Scientific investigations
into fermentation of
grape sugar were
pioneering studies
of glycolysis
Anaerobic
glycolysis
Glycolysis is the earliest discovered and most important
process of carbohydrates metabolism.
Glycolysis – metabolic pathway in which glucose is
transformed to pyruvate with production of a small
amount of energy in the form of ATP or NADH.
Glycolysis is an anaerobic process (it does not require
oxygen).
Glycolysis pathway is used by anaerobic as well as aerobic
organisms.
In glycolysis one molecule of glucose is converted into two
molecules of pyruvate.
In eukaryotic cells, glycolysis takes place in the cytosol.
Pyruvate can be further metabolized to:
(1) Lactate or ethanol (anaerobic conditions)
(2) Acetyl CoA (aerobic conditions)
• Acetyl CoA is further oxidized to CO2 and H2O via
the citric acid cycle
• Much more ATP is generated from the citric acid
cycle than from glycolysis
Acetyl CoA
• Catabolism of glucose in
aerobic conditions via
glycolysis and the citric
acid cycle
The glycolytic pathway consist of ten enzymecatalyzed reactions that begin with a glucose and
split it into two molecules of pyruvate
Glycolysis
(10 reactions)
can be
divided into
three stages
• In the 1st stage
(hexose stage)
2 ATP are
consumed per
glucose
• In the 3rd stage
(triose stage)
4 ATP are
produced per
glucose
• Net: 2 ATP
produced per
glucose
Stage 1, which is the conversion of glucose into fructose
1,6-bisphosphate, consists of three steps: a phosphorylation,
an isomerization, and a second phosphorylation reaction.
The strategy
of these
initial steps
in glycolysis
is to trap
the glucose
in the cell
and form a
compound
that can be
readily
cleaved into
phosphorylated
threecarbon units.
Stage 2 is the cleavage of the fructose
1,6-bisphosphate into two three-carbon fragments
dihydroxyacetone phosphate and glyceraldehyde 3phosphate.
Dihydroxyacetone phosphate and glyceraldehyde 3phosphate are readily interconvertible.
In stage 3,
ATP is
harvested
when the
threecarbon
fragments
are
oxidized to
pyruvate.
Glycolysis Has 10 Enzyme-Catalyzed Steps
• Each chemical reaction prepares a substrate for the next
step in the process
1. Hexokinase
• Transfers the g-phosphoryl of ATP to glucose C-6 oxygen to
generate glucose 6-phosphate (G6P)
• Four kinases in glycolysis: steps 1,3,7, and 10
• All four kinases require Mg2+ and have a similar mechanism
Properties of hexokinases
• Broad substrate specificity - hexokinases can
phosphorylate glucose, mannose and fructose
• Isozymes - multiple forms of hexokinase occur in
mammalian tissues and yeast
• Hexokinases I, II, III are active at normal glucose
concentrations
• Hexokinase IV (Glucokinase) is active at higher glucose
levels, allows the liver to respond to large increases in
blood glucose
• Hexokinases I, II and III are allosterically inhibited by
physiological concentrations of their immediate product,
glucose-6-phosphate, but glucokinase is not.
2. Glucose 6-Phosphate Isomerase
• Converts glucose 6-phosphate (G6P) (an aldose) to
fructose 6-phosphate (F6P) (a ketose)
• Enzyme preferentially binds the a-anomer of G6P
(converts to open chain form in the active site)
• Enzyme is highly stereospecific for G6P and F6P
• Isomerase reaction is near-equilibrium in cells
3. Phosphofructokinase-1 (PFK-1)
• Catalyzes transfer of a phosphoryl group from ATP to the
C-1 hydroxyl group of F6P to form fructose 1,6bisphosphate (F1,6BP)
• PFK-1 is metabolically irreversible and a critical regulatory
point for glycolysis in most cells
• A second phosphofructokinase (PFK-2) synthesizes
fructose 2,6-bisphosphate (F2,6BP)
4. Aldolase
• Aldolase cleaves the hexose F1,6BP into two triose
phosphates: glyceraldehyde 3-phosphate (GAP) and
dihydroxyacetone phosphate (DHAP)
• Reaction is near-equilibrium, not a control point
5. Triose Phosphate Isomerase (TPI)
• Conversion of DHAP into GAP
• Reaction is very fast, only the D-isomer of GAP is formed
• Reaction is reversible. At equilibrium, 96% of the triose
phosphate is DHAP. However, the reaction proceeds readily
from DHAP to GAP because the subsequent reactions of
glycolysis remove this product.
