GLYCOLYSIS

Professor Nazmi Özer, PhD, MedSp

Department of Biochemistry
Faculty of Pharmacy
Girne American University
Kyrenia / Girne / TRNC
Spring 2024
Major pathways for glucose utilization
Entry of other saccharides into preparatory
phase of glycolysis
 Glycolysis – 1
Glycolysis = glykys (sweet or sugar) + lysis (splitting)

• A molecule of glucose is degraded in a series of enzyme-

catalyzed reactions to yield two molecules of three-carbon
compound pyruvate.

• During the sequential reactions of glycolysis, some of the free

energy released from glucose is conserved in the form of ATP
and NADH.

• Glycolysis is the only source of metabolic energy in some

mammalian tissues and cell types (erythrocytes, renal medulla,
brain and sperm).
 Glycolysis – 2
Glycolysis can be understood on four levels:

1. The chemical interconversion steps, that is, the sequence of

reactions by which glucose is converted to two pyruvate.

2. The mechanism of the enzymatic conversion of each pathway

intermediate to its successor.

3. Through the energetics of the conversions.

4. The mechanisms controlling the flux (rate of flow) of metabolites

through the pathway.

In glycolysis no O2 consumed
 History - 1
• Glycolysis is the first metabolic pathway to be
elucidated and fully understood.

• Louis Pasteur, in the years 1854-1864, established

that fermentation is caused by microorganisms
(Vitalistic theory).

• Eduard Buchner in 1897 demonstrated that cell-free

yeast extracts can also carry out fermentation
(collapse of Vitalistic theory), by this work he got
Nobel Prize in Chemistry, in 1907.
 History - 2
• Arthur Harden and William Young, in the years 1905-1910 made
two important discoveries:
i. Inorganic phosphate (Pi) is required for fermentation.
ii. Pi is incorporated into fructose-1,6-bisphosphate (a hexose
phosphate), an intermediate in the fermentation process.
• A cell-free extract can be separated into two fractions; Zymase
and cozymase. Zymase is a mixture of enzymes and cozymase is a
mixture of cofactors and coenzymes such as NAD+, ATP and metal
ions.
• By the time of 1940, the elucidation of glycolytic pathway was
completed and called as Embden-Meyerhof-Parnas pathway.
 Properties of Glycolysis
• Glycolysis, as an energy source, is a universal metabolic pathway.

• All glycolytic enzymes are found in cytoplasm.

• All glycolytic enzymes require Mg++ but, in some organisms, some

glycolytic enzymes require Mn++.

• All intermediate molecules in glycolysis are phosphorylated except

Glucose and Pyruvate.

• Phosphate groups, at pH=7.4, are ionised and negatively (-)

charged.
Importance of Phosphorylated Intermediates
• Phosphate groups, at pH=7.4, are ionised and negatively (-) charged and inner face
of plasma membrane is also negatively (-) charged and plasma membrane lacks
transporters for phosphorylated sugars. Thus, no more energy is needed to retain
the molecules in cells.

• Phosphate groups are essential components in the enzymatic conservation of

metabolic energy. High-energy phosphate compounds donate phosphoryl groups to
ADP to synthesize ATP.

• Phosphates on molecules function as identification card for enzymes and enable

the discrimination of different substrates by enzymes.

• Phosphate groups bind to the active site of the enzymes thus, lowers the activation
energy. The phosphate groups of ADP, ATP and glycolytic intermediates form
complexes with Mg++, and the substrate binding sites of many glycolytic enzymes
are specific for these Mg++ complexes.
 Hexokinase
GLUCOSE + ATP GLUCOSE-6-P
 An Overview of Glucose Metabolism - 1
• Glucose usually arises in the blood
as a result of breakdown of higher
polysaccharides or from its
synthesis from noncarbohydrate
sources.

• Glucose enters most cells by

specific carriers that transport it
from the exterior of the cell into
the cytosol.

