GLYCOLYSIS

INTRODUCTION

• The glycolytic or the Embden-Meyerhof-Parnas

(EMP) pathway is used by all tissues for the
oxidation of glucose to provide energy (as ATP)
and intermediates for other metabolic
pathways.
• Glycolysis is defined as a sequence of reactions
by which glucose is broken down to yield
pyruvate, energy(ATP) and NADH and takes
place in the cell cytosol.
• INTRODUCTION CTD

• Under different conditions, glycolysis may go into:

Aerobic pathway which sets the stage for the
oxidative decarboxylation of pyruvate to acetyl CoA,
a major component of the TCA cycle in the
mitochondria.
OR
Anaerobic pathways when pyruvate is reduced to
lactate
OR
 Alcoholic fermentation pathway occurring mainly in
prokaryotes e.g yeast and some strains of bacteria in which
pyruvate is converted to ethanol and carbon dioxide
 REACTIONS OF GLYCOLYSIS

• The conversion of glucose to pyruvate occurs in two stages

.
• The first five reactions of glycolysis correspond to an
energy-investment phase in which the phosphorylated
forms of intermediates are synthesized at the expense of
ATP. (This phase is followed by a cleavage phase)
• The subsequent reactions of glycolysis constitute an
energy-generation phase in which a net of two molecules
of ATP are formed by substrate-level phosphorylation per
glucose molecule metabolized.
• The reactions of glycolysis occur in the cytosolic
compartment of the cell where the enzymes are located.
• The sequence of reactions of the glycolytic pathway which
occurs in ten (10) steps are shown below:
• REACTION 1 : Glucose phosphorylation - Conversion of
glucose to glucose-6-phosphate
• Glucose enters the glycolytic pathway by phosphorylation
to glucose -6- phosphate at the expense of ATP by
hexokinase or glucokinase.
• Note:Mammals have four isozymes (I–IV) of the enzyme hexokinase
that catalyze the phosphorylation of glucose to glucose 6-phosphate.
• Phosphorylated sugar molecules do not readily penetrate
cell membranes because there are no specific
transmembrane carriers for these compounds and
because they are too polar to diffuse through the lipid
bilayer of membranes.
• Therefore, the irreversible phosphorylation of glucose
effectively traps the sugar as cytosolic glucose 6-
phosphate and commits it to further metabolism in the
REACTION 1 : Glucose phosphorylation - Conversion of glucose to glucose-6-phosphate
 Properties of hexokinase and glucokinase
• HEXOKINASE (There are three isoforms - Hexokinases I–III):
1.Hexokinase is present in all cells/tissues(ubiquitous) and
in these tissues, glucose phosphorylation is catalyzed by
one of these isozymes of hexokinase.
2. It is one of the regulatory enzymes of glycolysis; being
inhibited by the reaction product, glucose-6-phosphate,
which accumulates when further metabolism of this
hexose phosphate is reduced by any product.(The inhibition of
hexokinase by glucose-6-phosphate is to ensure that some feedback control
may be exerted on glucose uptake in the extrahepatic tissues which are
dependent on glucose).
3. They have a low Km (and, therefore, a high affinity for
glucose) and this permits the efficient phosphorylation and
subsequent metabolism of glucose even when tissue
concentrations of glucose are low.
4. They have a low maximal velocity(Vmax) for
glucose and thus do not phosphorylate more
glucose than the cell can use.
5. These isozymes have broad substrate specificity
and are able to phosphorylate several hexoses in
addition to glucose such as fructose, mannose
and galactose (lack of specificity) but at a much
slower rate.
6. They can act on both the - and - anomers of
glucose.
7. Non-inducible enzyme, not affected by diabetes
or insulin
• GLUCOKINASE(also called hexokinase 1V)
• 1. It is found predominantly in liver parenchymal
cells and pancreatic β cells, where they are
responsible for glucose phosphorylation. In β cells,
glucokinase functions as a glucose sensor,
determining the threshold for insulin secretion and
also serves as a glucose sensor in hypothalamic
neurons, playing a key role in the adrenergic
response to hypoglycemia.
• 2. Glucokinase activity is not directly inhibited by
glucose 6-phosphate as are the other hexokinases.
 3. It has a much higher Km, requiring a higher glucose
concentration for half-saturation and therefore functions only
when the intracellular concentration of glucose in the
hepatocyte is elevated such as during the brief period
following consumption of a carbohydrate-rich meal, when
high levels of glucose are delivered to the liver via the portal
vein.
4.Glucokinase has a high Vmax, allowing the liver to effectively
remove the flood of glucose delivered by the portal blood.
• This prevents large amounts of glucose from entering the
systemic circulation following such a meal, thereby
minimizing hyperglycemia during the absorptive period.
• [Note: GLUT-2 insures that blood glucose equilibrates rapidly
across the hepatocyte membrane.]
• 5.Glucokinase has absolute specificity
• 6. Inducible, synthesis induced by insulin & repressed in
• REACTION 2
• Glucose 6-phosphate isomerization
• The isomerization of glucose 6-phosphate to
fructose 6-phosphate is catalyzed by
phosphohexoseisomerase.
(phosphoglucoseisomerase)
• The reaction is readily reversible and is not a rate-
limiting or regulated step.
Glucose-6-phosphate
 Reaction 3: Fructose 6-phosphate phosphorylation

