A metabolic pathway. The breakdown of glucose by enzymes, releasing energy and pyruvic acid.

1. Glycolysis
A Metabolic Pathway
Gobinda Panigrahi
M. Sc Part-I (2015-16)
P. G. DEPARTMENT OF BOTANY
BERHAMPUR UNIVERSITY,
BHANJA BIHAR, BERHAMPUR- 760007
GANJAM, ODISHA, INDIA
e-mail-

2. GENERAL INFORMATION
 The word GLYCOLYSIS is combination of GLYCOSE means GLUCOSE and LYSIS means
DEGRADATION, which together means the degradation of glucose.
 Glycolysis is the metabolic pathway that converts glucose (C6H12O6), into pyruvate
(CH3COCOO- + H+ ).
 The free energy released in this process is used to form the high energy compounds ATP
(ADENOSINE TRI PHOSPHATE) and NADH (reduced NICOTINAMIDE ADENINE
DINUCLEOTIDE).
 Glycolysis is a determined sequence of ten enzyme catalyzed reactions.

3.  Glycolysis is an oxygen independent metabolic pathway, meaning that it doesn’t use
molecular oxygen (i.e. atmospheric oxygen) for any of its reactions.
 However the products of glycolysis (pyruvate and NADH+H+) are sometimes disposed of
using atmospheric oxygen.
 When molecular oxygen is used in the disposal of the products of glycolysis the process
is usually referred to as aerobic, whereas if the disposal uses no oxygen the process is
said to be anaerobic.

4.  Thus, glycolysis occurs, with variations, in nearly all organisms, both aerobic
and anaerobic.
 The wide occurrence of glycolysis indicates that it is one of the most ancient
metabolic pathways.
 Indeed, the reactions that constitute glycolysis and its parallel pathway, the
pentose phosphate pathway, occur metal-catalyzed under the oxygen free
conditions of the archean oceans, also in the absence of oxygen.

5.  Glycolysis could thus have originated from chemical constraints of the
prebiotic world.
 Glycolysis occurs in most organisms in the cytosol of the cell.
 The most common type of glycolysis is the Embden-Meyerhof-Parnas
(EMP pathway), which was discovered by Gustav Embden, Otto Meyerhof,
and Jacob Karol Parnas.

6. Sequence of reactions
The entire glycolytic pathway can be separated into two phases:
 The Preparatory Phase – constituted by the reactions in which ATP is consumed and is
hence also known as the investment phase.
 The Pay Off Phase – constituted by the reactions in which ATP is produced.

7. Preparatory Phase
 The first five steps are regarded as the preparatory phase, since they consume energy to
convert the glucose into two three-carbon sugar phosphates.
A. Glucose → glucose-6-phosphate (G6P), first ATP utilization
Enzyme: hexokinase (HK)
Reaction: Transfer phosphoryl group
∆G0’= -20.9 kJ/mol; ∆G = -27.2 kJ/mol (physiological condition)

8. Reaction mechanism
 C6 – OH group of glucose nucleophilic attacks on the у-phosphate of an Mg2+ - ATP complex

9. B. Glucose 6 –phosphate (G6P) to Fructose 6 –phosphate (F6P)
Enzyme: Phosphoglucose isomerase (PGI)
Reaction: Isomerization of an aldose to ketose
∆G0’ = +2.2 kJ/mol; ∆G = -1.4 kJ/mol

10. Reaction mechanism
The reaction requires ring
opening, followed by
isomerization, and subsequent
ring closure.
1. An acid (ε-amino group of
Lys) donates a proton to O5
to open the ring.
2. A base (carboxylate of Glu)
abstracts the acidic proton
from C2.
3. The proton is replaced on
C1.
4. The proton on O5 is
returned to the acid (Lys)
and the O5 nucleophilic
attacks on C2 to close the
ring.

11. C. Fructose 6-phosphate (F6P) to Fructose-1,6-bisphosphate (FBP), Second ATP Utilization
Enzyme: Phosphofructokinase (PFK)
Reaction: Transfer phosphoryl group
∆G0’= -17.2 kJ/mol ∆G = -25.9 kJ/mol
Reaction mechanism
Similar to the hexokinase reaction.