• The enzymes of glycolysis are

located in the cytosol.
An Overview of Glucose Metabolism - 2
Glycolysis is the degradation of glucose by glycolytic enzymes to give two
pyruvate molecules at 10 steps. During these reactions, no O2 consumed. All
these reactions can be collected under two groups.

A) Activation section
In first five (5) reactions, two moles ATP per mole of glucose is comsumed
and the glycolytic intermediate molecules are activated. At this phase no
energy is obtained. At this phase, two (2) reactions are irreversible.

B) Phosphorylation section
With the following five (5) reactions 4 moles of ATP are synthesized.
At this phase, the last reaction, Pyruvate Kinase reaction, is irreversible.
 An Overview of Glucose Metabolism - 3
Glucose 2x Glyceraldehyde-3-phosphate

ATP à 1 ADP à Pi àà 6
Glucose-6-phosphate 2x 1,3-Diphosphoglycerate

2 ADP à à 7 ATP à à

Fructose-6-phosphate 2x 3-Phosphoglycerate

ATP à 3 ADP à
8
Fructose-1,6-diphosphate 2x 2-Phosphoglycerate

9
4 2x PEP
Dihydroxyacetone Glyceraldehyde-3-
phosphate phosphate ADP à à 10 ATP à à
5 2x Pyruvate

Activation Phosphorylation
 The first enzyme in Glycolysis: Hexokinase
An irreversible reaction.

hexokinase

Glucose ΔGo’= -4 Kcal/mole (-16.7 kJ/mole) Glucose-6-phosphate

At the first step, ATP is used as phosphate donor and

glucose is activated (converted to Glucose-6-phosphate).
 Hexokinase and Glucokinase
There are four hexokinase isozymes in the body. Type IV hexokinase in
the liver is called as glucokinase. The differences in the kinetic behaviors
of these isozymes plays an important role in the regulation of
metabolism. One or more isozymes of hexokinase (except hexokinase IV)
are found in all cell types, including liver, which makes glycolysis.
Hexokinase Glucokinase
Km 10-4 M 10-2 M
Substrate specificity Glucose Glucose
Mannose
2-Deoxyglucose
Glucosamine

G-6-P inhibition + -
 Hexokinase and Glucokinase
100

80

60
% Vmax

40

20

0
0 10 20 30 40
Glukoz, mM
Glucose, mM
Hexokinase
Hekzokinaz Glucokinase
Glukokinaz
 De Bruyn-van Eckenstein transformation
O=C-H OH-C-H O=C-H
| || |
H-C-OH H-C-OH OH-C-H
| | |
OH-C-H OH-C-H OH-C-H
| | |

Glucose enediol Mannose

OH-CH2
|
C=O
|
H-C-H
|
Fructose
Phosphohexose isomerase
Second reaction is catalysed by an
isomerase; an aldose phosphate is
converted to a ketose phosphate.
Glucose-6-P Fructose-6-P

ΔGo’= +0.4 Kcal/mole (+1.7 kJ/mol)

Phosphohexose
isomerase

Glucose-6-phosphate Fructose-6-phosphate
 Phosphofructokinase - I
It is the first “committed” and “irreversible” step in the glycolytic
pathway and here, fructose 1,6-bisphosphate (F1,6BP) is targeted to
glycolysis. This step is one of the important step for the “regulation”
of Glycolysis. Phosphofructokinase is an allosteric enzyme.

Phosphofructokinase
(PFK-I)

Mg2+
ATP ADP

Fructose-6-phosphate Fructose-1,6-
bisphosphate
ΔGo’ = (-3.4 Kcal/mole (-14.2 kJ/mole)
 Naming of Phosphorylated compouds

Compounds that contain two phosphate or phosphoryl groups

attached at different positions in the molecule are named
bisphosphates (or bisphospho compounds); for example,
fructose 1,6-bisphosphate and 1,3-bisphosphoglycerate.
Compounds with two phosphates linked together as a
pyrophosphoryl group are named diphosphates; for example,
adenosine diphosphate (ADP). Similar rules apply for the
naming of trisphosphates (such as inositol 1,4,5-trisphosphates)
and triphosphates (such as adenosine triphosphate, ATP)
 Aldolase
Aldolase catalyze the aldole cleavage reaction. Fructose-1,6-bisphosphate is cleaved to give
two three carbon sugar phosphates; One is an aldose phosphate, Glyceraldehyde-3-
phosphate, (GAL3P), the other one is a ketose phosphate, Dihydroxyacetone phosphate
(DHAP). Class I aldolase: animals and plants and Class II aldolase: fungi and bacteria.

