Glycolysis, from Greek word 

glykys, meaning “sweet”, and lysis, meaning “dissolution

or breakdown”, can be defined as the sequence of enzymatic reactions that, in
the cytosol, also in the absence of oxygen, leads to the conversion of one molecule
of glucose, a six carbon sugar, to two molecules of pyruvate, a three carbon
compound, with the concomitant production of two molecules of ATP, the universal
energy currency in biological systems.
The Glycolytic Pathway
Glycolysis, which evolved before a substantial amount of oxygen had accumulated in
the atmosphere, is the metabolic pathway with the largest flux of carbon in most
living cells, and is present in almost all organisms.
This pathway, not requiring oxygen, played a crucial role in metabolic processes
during the first 2 billion years of evolution of life, and probably represents the most
ancient biological mechanism for extracting energy from organic molecules when
oxygen availability is low. Moreover, it is a source of precursors for aerobic
catabolism and for various biosynthetic processes.
Note: glycolysis is also known as the Embden-Meyerhof pathway, named after
Gustav Embden and Otto Meyerhof, the two researchers who elucidated the entire
pathway in the muscle.
CONTENTS

 Glycolysis: the first metabolic pathway to be elucidated

 Why is glycolysis so important?
 The steps of glycolysis
 Reaction 1: glucose phosphorylation to glucose 6-phosphate
 The importance of glucose phosphorylation
 Other possible fates of glucose 6-phosphate
 Reaction 2: isomerization of glucose 6-phosphate to fructose 6-phosphate
 Reaction 3: phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate
 Reaction 4: cleavage of fructose 1,6-bisphosphate into two three-carbon fragments
 Reaction 5: interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-
phosphate
 Reaction 6: oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate
 Reaction 7: phosphoglycerate kinase and the first ATP forming reaction
 What is substrate-level phosphorylation?
 Reaction 8: from 3-phosphoglycerate to 2-phosphoglycerate
 Synthesis of 2,3-bisphosphoglycerate and the Rapoport-Luebering pathway
 Reaction 9: formation of phosphoenolpyruvate
 Reaction 10: pyruvate kinase and the transfer of the phosphoryl group from the PEP to
the ADP
 The fate of NADH and pyruvate produced in glycolysis
 Lactic acid fermentation
 Alcoholic fermentation
 Fate of pyruvate and NADH under aerobic conditions
 Anabolic fates of pyruvate
 Glycolysis and ATP production
 Glycolysis and ATP production under anaerobic conditions
 Glycolysis and ATP production under aerobic conditions
 Feeder pathways for glycolysis
 Glycogen and starch
 Fructose
 Fructose and hexokinase
 Sorbitol
 Galactose
 Mannose
 Regulation of glycolysis
  Hexokinase
 Comparison of the kinetic properties of hexokinase isoenzymes
 Regulation of the activity of hexokinases I-III
 Regulation of the activity of hepatic glucokinase
 Regulation of phosphofructokinase 1 activity
 Regulation of pyruvate kinase activity
 References

Glycolysis: the first metabolic pathway to be

elucidated
The development of biochemistry has gone hand in hand with the elucidation of
glucose metabolism, especially glycolysis, the first metabolic pathway to have been
elucidated.
Though the elucidation of this metabolic pathway was worked out in the ‘40 of the
last century, the key discovery about glucose metabolism was made in 1897, quite
by accident, following a problem arose a year earlier, when a German chemist, M.
Hahn, in attempting to obtain and preserve cell-free protein extracts of yeast,
encountered difficulties in its conservation. A colleague, Hans Buchner,
remembering a method for preserving jams, suggested to add sucrose to the extract.
Eduard Buchner, Hans’s brother, put the idea of Hans into practice, and observed
that the solution produced bubbles. This prompted Eduard to conclude that a
fermentation was occurring, a quite surprising discovery. Indeed fermentation,
according to Pasteur’s assertion in 1860, was inextricably tied to living cells, whereas
it was now demonstrated that it could also occur outside them. Briefly, these two
researchers refuted the vitalist dogma and had a pivotal role in starting modern
biochemistry.
Eduard Buchner was awarded the Nobel Prize in Chemistry in 1907 for this
research, and was the first of several researchers who won the award for their
discoveries concerning the glycolytic pathway.
It was later demonstrated, working with muscle extracts, that many of the reactions
of lactic fermentation  were the same of those of alcoholic fermentation , thus
revealing the underlying unity in biochemistry.
As previously mentioned, glycolysis was then fully elucidated in the first half of the
last century largely due to the work of researchers such as Gerty and Carl Cori, Carl
Neuberg, Arthur Harden, William Young, Jacob Parnas, Otto Warburg, Hans von
Euler-Chelpin, Gustav Embden and Otto Meyerhof. In particular, Warburg and von
Euler-Chelpin elucidated the whole pathway in yeast, and Embden and Meyerhof in
muscle in the 30’s.

Why is glycolysis so important?

Glycolysis is essential to most living cells both from the energy point of view and as a
source of precursors for many other metabolic pathways. And the rate of carbon flow
through glycolysis, namely, the amount of glucose converted to pyruvate per unit
time, is regulated to meet these two basic needs for the cell.
 From the energetic point of view, although glycolysis is a relatively inefficient
pathway, it can occur in the absence of oxygen, the condition in which life evolved on
Earth and that many contemporary cells, both eukaryotic and prokaryotic,
experience. Here are some examples.
 In most animals, muscles exhibit an activity-dependent anaerobiosis, namely, they can
work anaerobically for short periods. For example, when animals, but also athletes,
perform high intense exercises, their need for ATP exceeds body’s ability to supply
oxygen to the muscle. In such situation, muscles function, albeit for a short period of time,
anaerobically.
 Another example is the cornea of the eye, a poorly vascularized tissue.
 Many microorganisms live in environments where oxygen is low or absent, such as deep
water, soil, but also skin pores. And a variety of microorganisms called obligate anaerobes
cannot survive in the presence of oxygen, a highly reactive molecule. Examples
are Clostridium perfringens, Clostridium tetani, and Clostridium botulinum, that cause
gangrene, tetanus and botulism, respectively.
It should also be underlined that glycolysis also plays a key role in those cells and
tissues in which glucose is the sole source of energy, such as:
 red blood cells, lacking mitochondria,
 sperm cells;
 the brain, which can also use ketone bodies for fuel in times of low glucose;
 the adrenal medulla.
A similar situation is also found in the plant world where many aquatic plants and
some plant tissues specialized in starch accumulation, such as potato tubers, use
glucose as the main source of energy.
Note: There are organisms that are facultative anaerobes, namely organisms that can
survive in the presence and in the absence of oxygen, acting aerobically or
anaerobically, respectively. Examples are animals belonging to the genus Mytilus,
which display an habitat-dependent anaerobiosis, a condition similar to the activity-
dependent anaerobiosis seen in muscle.
Finally, it should not be forgotten that under aerobic conditions, in cells with
mitochondria, glycolysis constitutes the upper part of the metabolic pathway leading
to the complete oxidation of glucose to carbon dioxide (CO 2) and water for energy
purposes.
Glycolysis as a Source of Building Blocks
Some glycolytic intermediates, for example glucose 6-phosphate (G-6-P), fructose 6-
phosphate (F-6-P) or dihydroxyacetone phosphate (DHAP), may be used as building
blocks in several metabolic pathways, such as those leading to the synthesis
of glycogen, fatty acids, triglycerides, nucleotides, of some amino acids, or 2,3-
bisphosphoglycerate (2,3-BPG).

The steps of glycolysis

The 10 steps that make up glycolysis can be divided into two phases.
The first, called the preparatory phase, consists of 5 steps and starts with the
conversion of glucose to fructose 1,6-bisphosphate (F-1,6-BP) through three
enzymatic reactions, namely, a phosphorylation at C-1, an isomerization, and a
second phosphorylation, this time at C-6, with consumption of 2 ATP. Fructose 1,6-
bisphosphate is then cleaved into two phosphorylated three-carbon compounds,
glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Finally, the
isomerization of DHAP to a second molecule of glyceraldehyde-3- phosphate occurs.
In the preparatory phase therefore a glucose is split into two molecules of
glyceraldehyde 3-phosphate, and two ATP are consumed.
In the second phase, called the payoff phase, consisting of the remaining 5 steps of
the pathway, the two molecules of glyceraldehyde 3-phosphate are converted into
two molecules of pyruvate, with the concomitant production of 4 ATP. So, in this
phase, part of the energy present in the chemical bonds of glucose is extracted and
conserved in the form of ATP. Furthermore, reducing equivalents are extracted and
conserved in the form of the reduced coenzyme NADH. The metabolic fate of NADH
will depend on the cell type and aerobic or anaerobic conditions.
Note: Glucose metabolized in the glycolytic pathway derives both from glucose that
enters the cell through specific membrane transporters, that in turn derives from the
bloodstream, and glucose 6-phosphate produced by glycogen degradation.

