14.01 Glycolysis

14.
1: GLYCOLYSIS
14.1.1: INTRODUCTION
In this chapter, we will provide you with a historical overview of glycolysis and introduce you to the 10 enzymatic reactions in the pathway.
Our main goal is to understand how the oxidation of our major food molecules, sugars in the case of glycolysis, can lead to ATP synthesis.
Before we begin our journey into the glycolytic pathway, it is useful to review the concept of free energy within reactions. For a reaction to
be spontaneous, the change in the free energy within the system must be negative. From the equation in Figure 14.1.1 you can see that the
change in free energy of a reaction is dependent on concentration of the reactants and the products, as well as the temperature within the
system.
Figure 14.1.1 : The Change in Free Energy of a Reaction. The change in the free energy of a reaction is equal to the standard free
energy change for the reaction plus the gas constant (R) multiplied with the Temperature in Kelvin (T) and the natural log (ln) of the
concentrations of the products of the reaction (noted as C and D in the example) over the concentration of the reactants (noted as A and B in
the example).
You will note that some reactions of glycolysis and other metabolic pathways that we will investigate are not favored. Thus, it is necessary
to drive the reaction to become spontaneous by either coupling the reaction with a spontaneous reaction that can generate enough free
energy to drive the nonspontaneous reaction forward, or by using Le Chatelier’s principle and remove the products from the enzyme’s area
as soon as they are made. This will help drive the reaction in the forward direction as it will reestablish equilibrium. In this way, a small
amount of product can be formed spontaneously. Essentially, the continued removal of product or addition of excess reactant, will drive the
reaction forward. It is of note that the temperature of the reaction can also influence the change in free energy. However, in biological
systems, temperature changes that will significantly affect the change in free energy usually are not compatible with maintaining most life
forms. Thus, it will not be a large consideration in the context of our metabolic discussions, here.
When reactions are coupled together to obtain a spontaneous reaction, the overall free energy change for a chemically coupled series of
reactions is equal to the sum of the free energy changes of the individual steps, as noted in Figure 14.1.2 .
Figure 14.1.2 : The Coupling of Two Reactions. The top reaction is nonspontaneous with a positive change in free energy of 21 kJ/mol.
This reaction can be driven in the forward direction, by coupling it with a strongly spontaneous reaction that releases -34 kJ/mol. The
overall net reaction has a negative change in free energy and is spontaneous
The metabolic reactions of carbohydrates and other food molecules play an important role in generating energy within living systems in the
form of ATP. For carbohydrates, this begins with the metabolic process known as glycolysis (or the breakdown,’lysis’, of sugars, ‘glyco’) At
the end of the 1910s Otto Meyerhof mapped some of these metabolic conversions by measuring heat trends and oxygen consumption in frog
muscles. When the muscle is working, lactic acid is formed from carbohydrates, and Otto Meyerhof showed that during recovery, this is
followed partly by the burning of lactic acid and partly by reprocessing of lactic acid to carbohydrates.
Concurrently, Archibald Hill was also outlining these processes in the muscles of frogs. In opposition to the prevailing view that mechanical
movement and chemical processes were parallel sequences, Hill was able to show through measurements of heat generated by the
mechanical processes that these were delayed in relation to the movements. The chemical sequence consists of a work phase, which is not
dependent on oxygen supply, and a recovery phase, when oxygen is required. Together, their work has opened the door to understanding
aerobic and anaerobic metabolism beginning with the process of glycolysis. They both shared the Nobel Prize in Physiology and Medicine
in 1922 for their work in these processes Figure 14.1.3 .
14.1.1 https://bio.libretexts.org/@go/page/15004
Figure 14.1.3 : The Nobel Prize for Physiology and Medicine in 1922. Archibald V. Hill and Otto Fritz Meyerhof received this award
for their work on sugar metabolism and breakdown. Photos from Nobel Prize.Org
The glycolytic pathways consists of 10 enzymatic steps that convert glucose to pyruvate. This conversion generates a small amount of
energy. The pyruvate can then be converted to lactic acid (lactate) in vertebrates or to ethanol in yeast in an anaerobic (or oxygen-
independent) pathway or it can be fully oxidized to carbon dioxide in an aerobic (or oxygen-requiring) pathway that takes place within the
mitochondria (which is shown in green in Figure 14.1.4 : ). Aerobic oxidation yields about 18 times as much energy as the anaerobic
pathways causing them to be favored over anaerobic pathways. In fact, most animal tissues can only survive short anaerobic bursts that
occur in isolation and don’t involve the entire organism. The aerobic pathway is required to sustain life. Yeast also prefer to grow using the
aerobic, mitochondrial pathway. However, if oxygen is unavailable, yeast and other fungi can switch to anaerobic growth and produce
ethanol as a byproduct. The production of alcoholic beverages through this fermentation process is quite popular.
Figure 14.1.4 : Overview of Aerobic and Anaerobic Oxidation of Glucose. Figure modified from Kim, Y. et al (2011) PLoS ONE
6(12):e28293
Figure 14.1.5 provides a summary of the glycolytic pathway coupled to the oxidative phosphorylation pathway that occurs within the
mitochondria.
Figure 14.1.5 : A Summary of the Glycolytic and Oxidative Phosphorylation Pathways. The glycolytic pathway is shown on the left
hand side in blue. During aerobic metabolism, pyruvate would be converted to acetyl-CoA where it would then enter into the Kreb cycle
within the matrix of the mitochondria. Future chapters will focus on the reactions inside the mitochondria. Our focus in this chapter will be
on the cytosolic reactions of the glycolytic pathway.
The major reactions of glycolysis are shown in Figure 14.1.6 . The pathway can be broken down into two major sections: (1) the energy
consuming reactions and the (2) energy generating reactions. People always say the old adage that ‘It takes money to make money’. The
same can be thought about the glycolytic pathway. The first section requires an investment of energy, in order to generate energy in the
second half of the reaction pathway. In this pathway, glucose, a 6 carbon hexose, is converted to two, 3C molecules - pyruvate. Note that
Figure 14.1.6 shows the entire pathway using Lewis wedge/dash representations plus the anaerobic conversion of pyruvate to lactate.
OH
O
glucose HO OH
HO OH
OH glyeraldehyde 3-phosphate -2
O3PO O
G3P
ATP
1. hexokinase NAD+ Pi
6. glyceraldehyde-3P
ADP
dehydrogenase
NADH + H+
CH2OPO3-2
HO O
O
glucose-6-P HO O
G6P HO OH
1,3-bisphosphoglycerate -2
O3PO O P O-
OH
1,3-BPG
-
O
2. phosphoglucose ADP
isomerase
-2 HO 7. phosphglycerate ATP
O3PO kinase
O 1
6 OH
fructose-6-P -2
OH 3-phosphoglycerate O3PO O
F6P
3PG
HO OH O -
ATP
3. phosphofructokinase 8. phosphglycerate
mutase
ADP
OPO3-2
-2
-2 O3PO HO O
O3PO 2-phosphoglycerate
O 1 2PG
fructose-1,6- 6
O-
bisP
F-1,6-BP OH
HO OH 9. enolase
H2O
-
4. aldolase -
O O
P
phosphoenol O O
O OH
-2 pyruvate PEP
O3PO OH -2
O3PO O O
1 -
glyeraldehyde 3-phosphate O
dihydroxyacetone phosphate G3P
DHAP ADP
5. triose phosphate 10. pyruvate
isomerase kinase
ATP
O O
pyruvate
C
lactate O
dehydrogenase
O- NADH
NAD+ + H+
HO O
lactate
Figure 14.1.6 : Glycolytic pathway. The chemical steps of the glycolytic pathway are shown using Lewis wedge/dash representations.
Enzymes required at each step are labeled in red. The energy consuming stage encompass reactions 1 - 3, whereas the energy producing
reactions occur in the second half of the pathway from reactions 4 - 10. The conversion of pyruvate to lactate by lactate dehydrogenase
represents anaerobic respiration as it occurs within mammalian species.
Glycolysis is the key anaerobic pathways for energy products in all organisms, except for lithotropes that use the oxidation of inorganic
molecules for energy production. In aerobic systems, glycolysis provides the release of fast energy within the body as glycogen metabolism
can quickly release free glucose for utilization. Given the centrality of glycolysis to all of life, we will explore each of the reactions in detail
below.
14.1.2: REACTION 1: GLUCOSE → GLUCOSE-6-PHOSPHATE. ΔGO=-4.0 KCAL/MOL.

