Editing Glycolysis (section)

== Sequence of reactions ==
===Summary of reactions===
<div class="skin-invert-image"> 
{{Glycolysis|navbox=no|style=border: solid 1px #aaa; margin: 0.5em; font-size:90%}}
</div>
===Preparatory phase===<!-- This section is linked from [[Cellular respiration]] -->

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<ref name="glycolysis_animation"/> ([[glyceraldehyde 3-phosphate|G3P]]).
<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Hexokinase]] [[glucokinase]] ('''HK''')<br />''a [[transferase]]''
|reverse_enzyme=
|substrate={{sm|d}}-[[Glucose]] ('''Glc''')
|product=α-{{sm|d}}-[[Glucose-6-phosphate]] ('''G6P''')
|reaction_direction_(forward/reversible/reverse)=forward
|minor_forward_substrate(s)=[[Adenosine triphosphate|ATP]]
|minor_forward_product(s)=[[adenosine diphosphate|ADP]] + P<sub>i</sub>
|minor_reverse_substrate(s)=
|minor_reverse_product(s)=
|substrate_image=D-glucose wpmp.svg
|product_image=Alpha-D-glucose-6-phosphate wpmp.svg
}}}}
</div>
Once glucose enters the cell, the first step is phosphorylation of glucose by a family of enzymes called [[hexokinase]]s 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 [[animal]]s, an [[isozyme]] of hexokinase called [[glucokinase]] is also used in the liver, which has a much lower affinity for glucose (K<sub>m</sub> 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:'' Mg<sup>2+</sup>
{{clear}}{{hr}}
<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Phosphoglucoisomerase]] ('''PGI''')<br />''an [[isomerase]]''
|reverse_enzyme=
|substrate=α-{{sm|d}}-[[Glucose 6-phosphate]] ('''G6P''')
|product=β-{{sm|d}}-[[Fructose 6-phosphate]] ('''F6P''')
|reaction_direction_(forward/reversible/reverse)=reversible
|minor_forward_substrate(s)=
|minor_forward_product(s)=
|minor_reverse_substrate(s)=
|minor_reverse_product(s)=
|substrate_image=Alpha-D-glucose-6-phosphate wpmp.svg
|product_image=Beta-D-fructose-6-phosphate wpmp.png
}}}}
</div>
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).
{{clear}}{{hr}}
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Phosphofructokinase 1|Phosphofructokinase]] ('''PFK-1''')<br />''a [[transferase]]''
|reverse_enzyme=
|substrate=β-{{sm|d}}-[[Fructose 6-phosphate]] ('''F6P''')
|product=β-{{sm|d}}-[[Fructose 1,6-bisphosphate]] ('''F1,6BP''')
|reaction_direction_(forward/reversible/reverse)=forward
|minor_forward_substrate(s)= ATP
|minor_forward_product(s)= ADP + 
P<sub>i</sub>
|minor_reverse_substrate(s)=
|minor_reverse_product(s)=
|substrate_image=Beta-D-fructose-6-phosphate wpmp.png
|product_image=beta-D-fructose-1,6-bisphosphate_wpmp.svg
}}}}

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 [[PFP (enzyme)|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.<ref>{{cite journal | vauthors = Reeves RE, South DJ, Blytt HJ, Warren LG | title = Pyrophosphate:D-fructose 6-phosphate 1-phosphotransferase. A new enzyme with the glycolytic function of 6-phosphofructokinase | journal = The Journal of Biological Chemistry | volume = 249 | issue = 24 | pages = 7737–7741 | date = December 1974 | pmid = 4372217 | doi = 10.1016/S0021-9258(19)42029-2 | doi-access = free }}</ref> A rarer ADP-dependent PFK enzyme variant has been identified in archaean species.<ref>{{cite journal | vauthors = Selig M, Xavier KB, Santos H, Schönheit P | title = Comparative analysis of Embden-Meyerhof and Entner-Doudoroff glycolytic pathways in hyperthermophilic archaea and the bacterium Thermotoga | journal = Archives of Microbiology | volume = 167 | issue = 4 | pages = 217–232 | date = April 1997 | pmid = 9075622 | doi = 10.1007/BF03356097 | bibcode = 1997ArMic.167..217S | s2cid = 19489719 }}</ref>