Reaction 1: glucose phosphorylation to glucose 6-

phosphate
In the first step of the glycolytic pathway glucose is phosphorylated to glucose 6-
phosphate at the expense of one ATP.
Glucose + ATP → Glucose 6-phosphate + ADP + H+
In most cells this reaction is catalyzed by hexokinase (EC 2.7.1.1), enzyme present
in the cells of all organisms, and in humans with four isozyme).
Hexokinase and pyruvate kinase, the other kinase of the glycolysis, like many other
kinases, require the presence of magnesium ion, Mg2+, or of another bivalent metal
ion such as manganese, Mn2+, for their activity. Mg2+ binds to the ATP to form the
complex MgATP2-, and in fact the true substrate of the enzyme is not ATP but this
complex. It should be emphasized that the nucleophilic attack by a hydroxyl group (-
OH) of glucose at the terminal phosphorus atom of the ATP is facilitated by the
action of Mg2+ that interacts with the negative charges of the phosphoryl groups of the
nucleoside triphosphate.
The formation of the phosphoester bond between a phosphoryl group and the
hydroxyl group at C-6 of glucose is thermodynamically unfavorable and requires
energy to proceed, energy that is provided by the ATP. Indeed, while the
phosphorylation of glucose at C-6 by inorganic phosphate has a ΔG°’ of 13.8 kJ/mol
(3.3 kcal/mol), namely, it is an endergonic reaction, the hydrolysis of ATP to ADP
and Pi has ΔG°’ of -30.5 kJ/mol (-7.3 kcal/mol), namely, it is an exergonic reaction.
The net reaction has a ΔG°’ of (-30.5 + 13.8) = -16.7 kJ/mol (-7.3 + 3.3 = -4.0
kcal/mol). Under cellular conditions the reaction is even more favorable, with a ΔG
equal to -33.5 kJ/mol (-8.0 kcal/mol).
Therefore, this is an essentially irreversible reaction.
Note: In biochemistry, phosphorylations are fundamental reactions catalyzed by
enzymes called kinases, a subclass of transferases. Kinases catalyze the transfer of
the terminal phosphoryl group, or γ-phosphoryl group, of a nucleoside triphosphate
to an acceptor nucleophile to form a phosphoester bond. Specifically, hexokinase
catalyzes the transfer of the γ-phosphoryl group of ATP to a variety of hexoses, that
is, sugars with six carbons, such as fructose and mannose), in addition to glucose.

The importance of glucose phosphorylation

The phosphorylation of the glucose has some functions.

 Glucose 6-phosphate, due to its negative charge and because there are no transporters for
phosphorylated sugars in the plasma membrane, cannot diffuse out of the cell. Thus,
after the initial phosphorylation, no further energy is needed to keep the phosphorylated
molecule within the cell, despite the large difference between its intra- and extracellular
concentrations.
Similar considerations are valid for each of the eight phosphorylated intermediates
between glucose 6-phosphate and pyruvate.
 The rapid phosphorylation of glucose maintains a low intracellular concentration of the
hexose, thus favoring its facilitated diffusion into the cell.
 Phosphorylation causes an increase in the energy content of the molecule, that is, it
starts to destabilize it, thus facilitating its further metabolism.

Other possible fates of glucose 6-phosphate

Glucose 6-phosphate is a key metabolite of glucose metabolism. In fact, in addition to
be metabolized in the glycolytic pathway, in anabolic conditions it can have other
fates (see Fig. 3). Here are some examples.
 It can be used in the synthesis of:
glycogen, a polysaccharide stored mainly in the liver and muscle;
complex polysaccharides present in the extracellular matrix;
galactose;
glucosamine and other sugars used for protein glycosylation.
 It can be metabolized by the pentose phosphate pathway, that provides cells with:
NADPH, needed for reductive biosynthesis, such as fatty acid, cholesterol, steroid
hormone, and deoxyribonucleotide biosynthesis, and for preventing oxidative
damage in cells such as erythrocytes;
ribose 5-phosphate, used in nucleotide synthesis but also in NADH, FADH 2 and
coenzyme A synthesis.

Reaction 2: isomerization of glucose 6-phosphate to

fructose 6-phosphate
In the second step of the glycolytic pathway, the isomerization of glucose 6-
phosphate, an aldose, to fructose 6-phosphate, a ketose, occurs. This reaction is
catalyzed by phosphoglucose isomerase, also known as phosphohexose isomerase
or glucose phosphate isomerase (EC 5.3.1.9).
Glucose 6-phosphate ⇄ Fructose 6-phosphate
Like hexokinase, phosphoglucose isomerase requires the presence of Mg 2+.
The ΔG°’ of the reaction is 1.7 kJ/mol (0.4 kcal/mol), while the ΔG is -2.5 kJ/mol (-0.6
kcal/mol). These small values indicate that the reaction is close to equilibrium and is
easily reversible.
The reaction essentially consists in the shift of the carbonyl group at C-1 of the open-
chain form of glucose 6-phosphate to C-2 of the open-chain form of fructose 6-
phosphate.

Phosphoglucose Isomerase Reaction

The enzymatic reaction can be divided at least into three steps. Since in aqueous
solution both hexoses are primarily present in the cyclic form, the enzyme must first
open the ring of G-6P, catalyze the isomerization, and, finally, the formation of the
five-membered ring of F-6-P.
This isomerization is a critical step for glycolytic pathway, as it prepares the molecule
for the subsequent two steps.
Why?

 The phosphorylation that occurs in the third step requires the presence of an alcohol group
at C-1, and not of a carbonyl group.
 In the fourth step, the covalent bond between C-3 and C-4 is cleaved, and this reaction is
facilitated by the presence of the carbonyl group at C-2.
Reaction 3: phosphorylation of fructose 6-phosphate to
fructose 1,6-bisphosphate
In the third step of the glycolytic pathway, a second phosphorylation reaction
occurs. Phosphofructokinase 1 or PFK-1 (EC 2.7.1.11) catalyzes the phosphorylation
of fructose 6-phosphate at C-1 to form fructose 1,6-bisphosphate, at the expense of
one ATP.
Fructose 6-phosphate + ATP → Fructose 1,6-bisphosphate + ADP + H +
PFK-1 is so named to distinguish it from phosphofructokinase 2 or PFK-2 (EC
2.7.1.105), the enzyme that catalyzes the phosphorylation of fructose 6-phosphate to
fructose 2,6-bisphosphate.
Like the reaction catalyzed by hexokinase/glucokinase, this phosphorylation, too, is
an essentially irreversible step, irreversibility, once again, achieved by coupling, by
phosphofructokinase 1, with the hydrolysis of ATP. In fact, phosphorylation of
fructose 6-phosphate by inorganic phosphate is endergonic, with a ΔG°’ of 16.3
kJ/mol (3.9 kcal/mol), whereas, when the reaction is coupled to the hydrolysis of
ATP, the overall equation becomes exergonic, with a ΔG°’ of -14.2 kJ/mol (-3.4
kcal/mol) and a ΔG of -22.2 kJ/mol (-5.3 kcal/mol).
While hexokinase allows to trap glucose inside the cell, phosphofructokinase 1
prevents glucose to be used for glycogen synthesis or the production of other
sugars, but is instead metabolized in the glycolytic pathway. In fact, unlike glucose 6-
phosphate, fructose 1,6-bisphosphate cannot be used directly in other metabolic
pathways than glycolysis/gluconeogenesis, that is, phosphofructokinase 1
catalyzes the first “committed” step of the glycolytic pathway. Such reactions are
usually catalyzed by enzymes regulated allosterically, that prevent the accumulation
of both intermediates and final products. PFK-1 is no exception, being subject to
allosteric regulation by positive and negative effectors that signal the energy level
and the hormonal status of the organism.
Some protists and bacteria, and perhaps all plants, have a phosphofructokinase that
uses pyrophosphate (PPi) as a donor of the phosphoryl group in the synthesis of F-
1,6-BP. This reaction has a ΔG°’ of -2.9 kJ/mol (-12.1 kcal/mol).
Fructose 6-phosphate + PPi → Fructose 1,6-bisphosphate + Pi
Note: The prefix bis– in bisphosphate, as fructose 1,6-bisphosphate, indicates that
there are two phosphoryl groups are bonded to different atoms.
The prefix di– in diphosphate, as in adenosine diphosphate, indicates that there are
two phosphoryl groups connected by an anhydride bond to form a pyrophosphoryl
group, namely, they are directly bonded to one another.
Similar rules also apply to the nomenclature of molecules that have three phosphoryl
groups standing apart, such as inositol 1,4,5-trisphosphate, or connected by
anhydride bonds, such as ATP or guanosine triphosphate or GTP.

