hydrogen is used to reduce two molecules of NAD+, a hydrogen carrier, to give NADH + H+ for each triose.
Hydrogen atom balance and charge balance are both maintained because the phosphate (Pi) group actually exists in the form of a hydrogen phosphate anion (HPO2−4),[6] which dissociates to contribute the extra H+ ion and gives a net charge of -3 on both sides.
Here, arsenate ([AsO43−), an anion akin to inorganic phosphate may replace phosphate as a substrate to form 1-arseno-3-phosphoglycerate. This, however, is unstable and readily hydrolyzes to form 3-phosphoglycerate, the intermediate in the next step of the pathway. As a consequence of bypassing this step, the molecule of ATP generated from 1-3 bisphosphoglycerate in the next reaction will not be made, even though the reaction proceeds. As a result, arsenate is an uncoupler of glycolysis.[19]
| 1,3-Bisphosphoglycerate (1,3BPG) | Phosphoglycerate kinase (PGK) a transferase |
3-Phosphoglycerate (3PG) | |
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| ADP | ATP | ||
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| Phosphoglycerate kinase (PGK) | |||
This step is the enzymatic transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP by phosphoglycerate kinase, forming ATP and 3-phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have now 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.
ADP actually exists as ADPMg−, and ATP as ATPMg2−, balancing the charges at −5 both sides.
Cofactors: Mg2+