DHAP
Adolase

F-1,6,DP
GAL3P
ΔGo’ = + 5.7 Kcal/mole (+23.9 kJ/mol)
 Phosphotriose Isomerase
Phosphotriose isomerase catalyze enediole mediated
transformation of DHAP to GAL3P.

DHAP Enediol GAL3P

ΔGo’ = +1.8 Kcal/mole (+7.6 kJ/mole)

Thus, at the first half of glycolysis 2 moles of GAL3P is formed
from one mole of glucose.
Activation Steps End, Phosphorylation Steps Start
Glucose 2x Glyceraldehyde-3-phosphate

ATP à 1 ADP à Pi àà 6
Glucose-6-phosphate 2x 1,3-Diphosphoglycerate

2 ADP à à 7 ATP à à

Fructose-6-phosphate 2x 3-Phosphoglycerate

ATP à 3 ADP à
8
Fructose-1,6-diphosphate 2x 2-Phosphoglycerate

9
4 2x PEP
Dihydroxyacetone Glyceraldehyde-3-
phosphate phosphate ADP à à 10 ATP à à
5 2x Pyruvate

Activation Phosphorylation
Glyceraldehyde-3-Phosphate Dehydrogenase

Pi, NAD+ NADH + H+

Mg2+

GAL3P Dehydrogenase

GAL3P ΔGo’ = +1.5 Kcal/mole (+6.3 kJ/mole) 1,3-DPGA*, 3-PGP*

There are several reasons that makes the GAL3P dehydrogenase

reaction an important reaction:
1. It is the first enzyme that the inhibition mechanism was discovered.
2. In this reaction, (Pi) is directly utilized (from medium) and later used for ATP synthesis.
3. This is the only reaction in that electrons were taken from substrate using NAD+.
4. At the of the reaction a high energy compound, 1,3-DPGA*, is obtained.

*, 1,3-Diphosphoglyceric acid or 3-Phosphoglyceroyl phosphate

 Inhibitors of GAL3P Dehydrogenase

A -SH + Hg+ -S-Hg + H+

) Mercury

-COO-
-
-SH + Cl Hg- -S-Hg- -COO- + Cl
B)
Para-chloromercurybenzoate (PCMB)

-SH + ICH2CONH2 -S- CH2CONH2 + H+ + l-

C)
Iodoacetamide*

Arsenate
H2AsO3- NADH + H+
O-AsO3H-
D) Hydrolysis

NAD+
GAL3P 3-Phosphoglyceroyl Arsenate
*, Iodoacetate, like iodoacetamide, is also an inhibitor
 Phosphoglycerate Kinase
Substrate level phosphorylation

ADP ATP

Mg2+
Phosphoglycerate Kinase
ΔGo’ = -4.5 Kcal/mole (-18.8 kJ/mole)
1,3-DPGA 3-PGA
(3-Posphoglyceroyl Phosphate) (3-Posphoglyceric Acid)
 Phosphoglyceromutase
Phosphoryl group schift from 3rd carbon to 2nd carbon

Phosphoglyceromutase
ΔGo’= +1.1 Kcal/mole (+4.4 KJ/mole)

3-PGA 2-PGA
• 2,3-Bisphosphoglycerate (2,3-BPG) provides phosphorylation of the enzyme.

• 2,3-BPG is found in trace (catalytic) amounts in cells.