The first step in glycolysis is catalyzed by enzymes known as hexokinases. The hexokinase family enzymes typically have broad specificity
for various hexoses and catalyze the phosphorylation of six-carbon position. Hexokinase phosphorylates glucose using ATP as the source of
the phosphate, producing glucose-6-phosphate, a more reactive form of glucose. Notably, this reaction prevents the phosphorylated glucose
molecule from continuing to interact with the GLUT transport proteins that can shuttle glucose into and out of the cell. Thus, once glucose
is phosphorylated it can no longer leave the cell. Do note that both of these sugar forms (the free sugar and the phosphorylated version) can
shift back and forth between the ring-closed and ring-opened conformation. Hexokinases require glucose to be in the closed conformation
for the phosphorylation reaction. Figure 14.1.7 shows the reaction which is catalyzed by hexokinase (a kinase that transfers the γ-
phosphate from ATP to a hexose in the ring-closed conformation).
OH CH2OPO3-2
ATP ADP
O O
HO HO
HO OH HO OH
OH 1. hexokinase OH
glucose-6-P
glucose G6P
Figure 14.1.7 : Summary reaction - hexokinase

This reaction is a nucleophilic substitution reaction on the gamma phosphate of ATP. A phosphoanhydride bond is broken in ATP as a
phosphoester bond is made producing glucose 6-phosphate. Hence the reaction proceeds with a negative ΔGo.
Vertebrates have 4 different types of hexokinases, I-IV (also called A-D). The regulation of these enzymes are presented in more detail in
Chapter 15.5. Hexokinases I-III bind glucose more tightly as reflected by low KM values. Type IV or glucokinase, binds it less tightly, and is
found in high concentrations in vertebrate livers. Yeast have 3 isozymes, P1, PII or hexokinase B, and glucokinase. Most prokaryotic
hexokinases are in the glucokinase (Type IV) class.
In addition to hexokinases, there are also many other different types of sugar kinase enzymes. Notably, all sugar kinases must prevent the
spurious hydrolysis of ATP by water, which can also be considered a phosphotransfer to water, instead of the preferred transfer of the γ
phosphate of ATP to a sugar ROH (alcohol), an "alcoholysis" reaction. Hexokinase does this through an "induced fit" mechanism, in which
glucose binding triggers a large conformation change to close off the active site to water.
This conformational change is illustrated in Figure 14.1.8 below which shows an interactive iCn3D model of the yeast hexokinase PI in in
the absence (2YHX) and presence (3B8A) of glucose.
NCBI iCn3D
Figure 14.1.8 : Yeast hexokinase PI in in the absence (2YHX) and presence (3B8A) of glucose. (Copyright; author via
source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...84YGEd1ob3th7A
Within Figure 14.1.8 , the gray structure is hexokinase without glucose (2YHX). However, it actually has a glucose analog bound, o-
tolyoylglucosamine (spacefill, yellow highlights), which apparently binds at the glucose binding site but does not cause a subsequent
conformation change. The cyan structures shows the enzyme with glucose shown as colored sticks. Note the large conformational change on
the actual binding of glucose, a classical example of an "induced fit" mechanism.
Figure 14.1.9 shows the surface of both enzymes using the same color coding as in the above figure These images better show how the
active site of hexokinase is occluded in the glucose-bound form. This prevents water access and hydrolysis of bound ATP instead of
"alcoholysis" of ATP (transfer of phosphate to glucose). The gray structure on left has the glucose analog bound which doesn't alter the
global conformation of the protein.
Figure 14.1.9 : Surface of human hexokinase I with bound o-tolyoylglucosamine (spacefill, left, 2YHX) and with bound glucose
(spacefill, right, 3B8A)
Hexokinase I is the key form in the brain. It forms a complex with porin in the mitochondrial outer membrane and ATP/ADP translocase or
carrier protein in the mitochondrial inner membrane which facilitates the hexokinase reaction. The mechanism for the reaction of human
hexokinase I is shown in Figure 14.1.10 below.
H H
H O H
O O H
H Mg 2+
H
H O O
H H
ATP O-
- -
O R
N O P O- O
N Glucose
P O P O O H
H 2N O O O
N O
N O-
OH
HO H 2N NH2+ HO O
HN 603Ser Asp 657
Arg 539
H H
H O H
O O H
H Mg 2+ O-
H - Glucose-6-P
H O O P
O O
-
H H O R
-
-
O O
N
N H
P O P O-
H 2N O O O O H
N O H
N O
OH
HO H 2N NH2+ HO O
ADP
HN 603Ser Asp 657
Arg 539
Figure 14.1.10 : Mechanism of human hexokinase I.

Ribeiro AJM et al. (2017), Nucleic Acids Res, 46, D618-D623. Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme
reaction mechanisms and active sites. DOI:10.1093/nar/gkx1012. PMID:29106569. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/696/.
Creative Commons Attribution 4.0 International (CC BY 4.0) License.
Figure 14.1.11 below shows an interactive iCn3D model of human hexokinase I with glucose and ADP in the active site (1dgk).
NCBI iCn3D
Figure 14.1.11 : Human hexokinase I with glucose and ADP in the active site (1dgk). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...aQy4gf6A2bWtp8
The key catalytic residue are shown in colored sticks and labeled. Glucose, ADP and PO42- are shown as colored spheres.
Note that hexokinase I, as well as forms II and III, have spatially distinct halves similar to those in the yeast enzymes. Forms I-III have a
molecule weight of about 100K. Form IV (glucokinase) has a molecular weight of 50K and is similar to the distinct halves of I-III.
suggesting that I-III arose through gene duplication.
Each half also binds glucose and ADP but the N-terminal of I and III are catalytically inactive. Hexokinase II, with both halves active, is the
main enzyme involved in glycolysis in most mammalian tissues. Other difference include that facts that glucose-6-phosphate, a product,
inhibits I-III. PO42- relieves G6P product inhibition in form I but not the others. ADP binding at multiple sites in hexokinase I likely causes
conformational change that affect structure and activity.
14.1.3: SUGAR KINASES IN GENERAL
We will see many kinases that use ATP to phosphorylate sugars, so it useful to explore both their differences and similarities at the
beginning of our studies on carbohydrate metabolism.
Figure 14.1.12 below shows comparative structures of the five carbohydrate kinase classes.
Figure 14.1.12 : Structures of the five carbohydrate kinase classes. All images are shown in rainbow format (blue: N-terminus, red: C-
terminus). Roy, S.; Vivoli Vega, M.; Harmer, N.J. Carbohydrate Kinases: A Conserved Mechanism Across Differing Folds. Catalysts 2019,
9, 29. https://doi.org/10.3390/catal9010029. Creative Commons Attribution License
Panel (a) shows the structure of human glucokinase (hexokinase class; PDB (protein data bank) ID: 4IWV).
Panel (b) shows the structure of Bacillus subtilis fructokinase dimer: second molecule shown in raspberry (ROK kinase class; PDB ID:
1XC3 [17]).
Panel (c) shows the structure of Escherichia coli ribokinase (ribokinase class; PDB ID: 1RKD; [18]).
Panel (d) shows the structure of Aquifex aeolicus IspE (GHMP kinase class; PDB ID: 2V2Z; [19]).
Panel (e) shows the structure of human PIK3C3 (phosphatidylinositol phosphate kinase class; PDB ID: 3IHY).
Table 14.1.1 : Overview of the Five Carbohydrate Kinase Classes.
Carbohydrate Common Substrates Native Phosphate Donors (Minor Donors Pfam ID

