Glycolysis

thumb|400px|class=skin-invert-image|Summary of aerobic respiration<!-- Force TemplateStyle to get included. --><!-- So forced. -->Glycolysis is the metabolic pathway that converts glucose () 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). Glycolysis is a sequence of ten reactions catalyzed by enzymes.

thumb|375x375px|Summary of the 10 reactions of the glycolysis pathway

The wide occurrence of glycolysis in other species indicates that it is an ancient metabolic pathway. 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.

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.

The glycolysis pathway can be separated into two phases:

1. Investment phase – wherein ATP is consumed
2. Yield phase – wherein more ATP is produced than originally consumed

Overview

The overall reaction for glycolysis is:

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thumb|445x445px|class=skin-invert-image|Glycolysis pathway overview

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 (P_i) groups:

• Each exists in the form of a hydrogen phosphate anion (), dissociating to contribute overall
• Each liberates an oxygen atom when it binds to an adenosine diphosphate (ADP) molecule, contributing 2O 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 ATPMg³⁻. 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.

In high-oxygen (aerobic) conditions, eukaryotic cells can continue from glycolysis to metabolise the pyruvate through the citric acid cycle or the electron transport chain to produce significantly more ATP.

Importantly, under low-oxygen (anaerobic) conditions, glycolysis is the only biochemical pathway in eukaryotes that can generate ATP, and, for many anaerobic respiring organisms the most important producer of ATP. Therefore, many organisms have evolved fermentation pathways to recycle NAD⁺ to continue glycolysis to produce ATP for survival. These pathways include ethanol fermentation and lactic acid fermentation.

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! Metabolism of common monosaccharides, including glycolysis, gluconeogenesis, glycogenesis and glycogenolysis

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History

The modern understanding of the pathway of glycolysis took almost 100 years to fully learn. The combined results of many smaller experiments were required to understand the entire pathway.

The first steps in understanding glycolysis began in the 19th century. For economic reasons, the French wine industry sought to investigate why wine sometimes turned distasteful, instead of fermenting into alcohol. The French scientist Louis Pasteur researched this issue during the 1850s. His experiments showed that alcohol fermentation occurs by the action of living microorganisms, yeasts, and that glucose consumption decreased under aerobic conditions (the Pasteur effect).

left|thumb|Eduard Buchner discovered cell-free fermentation.

The component steps of glycolysis were first analysed by the non-cellular fermentation experiments of Eduard Buchner during the 1890s. 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. 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. 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.

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. 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. A rarer ADP-dependent PFK enzyme variant has been identified in archaean species.

Cofactors: Mg²⁺

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). Two classes of aldolases exist: class I aldolases, present in animals and plants, and class II aldolases, present in fungi and bacteria. The two classes use distinct 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 resonance and by a positively charged prosthetic group.

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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===<!-- This section is linked from Cellular respiration -->

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.

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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⁺

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Phosphoglycerate mutase isomerises 3-phosphoglycerate into 2-phosphoglycerate.

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Enolase next converts 2-phosphoglycerate to phosphoenolpyruvate. This reaction is an elimination reaction involving an E1cB mechanism.

Cofactors: 2 Mg²⁺, one "conformational" ion to coordinate with the carboxylate group of the substrate, and one "catalytic" ion that participates in the dehydration.

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A final substrate-level phosphorylation now forms a molecule of pyruvate and a molecule of ATP by means of the enzyme pyruvate kinase. This serves as an additional regulatory step, similar to the phosphoglycerate kinase step.

Cofactors: Mg²⁺

Biochemical logic

The existence of more than one point of regulation indicates that intermediates between those points enter and leave the glycolysis pathway by other processes. For example, in the first regulated step, hexokinase converts glucose into glucose-6-phosphate. Instead of continuing through the glycolysis pathway, this intermediate can be converted into glucose storage molecules, such as glycogen or starch. The reverse reaction, breaking down, e.g., glycogen, produces mainly glucose-6-phosphate; very little free glucose is formed in the reaction. The glucose-6-phosphate so produced can enter glycolysis after the first control point.