Reaction 4: cleavage of fructose 1,6-bisphosphate into

two three-carbon fragments
In the fourth step of the glycolytic pathway, fructose 1,6-bisphosphate aldolase, often
called simply aldolase (EC 4.1.2.13), catalyzes the reversible cleavage of fructose
1,6-bisphosphate into glyceraldehyde 3-phosphate, an aldose, and dihydroxyacetone
phosphate, a ketose. The enzyme cleaves the bond between C-3 and C-4.
Fructose 1,6-bisphosphate ⇄ Dihydroxyacetone phosphate + Glyceraldehyde 3-
phosphate

All glycolytic intermediates downstream to this reaction are three-carbon molecules,

instead of six-carbon molecules as the previous ones.
The ΔG°’ of the reaction in the direction of glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate production is of 23.8 kJ/mol (5.7 kcal/mol), and the
Km is approximately 10-4 M, values that would indicate that the reaction does not
proceed as written from left to right. However, under normal cellular conditions, due
to the lower concentrations of the reactants, the ΔG is -1.3 kJ/mol (-0.3 kcal/mol), a
very small value, thus the reaction is easily reversible, that is, essentially to
equilibrium.
Note: The name “aldolase” derives from the nature of the reverse reaction, from right
to left as written, that is, an aldol condensation.

Reaction 5: interconversion of dihydroxyacetone

phosphate and glyceraldehyde 3-phosphate
Of the two products of the previous reaction, glyceraldehyde 3-phosphate goes
directly into the second phase of the glycolytic pathway. Conversely, DHAP is not on
the direct pathway of glycolysis and must be converted, isomerized, to
glyceraldehyde 3-phosphate to continue through the pathway. This isomerization is
catalyzed by triose phosphate isomerase (EC 5.3.1.1).
Dihydroxyacetone phosphate ⇄ Glyceraldehyde 3-phosphate

Triose phosphate isomerase, in converting dihydroxyacetone phosphate into

glyceraldehyde 3-phosphate, catalyzes the transfer of a hydrogen atom from C-1 to
C-2, that is, catalyzes an intramolecular oxidation-reduction. And in essence, after
the enzyme reaction, the carbons C-1, C-2 and C-3 of the starting glucose to
become equivalent,  chemically indistinguishable, from the carbons C-6, C-5 and C-
4, respectively.
Therefore, the net result of the the last two steps of glycolysis is the production
of two molecules of glyceraldehyde 3-phosphate.
The ΔG°’ of the reaction is of 7.5 kJ/mol (1.8 kcal/mol), while the ΔG is 2.5 kJ/mol
(0.6 kcal/mol). Although at equilibrium dihydroxyacetone phosphate represent about
96% of the trioso phosphates, the reaction proceeds readily towards the formation of
glyceraldehyde 3-phosphate because of the subsequent step of the glycolytic
pathway that removes the glyceraldehyde 3-phosphate produced.
One of the distinguishing features of triose phosphate isomerase is the great
catalytic efficiency. The enzyme is in fact considered kinetically perfect. Why? The
enzyme enhances the isomerization rate by a factor of 10 10 compared with that
obtained with a catalyst such as acetate ion. Indeed, the K cat/KM ratio for the
isomerization of glyceraldehyde 3-phosphate is equal to 2×10 8 M-1s-1, value close to
the diffusion-controlled limit. Thus, the rate-limiting step in the reaction catalyzed by
triose phosphate isomerase is diffusion-controlled encounter of enzyme and
substrate.
From the energetic point of view, the last two steps of glycolysis are unfavorable,
with ΔG°’ of 31.3 kJ/mol (7.5 kcal/mol), whereas the net ΔG°’ of the first five
reactions is of 2.1 kJ/mol (0.5 kcal/mol), with a Keq of about 0.43. And it is the free
energy derived from the hydrolysis two ATP that, under standard-state conditions,
makes the value of the overall equilibrium constant close to one. If instead we
consider ΔG, it is quite negative, -56.8 kJ/mol (-13.6 kcal/mol).
Notice that dihydroxyacetone phosphate may also be reduced to glycerol 3-
phosphate (see Fig. 3) in the reaction catalyzed by cytosolic glycerol 3-phosphate
dehydrogenase (EC 1.1.1.8).
Dihydroxyacetone phosphate + NADH + H+ ⇄ Glycerol 3-phosphate + NAD+
The enzyme acts as a bridge between glucose and lipid metabolism because the
glycerol 3-phosphate produced is used in the synthesis of lipids such as
triacylglycerols.
This reaction is an important sources of glycerol 3-phosphate in adipose tissue and
small intestine.

Reaction 6: oxidation of glyceraldehyde 3-phosphate to

1,3-bisphosphoglycerate
In the sixth step of the glycolytic pathway, the first step of the second phase, the
payoff phase, glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) catalyses
the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate (1,3-BPG),
with the concomitant reduction of NAD+ to NADH.
Glyceraldehyde 3-phosphate + NAD+ + Pi ⇄ 1,3-Bisphosphoglycerate + NADH + H+
This is the first of the two glycolytic reactions in which the chemical energy needed for
the subsequent synthesis of ATP is harvested and made available; the other reaction
is catalyzed by enolase (EC 4.2.1.11). Why?
This reaction is the sum of two processes.
 In the first reaction, the oxidation of the aldehyde group to a carboxyl group occurs, step
in which NAD+ is used as oxidizing agent. The reaction is quite exergonic, with a ∆G’° of
-43 kJ/mol (-10.3 kcal/mol).
 In the second reaction, the formation of the bond between the carboxylic group at C-1 of
1,3-bisphosphoglycerate and orthophosphate occurs, to form an anhydride called acyl
phosphate. The reaction is quite endergonic, with a ∆G’° of 49.3 kJ/mol (11.8 kcal/mol).
These two chemical processes must not take place in succession but must be
coupled in order to allow the formation of the acyl phosphate because the oxidation
of the aldehyde group is used to drive the formation of the anhydride, with an overall
ΔG°’ of 6.3 kJ/mol (1.5 kcal/mol), and a ΔG of 2.5 kJ/mol (0.6 kcal/mol), both slightly
endergonic.
Therefore, the free energy that might be released as heat is instead conserved by
the formation of the acyl phosphate.

Note: The reversible reduction of the nicotinamide ring of NAD+ or NADP+ is due to

the loss of two hydrogen atoms by another molecule, in this case the aldehyde group
of glyceraldehyde 3-phosphate, that undergoes oxidation, and to the subsequent
transfer of a hydride ion, the equivalent of two electrons and a proton, to the
nicotinamide ring. The other proton removed from the substrate is released to the
aqueous solution. Below, the half reactions for both coenzymes.
NAD+ + 2 e– + 2 H+ → NADH + H+
NADP+ + 2 e– + 2 H+ → NADH + H+

Reaction 7: phosphoglycerate kinase and the first ATP

forming reaction
In the seventh step of the glycolytic pathway, phosphoglycerate kinase (EC 2.7.2.3)
catalyzes the transfer of the high-energy phosphoryl group from the acyl phosphate
of 1,3-BPG to ADP to form ATP and 3-phosphoglycerate (3-PG).
1,3-Bisphosphoglycerate + ADP + H+ ⇄ 3-Phosphoglycerate + ATP
The ΔG°’ of the reaction is of -18.5 kJ/mol (-4.4 kcal/mol), namely, it is an exergonic
reaction. The ΔG is 1.3 kJ/mol (0.3 kcal/mol).
The high phosphoryl-transfer potential of the acyl phosphate is used to
phosphorylate ADP. The production of ATP in this manner is called substrate-level
phosphorylation. In other words, part of the energy released during the oxidation of
the aldehyde group in the sixth step is now conserved by the synthesis of ATP from
the ADP and Pi.
The reaction catalyzed by phosphoglycerate kinase is the first reaction of glycolysis
in which part of the chemical energy present in glucose molecule is conserved as
ATP. And, because the reactions catalyzed by aldolase and triose phosphate
isomerase, step 4 and 5, respectively, lead to the formation of two molecules of
glyceraldehyde 3-phosphate per molecule of glucose, in this step two ATP are
produced and the ATP debt created by the preparatory phase, steps 1 and 3,
respectively, is “paid off”.
It should be noted that the enzyme is named for the reverse reaction, from right to
left as written, that is, the phosphorylation of 3-phosphoglycerate to form 1,3-
bisphosphoglycerate at the expense of one ATP.
Indeed, this enzyme, like all other enzymes, is able to catalyze the reaction in both
directions. And the direction leading to the synthesis of 1,3-bisphosphoglycerate
occurs during the photosynthetic CO2 fixation and gluconeogenesis.
The sixth and seventh reactions of glycolysis, are, as a whole, an energy-coupling
process in which the common intermediate is 1,3-bisphosphoglycerate. While the
reaction leading to the synthesis of 1,3-BPG is endergonic, with a ΔG°’ of 6.3 kJ/mol
(1.5 kcal/mol), the second reaction is strongly exergonic, with a ΔG°’ of -18.5 kJ/mol
(-4,4 kcal/mol). The overall ΔG°’ is -12.2 kJ/mol (-2.9 kcal/mol), namely, the reaction
catalyzed by phosphoglycerate kinase is sufficiently exergonic to pull even the
previous one, too, making the overall reaction exergonic.
Glyceraldehyde 3-phosphate + ADP + Pi + NAD+ ⇄ 3-Phosphoglycerate + ATP +
NADH + H+
In reality, phosphoglycerate kinase reaction is sufficiently exergonic to pull also the
reactions catalyzed by aldolase and triose phosphate isomerase.