• Fructose 6-phosphate is phosphorylated by

phosphofructokinase-1 (PFK-1) to Fructose 1,6-
bisphosphate.
• This is the second molecule of ATP consumed in
the pathway.
• This reaction catalyzed by PFK-1 is the most
important control point and the rate-limiting and
committed step of glycolysis.
• [Note:Fructose 2,6-bisphosphate is formed from
fructose 6-phosphate by phosphofructokinase-2
(PFK-2)].
• Regulation of Phosphofructokinase-1
• 1. Regulation of PFK-1 by intracellular energy levels:
• PFK-1 is inhibited allosterically by elevated levels of
ATP, which act as an energy-rich signal indicating an
abundance of high-energy compounds.
• Elevated levels of citrate, an intermediate in the
TCA cycle , also inhibit PFK-1. [Note: Inhibition by
citrate favors the use of glucose for glycogen
synthesis] .
• Conversely, PFK-1 is activated allosterically by high
concentrations of AMP, which signal that the cell’s
energy stores are depleted.
• 2. Regulation by fructose 2,6-bisphosphate:
• Fructose 2,6-bisphosphate is the most potent
activator of PFK-1.
• [Note: Fructose 2,6-bisphosphate is an inhibitor of fructose
• 1,6-bisphosphatase, an enzyme of gluconeogenesis (see later). The
reciprocal actions of fructose 2,6-bisphosphate on glycolysis
(activation) and gluconeogenesis (inhibition) insure that both
pathways are not fully active at the same time, preventing a futile
cycle of glucose oxidation to pyruvate followed by glucose
resynthesis from pyruvate.]
Regulation of PFK-1
• Other regulatory mechanisms - fed & fasted states-
(hormonal)
• a. During the well-fed state: Decreased levels of glucagon and
elevated levels of insulin (such as occur following a
carbohydrate-rich meal) cause an increase in hepatic fructose
2,6-bisphosphate (Because PFK-2 is dephosphorylated,
making it active) and, thus, increase in the rate of glycolysis
and inhibition of gluconeogenesis.
• Therefore, fructose 2,6-bisphosphate acts as an intracellular
signal of glucose abundance.
• b. During fasting: By contrast, the elevated levels of glucagon
and low levels of insulin that occur during fasting cause a
decrease in hepatic fructose 2,6-bisphosphate (Because PFK-2
is phosphorylated making it inactive).
• This results in inhibition of glycolysis and activation of
gluconeogenesis.
 REGULATION OF GLYCOLYSIS
• PFK-I is the major regulatory enzyme Rate of glycolysis is controlled primarily by
of glycolysis. . It is inhibited by citrate allosteric regulation of the 3 key enzymes
& ATP and activated by AMP and In (irreversible steps), hexokinase, PFK-1, and
the liver only, PFK-1 is activated by pyruvate kinase.
fructose-2,6-bisphosphate (F-2,6-
Inhibitor Activator Enzyme
bisP).
• PFK-II, the enzyme that synthesize G-6-P AMP, ADP, Pi Hexokinase
the activator F-2,6-bisP, is itself a
regulatory enzyme and inhibited by
phosphorylation. and activated by citrate, ATP AMP, F-2,6- PFK-1
bisP
dephosphorylation. The reverse
(liver only)
reaction is catalyzed by fructose-2,6-
bisphosphatase(F-2,6-bisPase in
gluconeogenesis). ATP, acetyl AMP, F-1,6- Pyruvate
• Hormones also regulate glycolysis CoA, bis P kinase
phosphorylation
e.g., glucagon inhibits glycolysis by
July 28, 2025 18
repressing the synthesis of F-2,6-bisP.
• Reaction 4- Fructose 1,6-bisphosphate
cleavage
• Aldolase cleaves fructose 1,6-bisphosphate to
dihydroxyacetone phosphate (DHAP) and
glyceraldehyde 3-phosphate.
• The reaction is reversible and not regulated.
[Note: Aldolase B, the hepatic isoform, also cleaves fructose 1-phosphate
and functions in dietary fructose metabolism].
Reaction 4 : Fructose 1,6-bisphosphate cleavage
• Reaction 5 : Dihydroxyacetone phosphate
isomerization
• Triose phosphate isomerase interconverts DHAP
and glyceraldehyde 3- phosphate.
• DHAP must be isomerized to glyceraldehyde 3-