Kinase Family in Parentheses)
Hexokinase Glucose, mannose, fructose ATP (ITP) PF00349, PF03727,

PF02685
ROK Kinase Glucose, allose, fructose, N-acetylglucosamine, N- ATP (polyphosphate) PF00480

acetylmannosamine
Ribokinase Ribose, 2-deoxy-d-ribose, adenosine ATP, ADP (GTP, ribonucleotide) PF00294
GHMP Kinase Galactose, N-acetylgalactosamine, ATP (GTP, ITP) PF00288
Phosphatidylinositol Phosphatidylinositol, phosphatidylinositol ATP (GTP) PF00454, PF01504

kinase phosphates
ROK is bacterial Repressor, Open reading frame, Kinased are predominantly bacterial enzymes. Ribokinases include adenosine kinases,
fructokinases and phosphofructokinases. GHMP Kinases include Galactokinase, Homoserine kinase, Mevalonate kinase, and
Phosphomevalonate kinase. Roy et al. Ibid
Many of these kinases are regulated by binding of allosteric effectors.
Figure 14.1.13 shows a common mechanism for all, using glucose as an example substrate.
this, hexokinasesare generally agreed to show a random sequential m
may bind rst [55]. Five conservedmotifs bind to the nucleotide, in
ts [54], which also coordinate an Mg2+ ion binding to the nucleotide
Figure 14.1.13 : Generic reaction mechanism for carbohydrate kinases (hexokinase shown). Roy et al, Ibid. The generic reaction is
initiated by a catalytic base abstracting a proton from the reactive hydroxyl (left). The oxygen atom then attacks the -phosphate of ATP
(second left), forming a pentacoordinate transition state (second right). This is stabilized by a divalent cation, and by the protein (not
shown). This transition state resolves leaving ADP and the phosphorylated carbohydrate (right).
14.1.4: REACTION 2: GLUCOSE-6-PHOSPHATE ↔ FRUCTOSE-6-PHOSPHATE. ΔGO=+0.4 KCAL/MOL

In the second step of glycolysis, the phosphoglucose isomerase (PGI) converts glucose-6-phosphate into one of its isomers, fructose-6-
phosphate. Recall that an isomerase is an enzyme that catalyzes the conversion of a molecule into one of its isomers. In this reaction the
aldose, glucose-6-phosphate, is converted to the ketose, fructose-6-phosphate. This conversion is essential to allow the eventual split of the
sugar into two three-carbon molecules. We'll present this reaction, catalyzed by phosphoglucose isomerase (PGI), in several different types
of representations (chair, wedge/dash and Fischer projection), as shown in Figure \(\PageIndex{14}\) below.
HO H O CH2OH
CH2OPO3-2 2. phosphoglucose -2
O3PO
isomerase 1 O
6 O H OH
O HO H
HO glucose-6-P HO H fructose-6-P
OH H OH
HO OH G6P H OH F6P
OH 2. PHOSPHOGLUCOSE H OH O
HO OH H OH O ISOMERASE
H2C O P O
glucose-6-P H2C O P O
G6P fructose-6-P O
O
F6P
Figure 14.1.14 : Summary reaction, phosphoglucoisomerase

From a functional perspective, see more clearly from the linear Fischer structure, it is evident that the C=O has been moved to the C2
position to create the ketose structure. The carbonyl O is now positioned to be an electron sink facilitating electron flow for reaction 4. This
isomerization reaction would be expected to have a ΔGo of about 0.
It has been proposed that the mechanism of PGI requires the enzyme to open the Glucose-6-Phosphate ring before the actual isomerization
step, which proceeds through the formation of a cis-enediol intermediate and keto-enol tautomers before conversion back to the cyclic form.
Figure 14.1.15 below shows a proposed mechanism for rabbit phosphoglucose isomerase
Lys 518
210Lys Lys 518

O O-
P Arg 272
NH3+ -
O O NH2+
H O O-
+ P
O H2N -
O O NH2
NH
G6P HO O
H H2N
OH
HO OH HO O
His388
O- N
HO H OH H His388
O HN O- N+
-
O
Glu357 O HN
O
Glu357
Glu216
Lys 518
Lys 518
O O-
O O- P
P -
-
O O NH2
O O NH2
H OH
O HO O-
HO OH
HO O H
HO O H H
O His388
H His388
O- N+
N+
O
O HN
HN
Glu357
Glu357
Lys 518
O O-
P
-
O O NH3+
O
HO OH
HO OH
O - His388
N
O
HN
Glu357
Figure 14.1.15 : Mechanism for phosphoglucose isomerase. Ribeiro AJM et al. Ibid
Figure 14.1.16 below shows an interactive iCn3D model of rabbit phosphoglucose isomerase with bound 6-phosphogluconic acid
(1DQR)
NCBI iCn3D
Figure 14.1.16 : Rabbit phosphoglucose isomerase with bound 6-phosphogluconic acid (1DQR). (Copyright; author via
source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...xo1SgF1ZpAySm9
One monomer of the dimer is shown in gray and the other in cyan. The catalytic residues Lys518 and His388 are shown in both subunits in
colored sticks and labeled. Additional residues that contribute to specificity (Ser209, Ser159, Thr214, Thr217, and Thr211) are shown in the
gray subunit with color sticks and labeled. 6-phosphogluconic acid, a competitive inhibitor, is shown in color spacefill.
The enzyme also has other functions in addition to its role in glycolysis, so it is a member of a group called "moonlighting" (a term that
refers to working at a secondary job). proteins. Outside of the cell, phosphogluco isomerase acts as a nerve growth factor and cytokine. It is
also called autocrine motility factor (PGI/AMF) and its cytokine activity is associated with aggresive cancers.
14.1.5: REACTION 3: FRUCTOSE-6-PHOSPHATE → FRUCTOSE-1,6-BISPHOSPHATE. ΔGO=-3.4

