Lecture No.

8
Glycolysis

• At the end of this lecture, student will be able to

–Explain different steps in glycolysis

–Explain the importance of glycolysis
–Describe the role of different enzymes in glycolysis

1
Faculty of pharmacy ©M. S. Ramaiah University of Applied Sciences
 Glycolysis

2
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 6 CH OPO 2
2 3
5 O
H H
H
4 H 1
OH
OH OH
3 2
H OH
glucose-6-phosphate

Glycolysis takes place in the cytosol of cells.

Glucose enters the Glycolysis pathway by conversion to
glucose-6-phosphate.
Initially there is energy input corresponding to cleavage of
two ~P bonds of ATP.

3
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 Types of glycolysis

4
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 6 CH 2 O H 6 CH O PO 2 
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
M g 2+
OH OH OH OH
3 2 3 2
H OH H ex ok inase H OH
glucose glucose -6 -ph osp hate

1. Hexokinase catalyzes:
Glucose + ATP  glucose-6-P + ADP
The reaction involves nucleophilic attack of the C6 hydroxyl O of
glucose on P of the terminal phosphate of ATP.
ATP binds to the enzyme as a complex with Mg++.

5
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 NH2

ATP N
N
adenosine triphosphate
O O O N N

O P O P O P O CH2 adenine
O
   H H
O O O
H H
OH OH
ribose

Mg++ interacts with negatively charged phosphate oxygen atoms,

providing charge compensation & promoting a favorable
conformation of ATP at the active site of the Hexokinase enzyme.

6
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 6 CH2OH 6 CH OPO 2
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 H 1
OH H OH
Mg2+
OH OH OH OH
3 2 3 2
H OH Hexokinase H OH
glucose glucose-6-phosphate

The reaction catalyzed by Hexokinase is highly spontaneous.

A phosphoanhydride bond of ATP (~P) is cleaved.
The phosphate ester formed in glucose-6-phosphate has a
lower G of hydrolysis.

7
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 6 CH 2 O H 6 CH O PO 2 
2 3
ATP ADP
5 O 5 O
Induced fit: H H H H
H H
4 1 4 H 1
OH H 2+ OH
Glucose binding OH OH
Mg
OH OH
3 2
to Hexokinase 3 2
H ex o k in ase
H OH H OH
stabilizes a glu co se glu cose -6 -ph osp hate
conformation
in which: glucose

 the C6 hydroxyl of the

bound glucose is close to the
terminal phosphate of Hexokinase ATP,
promoting catalysis.
 water is excluded from the active site
This prevents the enzyme from catalyzing ATP hydrolysis, rather
than transfer of phosphate to glucose
8
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 glucose

Hexokinase

It is a common motif for an enzyme active site to be located at an

interface between protein domains that are connected by a flexible
hinge region.
The structural flexibility allows access to the active site, while
permitting precise positioning of active site residues, and in some
cases exclusion of water, as substrate binding promotes a particular
conformation.

9
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 6 CH O PO 2 
2 3
6 CH 2
5 O 2 O PO 3 1 CH 2 O H
H H O
H
4 H 1 5 H HO 2
OH
OH OH H 4 3 OH
3 2
OH H
H OH
P h o s p h o g lu c o s e Is o m e ra s e
g lu c o s e -6 -p h o s p h a te fru c to s e -6 -p h o s p h a te

2. Phosphoglucose Isomerase catalyzes:

glucose-6-P (aldose)  fructose-6-P (ketose)
The mechanism involves acid/base catalysis, with ring opening,
isomerization via an enediolate intermediate, and then ring
closure. A similar reaction catalyzed by Triosephosphate
Isomerase will be presented in detail.
10
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 P h o s p h o fru c to k in a s e
2 2
6 CH
2 O PO 3 1 CH 2 O H 6 CH
2 O PO 3 1 CH 2 O PO 3 2 
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH M g2+ H 4 3 OH
OH H OH H
fru c to s e -6 -p h o s p h a te f r u c t o s e - 1 ,6 - b i s p h o s p h a t e

3. Phosphofructokinase catalyzes:
fructose-6-P + ATP  fructose-1,6-bisP + ADP
This highly spontaneous reaction has a mechanism similar to that of
Hexokinase
The Phosphofructokinase reaction is the rate-limiting step of
Glycolysis
The enzyme is highly regulated, as will be discussed later

Faculty of Pharmacy
11
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 2
1 CH 2 O PO 3

2C O
H O
2
HO 3 C H A ld o la se 3
CH 2 O PO 3 1C

H 4C OH 2C O + H 2C O H
2
H C OH 1 CH 2 O H 3 CH 2 O PO 3
5
2 d ih y d ro x y a c e to n e g ly c e ra ld e h y d e -3 -
6 CH 2 O PO 3
p h o sp h a te p h o sp h a te
fru c to se -1 ,6 -
b isp h o sp h a te
T rio se p h o sp h a te Iso m e ra se

4. Aldolase catalyzes: fructose-1,6-bisphosphate 

dihydroxyacetone-P + glyceraldehyde-3-P
The reaction is an aldol cleavage, the reverse of an aldol condensation
Note that C atoms are renumbered in products of Aldolase

Faculty of Pharmacy
12
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 lysine 1CH2OPO3
2
H
 2C NH (CH2)4 Enzyme
H 3N +
C COO +
HO 3
CH
CH2
H 4
C OH
CH2
H 5
C OH
CH2 2
6 CH 2OPO 3
CH2
Schiff base intermediate of
 NH3 Aldolase reaction

A lysine residue at the active site functions in catalysis.

