Chapter 14 (Part 1)

Glycolysis
Refer to: Lehninger Principles of Biochemistry (Chapter 14)

Dr Fawaz Awad; 2018

 Objectives
• To understand how the glycolytic pathway is used to
convert glucose to pyruvate (aerobically) and lactate
(anaerobically) with conservation of chemical potential
energy in the form of ATP and NADH.

• To learn the intermediates, enzymes, and cofactors of

the glycolytic pathway.
What are the fates of the absorbed glucose?
Major pathways of glucose utilization
 Glucose Usage
I. Oxidation: classified into two groups:
A. Major pathway ( for energy production): Glycolysis and TCA
B. Minor pathway (not for energy production): Hexose monophosphate shunt and
Uronic acid pathway.

II. Conversion to biologically active substances as:

– Galactose that is important for formation of lactose, glycolipids..etc.
– Fructose needed for nutrition of sperms.
– Amino sugars
– Non essential amino acids
– Fatty acids
– Ribose 5-P
– Glucuronic acid

III. Storage of glucose: As: 1. Glycogen in liver and muscle, 2. TG in adipose tissue.

IV. Excretion of glucose in urine: (renal sugar threshold) when blood glucose is above
180 mg/dl (glucosuria).
 Overview of cellular respiration

1 Glucose

2 ATP
1 GLYCOLYSIS
2 NADH + 2 H+

2 Pyruvic acid

2 FORMATION 2 CO2
OF ACETYL
COENZYME A
2 NADH + 2 H+
4 ELECTRON
TRANSPORT
2 Acetyl
CHAIN
coenzyme A
2 ATP Electrons 32 or 34 ATP
e–
4 CO2 e–
3
e–
KREBS 6 NADH + 6 H+
CYCLE
2 FADH2 6 O2

6 H2O
 Glucose catabolism / cellular
respiration
1. Glycolysis
• Anaerobic respiration – does not require oxygen
2. Formation of acetyl coenzyme A
3. Krebs cycle reactions
4. Electron transport chain reactions
• Aerobic respiration – requires oxygen
 Glycolysis
1. Glycolysis
– Splits 6-carbon glucose into 2 3-carbon molecules of pyruvic
acid
– Consumes 2 ATP but generates 4
– 10 reactions
– Fate of pyruvic acid (pyruvate) depends on oxygen
availability
• If oxygen is scarce (anaerobic), reduced to lactic acid
– Hepatocytes can convert it back to pyruvic acid
• If oxygen is plentiful (aerobic), converted to acetyl
coenzyme A
 Cellular respiration begins with
glycolysis
• Glycolysis is an almost universal central pathway of glucose
catabolism, the pathway with the largest flux of carbon in most
cells.

Glycolysis is the only ATP generating pathway in the brain and eye.

RBC Lack mitochondria, so cannot convert pyruvate to CO2.

Cornea and lens Lack mitochondria too (mitochondria could absorb

and scatter light).

• The thermodynamic principles and the types of regulatory

mechanisms that govern glycolysis are common to all pathways of
cell metabolism.
Cellular respiration begins with
glycolysis
In the sequential reactions of glycolysis, three types of
chemical transformations are particularly
noteworthy:

(1) Degradation of the carbon skeleton of glucose to

yield pyruvate.

(2) Phosphorylation of ADP to ATP by high-energy

phosphate compounds formed during glycolysis.

(3) Transfer of a hydride ion (anion of hydrogen, H−) to

NAD+, forming NADH.
Three possible fates for pyruvate
 ATP Formation Coupled to
Glycolysis

• Converts hexose to two pyruvates

• Generates 2 ATP and 2 NADH

Glucose+2ADP+2NAD++2Pi ->
2pyruvate+2ATP+2NADH+2H++2H20
 Energy Remaining in Pyruvate???
Energy Yield From Glycolysis

Glucose 6 CO2 = -2840 kJ/mole

2 ATPs produced = 2 x 30.5 = 61 kJ/mole glucose

Energy yield = 61/2840 = 2% recovered as ATP

(releases only a small fraction of the total available
energy of the glucose molecule).