KCAL/MOL
The third step is the phosphorylation of fructose-6-phosphate, catalyzed by the enzyme phosphofructokinase-1 (PFK1). A second ATP
molecule donates a high-energy phosphate to fructose-6-phosphate, producing fructose-1,6-bisphosphate. The term bisphosphate is used
when two phosphate groups are joined to a molecule at different positions on the molecule. In this case, one phosphate is at the 1-carbon
position and the other is at the 6-carbon position. This differs from the term diphosphate, that is used when the phosphate groups are joined
in a sequence, as in the molecule ADP. In ADP, both phosphate groups are joined in tandem from the 5-carbon position on the ribose ring
structure.
In the glycolytic pathway, PFK1 is a rate-limiting enzyme. The mechanism of PFK1 regulation is discussed in greater detail in Chapter 15.5
however, we will introduce the process here. Essentially, the enzyme is sensitive to the energy load within the cell. Recall that ATP is a
recycled molecule. There is a fixed amount of it within the body. Thus, it is constantly fluctuating between the states of ATP, ADP, and
AMP. Thus, the whole purpose of glucose oxidation, is to convert ADP back into ATP regenerating this energy rich molecule. PFK1
activity, therefore, is sensitive to the ATP:ADP ratio within the cell. PFK1 is more active when the concentration of ADP is high and the
concentration of ATP is low, and it is less active when ADP levels are low and the concentration of ATP is high. This later state is indicative
of energy availability within the cell. Thus, the cell does not currently need to produce more energy and reducing PFK1 activity slows the
glycolytic pathway down. This is a type of end product inhibition, since ATP is the end product of glucose catabolism. Note, however, that
some ATP must be present for the reaction to move forward, as ATP is also a substrate for PFK1. ATP serves as the phosphate donor in the
reaction and is required in the process. This is the second energy intensive step in the glycolytic pathway. At the end of the PFK1 step, a
total of 2 ATP molecules have been broken down in the glycolytic pathway.
The PFK1 enzymatic step is also important within the glycolytic pathway as it is the committed step in the pathway. Glucose-6-phosphate
may be used for other purposes within the cell, and the isomerase step that converts glucose-6-phosphate to fructose-6-phosphate is readily
reversible. The PFK1 enzyme only works in the forward direction to create fructose 1,6-bisphosphate. It cannot in the reverse direction and
recover the reactant. Thus, it is thought of as the committed step in the glycolytic pathway as the fructose-1,6-bisphosphate will
predominantly be converted into pyruvate through the remaining enzymatic steps.
The reaction catalyzed by PFK1, is shown in Figure 14.1.17 below.
-2 HO -2
O3PO
O3PO -2
O3PO
O 1 ATP 1
6 ADP O
6
OH OH
3. phosphofructokinase
HO OH HO OH
fructose-6-P fructose-1,6-bisP
F6P F-1,6-BP
Figure 14.1.17 : Summary reaction - phosphofructokinase

By phosphorylating this intermediate, both products of the cleavage of this 6C molecule will be phosphorylated, keeping both more readily
inside the cell. This reaction is a nucleophilic substitution reaction on the gamma phosphate of ATP. A phosphoanhydride bond is broken in
ATP as a phosphoester bond is made producing fructose-1,6-bisphosphate. As in reaction 1, this reaction proceeds with a negative ΔGo
The PFK1 enzyme exists as a tetramer and as with another "famous" tetramer, hemoglobin, it can exist in T and R symmetry states. Hence it
is an allosteric enzyme and has many allosteric regulators. One important allosteric activator of eukaryotic (but not prokaryotic) PFK is
fructose-2,6-bisphosphate. This is formed by a separate phosphofructokinase enzyme named PFK2. This regulatory pathway will be
described in greater detail in Chapter 15.5. Hence the number 1 is added to the name of the glycolytic enzyme, PFK1, that forms fructose
1,6-bisphosphate, to indicate the position of phosphorylation. Mammals have three isoform of PFK1, muscle (PFKM), liver (PFKL) and
platelet (PFKP) .
The mechanism for E. Coli phosphofructokinase is shown in Figure 14.1.18 below. A metal cofactor (Mg2+) coordinates substrate
positioning of the ATP and stabilizes the gamma phosphate for nucleophilic attack by the fructose alcohol group at position-1. Activation of
the fructose alcohol group is mediated by proton abstraction with a coordinated Asp127 of PFK1.
Arg72
Arg171
-
O NH
O- R Gly11
O P +
H2 N NH2
O NH O HN R
F6P O
+
H2 N NH2
Thr125
OH
HO HO
O O-
OH H O P O O O HO
P OH
O- O- ATP
-
O H O P O
O O-
O H O N
H N
Mg2+ O
Asp127 - H N
O
O O NH2
H O O- N
H
Asp129 Asp103
-
O
O-
O P
O F-1,6-BP
O
OH
HO ADP
O O
OH P - HO O HO
H H -
O O P O OH
O H
O O- O P O
H -
O O O
O H N N
H Mg2+ O
Asp127 - H N
O
O O NH2
H O O- N
H
Asp129 Asp103
-
O
O-
O P
O
O F1,6-BP
OH
HO O ADP
O
OH P - HO O HO
H H -
O O P O OH
O+ -
O O- O P O
H H
O O- O
O H N N
H Mg 2+ O
Asp127 - H N
O
O O NH2
H O O- N
H
Asp129 Asp103
Figure 14.1.18 : Proposed mechanism for E. Coli phosphofructokinase. Ribeiro AJM et al. Ibid
Figure 14.1.19 below shows an interactive iCn3D model of the E. Coli phosphofructokinase with bound F1,6-bisphosphate and ADP
products (1PFK). (long load)
NCBI iCn3D
Figure 14.1.19 : E. Coli phosphofructokinase with bound F1,6-bisphosphate and ADP products (1PFK). (Copyright;
author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...t4KKgLAGzHrzo8
Each monomer of the tetramer is shown in a different color. The gray monomers shows key binding and catalytic residues described in the
mechanism above. F1,6-bisphosphate is shown as spacefill and ADP is shown as sticks.
The structure of the human platelet PFK1 tetramer has been determined in the presence of ATP and ADP. Figure 14.1.20 below shows an
interactive iCn3D model of the human phosphofructokinase-1 dimer (for clarity) in complex with ATP and Mg (4XYJ). (long load)
NCBI iCn3D
Figure 14.1.20 : Human phosphofructokinase-1 dimer in complex with ATP and Mg (4XYJ). (Copyright; author via
source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...AKfonyGsNq6Cs9 (long load). Here is a
link to the ADP bound structure: https://structure.ncbi.nlm.nih.gov/i...N81MBS4PZfSQp9
The monomers are shown in colored surface with secondary structure underneath. Note that side chains from each monomer in the dimer
contribute to binding and catalysis. The actual PDB structure is tetrameric. The structures have an E173S mutation.
Figure 14.1.21 shows the differences in orientation of the key side chains in the ADP structure (left) and the ATP structure (right) that are
key in the conformation changes in the enzyme on binding substrate.
ADP bound form of human PFK (4XKJ) ADP bound form of human PFK (4XYJ)
Figure 14.1.21 : Conformation changes in the active site of PFK on ATP hydrolysis
14.1.6: REACTION 4: FRUCTOSE-1,6-BISPHOSPHATE → DIHYDROXYACETONE PHOSPHATE (DHAP)