In the second regulated step (the third step of glycolysis), phosphofructokinase converts fructose-6-phosphate into fructose-1,6-bisphosphate, which then is converted into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The dihydroxyacetone phosphate can be removed from glycolysis by conversion into glycerol-3-phosphate, which can be used to form triglycerides. Conversely, triglycerides can be broken down into fatty acids and glycerol; the latter, in turn, can be converted into dihydroxyacetone phosphate, which can enter glycolysis after the second control point.

Free energy changes

{| align="right" class="wikitable"

|+ Concentrations of metabolites in erythrocytes

! Compound

! Concentration / mM

|-

|Glucose

|5.0

|-

|Glucose-6-phosphate

|0.083

|-

|Fructose-6-phosphate

|0.014

|-

|Fructose-1,6-bisphosphate

|0.031

|-

|Dihydroxyacetone phosphate

|0.14

|-

|Glyceraldehyde-3-phosphate

|0.019

|-

|1,3-Bisphosphoglycerate

|0.001

|-

|2,3-Bisphosphoglycerate

|4.0

|-

|3-Phosphoglycerate

|0.12

|-

|2-Phosphoglycerate

|0.03

|-

|Phosphoenolpyruvate

|0.023

|-

|Pyruvate

|0.051

|-

|ATP

|1.85

|-

|ADP

|0.14

|-

|P_i

|1.0

|}

The change in free energy, ΔG, for each step in the glycolysis pathway can be calculated using ΔG = ΔG°′ + RTln Q, where Q is the reaction quotient. This requires knowing the concentrations of the metabolites. All of these values are available for erythrocytes, with the exception of the concentrations of NAD⁺ and NADH. The ratio of NAD⁺ to NADH in the cytoplasm is approximately 1000, which makes the oxidation of glyceraldehyde-3-phosphate (step 6) more favourable.

Using the measured concentrations of each step, and the standard free energy changes, the actual free energy change can be calculated. (Neglecting this is very common—the delta G of ATP hydrolysis in cells is not the standard free energy change of ATP hydrolysis quoted in textbooks).

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|+ Change in free energy for each step of glycolysis The details of regulation for some enzymes are highly conserved between species, whereas others vary widely.

1. Gene Expression: Firstly, the cellular concentrations of glycolytic enzymes are modulated via regulation of gene expression via transcription factors, with several glycolysis enzymes themselves acting as regulatory protein kinases in the nucleus.
2. Allosteric inhibition and activation by metabolites: In particular end-product inhibition of regulated enzymes by metabolites such as ATP serves as negative feedback regulation of the pathway.
3. Allosteric inhibition and activation by Protein-protein interactions (PPI). Indeed, some proteins interact with and regulate multiple glycolytic enzymes.
4. Post-translational modification (PTM). In particular, phosphorylation and dephosphorylation is a key mechanism of regulation of pyruvate kinase in the liver.
5. Localization A rise in the blood glucose concentration causes them to release insulin into the blood, which has an effect particularly on the liver, but also on fat and muscle cells, causing these tissues to remove glucose from the blood. When the blood sugar falls the pancreatic beta cells cease insulin production, but, instead, stimulate the neighboring pancreatic alpha cells to release glucagon into the blood. The phosphorylation and dephosphorylation of these enzymes (ultimately in response to the glucose level in the blood) is the dominant manner by which these pathways are controlled in the liver, fat, and muscle cells. Thus the phosphorylation of phosphofructokinase inhibits glycolysis, whereas its dephosphorylation through the action of insulin stimulates glycolysis. The simultaneously phosphorylation of, particularly, phosphofructokinase, but also, to a certain extent pyruvate kinase, prevents glycolysis occurring at the same time as gluconeogenesis and glycogenolysis.