- Subsequent oxidation of pyruvate and NADH can recover

more of the free energy from glucose by oxidative reactions
in the citric acid cycle and oxidative phosphorylation
 Importance of Phosphorylated
Intermediates
Each of the nine glycolytic intermediates between glucose and pyruvate is
phosphorylated.

The phosphoryl groups have three functions:

1. The phosphorylated glycolytic intermediates cannot leave the cell (no
receptors for it on plasma membrane).

2. Phosphoryl groups are essential components in the enzymatic conservation of

metabolic energy. High-energy phosphate compounds formed in glycolysis
(1,3-bisphosphoglycerate and phosphoenolpyruvate) donate phosphoryl
groups to ADP to form ATP.

3. Binding energy resulting from the binding of phosphate groups to the active
sites of enzymes lowers the activation energy and increases the specificity of
the enzymatic reactions.
Glycolysis has two stages
A. An energy investment or preparatory phase
(Reactions, 1-5).
Glucose to two glyceraldehyde 3-phosphate
molecules. Two ATPs are invested.

B. An energy payoff phase (Reactions 6-10).

Two glyceraldehyde 3-phosphate molecules to two

pyruvate plus four ATP molecules.
A net of two ATP molecules overall plus two
NADH.
Detailed Reactions of glycolysis

Two phases of glycolysis

Take care or reversible and irreversible reaction

An energy investment or preparatory
phase
Reaction 1: Phosphorylation

First ATP Utilization

• This reaction, which is irreversible under intracellular conditions, is

catalyzed by hexokinase.
• 1st step in glycolysis; ΔG large, negative
• This is a priming reaction - ATP is consumed here in order to get
more later
• ATP makes the phosphorylation of glucose spontaneous
1. Hexokinase, like many other kinases, requires Mg2 for its
activity.
2. The true substrate of the enzyme is not ATP but the MgATP
complex.
3. Mg2 shields the negative charges of the phosphoryl groups in
ATP, making the terminal phosphorus atom an easier target for
nucleophilic attack by an -OH of glucose.
4. Hexokinase undergoes a profound change in shape, an induced
fit, when it binds glucose.
5. The movement of the two domains of the protein move closer
to each other when ATP binds brings bound ATP closer to a
molecule of glucose also bound to the enzyme and blocks the
access of water (from the solvent), which might otherwise enter
the active site and attack (hydrolyze) the phosphoanhydride
bonds of ATP.
 Different Hexokinase Isozymes
Hexokinase vs. glucokinase

Tissue-specific 
isozymes.
 Different Hexokinase Isozymes
• Two major forms hexokinase (all cells) &
glucokinase (liver).

• Km for hexokinase is 10-6 to 10-4 M; cell has 4 X

10-3 M glucose

• Km for glucokinase is 10-2 M only turns on when

cell is rich in glucose

• Glucokinase functions when glucose levels are

high to sequester glucose in the liver.
 Hexokinase vs. Glucokinase
Hexokinase
Found in the cytosol of most tissues
– Low specificity, mainly glucose. Also Fructose and Mannose.
– High affinity to glucose: Low Km: (Km = 0.1 mM).
– Inhibited by its product Glucose-6-phosphate (allosteric inhibitor).
– Insulin and Glucagon has No affect

Glucokinase
Found mainly in the Liver and pancreatic β cells
– Also known as hexokinase D or hexokinase IV
– Glucose is the only substrate
– Low affinity: High Km (Km ~10mM) High specificity for glucose
– Inhibited by fructose-6-phosphate (not glu-6-p).
– In fasting its activity is decreased.
– Glucagon act as repressor.
– Insulin is inducer
Reaction 2 (isomerization) Phosphoglucoisomerase
The enzyme phosphohexose isomerase catalyzes the
reversible isomerization of glucose 6-phosphate, an aldose,
to fructose 6-phosphate, a ketose:

• Near-equilibrium rxn (reversible)

• Enzyme is highly stereospecific (doesn’t work with epimers of
glucose-6-phosphate
Rx 2: Phosphoglucoisomerase

• Why does this reaction occur??