12. D. Fructose-1,6-bisphosphate to two trioses, Glyceraldehyde-3-phosphate (GAP) and
Dihydroxyacetone phosphate (DHAP)
Enzyme: Aldolase
Reaction: Aldol cleavage
∆G0’= +22.8 kJ/mol ∆G= -5.9 kJ/mol

13. E. Dihydroxyacetone phosphate to Glyceraldehyde-3-phosphate (GAP)
Enzyme: Triose phosphate isomerase (TIM)
Reaction: Isomerization (aldose to ketose)
∆G0’= +7.9 kJ/mol ∆G= +4.4 kJ/mol

14. Reaction mechanism
1. Glu-165 interacts with the proton on C2
of GAP, and the proton of His-95 interacts
with C1=O to form GAP:TIM Michaelis
complex.
2. at the first transition state, these two
protons participate in the low-barrier
hydrogen bonds (very short hydrogen
bonds).
3.the protons are moved to the carbonyl
oxygen of Glu-165 and to the carbonyl
oxygen of GAP.
4. At the second transition state, Glu-165
donates the proton to C1 of GAP, and GAP
donates the proton of O2-H to His-95
through the low-barrier hydrogen bonds.
5. After the proton transfer, DHAP:TIM
Michaelis complex is formed.

15. Pay-off Phase
 The second half of glycolysis is known as the pay-off phase, characterized by a net gain of
the energy-rich molecules ATP and NADH.
 Since glucose leads to two triose sugars in the preparatory phase, each reaction in the pay-
off phase occurs twice per glucose molecule.
 This yields 2 NADH molecules and 4 ATP molecules, leading to a net gain of 2 NADH
molecules and 2 ATP molecules from the glycolytic pathway per glucose.

16. F. Glyceraldehyde 3-phosphate (GAP) to 1,3-Bisphosphoglycerate (1,3-BPG), First “High
Energy” Intermediate Formation
Enzyme: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
Reaction: Oxidation and Phosphorylation
∆G0’= +6.7 kJ/mol ∆G= -1.1 kJ/mol

17. Reaction mechanism
The active site has the essential sulfhydryl
group (-SH), a base residue (-B:) and
cofactor NAD+.
1. GAP binds to the enzyme.
2. The base amino acid residue abstracts the
proton from –SH, and thus the activated –S-
attacks on the carbonyl carbon to form
thiohemiacetal intermediates.
3. By transferring the proton to NAD+ , the
thiohemiacetal becomes an acyl thioester.
4. NADH is replaced with another NAD+ .
5. A phosphate ion enters into the active site
and its nucleophilic attack on the carbonyl
carbon of acyl thioester forms 1,3-
bisphosphoglycerate. The –S receives a
proton from the base amino acid residue,
and mthe product is released.

18. G. 1,3-Bisphosphoglycerate (1,3-BPG) to 3-Phosphoglycerate (3PG), First ATP Generation
Enzyme: Phosphoglycerate kinase (PGK)
Reaction: Phosphorylation
∆G0’= -18.8 kJ/mol ∆G = ~0 kJ/mol

19. Reaction
mechanism
nucleophilic attack of the
terminal phosphoryl oxygen
of ADP on the C1
phosphorus atom of 1,3-
BPG forms the reaction
product.

20. H. 3-Phosphoglycerate (3PG) to 2-Phosphoglycerate (2PG)
Enzyme: Phosphoglycerate mutase (PGM)
Reaction: Intramolecular phosphoryl group transfer
∆G0’= +4.7 kJ/mol ∆G = -0.6 kJ/mol

21. Reaction
mechanism
1. 3PG binds to the
phosphorylated enzyme (
E-His-PO3
- )
2. The PO3
– is
transferred to the
substrate to form 2,3-
BPG:E complex.
3. The PO3
– attached on
the O3 is transferred to
the enzyme, and 2PG is
released.