What is substrate-level phosphorylation?

Substrate-level phosphorylation is defined as the production of ATP by the transfer of
a phosphoryl group from a substrate to ADP, a process involving chemical
intermediates and soluble enzymes.
There is also a second type of phosphorylation for the synthesis of ATP
called oxidative phosphorylation, a process involving not chemical intermediates and
soluble enzymes but transmembrane proton gradients and membrane-bound
enzymes.
Because the standard free energy of hydrolysis of the phosphoryl group of 3-
phosphoglycerate is equal to 12.5 kJ/mol (-3 kcal/mol), it is not sufficient to produce
ATP by phosphoryl group transfer. In the two subsequent reactions of glycolysis, 3-
phosphoglycerate is converted to phosphoenolpyruvate (PEP), a molecule with a
phosphoryl group transfer potential sufficiently elevated to allow the synthesis of
ATP.

Reaction 8: from 3-phosphoglycerate to 2-

phosphoglycerate
In the eighth step of the glycolytic pathway, 3-phosphoglycerate is converted into 2-
phosphoglycerate (2-PG), in a reversible reaction catalyzed by phosphoglycerate
mutase (EC 5.4.2.1). The reaction requires Mg2+, and has a very small ΔG, equal to
about 0.8 kJ/mol (0.2 kcal/mol) and a ΔG°’ of 4.4 kJ/mol (1.1 kcal/mol).
Phosphoglycerate mutase is a mutase, enzymes that catalyze intramolecular group
transfers, in this case the transfer of a phosphoryl group from C-3 to C-2 of the 3-
phosphoglycerate. Mutases, in turn, are a subclass of isomerases.
The mechanism by which this reaction takes place depends on the type of organism
studied. For example, in yeast or in rabbit muscle the reaction occurs in two steps
and involves the formation of phosphoenzyme intermediates. In the first step, a
phosphoryl group bound to a histidine residue in the active site of the enzyme is
transferred to the hydroxyl group at C-2 of 3-PG to form 2,3-bisphosphoglycerate. In
the next step, the enzyme acts as a phosphatase converting 2,3-BPG into 2-
phosphoglycerate; however, the phosphoryl group at C-3 is not released but linked
to the histidine residue of the active site to regenerate the intermediate enzyme-His-
phosphate. Schematically:
Enzyme-His-phosphate + 3-Phosphoglycerate ⇄ Enzyme-His + 2,3-
Phosphoglycerate

Enzyme-His + 2,3-Bisphosphoglycerate ⇄ Enzyme-His-phosphate + 2-

Phosphoglycerate

Notice that the phosphoryl group of 2-phosphoglycerate is not the same as that of
the substrate 3-phosphoglycerate.
Approximately once in every 100 catalytic cycles, 2,3-BPG dissociates from the
active site of the enzyme, leaving it unphosphorylated, that is, in the inactive form.
The inactive enzyme may be reactivated by binding 2,3-bisphosphoglycerate, which
must, therefore, be present in the cytosol to ensure the maximal activity of the
enzyme. And 2,3-BPG is present in small, but sufficient amounts in most cells,
except for red blood cells, where it acts as an allosteric inhibitor, too, reducing  the
affinity of hemoglobin for oxygen, and has a concentration of 4-5 mM.

Note: 3-Phosphoglycerate can also be used for the biosynthesis of serine, from which
glycine and cysteine derive (see Fig. 3). The biosynthesis of serine begins with the
reaction catalyzed by phosphoglycerate dehydrogenase (EC 1.1.1.95). The enzyme
catalyzes the oxidation of 3-phosphoglycerate to 3-phosphohydroxypyruvate and the
concomitant reduction of NAD+ to NADH. This reaction is also the rate-limiting step of
this biosynthetic pathway, because serine inhibits the activity of the enzyme.

Synthesis of 2,3-bisphosphoglycerate and the Rapoport-

Luebering pathway
1,3-Bisphosphoglycerate can be also converted into 2,3-bisphosphoglycerate (see
Fig. 3).
In red blood cells this reaction is catalyzed by the bisphosphoglycerate mutase, one
of the three isoforms of phosphoglycerate mutase found in mammals. The enzyme
requires the presence of 3-phosphoglycerate as it catalyzes the intermolecular
transfer of a phosphoryl group from C-1 of 1,3-bisphosphoglycerate to the C-2 of 3-
phosphoglycerate. Therefore, 3-phosphoglycerate becomes 2,3-BPG, while 1,3-BPG
is converted into 3-phosphoglycerate. The mutase enzyme activity has EC number
5.4.2.4.
Synthesis of 2,3-BPG
2,3-Bisphosphoglycerate can then be hydrolyzed to 3-phosphoglycerate in the
reaction catalyzed by the phosphatase activity of bisphosphoglycerate mutase, that
removes the phosphoryl group at C-2. This activity has EC number 3.1.3.13. The
enzyme is also able to catalyze the interconversion of 2-phosphoglycerate and 3-
phosphoglycerate, therefore, it is a trifunctional enzyme. 3-Phosphoglycerate can
then re-enter the glycolytic pathway. This detour from glycolysis, also
called Rapoport-Luebering pathway, that leads to the synthesis of 3-
phosphoglycerate without any ATP production.
The other two isoforms of phosphoglycerate mutase, phosphoglycerate mutase 1 or
type M, present in the muscle, and phosphoglycerate mutase 2 or type B, present in
all other tissues, are able to catalyze, in addition to the interconversion of the 2-
phosphoglycerate and 3-phosphoglycerate, the two steps of Rapoport-Luebering
pathway, although with less efficacy than the glycolytic reaction. Therefore they are
trifunctional enzymes.

Reaction 9: formation of phosphoenolpyruvate

In the ninth step of the glycolytic pathway, 2-phosphoglycerate is dehydrated to
form phosphoenolpyruvate, an enol, in a reversible reaction catalyzed by enolase.
2-Phosphoglycerate ⇄ Phosphoenolpyruvate + H2O
 The reaction requires Mg2+ that stabilizes the enolic intermediate that is formed
during the process.
The ΔG°’ of the reaction is 7.5 kJ/mol (1.8 kcal/mol), while ΔG -3.3 kJ/mol (-0.8
kcal/mol).
Like 1,3-BPG, phosphoenolpyruvate has a phosphoryl group transfer potential high
enough to allow ATP formation. Why does this phosphoryl group have a high free
energy of hydrolysis?
Although phosphoenolpyruvate and 2-phosphoglycerate contain nearly the same
amount of metabolic energy with respect to decomposition to CO 2, H20 and Pi, 2-PG
dehydration leads to a redistribution of energy such that the standard free energy of
hydrolysis of the phosphoryl groups vary as described below:
 -17.6 kJ/mol (-4.2 kcal/mol) for 2-phosphoglycerate, a phosphoric ester;
 -61.9 kJ/mol (-14.8 kcal/mol) for phosphoenolpyruvate, an enol phosphate.
What happens is that the phosphoryl group traps PEP in its unstable enol form.
When, in the last step of glycolysis, phosphoenolpyruvate donates the phosphoryl
group to ADP, ATP and the enol form of pyruvate are formed. The enol form of
pyruvate is unstable and tautomerizes rapidly and nonenzymatically to the more
stable keto form, that predominates at pH 7. So, the high phosphoryl-transfer
potential of PEP is due to the subsequent enol-keto tautomerization of pyruvate.