+ GLYCERALDEHYDE 3-PHOSPHATE (G3P). ΔGO = + 5.7 KCAL/MOL
The newly added high-energy phosphates further destabilize fructose-1,6-bisphosphate. The fourth step in glycolysis employs an enzyme,
aldolase, to cleave fructose-1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.
This reaction is shown in Figure 14.1.22 below in both wedge dash and Fischer projections
-2
-2 O3PO
O3PO
1 O
6 O -2 -2
O3PO O
O3PO OH +
fructose-1,6-bisP OH 4. aldolase
F-1,6-BP dihydroxyacetone phosphate glyeraldehyde 3-phosphate
HO OH DHAP G3P
O
CH2O P O
O H
O O O
HO H O3PO C CH2OH
fructose-1,6-bisP H2 + H OH
H OH
F-1,6-BP 4. ALDOLASE CH2OPO3
H OH O
H2C O P O
O dihydroxyacetone phosphate glyceraldehyde-3-P
DHAP G3P
Figure 14.1.22 : Summary reaction - aldolase

This is the first C-C bond cleavage within glucose on the path to complete cleavage during aerobic respiration and release of 6 carbon
dioxide molecules per glucose metabolized. This reaction is the reverse of an aldol condensation when an enol or enolate reacts with a
carbonyl C to form an adduct. Note that both products are phosphorylated. The reaction is not thermodynamically favored but is pulled in
the forward direction by the utilization of the product in subsequent reactions in the pathway.
Table 14.1.2 Characteristics of the Three Classes of Aldolases (I, IA, and II) and the Organisms in Which They are Found.
| y g y
Aldolase Organisms Characteristics

class
I Higher eukaryotes (plants, animals), some • Mechanism of action: Schiﬀ base forms
protozoans (e.g., Trypanosoma brucei, Leishmania substrate intermediate
donovani, Plasmodium falciparum), some bacteria, • Structure: Typically forms homo-tetramers,
some algae contains TPI β/α-barrel fold
IA Archaea • Mechanism of action: Schiﬀ base forms
substrate intermediate
• Structure: Typically forms homo-octamers/
decamers/higher oligomers, contains TPI
β/α-barrel fold
II “Lower” eukaryotes (some protozoans (e.g., • Mechanism of action: Divalent ion forms
Giardia lamblia), fungi, yeasts, algae), most substrate intermediate, metal-dependent
bacteria, some archaea • Structure: Typically forms homo-dimers,
contains TPI β/α-barrel fold
Pirovich er al, Frontiers in Molecular Biosciences , 8 (2021), https://www.frontiersin.org/article/...lb.2021.719678. AUTHOR=Pirovich

David B., Da’dara Akram A., Skelly Patrick J. Creative Commons Attribution License (CC BY).
Class I Aldolase
These enzymes proceed through a Schiff base intermediate between a reactive lysine and and the reactant/product. The enzyme is favored in
the reverse direction and its perhaps easier to see the mechanism presented in that fashion. In rabbits, the muscle Class I aldolase (RAMA)
uses Lys-229 to form a Schiff base with DHAP as shown in a mechanism presented in Figure 14.1.23 below.
F1,6-BP
DHAP -
- O O OH
O 1 2 3
P -2
O3PO 3 5 6
O OH 2 OPO3-2
O 4
O 1
OH OH
Enz-Lys-NH 2 Enz-Lys-NH 2
B:
Schiff base formation Enz

Lys +
Arg B H H3
Lys N
Arg N O-
H -
O 6
NH+ HN O 3 5 -2
H -
O N+ P 2 4 OPO 3
HN O OH H O- 1
N+ P 2 3 H N O OH OH
H O- 1
H
H N O H
H B:
Lys
Arg
NH Enz
- +
HN H O
O OH H2
N+ P
O- 1 2 3 H N
H N H O
6
H C 5
O 4 OPO3-2
B H OH G3P
Figure 14.1.23 : Class I aldolases - general mechanism (after Bolt et al., Arch Biochem Biophys. 2008 June 15; 474(2): 318–330)
Only the pro(S) proton of the dihydroxyacetone phosphate C3 carbon is removed and effectively exchanged with the glyceraldehyde-3-
phosphate substrate. Figure 14.1.24 below reviews how the pro(R) and pro(S) hydrogens can be visually differentiated by replacing one
with a deuterium and determining the stereochemistry of the now chiral C3.
DHAP
-
-
O
O D H - O- H D
1 2 O 1 2
P P
O (R) OH O (S) OH
O 3 O 3
O O
Figure 14.1.24 : Visualization of the Pro(R) and Pro(S) Hydrogens on DHAP

Figure 14.1.20 below shows key active site residues in the active site of a Class I aldolase from rabbit muscle. The first step in Schiff base
formation with Lys229 is shown.
Glu189
DHAP
-
Ser300 OH O O
OH O
O Tyr 363
O- H
O
NH2 P
O O H
O- H
Lys 229 H NH2
O O
Glu187 Lys 146
Figure 14.1.20 : Catalytic residues in rabbit muscle Class I enolase.

Figure 14.1.21 below shows an interactive iCn3D model of the dihydroxyacetone phosphate enamine intermediate in fructose-1,6-
bisphosphate aldolase from rabbit muscle (2QUT)
NCBI iCn3D
Figure 14.1.21 : Dihydroxyacetone phosphate enamine intermediate in fructose-1,6-bisphosphate aldolase from rabbit muscle
(2QUT). (Copyright; author via source). Click the image for a popup or use this external link:
https://structure.ncbi.nlm.nih.gov/i...uR5KZBsfN8QQh9
Only one monomer of the four in the homotetramer is shown. The ligand, 1,3-dihydroxyacetonephosphate, is shown in spacefill with CPK
colors. It is Schiff base linkage with Lys229. The key catalytic residues are shown and labeled.
The reaction proceeds between eneamine and iminium covalent intermediates. A K146M mutation (in the active site) decreases enzyme
activity and allows trapping of the K229 eneamine intermediate. A key tyrosine (Y363) in its deprotonated state formed in the presence of
the iminium phosphate and a local water appears to abstract the C3 pro(S) proton to form the enamine.
Class II Aldolase
Figure 14.1.22 :
O
O
Glu O- F1,6-BP
Glu O-
O-
- H O HO
O
1 2
P C3 -2
O3PO 3 5
O OH 2 4 6 OPO3-2
O 1
O OH OH
DHAP Zn2+
O O
H
Glu O Glu O
H OH G3P
O 4 -6 OPO3-2
C
- O- H O-
O 1 2 6 - 3
P C3 C 5 O
P
1
2 CH
O OH O 4 OPO3-2 O
O O O
O- OH G3P O
Zn2+ Zn2+
enediolate
Figure 14.1.22 : Class II aldolases - general mechanism (after Bolt et al, ibid)
14.1.7: REACTION 5: DHAP ↔ G3P. ΔGO= +1.8 KCAL/MOL

This reaction, catalyzed by triose phosphate isomerase (TPI), is shown in Figure 14.1.23 below.
Figure \(\PageIndex{23}\: Summary reaction - triose phosphate isomerase.