– next step (phosphorylation at C-1) would be
tough for hemiacetal -OH, but easy for primary -
OH
– isomerization activates C-3 for cleavage in
aldolase reaction
 Reaction 3: phosphorylation
The PFK-1 reaction is essentially irreversible under cellular conditions and
fructose 1,6-bisphosphate is targeted for glycolysis.

PFK1
Second ATP Utilization

PFK is the committed step in glycolysis!

• The second priming reaction of glycolysis
• -ΔG, means PFK is highly regulated
• Kinase : enzyme that is responsible for transfer of phosphate group
from ATP to nucleophile acceptor.
It is the major point of regulation in glycolysis
Phosphofructokinase is highly regulated
• PFK1 increases activity when energy status is low
• PFK1 decreases activity when energy status is high

• ATP inhibits, AMP reverses inhibition

• Citrate is also an allosteric inhibitor
• Fructose-2,6-bisphosphate is allosteric activator
• Ribulose 5-phosphate (intermediate in pentose
pathway), activate it
Reaction 4: cleavage
The enzyme fructose 1,6-bisphosphate aldolase, often called simply aldolase,
catalyzes a reversible aldol condensation. Fructose 1,6-bisphosphate is cleaved
to yield two different triose phosphates, glyceraldehyde 3-phosphate, an
aldose, and dihydroxyacetone phosphate, a ketose:

Aldolase acts in the reverse direction during the process of gluconeogenesis

Reaction 5: isomerization
Tracking carbons
Tracking carbons
 Production of two glyceraldehyde-3-phosphate
molecules from one glucose molecule with the
expenditure of two ATPs.

 Therefore: the energy yields of the following steps are

multiplied by two.
 Glycolysis - Second Phase

Metabolic energy produces 4 ATP

• Net two ATP yield for glycolysis.

• Second phase involves two very high
energy phosphate intermediates
• .

– 1,3 BPG (bisphosphoglycerate)

– Phosphoenolpyruvate
Fig 14-2
 Reaction 6: oxidation

• The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl

group but to a carboxylic acid anhydride with phosphoric acid.
• This type of anhydride, called an acyl phosphate, has a very high standard free
energy of hydrolysis that is conserved by formation of the acyl phosphate group at
C-1.
*A compound loses two electrons and two hydrogen ions (that is, two hydrogen
atoms); these reactions are commonly called dehydrogenations and the enzymes that
catalyze them are called dehydrogenases
1ST energy-conserving reaction of glycolysis that eventually lead to the formation of
ATP.
 Reaction 7: substrate level phosphorylation

ATP formation

Note: phosphoglycerate kinase (PGK) is named for the reverse reaction. it catalyzes the
reaction in both directions. This enzyme acts in the direction suggested by its name during
gluconeogenesis.
• The formation of ATP by phosphoryl group transfer from a
substrate such as 1,3-bisphosphoglycerate is referred to
as a substrate-level phosphorylation, to distinguish this
mechanism from respiration-linked phosphorylation.

• Substrate-level phosphorylations involves soluble

enzymes and chemical intermediates (1,3-
bisphosphoglycerate in this case).

• Respiration-linked phosphorylations, on the other hand,

involves membrane-bound enzymes and transmembrane
gradients of protons
Reaction 8: shift of phosphoryl group

Reversible shift to phosphoryl group between C2-C3 of glycerate

 Phosphoglycerate mutase is initially
phosphorylated by phosphoryl transfer from
2,3-BPG, which is required in small quantities to
initiate the catalytic cycle and is continuously
regenerated by that cycle

• First: The enzyme mutase is phosphorylated on a His residue.