''Cofactors:'' Mg<sup>2+</sup>
{{clear}}{{hr}}
{{Stack|margin=yes|{{Complex enzymatic reaction
|major_substrate_1=β-{{sm|d}}-[[Fructose 1,6-bisphosphate]] ('''F1,6BP''')
|major_substrate_1_stoichiometric_constant=
|major_substrate_1_image=beta-D-fructose-1,6-bisphosphate_wpmp.svg
|major_substrate_2=
|major_substrate_2_stoichiometric_constant=
|major_substrate_2_image=
|major_product_1={{sm|d}}-[[Glyceraldehyde 3-phosphate]] ('''GADP''')
|major_product_1_stoichiometric_constant=
|major_product_1_image=D-glyceraldehyde-3-phosphate wpmp.png
|major_product_2=[[Dihydroxyacetone phosphate]] ('''DHAP''')
|major_product_2_stoichiometric_constant=
|major_product_2_image=glycerone-phosphate_wpmp.png
|forward_enzyme=[[Fructose-bisphosphate aldolase]] ('''ALDO''')<br />''a [[lyase]]''
|reverse_enzyme=
|reaction_direction_(forward/reversible/reverse)=reversible
|minor_forward_substrate(s)=
|minor_forward_product(s)  =
|minor_reverse_product(s)  =
|minor_reverse_substrate(s)=
}}}}
Destabilizing the molecule in the previous reaction allows the hexose ring to be split by [[Fructose-bisphosphate aldolase|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.
{{clear}}{{hr}}
<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Triosephosphate isomerase]] ('''TPI''')<br />''an isomerase''
|reverse_enzyme=
|substrate=[[Dihydroxyacetone phosphate]] ('''DHAP''')
|product={{sm|d}}-[[Glyceraldehyde 3-phosphate]] ('''GADP''')
|reaction_direction_(forward/reversible/reverse)=reversible
|minor_forward_substrate(s)=
|minor_forward_product(s)  =
|minor_reverse_substrate(s)=
|minor_reverse_product(s)  =
|substrate_image=glycerone-phosphate_wpmp.png
|product_image=D-glyceraldehyde-3-phosphate wpmp.png
}}}}
</div>
[[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.
{{clear}}

===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.<ref name="glycolysis_animation"/> 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.

<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Glyceraldehyde phosphate dehydrogenase]] ('''GAPDH''')<br />''an [[oxidoreductase]]''
|reverse_enzyme=
|substrate=[[Glyceraldehyde 3-phosphate]] ('''GADP''')
|product={{sm|d}}-[[1,3-Bisphosphoglycerate]] ('''1,3BPG''')
|reaction_direction_(forward/reversible/reverse)=reversible
|minor_forward_substrate(s)=NAD<sup>+</sup> '''+''' P<sub>i</sub>
|minor_forward_product(s)=NADH '''+''' H<sup>+</sup>
|minor_reverse_substrate(s)=&nbsp;
|minor_reverse_product(s)=&nbsp;
|substrate_image=D-glyceraldehyde-3-phosphate wpmp.png
|product_image=1,3-bisphospho-D-glycerate.png
}}}}
</div>
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+|NAD<sup>+</sup>]], a hydrogen carrier, to give NADH '''+''' H<sup>+</sup> for each triose.

Hydrogen atom balance and charge balance are both maintained because the phosphate (P<sub>i</sub>) group actually exists in the form of a [[Phosphoric acid#Orthophosphoric acid chemistry|hydrogen phosphate]] anion ({{chem2|HPO4(2−)}}),<ref name="ImportanceBalance" /> which dissociates to contribute the extra H<sup>+</sup> ion and gives a net charge of -3 on both sides.

Here, [[arsenate]] ({{chem2|[AsO4](3-)}}), 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|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|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.<ref name = "Garrett_2012">{{Cite book|title=Biochemistry| vauthors = Garrett RH, Grisham CM |publisher=Cengage Learning | edition = 5th |year=2012|isbn=978-1-133-10629-6}}</ref>
{{clear}}{{hr}}
<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Phosphoglycerate kinase]] ('''PGK''')<br />''a [[transferase]]''
|reverse_enzyme=[[Phosphoglycerate kinase]] ('''PGK''')
|substrate=[[1,3-Bisphosphoglycerate]] ('''1,3BPG''')
|product=[[3-Phosphoglycerate]] ('''3PG''')
|reaction_direction_(forward/reversible/reverse)=reversible
|minor_forward_substrate(s)=ADP + H<sup>+</sup>
|minor_forward_product(s)=ATP
|minor_reverse_substrate(s)=&nbsp;
|minor_reverse_product(s)=&nbsp;
|substrate_image=1,3-bisphospho-D-glycerate.png
|product_image=3-phospho-D-glycerate wpmp.png
}}}}
</div>
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<sup>−</sup>, and ATP as ATPMg<sup>2−</sup>, balancing the charges at −5 both sides.