This is another simple isomerization reaction. Only one product, glyceraldehyde-3P continues on in glycolysis, so only one enzyme is
needed to metabolize the cleavage products of this reaction further. As in other isomerization reactions, the ΔGo is close to 0.
A proposed mechanism for this reaction is shown in Figure \(\PageIndex{24} below.
Figure 14.1.24 : Proposed mechanism for triose phosphate isomerase (after Bolt et al, ibid)
Note that the reaction employees the deprotonation of a neutral imidazole side chain to form an imidazolate anion. This would not likely to
be favorable, given its pKa value.
The are four possible conserved proton donors with more reasonable pKas in the active sites of TPIs, including K12, H95, E97 and E165.
Mutations of E97 to E97Q and E97D lead to a 4000-fold reduction in kcat (E97Q) but only a 100-fold reduction for E97D suggesting that
E97 is the key general acid/base.
Figure 14.1.25 below shows an interactive iCn3D model of the chicken triosephosphate isomerase-phosphoglycolohydroxamate complex
(1TPH)
NCBI iCn3D
Figure 14.1.25 : Chicken triosephosphate isomerase-phosphoglycolohydroxamate complex (1TPH) . (Copyright; author via
source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...AFXpt9kg5aki5A
The four key conserved residues are shown in the gray monomer of the homodimer.
14.1.8: REACTION 6: G3P ↔1,3 BPG. ΔGO= +1.5 KCAL/MOL

This reaction, catalyzed by glyceraldehyde-3-phosphate dehydrogenase, is shown in Figure 14.1.26 below
Figure \(\PageIndex{26}\: Summary reaction - glyceraldehyde-3-phosphate dehydrogenase

This is a big reaction! Note that the ΔGo is close to 0 but look at what really happened. The carbonyl O in G3P has been oxidized to the
form of a mixed anhydride which can donate a phosphate to ADP (in the next step) to form ATP. That this is an oxidation reaction should be
obvious from the fact the the carbonyl C in G3P has two bonds to O but 3 bonds in 1,3 BPG.
To carry out an oxidation reaction, you need an oxidizing agent. In comes NAD+, a modest put very prevalent oxidizing agent in biology.
When glucose is oxidized completely by O2 to CO2 during combustion, much energy is released so we can surmise that oxidation reactions,
if carried out by powerful oxidizing agents like O2, proceed with a large negative ΔGo. For every oxidation reaction, the oxidizing agent is
reduced. All reactions are potentially reversible so the products formed are new potential oxidizing and reducing agents. As in acid/base
reactions, which proceed from stronger acid to weaker conjugate acid, redox reactions proceed from stronger to weaker oxidizing agent. For
reaction 6, we must use tables of redox potentials to calculate the actual ΔGo. It turns out to be close to 0, which is great since in the same
reaction, a substrate-level phosphorylation reaction (using inorganic phosphate - Pi instead of ATP) occurs. In summary this reaction
catalyzes the first and only oxidation of glucose in glycolysis which has paid (thermodynamically) for the generation of a mixed anhydride
whose phosphorylating potential is higher that that of ATP.
Figure 14.1.27 below shows a proposed mechanism for glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi
Figure 14.1.27 : Proposed mechanism for glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi (after Reis et al. Phys.Chem.
Chem. Phys., 2013, 15, 3772. https://pubmed.ncbi.nlm.nih.gov/23389436/)
The reaction proceeds in two parts. The first (top section) is the oxidation of glyceraldehyde-3-phosphate (G3P) by NAD+ to the state of a
thioester attached to Cys166. The now reduced NADH dissociates, and is replaced by a new NAD+ for another cycle of catalysis. In the
meantime, inorganic phosphate, Pi, binds and reacts with the thioester to form 1,3-bisphosphoglycerate (1,3-BPG).
The mechanism is not entirely clear. There are two phosphate binding sites, Pi (red) and Ps (purple) which interact with phosphate groups
on the substrates. The oxidation part of the reactions appears to take place at the Pi site. Ps is composed of the side chains of Thr197,
Thr199 and the 2-hydroxyl group of the ribose in NAD+. Pi site seems more variable but in the T. cruzi enzyme appears to consist of Thr226
and Arg249, Gly227, Ser 247. The mechanism appears to involve a flip of orientation of substrates and intermediates after the dissociation
NADH from the enzyme.
Figure 14.1.28 below shows an interactive iCn3D model of the Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase with bound
NAD and a 1,3-bisphosphoglycerate analogue (1QXS)
NCBI iCn3D
Figure 14.1.28 : Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase with bound NAD and a 1,3-
bisphosphoglycerate analogue (1QXS) . (Copyright; author via source). Click the image for a popup or use this external link:
https://structure.ncbi.nlm.nih.gov/i...WnsNSgmA3AJyw6
Only two monomers of the homotetramer are shown for clarity. The Pi site is shown in magenta and the Ps site in purple. The phosphonic
acid analog is shown in spacefill and NAD in sticks. The active site His194 and Cys166 are also shown in sticks and labeled.
14.1.9: REACTION 7: 1,3 BPG + ADP + H+ → 3PG + ATP ΔGO= -4.5 KCAL/MOL
This reaction, catalyzed by phosphoglycerate kinase, is shown in Figure 14.1.29 below
Figure 14.1.29 : Summary reaction - phosphoglycerate kinase

It's finally happened! An ATP has been made for each of the two 1,3-BPG molecules derived from glucose. We've made back the ATP used
in steps 1 and 3. A mixed phosphoanhydride bond is broken in 1,3 BPG as a phosphoanhydride bond is made in ATP. As the mixed
phosphoanhydride has higher energy than its hydrolysis product compared to the phosphoanhydride in ATP, the reaction proceeds with a
negative ΔGo
A mechanism for the reaction is shown in Figure 14.1.30 below.
Figure 14.1.30 : Reaction mechanism for phosphoglycerate kinase
Figure 14.1.31 below shows an interactive iCn3D model of human phosphoglycerate kinase in complex with ADP, 3PG and magnesium
trifluoride (2WZB).
NCBI iCn3D
Figure 14.1.31 : Human phosphoglycerate kinase in complex with ADP, 3PG and magnesium trifluoride (2WZB). (Copyright;
author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...w5yvu2VuGhmQXA
3-phosphoglycerate is shown in spacefill with CPK colors. ADP plus the adjacent MgF3 is a mimetic for the transition state for ATP
synthesis. Hence this structure shows the products/transition state in a closed active site, which as we have seen before prevents spurious
hydrolysis of 1,3-BPG or ATP.
Figure 14.1.32 below shows an interactive iCn3D model of the alignment of the open form of human phosphoglycerate kinase (2XE7) with
bound 3PG and ADP with the closed form with bound 3PG, ADP, and MgF3 (2WZB).
NCBI iCn3D
Figure 14.1.32 : Alignment of the open form of human phosphoglycerate kinase (2XE7) with bound 3PG and ADP with the
closed form with bound 3PG, ADP, and MgF3 (2WZB). (Copyright; author via source). Click the image for a popup or use this external
link: https://structure.ncbi.nlm.nih.gov/i...n5FfsJeYitiWr9
Use the "a" key to toggle back between the open form (magenta) and closed forms (cyan). In the closed state most closely representing the
bond transition state of ATP, the two lobes of the enzyme clamp together.
14.1.10: REACTION 8: 3PG ↔ 2PG ΔGO= +1.1 KCAL/MOL