22. I. 2-Phosphoglycerate (2PG) to Phosphoenolpyruvate (PEP)
Enzyme: Enolase
Reaction: Dehydration
∆G0’= -3.2 kJ/mol ∆G = -2.4 kJ/mol

23. Reaction mechanism
- The enzyme forms a complex
with Mg2+ before the substrate
is bound.
- Fluoride ion (F- ) inhibits this
process, since F- forms a
complex with bound Mg2+ in
the active site, and block the
substrate binding.
- A water molecule is in the
active site, which hydrogen-
bonds to two Glu residues.
1. The water molecule bound
to the two carboxylates of Glu
residues abstracts a proton at
C2, and thus the carboanion is
formed (rapid reaction). The
abstracted proton is readily
exchanged with a proton in the
solvent.
2. Slow elimination of Mg2+ -
stabilized OH at C3 produces a
phosphoenolpyruvate (PEP)
and a water molecule. R

24. J. Phosphoenolpyruvate (PEP) to Pyruvate
Enzyme: Pyruvate kinase
Reaction: Hydrolysis to ATP synthesis
∆G0’= -23.0 kJ/mol ∆G = -13.9 kJ/mol

25. Reaction mechanism
- Monovalent (K+) and
divalent (Mg2+) cations are
required.
1. A nucleophilic attack of the
ADP β-phosphoryl oxygen
atom on the phosphorus atom
of PEP forms ATP and
enolpyruvate.
2. A tautomerization is taken
place to form a pyruvate from a
enolpyruvate.

27. Net ATP count in a glycolytic pathway

28. Regulation
 Glycolysis is regulated by slowing down or speeding up certain steps in the pathway by
inhibiting or activating the enzymes that are involved.
 The steps that are regulated may be determined by calculating the change in free energy, ∆G,
for each step.
 When ∆G is negative, a reaction proceed spontaneously in the forward direction only and is
considered irreversible.
 When ∆G is positive, the reaction is non-spontaneous and will not proceed in the forward
direction unless coupled with an energetically favorable reaction.
 When ∆G is zero, the reaction is at equilibrium, can proceed in either directions and is
considered reversible.
 If a step is at equilibrium (∆G is zero), the enzyme catalyzing the reaction will balance the
products and reactants and cannot confer directionality to the pathway.
 These steps (and associated enzymes) are considered unregulated.
 If a step is not at equilibrium, but spontaneous (∆G is negative), the enzyme catalyzing the
reaction is not balancing the products and reactants and is considered to be regulated.

29. Importance of glycolysis
 Nearly all of the energy used by living cells comes from the energy in the bonds of the sugar
glucose.
 Glycolysis is the first pathway used in the breakdown of glucose to extract energy.
 It takes place in the cytoplasm of both prokaryotic and eukaryotic cells.
 It was probably one of the earlier metabolic pathways to evolve since it is used by nearly all of
the organisms on earth.
 The process doesn’t use oxygen and is, therefore, anaerobic.
 Glycolysis is the first of the main metabolic pathways of cellular respiration to produce
energy in the form of ATP.
 Overall, the process of glycolysis produces a net gain of two pyruvate molecules, two ATP
molecules, and two NADH molecules for the cell to use for energy.

30. Glycolysis in disease
Genetic diseases
 glycolytic mutations are generally rare due to importance of the metabolic pathway, this means
that the majority of occurring mutations result in an inability for the cell to respire, and
therefore cause the death of the cell at an early stage.
 However some mutations are seen with one notable example being Pyruvate kinase deficiency,
leading to chronic hemolytic anemia.
Cancer
 Malignant rapidly growing tumor cells typically have glycolytic rates that are upto 200 times
higher than those of their normal tissues of origin. This phenomenon was first described in
1930 by Otto Warburg and is referred to as Warburg effect.
 This hypothesis claims that cancer is primarily caused by dysfunctionality in mitochondrial
metabolism, rather than because of uncontrolled growth of cells.

31. Thank you all for your benign presence