Hexokinase and glucokinase

thumb|right|[[Yeast hexokinase B ()]]

All cells contain the enzyme hexokinase, which catalyzes the conversion of glucose that has entered the cell into glucose-6-phosphate (G6P). Since the cell membrane is impervious to G6P, hexokinase essentially acts to transport glucose into the cells from which it can then no longer escape. Hexokinase is inhibited by high levels of G6P in the cell. Thus the rate of entry of glucose into cells partially depends on how fast G6P can be disposed of by glycolysis, and by glycogen synthesis (in the cells which store glycogen, namely liver and muscles).

Glucokinase, unlike hexokinase, is not inhibited by G6P. It occurs in liver cells, and will only phosphorylate the glucose entering the cell to form G6P, when the glucose in the blood is abundant. This being the first step in the glycolytic pathway in the liver, it therefore imparts an additional layer of control of the glycolytic pathway in this organ. but the concentration of ATP does not change more than about 10% under physiological conditions, whereas a 10% drop in ATP results in a 6-fold increase in AMP. Thus, the relevance of ATP as an allosteric effector is questionable. An increase in AMP is a consequence of a decrease in energy charge in the cell.

Citrate inhibits phosphofructokinase when tested in vitro by enhancing the inhibitory effect of ATP. However, it is doubtful that this is a meaningful effect in vivo, because citrate in the cytosol is utilized mainly for conversion to acetyl-CoA for fatty acid and cholesterol synthesis.

TIGAR, a p53 induced enzyme, is responsible for the regulation of phosphofructokinase and acts to protect against oxidative stress. TIGAR is a single enzyme with dual function that regulates F2,6BP. It can behave as a phosphatase (fructuose-2,6-bisphosphatase) which cleaves the phosphate at carbon-2 producing F6P. It can also behave as a kinase (PFK2) adding a phosphate onto carbon-2 of F6P which produces F2,6BP. In humans, the TIGAR protein is encoded by C12orf5 gene. The TIGAR enzyme will hinder the forward progression of glycolysis, by creating a build up of fructose-6-phosphate (F6P) which is isomerized into glucose-6-phosphate (G6P). The accumulation of G6P will shunt carbons into the pentose phosphate pathway.

Pyruvate kinase

thumb|right|[[Yeast pyruvate kinase ()]]

The final step of glycolysis is catalysed by pyruvate kinase to form pyruvate and another ATP. It is regulated by a range of transcriptional, covalent and non-covalent regulation mechanisms, which can vary widely. For example, in the liver, pyruvate kinase is regulated based on glucose availability. During fasting (no glucose available), glucagon activates protein kinase A which phosphorylates pyruvate kinase to inhibit it. An increase in blood sugar leads to secretion of insulin, which activates protein phosphatase 1, leading to dephosphorylation and re-activation of pyruvate kinase. Use is therefore made of two "shuttles" to transport the electrons from NADH across the mitochondrial membrane. They are the malate-aspartate shuttle and the glycerol phosphate shuttle. In the former the electrons from NADH are transferred to cytosolic oxaloacetate to form malate. The malate then traverses the inner mitochondrial membrane into the mitochondrial matrix, where it is reoxidized by NAD⁺ forming intra-mitochondrial oxaloacetate and NADH. The oxaloacetate is then re-cycled to the cytosol via its conversion to aspartate which is readily transported out of the mitochondrion. In the glycerol phosphate shuttle electrons from cytosolic NADH are transferred to dihydroxyacetone to form glycerol-3-phosphate which readily traverses the outer mitochondrial membrane. Glycerol-3-phosphate is then reoxidized to dihydroxyacetone, donating its electrons to FAD instead of NAD⁺. This occurs via the conversion of pyruvate into acetyl-CoA in the mitochondrion. However, this acetyl CoA needs to be transported into cytosol where the synthesis of fatty acids and cholesterol occurs. This cannot occur directly. To obtain cytosolic acetyl-CoA, citrate (produced by the condensation of acetyl CoA with oxaloacetate) is removed from the citric acid cycle and carried across the inner mitochondrial membrane into the cytosol. Cholesterol can be used as is, as a structural component of cellular membranes, or it can be used to synthesize the steroid hormones, bile salts, and vitamin D.