This reaction, catalyzed by phosphoglycerate mutase (PGM), is shown in Figure 14.1.33 below
Figure 14.1.33 : Summary reaction - phosphoglycerate mutase
This isomerization reaction proceeds with little thermodynamic barrier. It's function is to locate the phosphate on C2 which on the next
reaction (dehydration) will form a molecule whose phosphoryl transfer potential is greater than ATP. It's seems so simple but the enzymes
that catalyse this reaction are diverse and quite complicated from a mechanistic perspective.
There are two types of PGMs, bisphosphoglycerate and monophosphoglycerate mutases that carry out three different reactions involving
shuffling of phosphates from one position to another in 3C sugars or cleavage of a phosphate from a sugar
3-phosphoglycerate ↔ 2-phosphoglycerate (reaction 8 of glycolysis) catalyzed by bisphosphoglycerate and monophosphoglycerate (the
glycolytic enzyme) mutases
1,3-bisphosphoglycerate ↔ 2,3-bisphosphoglycerate by bisphosphoglycerate mutases
2,3-phosphoglycerate ↔ 3-phosphoglycerate + Pi by bisphosphoglycerate mutases
Within the monophosphoglcyerate mutases, and more specifically the phosphoglycerate mutase (PGM) of glycolysis, there are two types
one that depends on the cofactor 2,3-phosphoglycerate. These are called cofactor-dependent phosphoglycerate mutase (dPGM) and are
found in mammals, yeast and some bacteria. They do not require metal ions.
one that does not depend on the cofactor 2,3-phosphoglycerate. These are called cofactor-independent phosphoglycerate mutase (iPGM)
and are found in plants and some bacteria. These can only interconvert 3PGA and 2PGA. One family of enzymes in the class requires
Mn2+ while the other requires Mg2+ or Zn2+. These enzyme are often structurally similar to alkaline phosphatases
The cofactor-independent and cofactor-dependent monophosphoglycerates (such as the phosphoglycerate mutase of glycolysis) are very
different structurally and mechanistically so we will look at both types of mechanisms. With each type the enzyme sequences are very
conserved.
Mechanism of cofactor (2,3-BPG) dependent phosphoglycerate mutase (dPGM)
The reaction is much less complicated than the cofactor independent PGM. It involves the transfer for the phosphate on the C3-OH to a the
nucleophilic nitrogen on histidine (His8) in the active site to form a covalent pHis8 intermediate. A proximal Glu86 presumably acts as
general acid to donate a protein to the dephosphorylated O on C3 of the substrate. An active site arginine (Arg 59) would stabilized the p-
His intermediate and its transition states. The phosphate on pHis could then be transferred to the O on carbon C2 of the substrate.
Figure 14.1.34 below shows an interactive iCn3D model of yeast phosphoglycerate mutase (cofactor dependent) bound to 3-
phosphoglycerate (1QHF)
NCBI iCn3D
Figure 14.1.34 : Yeast phosphoglycerate mutase (cofactor dependent) bound to 3-phosphoglycerte (1QHF). (Copyright; author
via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9F4oc6mmr5DFs9
Mechanism of cofactor independent phospoglycerate mutase (iPGM)
Let's consider the mechanism for the Mn2+-requiring iPGM from Geobacillus stearothermophilus. Two Mn2+ ions are in the active site. The
mechanism for the first half of the cofactor independent phosphoglycerate mutase (iPGM) reaction is shown in Figure 14.1.35 below.
Figure 14.1.35 : Part A - Mechanism for cofactor independent phosphoglycerate mutase (iPGM) from Geobacillus stearothermophilus (after
Bolt et al, ibid)
Arg 261 interacts with the substrate, stabilizing the negative charge in it and in its transition state. It also makes the target phosphorous more
electrophilic. Ser62 is activated by a Mn2+ ion to become more nucleophilic on abstraction of a proton by Lys336. Reaction 3 probably
proceeds through an SN2 mechanism.
The rest of the mechanism is shown in Figure 14.1.36 below.
Figure 14.1.36 : Part B - Mechanism for cofactor independent phosphoglycerate mutase (iPGM) from Geobacillus stearothermophilus (after
Bolt et al, ibid)
The iPGMs are monomers with two distinct domains (lobes) containing a substrate binding site separated from an active site. They are
connected by flexible sequence than bend to produce either an open or closed form of the enzymes (as we have seen before). In some
enzymes the two sites are merged at the interface between the domain. The active site Ser62 becomes phosphorylated and then transfers its
phosphate to the new site in the substrate which is oriented differently in the enzyme.
Figure 14.1.37 below shows an interactive iCn3D model of the Geobacillus stearothermophilus cofactor-Independent Phosphoglycerate
Mutase (iPGM) with bound 2-phosphoglycerate (product) (1O98)
NCBI iCn3D
Figure 14.1.37 : Cofactor-Independent Phosphoglycerate Mutase with bound 2-phosphoglycerate (product) (1O98) .
(Copyright; author via source). Click the image for a popup or use this external link:
https://structure.ncbi.nlm.nih.gov/i...71iYHXAEKMGsEA
14.1.11: REACTION 9: 2PG ↔ PEP + H2O ΔGO= +0.4 KCAL/MOL

This reaction, catalyzed by enolase, is shown in Figure 14.1.38 below
Figure \(\PageIndex{38}\: Summary reaction - enolase

Now you can see the rationale for reaction 8. By a simple and thermodynamically simple dehydration, a molecule with high phosphoryl
transfer potential (see dotted red box) has been produced which in the next and final step of glycolysis produces ATP.
This enzyme has an active site Mg2+ that is required for catalysis. Mammals have three forms of the enzyme, α-enolase (ENO-1) found in
most tissues, β-enolase (ENO-3) found mostly in muscle and γ-enolase (ENO-2) found in neurons. A possible mechanism for yeast enolase
is shown in Figure 14.1.39 below.
Figure 14.1.39 : A possible mechanism for yeast enolase

Figure 14.1.40 below shows an interactive iCn3D model of Yeast enolase with bound 2-phosphoglycerate (7ENL)
NCBI iCn3D
Figure 14.1.40 : Yeast enolase with bound 2-phosphoglycerate (7ENL) . (Copyright; author via source). Click the image for a
popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...TgwYV9vF8cYtPA
14.1.12: REACTION 10: PEP + ADP → PYR + ATP ΔGO= -7.5 KCAL/MOL
This reaction, catalyzed by pyruvate kinase, is shown in Figure 14.1.41 below
Figure 14.1.41 : Summary reaction - pyruvate kinase

In this step, 1 more ATP is made for each PEP consume (hence 2 ATPs for both 3C PEPs). The phosphoryl transfer potential for PEP is
higher than than for ATP, which allows this reaction to proceed with a large negative ΔGo (-7.5 kcal/mol).
The mechanism for rabbit pyruvate kinase is shown in Figure 14.1.42 below.
Figure 14.1.42 : Mechanism for rabbit pyruvate kinase

Figure 14.1.43 below shows an interactive iCn3D model of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate (1PKN)
NCBI iCn3D
Figure 14.1.43 : Rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate (1PKN). (Copyright; author via
source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...cZkm8DdEw2N3C6
We are done! Given that glycolysis is the central anaerobic energy extracting pathway in all life, it is important that we examined each
enzyme in detail.
This is the net reaction of the glycolytic pathway:
+ +
Glc + Pi + 2 ADP + 2 NAD ⟶ 2 Pyr + 2 ATP + 2 NADH + 2 H + 2 H2 O
14.1.13: UNCOUPLING GLYCOLYTIC OXIDATION AND PHOSPHORYLATION (ATP FORMATION)

