Editing Glycolysis (section)

===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>
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<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).
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{{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>
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{{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.
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<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.
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