In the citric acid cycle all the intermediates (e.g. citrate, iso-citrate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate) are regenerated during each turn of the cycle. Adding more of any of these intermediates to the mitochondrion therefore means that that additional amount is retained within the cycle, increasing all the other intermediates as one is converted into the other. Hence the addition of oxaloacetate greatly increases the amounts of all the citric acid intermediates, thereby increasing the cycle's capacity to metabolize acetyl CoA, converting its acetate component into and water, with the release of enough energy to form 11 ATP and 1 GTP molecule for each additional molecule of acetyl CoA that combines with oxaloacetate in the cycle.

• Pyruvate:ferredoxin oxidoreductase, which converts pyruvate into acetyl-CoA and CO₂ while reducing the ferredoxin.
• Hydrogenase, which converts H⁺ into H₂ while oxidizing the ferredoxin.
• Acetyl-CoA synthetase (in reverse), which converts acetyl-CoA and ADP + Pi into acetate, coenzyme A, and ATP. (A variant of the reaction uses acetate:succinate CoA transferase and succinyl-CoA synthatase in reverse.)

The net reaction is conversion of singular equivalents of pyruvate, ADP, and Pi into ATP, CO₂, acetate, and H₂.

The following metabolic pathways are all strongly reliant on glycolysis as a source of metabolites: and many more.

• Pentose phosphate pathway, which begins with the dehydrogenation of glucose-6-phosphate, the first intermediate to be produced by glycolysis, produces various pentose sugars, and NADPH for the synthesis of fatty acids and cholesterol.
• Glycogen synthesis also starts with glucose-6-phosphate at the beginning of the glycolytic pathway.
• Glycerol, for the formation of triglycerides and phospholipids, is produced from the glycolytic intermediate glyceraldehyde-3-phosphate.
• Various post-glycolytic pathways:

:* Fatty acid synthesis

:* Cholesterol synthesis

:* The citric acid cycle which in turn leads to:

::*Amino acid synthesis

::*Nucleotide synthesis

::*Tetrapyrrole synthesis

Although gluconeogenesis and glycolysis share many intermediates the one is not functionally a branch or tributary of the other. There are two regulatory steps in both pathways which, when active in the one pathway, are automatically inactive in the other. The two processes can therefore not be simultaneously active. Indeed, if both sets of reactions were highly active at the same time the net result would be the hydrolysis of four high energy phosphate bonds (two ATP and two GTP) per reaction cycle.

Genetic diseases

Glycolytic mutations are generally rare due to importance of the metabolic pathway; the majority of occurring mutations result in an inability of the cell to respire, and therefore cause the death of the cell at an early stage. However, some mutations (glycogen storage diseases and other inborn errors of carbohydrate metabolism) are seen with one notable example being pyruvate kinase deficiency, leading to chronic hemolytic anemia.

In combined malonic and methylmalonic aciduria (CMAMMA) due to ACSF3 deficiency, glycolysis is reduced by −50%, which is caused by reduced lipoylation of mitochondrial enzymes such as the pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex.