phosphate for further metabolism by the
glycolytic pathway.
• This isomerization results in the net production
of two molecules of glyceraldehyde 3-phosphate
from the cleavage products of fructose 1,6-
bisphosphate.
• [Note: DHAP is used for the synthesis of glycerol-3 phosphate utilized in
triacylglycerol synthesis.]
• Reaction 6 : Glyceraldehyde 3-phosphate oxidation
• This oxidation of glyceraldehyde 3-phosphate to 1,3-
bisphosphoglycerate (1,3-BPG) by glyceraldehyde 3-
phosphate dehydrogenase is the first oxidation-reduction
reaction of glycolysis and leads to the formation of 1,3-
Bisphosphoglycerate.
• The reaction requires NAD+ as the oxidizing agent and and
it is reduced to NADH (produced).
• [Note: Because there is a limited amount of NAD+ in the cell,
the NADH formed by the dehydrogenase reaction must be
oxidized back to NAD+ for glycolysis to continue.
• Two major mechanisms for oxidizing NADH to NAD+ are the
reduction of pyruvate to lactate by lactate dehydrogenase
(LDH) (anaerobic glycolysis-see later) and the electron
transport chain ([ETC]- aerobic].
Glyceraldehyde 3-phosphate oxidation
• The oxidation of the aldehyde group of glyceraldehyde 3-
phosphate to a carboxyl group is coupled to the attachment of Pi
to the carboxyl group.
• This phosphate group, linked to carbon 1 of the 1,3-BPG product
by a high-energy bond conserves much of the free energy
produced by the oxidation of glyceraldehyde 3-phosphate.
• This high-energy phosphate drives ATP synthesis in the next
reaction of glycolysis.
• Note:pentavalent arsenic (arsenate) can prevent net ATP and NADH
production by glycolysis without inhibiting the pathway itself by competing
with Pi as a substrate for glyceraldehyde 3-phosphate dehydrogenase,
forming a complex that spontaneously hydrolyzes to form 3-
phosphoglycerate
• By bypassing the synthesis of and phosphate transfer from 1,3-BPG, the cell
is deprived of energy usually obtained from the glycolytic pathway- this is
arsenate poisoning
• [Note: Arsenate also inhibit ATP synthase in ETC.]
• RXN 7 : 3-Phosphoglycerate synthesis and ATP production
• When 1,3-BPG is converted to 3-phosphoglycerate, the high-
energy phosphate group of 1,3-BPG is used to synthesize
ATP from ADP.
• This reaction is catalyzed by phosphoglycerate kinase, which,
unlike most other kinases, is physiologically reversible.
• Because two molecules of 1,3-BPG are formed from each
glucose molecule, this kinase reaction replaces the two ATP
molecules consumed by the earlier formation of glucose 6-
phosphate and fructose 1,6-bisphosphate.
• [Note: This reaction is an example of substrate-level
phosphorylation, in which the energy needed for the
production of a high-energy phosphate comes from a high
energy substrate rather than from the ETC].
Reaction 7: 2-ATP and Phosphoglycerate synthesis production
• Rapoport Luebering Glycolytic Shunt.
• 2,3-Bisphosphoglycerate synthesis in RBC:
• In the RBCs some of the 1,3-BPG formed in the above reaction 6 is
converted to 2,3-BPG by the action of bisphosphoglycerate mutase.
• 2,3-BPG, which is found in only trace amounts in most cells, is
present at high concentration in RBC and serves to increase O 2
delivery.
• It enables the oxyhaemoglobin of the RBC to unload oxygen to the
tissues.
• 2,3-BPG is hydrolyzed by a phosphatase to
3-phosphoglycerate, which is also an intermediate in glycolysis.
• There is no production of ATP when glycolysis takes this route. This
may be an advantage to the RBCs, since it would allow the
accumulation of 2,3-BPG and glycolysis to proceed when the need
for ATP is minimal.
• In the RBC, glycolysis is modified by inclusion of these shunt
reactions called Rapoport Luebering Glycolytic Shunt .
Rapoport Luebering Glycolytic Shunt in RBCs
REACTIONS 8,9, AND 10