Fate of carbon atoms from hexose stage
to triose stage
6. Glyceraldehyde 3-Phosphate
Dehydrogenase (GAPDH)
• Conversion of GAP to 1,3-bisphosphoglycerate (1,3BPG)
• Molecule of NAD+ is reduced to NADH
• Energy from oxidation of GAP is conserved in acidanhydride linkage of 1,3BPG
• Next step of glycolysis uses the high-energy phosphate
of 1,3BPG to form ATP from ADP
7. Phosphoglycerate Kinase (PGK)
• Transfer of phosphoryl group from the energy-rich mixed
anhydride 1,3BPG to ADP yields ATP and
3-phosphoglycerate (3PG)
• Substrate-level phosphorylation - Steps 6 and 7 couple
oxidation of an aldehyde to a carboxylic acid with the
phosphorylation of ADP to ATP
8. Phosphoglycerate Mutase
• Catalyzes transfer of a phosphoryl group from one part
of a substrate molecule to another
• Reaction occurs without input of ATP energy
9. Enolase: 2PG to PEP
• 2-Phosphoglycerate (2PG) is dehydrated to
phosphoenolpyruvate (PEP)
• Elimination of water from C-2 and C-3 yields the enolphosphate PEP
• PEP has a very high phosphoryl group transfer potential
because it exists in its unstable enol form
Net reaction of glycolysis
During the convertion of glucose to pyruvate:
• Two molecules of ATP are produced
• Two molecules of NAD+ are reduced to NADH
Glucose + 2 ADP + 2 NAD+ + 2 Pi
2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O
Other Sugars Can Enter Glycolysis
• Glucose is the main metabolic
fuel in most organisms
• Other sugars convert to
glycolytic intermediates
• Fructose and sucrose (contains
fructose) are major sweeteners
in many foods and beverages
• Galactose from milk lactose (a
disaccharide)
• Mannose from dietary
polysaccharides, glycoproteins
The Entry of Fructose into Glycolysis
Much of the ingested fructose is metabolized by the liver, using
the fructose 1-phosphate pathway.
The first step is the phosphorylation of fructose to fructose 1phosphate by fructokinase.
Fructose 1-phosphate is then
split into glyceraldehyde and
dihydroxyacetone phosphate, an
intermediate in glycolysis, by a
specific fructose 1 -phosphate
aldolase.
Glyceraldehyde is then
phosphorylated to glyceraldehyde
3-phosphate, a glycolytic
intermediate, by triose kinase.
10. Pyruvate Kinase (PK)
PEP + ADP  Pyruvate + ATP
• Catalyzes a substrate-level
phosphorylation
• Metabolically irreversible
reaction
• Regulation both by
allosteric modulators and
by covalent modification
• Pyruvate kinase gene can be
regulated by various
hormones and nutrients
Fructose Is Converted to Glyceraldehyde 3-Phosphate
Fructose can be phosphorylated to fructose 6-phosphate by
hexokinase.
However, the affinity of hexokinase for glucose is 20 times
as great as it is for fructose.
Little fructose 6-phosphate is formed in the liver because
glucose is so much more abundant in this organ.
Glucose, as the preferred fuel, is also trapped in the muscle
by the hexokinase reaction.
Because liver and muscle phosphorylate glucose rather than
fructose, adipose tissue is exposed to more fructose than
glucose.
Hence, the formation of fructose 6-phosphate in the adipose
tissue is not competitively inhibited to a biologically significant
extent, and most of the fructose in adipose tissue is
metabolized through fructose 6-phosphate.
The Entry of Galactose into Glycolysis
Galactose is converted into glucose 6-phosphate in four
steps.