• 2,3-BPG is found at high concentration in erythrocytes because it plays an

important role in oxygen transport from lungs to tissues.
 Enolase
Dehydration reaction

F-
H2O
Mg2+

Enolase
ΔGo‘= +0.4 Kcal/mole (+1.7 kJ/mole)

2-PGA PEP
(2-Phosphoglyceroyl phosphate) (Phosphoenolpyruvate)
 Pyruvate Kinase
Substrate-level phosphorylation

ADP ATP
Mg2+
Pyruvate Kinase Nonezymatic reaction

ΔGo’ = 0 Kcal/mole ΔGo‘=-7.5 Kcal/mole (31.4 kJ/mole)

PEP Enol-Pyruvate Pyruvate

1. Pyruvate kinase reaction is the last step of Glycolysis.

2. Reaction type is “substrate-level phosphorylation”.
3. Pyruvate kinase reaction is the most important irreversible reaction
because very large free energy released.
Types of Reactions in Glycolysis
1. Phosphoryl group transfer
Hexokinase
Phosphofructokinase (PFK - 1)

2. Phosphoryl group shift

Phosphoglucomutase
Phosphoglyceromutase

3. Isomerization
Phosphohexose isomerase
Phosphotriose isomerase

4. Dehydration
Enolase

5. Aldol cleavage
Aldolase

6. Oxidation
Glyceraldehyde-3-phosphate dehydrogenase
 Energy Balance Sheet of Glycolysis - 1

Reaction Input Output

1 Hexokinase 1 ATP -
2 Phosphohexose isomerase - -
3 Phosphofructokinase (PFK) 1 ATP -
4 Aldolase - -
5 Phosphotriose isomerase - -
6 Glyceraldehyde-3-phosphate dehydrogenase 2 Pi, 2 NAD+ 2 NADH

7 Phosphoglycerate kinase 2 ADP 2 ATP

8 Phosphoglucomutase - -
9 Enolase - -
10 Pyruvate kinase 2 ADP 2 ATP
NET RESULT 2 ADP, 2 Pi 2 ATP
2 NAD+ 2 NADH
 Energy Balance Sheet of Glycolysis - 2
Anaerobic glycolysis:
Glucose+2ATP+2NAD++4ADP+2Pi → 2Pyruvate+2ADP+2NADH+2H++4ATP+2H2O

Summary of anaerobic glycolysis:

Glucose + 2ADP + 2Pi → 2Lactate + 2ATP + 2H2O

Aerobic glycolysis:
Glucose+2ATP+2NAD++4ADP+2Pi → 2Pyruvate+2ADP+2NADH+2H++4ATP+2H2O

Summary of anaerobic glycolysis:

Glucose + 2NAD++ 2ADP + 2Pi → 2Pyruvate + 2NADH + 2H+ + 2ATP + 2H2O ;

Then, NADH is oxidized in Electron Transport Chain (ETC)

2NADH + 2H+ + O2 → 2NAD+ + 2H2O (in mitochondria)

 Regulation of Glycolysis
Louis Pasteur: He was studying the fermentation of glucose in yeast and he
showed that the rate and total consumption of glucose under anaerobic
conditions was much higher (about 10 times) than the glucose consumed
under aerobic condition. This is called as Pasteur Effect.

ATP yield in anaerobic and aerobic glycolysis ;

- Anaerobic conditions: Net, 2 moles of ATP is obtained per oxidized glucose.
- Aerobic conditions: Net, 30-32 moles ATP is obtained per oxidized glucose.

Factors regulating glycolysis;

- ATP, ADP, AMP, F2,6DP, F1,6DP
*NADH, NAD+
*Hexokinase, PFK-1, Pyruvate kinase
*Hormones: Glucagon, Epinephrine, Insulin
Glucose is Metabolized Differently in Various
Cells - 1
Red Blood Cells Brain Tissue Cells
Glucose
Glucose
GLUT1 GLUT3
a a
Glucose
Glucose
b
c
b
c PPP Glucose-6-P
PPP Glucose-6-P d
d (2) Pyruvate