3-Phosphoglycerate
• Reaction 8 : Phosphate group shift and formation of 2-Phosphoglycerate
• This reaction involves the shift of phosphate group of phosphoglycerate
from carbon 3 to carbon 2.
• This shift of the phosphate group is catalysed by phosphoglycerate mutase
and the reaction is freely reversible.

• Reaction 9: 2-Phosphoglycerate dehydration and formation of

phosphoenolpyruvate(PEP)
• PEP is formed by the removal of a molecule of water(-dehydation).
• The dehydration of 2-phosphoglycerate by enolase redistributes the
energy within the substrate, forming phosphoenolpyruvate (PEP), which
contains a high-energy enol phosphate.
• The reaction is reversible, despite the high-energy nature of the product.
• [Note: Fluoride inhibits enolase, and water fluoridation reduces lactate production by
mouth bacteria, decreasing dental caries]. Fluoride/oxalate bottles are used for collection
of blood for blood glucose estimation to preserve the carbons of glucose for estimation.
• Reaction 10 : Pyruvate synthesis and ATP production
• The conversion of PEP to pyruvate, catalyzed by
pyruvate kinase (PK), is the third irreversible
reaction of glycolysis.
• The high-energy enol phosphate in PEP is used to
synthesize ATP from ADP by pyruvate kinase and
is another example of substrate-level
phosphorylation.
• Pyruvate is the normal end product of glycolysis
under aerobic conditions.
• REGULATION OF PYRUVATE KINASE
• 1. Feedforward regulation: PK is activated by
fructose 1,6-bisphosphate, the product of the
PFK-1 reaction.
• This feedforward (instead of the more usual
feedback) regulation has the effect of linking the
two kinase activities: increased PFK-1 activity
results in elevated levels of fructose 1,6-
bisphosphate, which activates PK.
• [Note: PK is also inhibited by ATP and alanine.]
• 2. Covalent regulation in the liver:
• Phosphorylation by cAMP-dependent PKA(protein kinase
A) leads to inactivation of the hepatic isozyme of PK .
• When blood glucose levels are low, elevated glucagon
increases the intracellular level of cAMP, which causes the
phosphorylation and inactivation of PK in the liver only.
• Therefore, PEP is unable to continue in glycolysis and,
instead, enters the gluconeogenesis pathway.
• This partly explains the observed inhibition of hepatic
glycolysis and stimulation of gluconeogenesis by glucagon.
• Dephosphorylation of PK by a phosphatase results in
reactivation of the enzyme.
• OTHER HORMONAL REGULATION OF GLYCOLYSIS
• Regulation of the activity of the irreversible glycolytic enzymes
by allosteric activation/inhibition or covalent
phosphorylation/dephosphorylation is short term (that is, the
effects occur over minutes or hours).
• The long-term hormonal effects is on the synthesis of new
enzyme molecules which can result in 10- to 20-fold increases in
enzyme synthesis that typically occur over hours to days.
• Regular consumption of meals rich in carbohydrate or
administration of insulin initiates an increase in the amount of
glucokinase,PFK-1, and PK in the liver.
• The change reflects an increase in gene transcription, resulting
in increased enzyme synthesis.
• Increased availability of these three enzymes favors the
conversion of glucose to pyruvate, a characteristic of the
absorptive state.
 Clinical correlates-Pyruvate kinase deficiency:

• Because mature RBC lack mitochondria, they are completely

dependent on glycolysis for ATP production.
• ATP is required to meet the metabolic needs of RBC and to fuel the
ion pumps necessary for the maintenance of the flexible, biconcave
shape that allows them to squeeze through narrow capillaries.
• The anemia observed in glycolytic enzyme deficiencies such as
seen in pyruvate kinase deficiency is a consequence of the reduced
rate of glycolysis, leading to decreased ATP production by
substrate-level phosphorylation.
• The resulting alterations in the RBC membrane lead to changes in
cell shape and, ultimately, to phagocytosis by cells of the
mononuclear phagocyte system, particularly splenic macrophages.
• The premature death and lysis of RBC result in mild-to-severe
nonspherocytic hemolytic anemia, with the severe form requiring
regular transfusions.
• Among patients with rare genetic defects of glycolytic enzymes,
the majority has a deficiency in PK.
• [Note: Liver PK is encoded by the same gene as the RBC isozyme.
However, liver cells show no effect because they can synthesize
more PK and can also generate ATP by oxidative phosphorylation.]
• Severity therefore depends both on the degree of enzyme
deficiency (generally 5%–35% of normal levels) and on the extent
to which RBC compensate by synthesizing increased levels of 2,3-
BPG.
• Almost all individuals with PK deficiency have a mutant enzyme
that shows altered kinetics or decreased stability.
• Individuals heterozygous for PK deficiency have resistance to the
most severe forms of malaria.
• Utilization (Fate) of Pyruvate
• The disposition of the pyruvate formed as the end product of the
glycolytic pathway depends upon the following:
• The organism involved.
• The tissue in question.
• The redox state of the tissue.
• Anaerobic glycolysis
• In the erythrocytes which have no mitochondrion, the skeletal
muscle during strenuous exercise and other anaerobic systems,
pyruvate undergoes a reduction reaction to form lactate.
• The reaction is catalyzed by the enzyme lactate dehydrogenase
and is reversible.
• The reducing agent in the reaction is NADH+H + which is
reoxidized to NAD+.
• The regeneration of NAD+ ensures the continuity of glycolysis
(reaction 6) under the anaerobic process.
Reduction of pyruvate to lactate
 LACTATE FORMATION AND UTILIZATION AND LACTIC ACIDOSIS

• Lactate formation in muscle:

• In exercising skeletal muscle, NADH production (by
glyceraldehyde 3-phosphate dehydrogenase and by the
three NAD+-linked dehydrogenases of the TCA cycle, exceeds
the oxidative capacity of the ETC.
• This results in an elevated NADH/NAD+ ratio, favoring
reduction of pyruvate to lactate by LDH.
• Therefore, during intense exercise, lactate accumulates in
muscle, causing a drop in the intracellular pH, potentially
resulting in cramps.
• Much of this lactate eventually diffuses into the
bloodstream and can be used by the liver to make glucose
when the cell resumes full aerobic metabolism(see cori
cycle later).
• Lactate utilization:
• The direction of the LDH reaction depends on the
relative intracellular concentrations of pyruvate
and lactate and on the ratio of NADH/NAD+.
• For example, in the liver and heart, this ratio is
lower than in exercising muscle.
• Consequently, the liver and heart oxidize lactate
(obtained from the blood) to pyruvate.
• In the liver, pyruvate is either converted to
glucose by gluconeogenesis or converted to acetyl
CoA that is oxidized in the TCA cycle.
• Heart muscle exclusively oxidizes pyruvate to
carbon dioxide and water via the TCA cycle.
 Clinical correlates-Lactic acidosis:
• Elevated concentrations of lactate in the plasma, termed lactic acidosis (a type
of metabolic acidosis), occurs not only as seen above in excersing muscle but also
when there is a collapse of the circulatory system, such as with myocardial
infarction, pulmonary embolism, and uncontrolled hemorrhage, or when an
individual is in shock.
• The failure to bring adequate amounts of O2 to the tissues results in impaired
oxidative phosphorylation and decreased ATP synthesis.
• To survive, the cells rely on anaerobic glycolysis for generating ATP, producing
lactic acid as the end product. [Note: Production of even meager amounts of ATP
may be lifesaving during the period required to reestablish adequate blood flow
to the tissues.]
• The additional O2 required to recover from a period when O2 availability has
been inadequate is termed the O2 debt.
• [Note: The O2 debt is often related to patient morbidity or mortality. In many
clinical situations, measuring the blood levels of lactic acid allows the rapid,
early detection of O2 debt in patients and the monitoring of their recovery.]
 Energy yield from glycolysis