Reaction 10: the transfer of the phosphoryl group from

the phosphoenolpyruvate to the ADP
In the final step of the glycolytic pathway, pyruvate kinase (EC 2.7.1.40) catalyzes
the transfer of the phosphoryl group from phosphoenolpyruvate to ADP to
form pyruvate and ATP. This is the second substrate-level phosphorylation of
glycolysis.
Phosphoenolpyruvate + ADP + H+ → Pyruvate + ATP
The enzyme is a tetramer and, like PFK-1, is a highly regulated. Indeed, it has
binding sites for numerous allosteric effectors. Moreover, in vertebrates, there are at
least three isozymes of pyruvate kinase, of which the M type predominates in muscle
and brain, while the L type in liver. These isozymes have many properties in
common, whereas differ in the response to hormones such as glucagon, epinephrine
and insulin.
The enzyme activity is stimulated by potassium ion (K +) and some other monovalent
cations.
The reaction is essentially irreversible, with a ΔG°’ of -31.4 kJ/mol (-7.5 kcal/mol), and
a ΔG of -16.7 kJ/mol (-4.0 kcal/mol), largely due, as anticipated in the previous
paragraph, to the tautomerization of the pyruvate from the enol form to the more
stable keto form.
 Spontaneous
Tautomerization of Pyruvate
And, of the -61.9 kJ/mol (14.8 kcal/mol) released from the hydrolysis of the
phosphoryl group of PEP, nearly half is conserved in the formation of the
phosphoanhydride bond between ADP and P i, whose ΔG°’ is of -30.5 kJ/mol (-7.3
kcal/mol). The remaining energy, -31.4 kJ/mol (-7.5 kcal/mol), is the driving force that
makes the reaction proceed towards ATP production.
While the reaction catalyzed by phosphoglycerate kinase, in the seventh step of the
glycolytic pathway, pays off the ATP debt of the preparatory phase, the reaction
catalyzed by pyruvate kinase allows a net gain of two ATP.

The fate of NADH and pyruvate produced in

glycolysis
Glycolysis produces 2 NADH, 2 ATP, and 2 pyruvate molecules per molecule of
glucose.
NADH must be reoxidized to NAD+ to allow glycolysis to proceed. NAD+, a coenzyme
that is produced from the vitamin B3, also known as niacin, is present in limited
amounts in the cytosol, ≤ 10-5M, a value well below than that of glucose metabolized
in a few minutes, and must be continuously regenerated. Therefore, the final step of
the glycolytic pathway is the regeneration of NAD + from NADH through aerobic or
anaerobic pathways, each of which involves pyruvate. Such pathways allow,
therefore, maintenance of the redox balance of the cell.
Pyruvate is a versatile metabolite that can enter several metabolic pathways, both
anabolic and catabolic, depending on the type of cell, the energy state of the cell and
the availability of oxygen.

Catabolic
Fates of Pyruvate
With the exception of some variations encountered in bacteria, exploited, for
example, in food industry for the production of various foods such as many cheeses,
there are essentially three pathways in which pyruvate may enter:

 reduction to lactate, through lactic acid fermentation;

 reduction to ethanol or ethyl alcohol, through alcoholic fermentation;
 aerobic oxidation.
This allows glycolysis to proceed in both anaerobic and aerobic conditions.
It is therefore possible to state that the catabolic fate of the carbon skeleton of
glucose is influenced by the cell type, the energetic state of the cell, and the
availability of oxygen.

Lactic acid fermentation

In animals, with few exceptions, and in many microorganisms when oxygen
availability is insufficient to meet the energy requirements of the cell, or if the cell is
 without mitochondria, the pyruvate produced by glycolysis is reduced to lactate in the
cytosol, in a reaction catalyzed by lactate dehydrogenase (EC 1.1.1.27).
Pyruvate + NADH + H+ ⇄ Lactate + NAD+
In the reaction, pyruvate, by accepting electrons from NADH, is reduced to lactate,
while NAD+ is regenerated. And the overall equilibrium of the reaction strongly favors
the formation of lactate, as shown by the value of ΔG°’ of -25.1 kJ/mol (-6 kcal/mol).
The conversion of glucose to lactate is called lactic acid fermentation. The overall
equation of the process is:
Glucose + 2 Pi + 2 ADP + 2H+ → 2 Lactate + 2 ATP + 2 H2O
Notice that fermentation, discovered by Louis Pasteur who defined it “la vie sans
l’air”, is a metabolic pathway that:
 extracts energy from glucose and stores it as ATP;
 does not consume oxygen;
 does not change the concentration of NAD+ or NADH.
With regard to coenzymes, neither NAD+ nor NADH appears in the overall equation,
although both are crucial in the process, that is, no net oxidation-reduction occurs. In
other words, in the conversion of glucose, C 6H12O6, to lactate, C3H6O3, the ratio of
hydrogen to carbon atoms of the reactants and products does not change.
From an energy point of view, it should however be emphasized that fermentation
extracts only a small amount of the chemical energy of glucose.
In humans, much of the lactate produced enters the Cori cycle for glucose production
via gluconeogenesis. We can also state that lactate production shifts part of the
metabolic load from the extrahepatic tissues, such as skeletal muscle during intense
bouts of exercise, like a 200-meter, when the rate of glycolysis can almost instantly
increase 2,000-fold, to the liver.
In contrast to skeletal muscle that releases lactate into the venous blood, the heart
muscle is able to take up and use it for fuel, due to its completely aerobic metabolism
and to the properties of the heart isozyme of lactate dehydrogenase, referred to as
H4. Therefore, portion of the lactate released by skeletal muscle engaged in intense
exercise is used by the heart muscle for fuel.
Note: Lactate produced by microorganisms during lactic acid fermentation is
responsible for both the scent and taste of sauerkraut, namely, fermented cabbage,
as well as for the taste of soured milk.

Alcoholic fermentation
In microorganisms such as brewer’s and baker’s yeast, in certain plant tissues, and
in some invertebrates and protists, pyruvate, under hypoxic or anaerobic conditions,
may be reduced in two steps to ethyl alcohol or ethanol, with release of CO 2.
The first step involves the non-oxidative decarboxylation of pyruvate to form
acetaldehyde, an essentially irreversible reaction. The reaction is catalyzed by
pyruvate decarboxylase (EC 4.1.1.1), an enzyme that requires Mg 2+ and thiamine
pyrophosphate, a coenzyme derived from vitamin thiamine or vitamin B 1. The
enzyme is absent in vertebrates and in other organisms that perform lactic acid
fermentation.
In the second step, acetaldehyde is reduced to ethanol in a reaction catalyzed by
alcohol dehydrogenase (EC 1.1.1.1), an enzyme that contains a bound zinc atom in
its active site. In the reaction, NADH supplies the reducing equivalents and is
oxidized to NAD+. At neutral pH, the equilibrium of the reaction lies strongly toward
ethyl alcohol formation.
The conversion of glucose to ethanol and CO2 is called alcoholic fermentation. The
overall reaction is:
Glucose + 2 Pi + 2 ADP + 2 H+ → 2 Ethanol + 2 CO2 + 2 ATP + 2 H2O
And, as for lactic fermentation, even in alcoholic fermentation no net oxidation-
reduction occurs.

Alcoholic fermentation is the basis of the production of beer and wine. Notice that the
CO2 produced by brewer’s yeast is responsible for the characteristics “bubbles” in
beer, champagne and sparkling wine, while that produced by baker’s yeast causes
dough to rise.

Fate of pyruvate and NADH under aerobic conditions

In cells with mitochondria and under aerobic conditions, the most common situation
in multicellular and many unicellular organisms, the oxidation of NADH and pyruvate
catabolism follow distinct pathways.
In the mitochondrial matrix, pyruvate is first converted to acetyl-CoA in the reactions
catalyzed by the pyruvate dehydrogenase complex, a mitochondrial multienzyme
complex. In the reaction, a oxidative decarboxylation, pyruvate loses a carbon atom
as CO2, and the remaining two carbon unit is bound to Coenzyme A to form acetyl-
coenzyme A or acetyl-CoA.
Pyruvate + NAD+ + CoA → acetyl-CoA + CO2 + NADH + H+
The acetyl group of acetyl-CoA is then completely oxidized to CO 2 in the citric acid
cycle, with production of NADH and FADH2. The pyruvate dehydrogenase complex
therefore represents a bridge between glycolysis, which occurs in the cytosol, and
the citric acid cycle, which occurs in the mitochondrial matrix.
In turn, electrons derived from oxidations that occur during glycolysis are transported
into mitochondria via the reduction of cytosolic intermediates. In this way, in the
cytosol NADH is oxidized to NAD+, while the reduced intermediate, once in the
mitochondrial matrix, is reoxidized through the transfer of its reducing equivalents to
Complex I of the mitochondrial electron transport chain. Here the electrons flow to
oxygen to form H2O, a transfer that supplies the energy needed for the synthesis of
ATP through the process of oxidative phosphorylation. Of course, also the electrons
carried by NADH formed by pyruvate dehydrogenase complex reactions and citric
acid cycle and by FADH2 formed by citric acid cycle meet a similar fate.
Note: FADH2 transfers its reducing equivalents not to Complex I but to Complex II.