Since we most interested in energy transduction at this point, let's consider just two important step in glycolysis that directly lead to ATP
synthesis. Only one oxidative step is found in this pathway, namely the oxidative phosphorylation of the 3C glycolytic intermediate
glyceraldehyde-3-phosphate, to 1,3-bisphosphoglycerate, a mixed anhydride (see link below for mechanism). The oxidizing agent is NAD+
and the phosphorylating agent is NOT ATP but rather Pi. The enzyme is named glyceraldehyde-3-phosphate dehydrogenase. It contains an
active site Cys, which helps explain how the enzyme can be inactivated with a stoichiometric amounts of iodoacetamide. A general base in
the enzyme abstracts an H+ from Cys, which attacks the carbonyl C of the glyceraldehyde, forming a tetrahedral intermediate. Instead of the
expected reaction (which would be the protonation of the alkoxide in an overall nucleophilic addition reaction at the aldehyde), a hydride
leaves from the former carbonyl C to NAD+ in an oxidation step. Notice, this is a two electron oxidation reaction similar to seen in alcohol
dehydrogenase. An acyl-thioester intermediate has formed, much like the acyl intermediate that formed in Ser proteases. Next inorganic
phosphorous, Pi, attacks the carbonyl C of the intermediate in a nucleophilic substitution reaction to form the mixed anhydride product, 1,3-
bisphosphoglycerate. Although we have formed a mixed anhydride, we cleaved a sulfur ester, which is destabilized with respect to its
hydrolysis products (since the reactant, the thioester, is not stabilized by resonance to the extent of regular esters owing to the poor donation
of electrons from the larger S to the carbonyl-like C.) In the next step, catalyzed by the enzyme phosphoglycerate kinase, ADP acts an a
nucleophile which attacks the mixed anhydride of the 1,3-bisphosphoglycerate to form ATP. Note that the enzyme is name for the reverse
reaction. We have coupled oxidation of an organic molecule (glyceraldehyde-3-phosphate) to phosphorylation of ADP through the
formation of a "high" energy mixed anhydride, 1,3-bisphosphoglycerate.
The linkage between oxidation of glyceraldehyde-3-phosphate and the phosphorylation of ADP by 1,3-bisphosphoglycerate can be
artificially uncoupled by adding arsenate, which has a similar structure as phosphate. The arsenate can form a mixed anhydride at C1 of
glyceraldehyde-3-phosphate, but since the bringing O-As bond is longer and not as strong as in the mixed anhydride, it is easily hydrolyzed.
This prevents subsequent transfer of phosphate to ADP to form ATP.
Figure 14.1.44 shows a summary of oxidation and substrate-level phosphorylation in glyceraldehyde-3-phosphate dehydrogenase.
Figure 14.1.44 : Oxidation and substrate-level phosphorylation in glyceraldehyde-3-phosphate dehydrogenase (after Voet and Voet)
Summary: Under anaerobic conditions, glucose (6Cs) is metabolized through glycolysis which converts it to two molecules of pyruvate
(3Cs). Only one oxidation step has been performed when glyceraldehyde 3-phospate is oxidized to 1,3-bisphosphoglycerate. To regenerate
NAD+ so glycolysis can continue, pyruvate is reduced to lactate, catalyzed by lactate dehydrogenase. These reactions take place in the
cytoplasm of cells actively engaged in anaerobic oxidation of glucose (muscle cells for examples during sprints). Note that the enzyme is
named for the reverse reaction, the oxidation of lactate by NAD+. This reaction is shown in Figure 14.1.45 below.
Figure 14.1.45 : Conversion of pyruvate to lactate

We will explore the fate of pyruvate under anaerobic conditions more in the next chapter section.
This page titled 14.1: Glycolysis is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.

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14.

14.1.2: REACTION 1: GLUCOSE → GLUCOSE-6-PHOSPHATE. ΔGO=-4.0 KCAL/MOL.

Figure 14.1.7 : Summary reaction - hexokinase

HN 603Ser Asp 657

Figure 14.1.10 : Mechanism of human hexokinase I.

Table 14.1.1 : Overview of the Five Carbohydrate Kinase Classes.

Carbohydrate Common Substrates Native Phosphate Donors (Minor Donors Pfam ID

Hexokinase Glucose, mannose, fructose ATP (ITP) PF00349, PF03727,

ROK Kinase Glucose, allose, fructose, N-acetylglucosamine, N- ATP (polyphosphate) PF00480

Ribokinase Ribose, 2-deoxy-d-ribose, adenosine ATP, ADP (GTP, ribonucleotide) PF00294

GHMP Kinase Galactose, N-acetylgalactosamine, ATP (GTP, ITP) PF00288

Phosphatidylinositol Phosphatidylinositol, phosphatidylinositol ATP (GTP) PF00454, PF01504

14.1.4: REACTION 2: GLUCOSE-6-PHOSPHATE ↔ FRUCTOSE-6-PHOSPHATE. ΔGO=+0.4 KCAL/MOL

Figure 14.1.14 : Summary reaction, phosphoglucoisomerase

210Lys Lys 518

14.1.5: REACTION 3: FRUCTOSE-6-PHOSPHATE → FRUCTOSE-1,6-BISPHOSPHATE. ΔGO=-3.4

Figure 14.1.17 : Summary reaction - phosphofructokinase

14.1.6: REACTION 4: FRUCTOSE-1,6-BISPHOSPHATE → DIHYDROXYACETONE PHOSPHATE (DHAP)

Figure 14.1.22 : Summary reaction - aldolase

Aldolase Organisms Characteristics

Pirovich er al, Frontiers in Molecular Biosciences , 8 (2021), https://www.frontiersin.org/article/...lb.2021.719678. AUTHOR=Pirovich

Schiff base formation Enz

Figure 14.1.24 : Visualization of the Pro(R) and Pro(S) Hydrogens on DHAP

Glu187 Lys 146

Figure 14.1.20 : Catalytic residues in rabbit muscle Class I enolase.

14.1.7: REACTION 5: DHAP ↔ G3P. ΔGO= +1.8 KCAL/MOL

Figure \(\PageIndex{23}\: Summary reaction - triose phosphate isomerase.

14.1.8: REACTION 6: G3P ↔1,3 BPG. ΔGO= +1.5 KCAL/MOL

Figure \(\PageIndex{26}\: Summary reaction - glyceraldehyde-3-phosphate dehydrogenase

Figure 14.1.29 : Summary reaction - phosphoglycerate kinase

14.1.10: REACTION 8: 3PG ↔ 2PG ΔGO= +1.1 KCAL/MOL

Figure 14.1.33 : Summary reaction - phosphoglycerate mutase

14.1.11: REACTION 9: 2PG ↔ PEP + H2O ΔGO= +0.4 KCAL/MOL

Figure \(\PageIndex{38}\: Summary reaction - enolase

Figure 14.1.39 : A possible mechanism for yeast enolase

Figure 14.1.41 : Summary reaction - pyruvate kinase

Figure 14.1.42 : Mechanism for rabbit pyruvate kinase

14.1.13: UNCOUPLING GLYCOLYTIC OXIDATION AND PHOSPHORYLATION (ATP FORMATION)

Figure 14.1.45 : Conversion of pyruvate to lactate

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