''Cofactors:'' Mg<sup>2+</sup>
{{clear}}{{hr}}
<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Phosphoglycerate mutase]] ('''PGM''')<br />''a [[mutase]]''
|reverse_enzyme=
|substrate=[[3-Phosphoglycerate]] ('''3PG''')
|product=[[2-Phosphoglycerate]] ('''2PG''')
|reaction_direction_(forward/reversible/reverse)=reversible
|minor_forward_substrate(s)=
|minor_forward_product(s)=
|minor_reverse_substrate(s)=
|minor_reverse_product(s)=
|substrate_image=3-phospho-D-glycerate wpmp.png
|product_image=2-phospho-D-glycerate_wpmp.png
}}}}
</div>
[[Phosphoglycerate mutase]] isomerises [[3-phosphoglycerate]] into [[2-phosphoglycerate]].
{{clear}}{{hr}}
<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Enolase]] ('''ENO''')<br />''a [[lyase]]''
|reverse_enzyme=[[Enolase]] ('''ENO''')
|substrate=[[2-Phosphoglycerate]] ('''2PG''')
|product=[[Phosphoenolpyruvate]] ('''PEP''')
|reaction_direction_(forward/reversible/reverse)=reversible
|minor_forward_substrate(s)=
|minor_forward_product(s)= H<sub>2</sub>O
|minor_reverse_substrate(s)= &nbsp;
|minor_reverse_product(s)=
|substrate_image=2-phospho-D-glycerate_wpmp.png
|product_image=phosphoenolpyruvate_wpmp.png
}}}}
</div>
[[Enolase]] next converts [[2-phosphoglycerate]] to [[phosphoenolpyruvate]]. This reaction is an elimination reaction involving an [[E1cB-elimination reaction|E1cB]] mechanism.

''Cofactors:'' 2 Mg<sup>2+</sup>, one "conformational" ion to coordinate with the carboxylate group of the substrate, and one "catalytic" ion that participates in the dehydration.
{{clear}}{{hr}}
<div>
{{Stack|margin=yes|{{Enzymatic Reaction
|forward_enzyme=[[Pyruvate kinase]] ('''PK''')<br />''a [[transferase]]''
|reverse_enzyme=
|substrate=[[Phosphoenolpyruvate]] ('''PEP''')
|product=[[Pyruvate]] ('''Pyr''')
|reaction_direction_(forward/reversible/reverse)=forward
|minor_forward_substrate(s)=ADP + H<sup>+</sup>
|minor_forward_product(s)=ATP
|minor_reverse_substrate(s)=
|minor_reverse_product(s)=
|substrate_image=phosphoenolpyruvate_wpmp.png
|product_image=pyruvate_wpmp.png
}}}}
</div>

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:'' Mg<sup>2+</sup>
{{clear}}

=== 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.<ref>{{Cite book | vauthors = Berg JM, Tymoczko JL, Stryer L | title = Biochemistry | place = New York | publisher = Freeman | year = 2007 | edition = 6th | page = 622 | isbn = 978-0-7167-8724-2 }}</ref>  Conversely, [[triglyceride]]s can be broken down into fatty acids and glycerol; the latter, in turn, can be [[Glycerol#Metabolism|converted]] into dihydroxyacetone phosphate, which can enter glycolysis ''after'' the second control point.