The first reaction is the phosphorylation of galactose to
galactose 1-phosphate by galactokinase.
Galactose 1-phosphate react with uridine
diphosphate glucose (UDP-glucose).
UDP-galactose and glucose 1-phosphate are
formed.
Enzyme - galactose 1-phosphate uridyl
transferase.
The galactose moiety of UDP-galactose is then
epimerized to glucose.
The configuration of the hydroxyl group at carbon 4
is inverted by UDP-galactose 4-epimerase.
Glucose 1-phosphate, formed from galactose, is
isomerized to glucose 6-phosphate by
phosphoglucomutase.
The Entry of Mannose into Glycolysis
Mannose is converted to Fructose 6-Phosphate in two
steps.
Hexokinase catalyzes the convertion of mannose into
mannose 6-phosphate.
Isomerase converts mannose 6-phosphate into fructose
6-phosphate (metabolite of glycolysis).
Intolerance to Milk
Many people are unable to metabolize the
milk sugar lactose and experience gastrointestinal disturbances if they drink milk.
Lactose intolerance, or hypolactasia, is caused by a deficiency of the
enzyme lactase, which cleaves lactose into glucose and galactose.
Microorganisms in the colon ferment undigested lactose to lactic acid
generating methane (CH4) and hydrogen gas (H2). The gas produced
creates the uncomfortable feeling of gut distention and the annoying
problem of flatulence.
The lactic acid is osmotically active and draws water into the intestine,
as does any undigested lactose, resulting in diarrhea.
The gas and diarrhea hinder the absorption of other nutrients (fats
and proteins).
Treatment:
- to avoid the products containing lactose;
- the enzyme lactase can be ingested.
Galactosemia
The disruption of galactose metabolism is referred to as
galactosemia.
Classic galactosemia is an inherited deficiency in galactose
1-phosphate uridyl transferase activity.
Symptoms:
- vomiting, diarrhea after consuming milk,
- enlargement of the liver, jaundice,
sometimes cirrhosis,
- cataracts,
- lethargy and retarded mental
development,
- markedly elevated blood-galactose level
- galactose is found in the urine.
The absence of the transferase in red blood cells is a
definitive diagnostic criterion.
The most common treatment is to remove galactose (and
lactose) from the diet.
Regulation of Glycolysis
The rate glycolysis is regulated to meet two major cellular needs:
(1) the production of ATP, and
(2) the provision of building blocks for synthetic reactions.
There are three control sites in glycolysis - the reactions catalyzed by
hexokinase,
phosphofructokinase 1, and
pyruvate kinase
These reactions are irreversible.
Their activities are regulated
by the reversible binding of allosteric effectors
by covalent modification
by the regulation of transcription (change of the enzymes amounts).
The time required for allosteric control, regulation by phosphorylation,
and transcriptional control is typically in milliseconds, seconds, and
hours, respectively.
Phosphofructokinase 1 Is the Key Enzyme in
the Control of Glycolysis
Phosphofructokinase 1 is the most important control element
in the mammalian glycolytic pathway.
Phosphofructokinase 1
in the liver is a tetramer
of four identical
subunits.
The positions of the
catalytic and allosteric
sites are identical.
High levels of ATP allosterically inhibit the
phosphofructokinase 1 in the liver lowering its affinity for
fructose 6-phosphate.
AMP reverses the inhibitory action of ATP, and so the
activity of the enzyme increases when the ATP/AMP ratio
is lowered (glycolysis is stimulated as the energy charge
falls).
A fall in pH also inhibits phosphofructokinase 1 activity.
The inhibition of phosphofructokinase by H+ prevents
excessive formation of lactic acid and a precipitous drop in
blood pH (acidosis).
Phosphofructokinase 1 is inhibited by citrate, an early
intermediate in the citric acid cycle.
A high level of citrate means that biosynthetic precursors are
abundant and additional glucose should not be degraded for this
purpose.
Fructose 2,6-bisphosphate (F-2,6-BP) is a
potent activator of phosphofructokinase 1.