(2) Lactate- (2) Lactate- (2) CO2 f

Symport
(2) H+ (2) H+ (2) Acetyl CoA (4) CO2
e
g

a. Glucose transporter (GLUT); b. Hexokinase; c. Pentose Phosphate Pathway (PPP)

d. Glycolysis; e. Lactic acid transport; f. Pyruvate dehydrogenase; g. TCA cycle
 Glucose is Metabolized Differently in Various
Cells - 2
Muscle & Heart Tissue Cells Adipose Tissue Cells
Glucose Glucose

GLUT4 GLUT4
a a

Glucose Glucose

b h
b h
c c
PPP Glucose-6-P Glycogen Glycogen
PPP Glucose-6-P
d i i
d
(2) Pyruvate- (2) Lactate- (2) Lactate-
Symport (2) Pyruvate-
(2) CO2 f (2) H+ (2) H+
e (2) CO2 f
(2) Acetyl CoA (4) CO2
(2) Acetyl CoA
g j
FAT
a. Glucose transporter (GLUT); b. Hexokinase; c. Pentose Phosphate Pathway (PPP); d. Glycolysis;
e. Lactic acid transport; f. Pyruvate dehydrogenase; g. TCA cycle; h. Glycogenesis; i. Glycogenolysis;
j. Lipogenesis
Insulin Stimulates Glucose Uptake by Increasing GLUT4
in the Plasma Membrane of Adipose & Muscle Cells
Insulin
+

IR Plasma Membrane GLUT4 GLUT4 GLUT4 GLUT4

+
+ +
Kinase-mediated signalling cascade

4 GLUT
GLUT 4 GL
UT
T4 4
G LU
Glucose is Metabolized Differently in Various
Cells - 3
Glucose
Liver Parenchymal Cells
GLUT2
a
Glucose

PPP b h
c
Glucose Glucose-6-P Glycogen
m
n d l i
GAP
(Glucuronides) Pyruvate- Lactate- LIS Lactate-
e
(2) CO2 f

(2) Acetyl CoA (4) CO2

k g
VLDL VLDL Fat j

a. Glucose transporter (GLUT); b. Hexokinase; c. Pentose Phosphate Pathway (PPP);

d. Glycolysis; e. Lactate import system; f. Pyruvate dehydrogenase; g. TCA cycle;
h. Glycogenesis; i. Glycogenolysis; j. Lipogenesis; k. VLDL formation and release;
l. Gluconeogenesis; m. Glucose release to circulation; n. Formation of glucuronides
Glucose Catabolism is Different in Cancerous
Tissues
• Glucose uptake and glycolysis proceed about 10 times faster in
most solid tumors.

• Tumor cells commonly experience hypoxia (lack of O2).

• They produce lactate by lactic acid fermentation.

• In some tumors, several glycolytic enzymes are overproduced.

• Hypoxia-inducible factor (HIF-1) acts at the level of mRNA

synthesis, to stimulate the synthesis of at least eight of the
glycolytic enzymes.
 Pyruvate Transformation Pathways
NADH produced by GAL3P dehydrogenase enzyme in glycolysis, is oxidized by
different mechanisms in different living organisms.

A) Under anaerobic conditions

1. Formation of lactic acid (in RBCs, Muscle and Heart Cells)
LDH
Pyruvate + NADH + H+ ↔ Lactate + NAD+
2. Formation of ethanol (in liver and kidney)
Pyruvate decarboxylase
Pyruvate → Acetaldehyde + CO2
Alchol dehydrogenase
Acetaldehyde + NADH + H+ ↔ Ethanol + NAD+

B) Under aerobic conditions

Electrons on NADH are transferred by “glycerophosphate” or/and “malate-
aspartate” shutles to mitochondria. Thus, NAD+ regenerated at ETC.
 The Fate of Pyruvate

Pyruvate

Fermentation Aerobic glycolysis Anaerobic glycolysis

Pyruvate Pyruvate Pyruvate
Pyruvate CoA Pyruvate
decarboxylase dehydrogenase NADH Lactate
Acetaldehyde + CO2 CO2 dehydrogenase
NAD+
NADH alcohol Acetyl-CoA Lactate
dehydrogenase
NAD+
CH3CH2OH
THANK YOU !!!!