=== Free energy changes ===
{| align="right" class="wikitable"
|+ Concentrations of metabolites in [[Red blood cell|erythrocytes]]<ref name = "Garrett_2005">{{Cite book | vauthors = Garrett R, Grisham CM | title = Biochemistry | place = Belmont, CA | publisher = Thomson Brooks/Cole | year = 2005 | edition = 3rd | page = 584 | isbn = 978-0-534-49033-1 }}</ref>{{rp|584}}
! 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<sub>i</sub>
|1.0
|}

The change in free energy, Δ''G'', for each step in the glycolysis pathway can be calculated using Δ''G'' = Δ''G''°′ + ''RT''ln ''Q'', where ''Q'' is the [[reaction quotient]].  This requires knowing the concentrations of the [[Metabolomics|metabolites]].  All of these values are available for [[Red blood cell|erythrocytes]], with the exception of the concentrations of NAD<sup>+</sup> and NADH.  The ratio of [[NADH|NAD<sup>+</sup> 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).

{| class="wikitable"
|+ Change in free energy for each step of glycolysis<ref name = "Garrett_2005" />{{rp|582–583}}
! Step
! Reaction
! colspan=2|Δ''G''°′ <br> (kJ/mol)
! colspan=2|Δ''G'' <br> (kJ/mol)
|-
| 1
| Glucose + ATP<sup>4−</sup> → Glucose-6-phosphate<sup>2−</sup> + ADP<sup>3−</sup> + H<sup>+</sup>
| {{decimal cell|−16.7}}
| {{decimal cell|−34}}
|-
| 2
| Glucose-6-phosphate<sup>2−</sup> → Fructose-6-phosphate<sup>2−</sup>
| {{decimal cell|1.67}}
| {{decimal cell|−2.9}}
|-
| 3
| Fructose-6-phosphate<sup>2−</sup> + ATP<sup>4−</sup> → Fructose-1,6-bisphosphate<sup>4−</sup> + ADP<sup>3−</sup> + H<sup>+</sup>
| {{decimal cell|−14.2}}
| {{decimal cell|−19}}
|-
| 4
| Fructose-1,6-bisphosphate<sup>4−</sup> → Dihydroxyacetone phosphate<sup>2−</sup> + Glyceraldehyde-3-phosphate<sup>2−</sup>
| {{decimal cell|23.9}}
| {{decimal cell|−0.23}}
|-
| 5
| Dihydroxyacetone phosphate<sup>2−</sup> → Glyceraldehyde-3-phosphate<sup>2−</sup>
| {{decimal cell|7.56}}
| {{decimal cell|2.4}}
|-
| 6
| Glyceraldehyde-3-phosphate<sup>2−</sup> + P<sub>i</sub><sup>2−</sup> + NAD<sup>+</sup> → 1,3-Bisphosphoglycerate<sup>4−</sup> + NADH + H<sup>+</sup>
| {{decimal cell|6.30}}
| {{decimal cell|−1.29}}
|-
| 7
| 1,3-Bisphosphoglycerate<sup>4−</sup> + ADP<sup>3−</sup> → 3-Phosphoglycerate<sup>3−</sup> + ATP<sup>4−</sup>
| {{decimal cell|−18.9}}
| {{decimal cell|0.09}}
|-
| 8
| 3-Phosphoglycerate<sup>3−</sup> → 2-Phosphoglycerate<sup>3−</sup>
| {{decimal cell|4.4}}
| {{decimal cell|0.83}}
|-
| 9
| 2-Phosphoglycerate<sup>3−</sup> → Phosphoenolpyruvate<sup>3−</sup> + H<sub>2</sub>O
| {{decimal cell|1.8}}
| {{decimal cell|1.1}}
|-
| 10
| Phosphoenolpyruvate<sup>3−</sup> + ADP<sup>3−</sup> + H<sup>+</sup> → Pyruvate<sup>−</sup> + ATP<sup>4−</sup>
| {{decimal cell|−31.7}}
| {{decimal cell|−23.0}}
|}

From measuring the physiological concentrations of metabolites in an erythrocyte it seems that about seven of the steps in glycolysis are in equilibrium for that cell type. Three of the steps—the ones with large negative free energy changes—are not in equilibrium and are referred to as ''irreversible''; such steps are often subject to regulation.

Step 5 in the figure is shown behind the other steps, because that step is a side-reaction that can decrease or increase the concentration of the intermediate glyceraldehyde-3-phosphate.  That compound is converted to dihydroxyacetone phosphate by the enzyme triose phosphate isomerase, which is a [[kinetic perfection|catalytically perfect]] enzyme; its rate is so fast that the reaction can be assumed to be in equilibrium.  The fact that Δ''G'' is not zero indicates that the actual concentrations in the erythrocyte are not accurately known.