• Second: The phosphoenzyme transfers its phosphoryl group to 3-
phosphoglycerate, forming 2,3-BPG.
• Third: The phosphoryl group at C-3 of 2,3-BPG is transferred to
the same His residue on the enzyme, producing 2-
phosphoglycerate and regenerating the phosphoenzyme.
 2,3-BPG (for hemoglobin) is made by
circumventing the PGK reaction
• In RBC 1,3 BPG is converted to 2,3 BPG that unites with
oxy Hb and help release of oxygen at tissues.
• 2,3-BPG acts to maintain Hb in low oxygen affinity form.
• RBC contain high levels of 2,3 BPG (4 to 5 mM)

2,3BPG required in small quantities to initiate catalytic cycle.

Reaction 9: dehydration

(PEP)

2nd energy-conserving reaction of glycolysis that

eventually lead to the formation of ATP.
Reaction 10: substrate level phosphorylation
ATP formation

The pyruvate kinase reaction is essentially irreversible under

intracellular conditions and is an important site of regulation.
Rx 10: Pyruvate Kinase

• Substrate level phosphorylation generates second

ATP
• Large, negative delta G - regulation!
• Allosterically activated by AMP, F-1,6-bisP
• Allosterically inhibited by ATP and acetyl-CoA
 The Overall Balance Sheet Shows a
Net Gain of ATP
Glucose + 2ATP + 2NAD + 4ADP + 2Pi
2 pyruvate + 2ADP + 2NADH + 2H + 4ATP + 2H2O

the overall equation for glycolysis under aerobic conditions:

Glucose + 2NAD+ + 2ADP + 2Pi
2 pyruvate + 2NADH + 2H + 2ATP + 2H2O

The two molecules of NADH provides the energy for synthesis of

ATP by respiration linked phosphorylation (oxidative
phosphorylation).
 Remember:
Pyruvate can go in three major
directions after glycolysis
• Under aerobic conditions pyruvate is oxidized to Acetyl-CoA
which can enter Citric acid (TCA) cycle.

• Under anaerobic conditions pyruvate can be reduced to

ethanol (fermentation) or lactate

• Under anaerobic conditions formation of ethanol and lactate is

important in the oxidization NADH back to NAD+

• Under aerobic conditions NADH is oxidized to NAD+ by the

respiratory electron transport chain.
Fates of pyruvate under anaerobic conditions
Fermentation
Some cells such as RBC ferment pyruvic acid to lactic acid by
lactate dehydrogenase

Lactate is recycled ,carried from blood to liver to be glucose

Fermentation other cells (yeast)
ferment pyruvic acid to alcohol

???? disease
Pyruvate decarboxylase

Alcohol dehydrogenase
 Beriberi disease
Pyruvate decarboxylase depends
on cofactors thiamine pyrophosphate (TPP) and
magnesium.

Beriberi is a disease caused by a lack of vitamin

B1 (thiamine) in the body.

Accumulate of body fluids , pain, paralysis

…death
The 11 steps of anaerobic glycolysis include the lactate dehydrogenase
reaction. Which of the following correctly describes this reaction or its
significance to the overall process of glucose breakdown?

A. It is necessary to generate NAD+ from NADH to keep glyceraldehyde 3-P

dehydrogenase (GAPDH) active
B. It converts phosphoenolpyruvate to lactate
C. During strenuous exercise, the product is exported from the liver in the
Cori cycle
D. It catalyzes an essentially irreversible reaction
E. The reaction takes place in the matrix of the mitochondria
Other than hexokinase, where is ATP
consumed in glycolysis?
A. pyruvate kinase
B. phosphoglycerate kinase
C. glyceraldehyde 3-phosphate dehydrogenase
D. enolase
E. Phosphofructokinase