Anabolic fates of pyruvate

Under anabolic conditions, the carbon skeleton of pyruvate may have fates other
than complete oxidation to CO2 or conversion to lactate. In fact, after its conversion
to acetyl-CoA, it may be used, for example, for the synthesis of fatty acids, or of the
amino acid alanine (see Fig. 3).
Glycolysis and ATP production
In the glycolytic pathway the glucose molecule is degraded to two molecules of
pyruvate.
In the first phase, the preparatory phase, two ATP are consumed per molecule of
glucose in the reactions catalyzed by hexokinase and PFK-1. In the second phase,
the payoff phase, 4 ATP are produced through substrate-level phosphorylation in the
reactions catalyzed by phosphoglycerate kinase and pyruvate kinase. So there is
a net gain of two ATP per molecule of glucose used. In addition, in the reaction
catalyzed by glyceraldehyde 3-phosphate dehydrogenase, two molecules of NADH
are produced for each glucose molecule.
Energy Changes of Glycolytic Reactions
The overall ΔG°’ of glycolysis is -85 kJ/mol (-20.3 kcal/mol), value resulting from the
difference between the ΔG°’ of the conversion of glucose into two pyruvate
molecules, -146 kJ/mol (-34,9 kcal/mol), and the ΔG°’ of the formation of ATP from
ADP and Pi, 2 x 30.5 kJ/mol = 61 kJ / mol (2  x 7.3 kcal/mol = 14.6 kcal/mol). Here
are the two reactions.
Glucose + 2 NAD+ → 2 Pyruvate + 2 NADH + 2 H+
2 ADP + 2 Pi → 2 ATP + 2 H2O
The sum of the two reactions gives the overall equation of glycolysis.
 Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H20
Thus, under standard conditions, the amount of released energy stored within ATP is
(61/146) x 100 = 41.8%.
Notice that the overall equation of glycolysis can also be derived by considering all
the reagents, ATP, NAD+, ADP, and Pi and all the products.
Glucose + 2 ATP + 2 NAD+ + 4 ADP + 2 Pi → 2 Pyruvate + 2 ADP + 2 NADH + 2
H+ + 4 ATP + 2 H20
Cancelling the common terms on both sides of the equation, we obtain the overall
equation shown above.

Glycolysis and ATP production under anaerobic

conditions
Under anaerobic conditions, regardless of what is the metabolic fate of pyruvate,
conversion to lactate, ethanol or other molecules, there is no additional production of
ATP downstream of glycolysis.
Therefore under these conditions, glycolysis extracts only a small fraction of the
chemical energy of the glucose molecule, energy equal to 2840 kJ/mol (679
kcal/mol) released as a result of its conversion to CO 2 and H2O. Indeed, only 146
kJ/mol are released in the conversion of a glucose molecule to two pyruvate
molecules, equal to 5%, [(146/2,840) x 100], of the available chemical energy.
Therefore,  pyruvate still contains most of the chemical energy of the hexose.
Similarly, the 4 electrons carried by NADH produced in step 6 of glycolysis cannot be
used for ATP production.
In lactic acid fermentation, the ΔG°’ associated with the conversion of a glucose
molecule to two molecules of lactate is -183.6 kJ/mol (-43.9 kcal/mol), and 33.2% of
such free energy, [(61/183.6) x 100] is stored within ATP, whereas it is 41.8% in the
conversion of a glucose molecule to two molecules of pyruvate.
It should be noted that under actual conditions the amount of free energy required for
the synthesis of ATP from ADP and Pi is much higher than that required under
standard conditions, namely, approximately 50%  of the energy released is stored
within ATP.

Glycolysis and ATP production under aerobic conditions

Under aerobic conditions, in cells with mitochondria, the amount of chemical energy
that can be extracted from glucose and stored within ATP is much greater than under
anaerobic conditions.
If we consider the two NADH produced during glycolysis, the flow of their 4 reducing
equivalents along the mitochondrial electron transport chain allows the production of
2-3 ATP per electron pair through oxidative phosphorylation. Therefore, 6 to 8
ATP are produced when one molecule of glucose is converted into two molecules of
pyruvate, 2 from glycolysis and 4-6 from oxidative phosphorylation.
Note: The amount of ATP produced from the reducing equivalents of NADH depends
upon the mechanism by which they are shuttled into mitochondria.
On the other hand, if we analyze the coordinated and consecutive action of
glycolysis, the pyruvate dehydrogenase complex, citric acid cycle,  mitochondrial
electron transport chain and oxidative phosphorylation, much more energy can be
extracted from glucose and stored within ATP. In this case, according to what
reported by Lehninger, 30 to 32 ATP are produced for each glucose molecule,
although recent estimates suggest a net production equal to 29.85 ATP/glucose, or
29.38 ATP/glucose if also ATP formed from GTP, in turn produced by the citric acid
cycle, is exported. Considering both estimates, the production of ATP is about 15
times greater than under anaerobic condition.

Feeder pathways for glycolysis

Other carbohydrates besides glucose, both simple and complex, can be catabolized
via glycolysis, after enzymatic conversion to one of the glycolytic intermediates.
Among the most important are:

 glycogen and starch, two storage polysaccharides;

 some disaccharides such as sucrose, maltose, lactose and trehalose;
 the monosaccharides galactose, fructose, and the less common mannose.
Feeder Pathways for Glycolysis
Dietary starch and disaccharides must be hydrolyzed in the intestine to the respective
monosaccharides before being absorbed. Once in the venous circulation,
monosaccharides reach the liver through the portal vein; this organ is the main site
where they are metabolized.
Glycogen and starch
Regarding the phosphorolytic breakdown of starch and endogenous glycogen refer
to the corresponding articles.

Fructose
Under physiological conditions, the liver removes much of the ingested fructose from
the bloodstream before it can reach extrahepatic tissues.
The hepatic pathway for the conversion of the monosaccharide to intermediates of
glycolysis consists of several steps.
In the first step fructose is phosphorylated to fructose 1-phosphate at the expense of
one ATP. This reaction is catalyzed by fructokinase (EC 2.7.1.4), and requires the
presence of Mg2+.
Fructose + ATP → Fructose 1-phosphate + ADP + H+
As for glucose, fructose phosphorylation traps the molecule inside the cell.
In the second step fructose 1-phosphate aldolase catalyzes the hydrolysis, an aldol
cleavage, of fructose 1-phosphate to dihydroxyacetone phosphate and
glyceraldehyde.
Fructose 1-phosphate → Dihydroxyacetone Phosphate + Glyceraldehyde

Dihydroxyacetone phosphate is an intermediate of the glycolytic pathway and, after

conversion to glyceraldehyde 3-phosphate, may flow through the pathway.
Conversely, glyceraldehyde is not an intermediate of the glycolysis, and is
phosphorylated to glyceraldehyde 3-phosphate at the expense of one ATP. The
reaction is catalyzed by triose kinase (EC 2.7.1.28), and requires the presence of
Mg2+.
Glyceraldehyde + ATP → Glyceraldehyde 3-phosphate + ADP + H +
In hepatocytes, therefore, a molecule of fructose is converted to two molecules of
glyceraldehyde 3-phosphate, at the expense of two ATP, as for glucose.
Fructose + 2 ATP → 2 Glyceraldehyde 3-phosphate +2 ADP +2  H+

Fructose and hexokinase

In extrahepatic sites, such as skeletal muscle, kidney or adipose tissue, fructokinase
is not present, and fructose enters the glycolytic pathway as fructose 6-phosphate. In
fact, as previously seen, hexokinase can catalyzes the phosphorylation of fructose at
C-6.
Fructose + ATP → Fructose 6-phosphate + ADP + H+
However, the affinity of the enzyme for fructose is about 20 times lower than for
glucose, so in the hepatocyte, where glucose is much more abundant than fructose,
or in the skeletal muscle under anaerobic conditions, that is, when glucose is the
preferred fuel, little amounts of fructose 6-phosphate are formed.
Conversely, in adipose tissue, fructose is more abundant than glucose, so that its
phosphorylation by hexokinase is not competitively inhibited to a significant extent by
glucose. In this tissue, therefore, fructose 6-phosphate synthesis is the entry
point into glycolysis for the monosaccharide.
With regard to the metabolic effects of fructose, it is important to underline that in the
liver the monosaccharide, being phosphorylated at C-1, enters glycolysis at triose
phosphate level, thus downstream to the reaction catalyzed by PFK-1, an enzyme
that plays a key role in the regulation of the flow of carbon through this metabolic
pathway. Conversely, when fructose is phosphorylated at C-6, it enters the glycolytic
pathway upstream of PFK-1.

Sorbitol
Fructose is the entry point into glycolysis for sorbitol, a sugar present in many fruits
and vegetables, and used as a sweetener and stabilizer, too. In the liver, sorbitol
dehydrogenase (EC 1.1.99.21) catalyzes the oxidation of sorbitol to fructose.
Sorbitol + NAD+ → Fructose + NADH + H+
The reaction requires the presence of zinc ion, and occurs in the cytosol.