F-2,6-BP activates phosphofructokinase I by
increasing its affinity for fructose 6-phosphate
and diminishing the inhibitory effect of ATP.
Fructose 2,6-bisphosphate is formed in a reaction catalyzed by
phosphofructokinase 2 (PFK2), a different enzyme from
phosphofructokinase 1.
Fructose 2,6-bisphosphate
is hydrolyzed to fructose 6phosphate by a specific
phosphatase, fructose
bisphosphatase 2 (FBPase2).
Both PFK2 and FBPase2 are
present in a single
polypeptide chain
(bifunctional enzyme).
Regulation of Glycolysis by Fructose 2,6-bisphosphate
 When blood glucose
level is low the glucagon
is synthesized by
pancreas
 Glucagon binds to cell
receptors, stimulates
the protein kinase A
activity
 Protein kinase A
phosphorylates the
PFK-2 inhibiting its
kinase activity and
stimulating its
phosphatase activity
As result the amount
of F-2,6-BP is decreased and glycolysis is
slowed.
Regulation of Hexokinase
Hexokinase is inhibited by its
product, glucose 6-phosphate
(G-6-P).
High concentrations of G-6-P signal that the cell no longer
requires glucose for energy, for glycogen, or as a source of
biosynthetic precursors.
Glucose 6-phosphate levels increase when glycolysis is inhibited at
sites further along in the pathway.
Glucose 6-phosphate inhibits hexokinase isozymes I, II and III.
Glucokinase (isozyme IV) is not inhibited by glucose 6-phosphate.
The role of glucokinase is to provide glucose 6-phosphate for the
synthesis of glycogen.
Regulation of Pyruvate Kinase (PK)
Several isozymic
forms of pyruvate
kinase are present in
mammals (the L type
predominates in liver,
and the M type in
muscle and brain).
Fructose 1,6-bisphosphate allosterically activates pyruvate kinase.
ATP allosterically inhibits pyruvate kinase to slow glycolysis
when the energy charge is high.
Finally, alanine (synthesized in one step from pyruvate) also
allosterically inhibits the pyruvate kinases (signal that
building blocks are abundant).
The isozymic forms of pyruvate kinase differ in their
susceptibility to covalent modification.
The catalytic properties of the L (liver) form—but not of the
M (brain) form controlled by reversible phosphorylation.
When the
blood-glucose
level is low, the
glucagon leads
to the
phosphorylation of
pyruvate
kinase, which
diminishes its
activity.
Inhibition
1) PFK-1 is
inhibited by ATP
and citrate
2) Pyruvate
kinase is
inhibited by ATP
and alanine
3) Hexokinase is
inhibited by
excess glucose
6-phosphate
Regulation of
Glycolysis
Stimulation
1) AMP and fructose 2,6bisphosphate (F2,6BP) relieve
the inhibition of PFK-1 by ATP
2) F1,6BP stimulate the activity
of pyruvate kinase
Alanine
Regulation of Hexose Transporters
Several glucose transporters (GluT) mediate the thermodynamically downhill
movement of glucose across the plasma membranes of animal cells.
GluT is a family of 5 hexose transporters.
Each member of this protein family consists of a single polypeptide chain
forming 12 transmembrane segments.
GLUT1 and GLUT3,
present in erythrocytes,
endothelial, neuronal
and some others
mammalian cells, are
responsible for basal
glucose uptake. Their Km
value for glucose is about
1 mM.
GLUT1 and GLUT3
continually transport
glucose into cells at an
essentially constant rate.
GLUT2, present in liver and pancreatic -cells has a very
high Km value for glucose (15-20 mM).
Glucose enters these tissues at a biologically significant
rate only when there is much glucose in the blood.
GLUT4, which has a Km value of 5 mM, transports glucose
into muscle and fat cells.
The presence of insulin leads to a rapid increase in the
number of GLUT4 transporters in the plasma membrane.
Insulin promotes the uptake of glucose by muscle and fat.
The amount of this transporter present in muscle
membranes increases in response to endurance exercise
training.
GLUT5, present in the small intestine, functions primarily
as a fructose transporter.