Cancer

Malignant tumor cells perform glycolysis at a rate that is ten times faster than their noncancerous tissue counterparts. During their genesis, limited capillary support often results in hypoxia (decreased O2 supply) within the tumor cells. Thus, these cells rely on anaerobic metabolic processes such as glycolysis for ATP (adenosine triphosphate). Some tumor cells overexpress specific glycolytic enzymes which result in higher rates of glycolysis. Often these enzymes are Isoenzymes, of traditional glycolysis enzymes, that vary in their susceptibility to traditional feedback inhibition. The increase in glycolytic activity ultimately counteracts the effects of hypoxia by generating sufficient ATP from this anaerobic pathway. This phenomenon was first described in 1930 by Otto Warburg and is referred to as the Warburg effect. The Warburg hypothesis claims that cancer is primarily caused by dysfunctionality in mitochondrial metabolism, rather than because of the uncontrolled growth of cells.

A number of theories have been advanced to explain the Warburg effect. One such theory suggests that the increased glycolysis is a normal protective process of the body and that malignant change could be primarily caused by energy metabolism.

This high glycolysis rate has important medical applications, as high aerobic glycolysis by malignant tumors is utilized clinically to diagnose and monitor treatment responses of cancers by imaging uptake of 2-¹⁸F-2-deoxyglucose (FDG) (a radioactive modified hexokinase substrate) with positron emission tomography (PET).

There is ongoing research to affect mitochondrial metabolism and treat cancer by reducing glycolysis and thus starving cancerous cells in various new ways, including a ketogenic diet.

Interactive pathway map

The diagram below shows human protein names. Names in other organisms may differ, and the numbers of isozymes (such as HK1, HK2, ...) likely differ also.

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.

{| class="wikitable"

!

!colspan="2"|This article

!colspan="2"|Alternative

|-

|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

|Glycerone phosphate

|

|-

|6

|Glyceraldehyde-3-phosphate

|GADP

|3-Phosphoglyceraldehyde

|PGAL; G3P; GALP; GAP; TP

|-

|7

| 1,3-Bisphosphoglycerate

|1,3BPG

|Glycerate-1,3-bisphosphate,<br />glycerate-1,3-diphosphate,<br />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

|Pyruvic acid conjugate base

|

|}

Structure of glycolysis components in Fischer projections and polygonal model

The intermediates of glycolysis depicted in Fischer projections show the chemical changing step by step. Such image can be compared to polygonal model representation.

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Structure of glycolysis components in skeletal diagram and conservation-of-matter model

thumb|1200px|The Glycolysis pathway diagram illustrates the metabolic reactions that allow for the breakdown of glucose into pyruvate, often as preparation for further catabolic reactions.

The intermediates of glycolysis depicted in skeletal diagram show the chemical structures changing step by step, with cofactors such as NADH, ATP, and water and phosphates to balance reactions' stoichiometry. Each enzyme that mediates each reaction is indicated in the reversible arrow model of chemical reactions, as most enzymes catalyze bidirectional chemical reactions. Duplicates, such as the reversible re-arrangement between dihydroxyacetone and glyceraldehyde on the bottom row of reactions, represent two moles of C3 fragments derived from a single mole of the preceding C6 fragment of fructose bisphosphate, giving a net of two ATP generated. Thus the diagram must be read with rules of stoichiometry and balance-of-matter principles in mind. Follow the green "START" button to the red "END" button to trace the pathway through the structural pathway diagram.

See also

• Carbohydrate catabolism
• Citric acid cycle
• Cori cycle
• Fermentation (biochemistry)
• Gluconeogenesis
• Glycolytic oscillation
• Glycogenoses (glycogen storage diseases)
• Inborn errors of carbohydrate metabolism
• Pentose phosphate pathway
• Pyruvate decarboxylation
• Triose kinase

References

External links

• A Detailed Glycolysis Animation provided by IUBMB (Adobe Flash Required)
• The Glycolytic enzymes in Glycolysis at RCSB PDB
• Glycolytic cycle with animations at wdv.com
• Metabolism, Cellular Respiration and Photosynthesis – The Virtual Library of Biochemistry, Molecular Biology and Cell Biology
• The chemical logic behind glycolysis at ufp.pt
• Expasy biochemical pathways poster at ExPASy
• metpath: Interactive representation of glycolysis