Galactose
Galactose, for the most part derived from intestinal digestion of the lactose, once in
the liver is converted, via the Leloir pathway, to glucose 1-phosphate.
For a more in-depth discussion of the Leloir pathway, see the article on galactose.
The metabolic fate of glucose 1-phosphate depends on the energy status of the cell.
Under conditions promoting glucose storage, glucose 1-phosphate can be channeled
to glycogen synthesis. Conversely, under conditions that favor the use of glucose as
fuel, glucose 1-phosphate is isomerized to glucose 6-phosphate in the reversible
reaction catalyzed by phosphoglucomutase (EC 5.4.2.2).
Glucose 1-phosphate ⇄ Glucose 6-phosphate

In turn, glucose 6-phosphate can be channeled to glycolysis and be used for energy
production, or dephosphorylated to glucose in the reaction catalyzed by glucose 6-
phosphatase, and then released into the bloodstream.

Mannose
Mannose is present in various dietary polysaccharides, glycolipids and glycoproteins.
In the intestine, it is released from these molecules, absorbed, and, once reached
the liver, is phosphorylated at C-6 to form mannose 6-phosphate, in the reaction
catalyzed by hexokinase.
Mannose + ATP → Mannose 6-phosphate + ADP + H +
Mannose 6-phosphate is then isomerized to fructose 6-phosphate in the reaction
catalyzed by mannose 6-phosphate isomerase (EC 5.3.1.8.).
Mannose 6-phosphate ⇄ Fructose 6-phosphate

Regulation of glycolysis
 The flow of carbon through the glycolytic pathway is regulated in response to
metabolic conditions, both inside and outside the cell, essentially to meet two needs:
the production of ATP and the supply of precursors for biosynthetic reactions.
And in the liver, to avoid wasting energy, glycolysis and gluconeogenesis are
reciprocally regulated so that when one pathway is active, the other slows down. As
explained in the article on gluconeogenesis, during evolution this was achieved by
selecting different enzymes to catalyze the essentially irreversible reactions of the
two pathways, whose activity are regulated separately. Indeed, if these reactions
proceeded simultaneously at high speed, they would create a futile cycle or
substrate cycle. A such fine regulation could not be achieved if a single enzyme
operates in both directions.
The control of the glycolytic pathway involves essentially the reactions catalyzed
by hexokinase, PFK-1, and pyruvate kinase, whose activity is regulated through:
 allosteric modifications, that occur on a time scale of  milliseconds and are instantly
reversible;
 covalent modifications, that is, phosphorylations and dephosphorylation, that occur on a
time scale of seconds;
 changes in enzyme concentrations, resulting from changes in the rate of their synthesis
and/or degradation, that occur on a time scale of hours.
Note: The main regulatory enzymes of gluconeogenesis are pyruvate carboxylase
(EC 6.4.1.1) and fructose 1,6-bisphosphatase (EC 3.1.3.11).

Hexokinase
In humans, hexokinase has four tissue specific isozymes, designated as hexokinase
I, II, III, and IV, encoded by as many genes.
Hexokinase I is the predominant isozyme in the brain, whereas in skeletal muscle
hexokinase I and II are present, accounting for 70-75% and 25-30% of the isozymes,
respectively.
Hexokinase IV, also known as glucokinase (EC 2.7.1.2), is mainly present in
hepatocytes and β cells of the pancreas, where it is the predominant isozyme. In the
liver it catalyzes, with glucose 6-phosphatase, the substrate cycle between glucose
and glucose 6-phosphate. Glucokinase differs from the other hexokinase isozymes
in kinetic and regulatory properties.
Note: Isoenzymes or isozymes are different proteins that catalyze the same reaction,
and that generally differ in kinetic and regulatory properties, subcellular distribution,
or in the cofactors used. They may be present in the same species, in the same
tissue or even in the same cell.

Comparison of the kinetic properties of hexokinase isozymes

The kinetic properties of hexokinase I, II, and III are similar.
Hexokinase I and II have a Km for glucose of 0.03 mM and 0.1 mM, respectively.
Therefore these isoenzymes work very efficiently at normal blood glucose levels, 4-5
mM.
Conversely, glucokinase has a high Km for glucose, approximately 10 mM; this
means that the enzyme works efficiently only when blood glucose concentration is
high, for example after a meal rich in carbohydrates with a high glycemic index.

Regulation of the activity of hexokinases I-III

Hexokinases I-III are allosterically inhibited by glucose 6-phosphate, the product of
their reaction. This ensures that glucose 6-phosphate does not accumulate in the
cytosol when glucose is not needed for energy, for glycogen synthesis, for the
pentose phosphate pathway, or as a source of precursors for biosynthetic pathways,
leaving, at the same time, the monosaccharide in the blood, available for other
organs and tissues. For example, when PFK-1 is inhibited, fructose 6-phosphate
accumulates and then, due to phosphoglucose isomerase reaction, glucose 6-
phosphate accumulates. Therefore, inhibition of PFK-1 leads to inhibition of
hexokinases I-III.
In skeletal muscle, the activity of hexokinase I and II is coordinated with that
of GLUT4, a low Km glucose transporter (5mM), whose translocation to the plasma
membrane is induced by both insulin and physical activity. The combined action of
GLUT4 on plasma membrane and hexokinase in the cytosol maintains
a balance between glucose uptake and its phosphorylation. Because blood glucose
concentration is between 4 and 5 mmol/L, its entry into the myocyte through GLUT4
may cause an increase in its concentration sufficient to saturate, or near saturate the
enzyme, which therefore operates at or near its V max.

Regulation of the activity of hepatic glucokinase

Glucokinase differs in three respects from hexokinases I-III, and is particularly
suitable for the role that the liver plays in glycemic control. Why?
 As previously said, glucokinase has a Km for glucose of about 10 mM, much higher than
the Km for glucose of hexokinases I-III, and higher than the value of fasting blood glucose
levels (4-5 mM) as well. In the liver, where it is the predominant hexokinase isoenzyme,
its role is to provide glucose 6-phosphate for the synthesis of glycogen and fatty acids.
The activity of glucokinase is linked to that of GLUT2, the major glucose transporter in
hepatocytes, with a high Km for glucose, approximately 10 mM. Hence, GLUT2 is very
active when blood glucose concentration is high, rapidly equilibrating sugar
concentrations in cytosol of hepatocytes and blood. Under such conditions glucokinase is
active and converts glucose to glucose 6-phosphate, and, due to high Km for glucose, its
activity continues to increase even when the intracellular concentration of the
monosaccharide reaches or exceeds 10 mM.  Therefore, the rate at which glucose uptake
and phosphorylation occurs are determined by the value of blood glucose level itself. On
the other hand, when glucose availability is low, its concentration in the cytosol of
hepatocytes is just as low, much lower than the Km for glucose of glucokinase, so that
glucose produced through gluconeogenesis and/or glycogenolysis is not phosphorylated
and can leave the cell.
A similar situation also occurs in pancreatic β cells, where the GLUT2/glucokinase
system causes the intracellular G-6-P concentration to equalize with glucose concentration
in the blood, allowing the cells to detect and respond to hyperglycemia.
 Unlike hexokinases I-III, glucokinase is not inhibited by glucose 6-phosphate, that is, is
not product inhibited, and catalyzes its synthesis even when it accumulates.
 Glucokinase is inhibited by the reversible binding of glucokinase regulatory protein or
GKRP, a liver-specific regulatory protein. The mechanism of inhibition by GKRP occurs
via the anchorage of glucokinase inside the nucleus, where it is separated from the other
glycolytic enzymes.

Regulation
Glucokinase Activity
The binding between glucokinase and GKRP is much tighter in the presence of fructose
6-phosphate, whereas it is weakened by glucose and fructose 1-phosphate.
In the absence of glucose, glucokinase is in its super-opened conformation that has low
activity. The rise in cytosolic glucose concentration causes a concentration dependent
transition of glucokinase to its close conformation, namely, its active conformation that is
not accessible for glucokinase regulatory protein. Hence, glucokinase is active and no
longer inhibited.
Notice that fructose 1-phosphate is present in the hepatocyte only when fructose is
metabolized. Hence, fructose relieves the inhibition of glucokinase by glucokinase
regulatory protein.
Example
After a meal rich in carbohydrates, blood glucose levels rise, glucose enters the hepatocyte
through GLUT2, and then moves inside the nucleus through the nuclear pores. In the
nucleus glucose determines the transition of glucokinase to its close conformation, active
and not accessible to GKRP, allowing glucokinase to diffuse in the cytosol where it
 phosphorylates glucose.
Conversely, when glucose concentration declines, such as during fasting when blood
glucose levels may drop below 4 mM, glucose concentration in hepatocytes is low, and
fructose 6-phosphate binds to GKRP allowing it to bind tighter to glucokinase. This
results in a strong inhibition of the enzyme. This mechanism ensures that the liver, at low
blood glucose levels, does not compete with other organs, primarily the brain, for glucose.
In the cell, fructose 6-phosphate is in equilibrium with glucose 6-phosphate, due to
phosphoglucose isomerase reaction. Through its association with GKRP, fructose 6-
phosphate allows the cell to decrease glucokinase activity, so preventing the accumulation
of intermediates.
To sum up, when blood glucose levels are normal, glucose is phosphorylated mainly
by hexokinases I-III, whereas when blood glucose levels are high glucose can be
phosphorylated by glucokinase as well.