• Despite the production of some ATP by substrate-

level phosphorylation during glycolysis, the end
product, pyruvate or lactate, still contains most of
the energy originally contained in glucose.
• The TCA cycle is required to release that energy
completely.
• 1. Anaerobic glycolysis:
• A net of two molecules of ATP are generated for
each molecule of glucose converted to two
molecules of lactate.
• There is no net production or consumption of
NADH.
• 2. Aerobic glycolysis:
• The generation of ATP is the same as in anaerobic
glycolysis (that is, a net gain of two ATP per
molecule of glucose).
• Two molecules of NADH are also produced per
molecule of glucose.
• Ongoing aerobic glycolysis requires the oxidation of
most of this NADH by the ETC, producing three ATP
for each NADH molecule entering the chain.
• [Note: NADH cannot cross the inner mitochondrial
membrane, and substrate shuttles are required]
 Summary-Energy yield from glycolysis
• Reaction ATP
• ATP Gain
• 1,3-bisphosphoglycerate to 3-PGA +2
• PEP to Pyruvate +2
• Gross ATP gain 4 ATP
• ATP Utilization
• Glucose G-6-Phosphate -1
• F-6-Phosphate F-1,6- bisphosphate -1
• Gross ATP loss -2 ATP

• Net gain of ATP in glycolysis = (4-2) = 2 moles of ATP

• Alcoholic fermentation
• In yeast and other anaerobic microorganisms, but not
in humans, glucose undergoes alcoholic fermentation.
• The reduction of pyruvate to ethanol occurs by the
two reactions :
• Pyruvate is first decarboxylated by thiamine-requiring
pyruvate decarboxylase to acetaldehyde and carbon
dioxide in an irreversible reaction.
• The acetaldehyde formed is reduced to ethanol by
alcohol/ethanol dehydrogenase which also utilizes
NADH (a way of reoxidizing NADH to NAD+).
• The reaction is reversible.
• The reaction equation is shown below:
Alcoholic fermentation in microorganisms (anaerobic process).
 Fate of pyruvate in aerobic systems
• Carboxylation to oxaloacetate
• Carboxylation of pyruvate to oxaloacetate by pyruvate
carboxylase is a biotin-dependent reaction.
• This irreversible reaction is important because it
replenishes the TCA cycle intermediate and provides
substrate for gluconeogenesis.
• Oxidation to Acetyl-CoA
• In aerobic systems the pyruvate which is formed in
glycolysis is neither reduced to lactate nor converted to
ethanol. In the mitochondrial matrix, the Pyruvate
Dehydrogenase complex converts pyruvate to acetyl
CoA and the reaction is a prelude to the TCA cycle-see
details later.
Oxidation of pyruvate to acetyl-CoA.
• Summary of Glycolysis
• Glycolysis is a set of reactions that convert glucose into pyruvate. In
aerobic organisms, glycolysis is the prelude to the Tricarboxylic acid (TCA)
cycle and the Electron Transport Chain (ETC), where most of the free
energy in glucose is harvested. The ten reactions of glycolysis occur in the
cytosol.
• Functions of Glycolysis
• The glycolytic pathway has 2 major functions.
• It degrades glucose to generate energy (ATP).
• It provides building blocks for the biosynthesis of cellular components.
For instance:
• Triose phosphate (DHAP) provides the glycerol moiety for triacylglycerol
(TAG) synthesis.
• Pyruvate can be used for the synthesis of the amino acid alanine and for
gluconeogenesis as well as for replenishing the substrates of the TCA
cycle.
• Glucose 6 phosphate is an important compound at the junction of
several pathways .