Regulation of phosphofructokinase 1 activity

Phosphofructokinase 1 is the key control point of carbon flow through the glycolytic
pathway.
The enzyme, in addition to substrate binding sites, has several binding sites for
allosteric effectors.
ATP, citrate, and hydrogen ions are allosteric inhibitors of the enzyme, whereas
AMP, Pi and fructose 2,6-bisphosphate are allosteric activators.
Regulation of PFK 1 and Fructose 1,6-bisphosphatase
It should be noted that ATP, an end product of glycolysis, is also a substrate of
phosphofructokinase 1. Indeed, the enzyme has two binding sites for the nucleotide:
a low-affinity regulatory site, and a high affinity substrate site.
What do allosteric effectors signal?

 ATP, AMP and Pi signal the energy status of the cell.

The activity of PFK-1 increases when the energy charge of the cell is low, namely, when
there is a need for ATP, whereas it decreases when the energy charge of the cell is high,
namely when ATP concentration in the cell is high. How?
When the nucleotide is produced faster than it is consumed, its cellular concentration is
high. Under such condition ATP, binding to its allosteric site, inhibits PFK-1 by reducing
the affinity of the enzyme for fructose 6-phosphate. From the kinetic point of view, the
increase in ATP concentration modifies the relationship between enzyme activity and
substrate concentration, chancing the hyperbolic fructose 6-phosphate velocity curve into
a sigmoidal one, and then, increasing Km for the substrate. However, under most cellular
conditions, ATP concentration does not vary much. For example, during a vigorous
exercise ATP concentration in muscle may lower of about 10% compared to the resting
state, whereas glycolysis rate varies much more than would be expected by such
reduction.
When ATP consumption exceeds its production, ADP and AMP concentrations rise, in
 particular that of AMP, due to the reaction catalyzed by adenylate kinase (EC 2.7.4.3), that
form ATP from ADP.
ADP + ADP ⇄ ATP + AMP

The equilibrium constant, Keq, of the reaction is:

Keq = [ATP][AMP]/[ADP]2= 0.44
Under normal conditions, ADP and AMP concentrations are about 10% and often
less than 1% of ATP concentration, respectively. Therefore, considering that the total
adenylate pool is constant over the short term, even a small reduction in ATP
concentration leads, due to adenylate kinase activity, to a much larger relative
increase in AMP concentration. In turn, AMP acts by reversing the inhibition due to
ATP.
Therefore, the activity of phosphofructokinase 1 depends on the cellular energy
status:
when ATP is plentiful, enzyme activity decreases;

when AMP levels increase and ATP levels fall, enzyme activity increases.

Why is not ADP a positive effector of PFK-1? There are two reasons.
When the energy charge of the cell falls, ADP is used to regenerate ATP, in the
reaction catalyzed by adenylate kinase Moreover, as previously said, a small
reduction in ATP levels leads to larger-percentage changes in ADP levels and,
above all, in AMP levels.

 Hydrogen ions inhibit PFK-1. Such inhibition prevents, by controlling the rate of

glycolysis, excessive lactate buildup and the consequent fall of blood pH.
 Citrate is an allosteric inhibitor of PFK-1 that acts by enhancing the inhibitory effect of
ATP.
It is the product of the first step of the citric acid cycle, a metabolic pathway that provides
building blocks for biosynthetic pathways and directs electrons into mitochondrial
electron transport chain for ATP synthesis via oxidative phosphorylation. High citrate
levels in the cytosol mean that, in the mitochondria, an overproduction of building blocks
is occurring and the current energy are met, namely, the citric acid cycle has reached
saturation. Under such conditions glycolysis, that feeds the cycle under aerobic
condition, can slow down, sparing glucose.
So, it should be noted that PFK-1 couples glycolysis and the citric acid cycle.
 In the liver, the central control point of glycolysis and gluconeogenesis is the substrate
cycle between F-6-P and F-1,6-BP, catalyzed by PFK-1 and fructose 1,6-bisphosphatase.
The liver plays a pivotal role in maintaining blood glucose levels within the normal range.
When blood glucose levels drop, glucagon stimulates hepatic glucose synthesis, via both
glycogenolysis and gluconeogenesis, and at the same time signals the liver to stop
consuming glucose to meet its needs.
Conversely, when blood glucose levels are high, insulin causes the liver to use glucose for
energy, and to synthesize glycogen, and triglycerides.
In this context, the regulation of glycolysis and gluconeogenesis is mediated by fructose
2,6-bisphosphate, a molecule that allows the liver to play a major role in regulating blood
glucose levels, and whose levels are controlled by insulin and glucagon.
As a result of binding to its allosteric site on PFK-1, fructose 2,6-
 bisphosphate increases the affinity of the enzyme for fructose 6-phosphate, its substrate,
while decreases its affinity for the allosteric inhibitors citrate and ATP. It is remarkable to
note that under physiological concentrations of the substrates and positive and negative
allosteric effectors, phosphofructokinase 1 would be virtually inactive in the absence of
fructose 2,6-bisphosphate.
On the other hand, the binding of fructose 2,6-bisphosphate to fructose 1,6-bisphosphatase
inhibits the enzyme, even in the absence of AMP, another allosteric inhibitor of the
enzyme.
Due to these effects, fructose 2,6-bisphosphate increases the net flow of glucose through
glycolysis.
For an more in-depth analysis of fructose 2,6-bisphosphate metabolism, refer to the article
on gluconeogenesis.
 Another metabolite involved in the control of the flow of carbon through glycolysis and
gluconeogenesis is xylulose 5-phosphate, a product of the pentose phosphate pathway,
whose concentration in hepatocytes rises after ingestion of a carbohydrate-rich meal. The
molecule, by activating protein phosphatase 2A, finally leads to an increase in the
concentration of fructose 2,6-bisphosphate, and then to an increase in the flow of carbon
through glycolysis and to a reduction in the flow of carbon through gluconeogenesis.

Regulation of pyruvate kinase activity

A further control point of carbon flow through glycolysis and gluconeogenesis is the
substrate cycle between phosphoenolpyruvate and pyruvate, catalyzed by pyruvate
kinase for glycolysis, and by the combined action of pyruvate carboxylase and
phosphoenolpyruvate carboxykinase (EC 4.1.1.32) for gluconeogenesis.
All isozymes of pyruvate kinase are allosterically inhibited by high concentrations
of ATP, long-chain fatty acids, and acetyl-CoA, all signs that the cell is in an optimal
energy status. Alanine, too, that can be synthesized from pyruvate through a
transamination reaction, is an allosteric inhibitor of pyruvate kinase; its accumulation
signals that building blocks for biosynthetic pathways are abundant.
 Regulation of Pyruvate Kinase Activity
Conversely, pyruvate kinase is allosterically activated by fructose 1,6-bisphosphate,
the product of the first committed step of glycolysis. Therefore, F-1,6-BP allows
pyruvate kinase to keep pace with the flow of intermediates. It should be underlined
that, at physiological concentration of PEP, ATP and alanine, the enzyme would be
completely inhibited without the stimulating effect of F-1,6-BP.
The hepatic isoenzyme, but not the muscle isoenzyme, is also subject to regulation
through phosphorylation by:
 protein kinase A or PKA, activated by the binding of glucagon to the specific receptor or
epinephrine to β-adrenergic receptors;
 calcium/calmodulin dependent protein kinase or CAMK, activated by the binding of
epinephrine to α1-adrenergic receptors.
Phosphorylation of the enzyme decreases its activity, by increasing the Km for
phosphoenolpyruvate, and slows down glycolysis.
For example, when the blood glucose levels are low, glucagon-induced
phosphorylation decreases pyruvate kinase activity. The phosphorylated enzyme is
also less readily stimulated by fructose 1,6-bisphosphate but more readily inhibited
by alanine and ATP. Conversely, the dephosphorylated form of pyruvate kinase is
more sensitive to fructose 1,6-bisphosphate, and less sensitive to ATP and alanine.
In this way, when blood glucose levels are low, the use of glucose for energy in the
liver slows down, and the sugar is available for other tissues and organs, such as the
brain. However, it should be noted that pyruvate kinase does not undergo glucagon-
induced phosphorylation in the presence of fructose 1,6-bisphosphate.
An increase in the insulin/glucagon ratio, on the other hand, leads to
dephosphorylation of the enzyme and then to its activation. The dephosphorylated
enzyme is more readily stimulated by its allosteric activators F-1,6-BP, and less
readily inhibited by allosteric inhibitors alanine and ATP.