The keto group of fructose-1,6-bisphosphate reacts with
the -amino group of the active site lysine, to form a
protonated Schiff base intermediate.
Cleavage of the bond between C3 & C4 follows.
Faculty of Pharmacy
13
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 2
1 CH 2 O PO 3

2C O
H O
2
HO 3 C H A ld o la se 3
CH 2 O PO 3 1C

H 4C OH 2C O + H 2C O H
2
H C OH 1 CH 2 O H 3 CH 2 O PO 3
5
2 d ih y d ro x y a c e to n e g ly c e ra ld e h y d e -3 -
6 CH 2 O PO 3
p h o sp h a te p h o sp h a te
fru c to se -1 ,6 -
b isp h o sp h a te
T rio se p h o sp h a te Iso m e ra se

5. Triose Phosphate Isomerase (TIM) catalyzes:

dihydroxyacetone-P  glyceraldehyde-3-P
Glycolysis continues from glyceraldehyde-3-P. TIM's Keq
favors dihydroxyacetone-P. Removal of glyceraldehyde-3-P
by a subsequent spontaneous reaction allows throughput.
Faculty of Pharmacy
14
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 T rio s e p h o s p h a te Is o m e ra s e
H H OH H O
+ + + +
H C OH H H C H H C
C O C OH H C OH
2 2 2
CH 2O PO 3 CH 2O PO 3 CH 2O PO 3

d ih y d ro x y a c e to n e e n e d io l g ly c e ra ld e h y d e -
p h o s p h a te in te rm e d ia te 3 -p h o s p h a te

The ketose/aldose conversion involves acid/base catalysis,

and is thought to proceed via an enediol intermediate, as
with Phosphoglucose Isomerase.
Active site Glu and His residues are thought to extract and
donate protons during catalysis.
Faculty of Pharmacy
15
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 OH
HC O O O
C C
CH 2 O PO 3 2  CH 2 O PO 3 2 
p ro p o se d p h o s p h o g ly c o la te
e n e d io la te tra n s itio n s ta te
in te rm e d ia te a n a lo g

2-Phosphoglycolate is a transition state analog that

binds tightly at the active site of Triose Phosphate
Isomerase (TIM).
This inhibitor of catalysis by TIM is similar in structure to
the proposed enediolate intermediate.
TIM is judged a "perfect enzyme." Reaction rate is limited
only by the rate that substrate collides with the enzyme.
Faculty of Pharmacy
16
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 G ly c e ra ld e h y d e -3 -p h o s p h a te
D e h y d ro g e n a se
2
H O + H+ O O PO 3
+
1 C NAD NADH 1 C
+ P i
H C OH H C OH
2 2
2 2
3 CH 2O PO 3 3 CH 2O PO 3

g ly c e ra ld e h y d e - 1 ,3 -b is p h o s p h o -
3 -p h o s p h a te g ly c e ra te

6. Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 
1,3-bisphosphoglycerate + NADH + H+
Faculty of Pharmacy
17
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 G ly c e ra ld e h y d e -3 -p h o s p h a te
D e h y d ro g e n a se
H O + H+ O O PO 3 2 
1 C NAD+ NADH 1 C
+ Pi
H C OH H C OH
2 2
2 2
3 CH 2 O PO 3 3 CH 2 O PO 3

g ly c e ra ld e h y d e - 1 ,3 -b is p h o s p h o -
3 -p h o s p h a te g ly c e ra te

Exergonic oxidation of the aldehyde in glyceraldehyde- 3-

phosphate, to a carboxylic acid, drives formation of an
acyl phosphate, a "high energy" bond (~P).
This is the only step in Glycolysis in which NAD+ is reduced
to NADH.
18
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 H O
H
1C
H3N+ C COO
H 2 C OH
CH2 2
3 CH2OPO3
SH glyceraldehyde-3-
cysteine phosphate

A cysteine thiol at the active site of Glyceraldehyde-

3-phosphate Dehydrogenase has a role in catalysis.
The aldehyde of glyceraldehyde-3-phosphate reacts
with the cysteine thiol to form a thiohemiacetal
intermediate.
19
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 Enz-Cys SH O OH

HC CH CH 2OPO 3 2
glyceraldehyde-3-
OH OH phosphate
Oxidation to a Enz-Cys S CH CH CH 2OPO 3 2
carboxylic acid NAD + thiohemiacetal
(in a ~ thioester) NADH intermediate
occurs, as NAD+ O OH
is reduced to Enz-Cys S C CH CH 2OPO 3 2
NADH. Pi
acyl-thioester
intermediate
O OH
2
Enz-Cys SH O 3PO C CH CH 2OPO 3 2
1,3-bisphosphoglycerate
The “high energy” acyl thioester is attacked by Pi to
yield the acyl phosphate (~P) product. 20
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 H O O
H H
C C
NH2 NH2

+
N  + N
2e + H
R R
NAD+ NADH

Recall that NAD+ accepts 2 e plus one H+ (a hydride)

in going to its reduced form.

Faculty of Pharmacy
21
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 P h o s p h o g ly c e ra te K in a s e
2 
O O PO 3 ADP ATP O O
1C 1
C
H 2
C OH H 2
C OH
2+
2 Mg 2
3 CH 2 O PO 3 3 CH 2 O PO 3

1 ,3 - b i s p h o s p h o - 3 -p h o s p h o g ly c e ra te
g ly c e ra te

7. Phosphoglycerate Kinase catalyzes:

1,3-bisphosphoglycerate + ADP 
3-phosphoglycerate + ATP
This phosphate transfer is reversible (low G), since
one ~P bond is cleaved & another synthesized.
The enzyme undergoes substrate-induced conformational
change similar to that of Hexokinase. 22
Faculty
Faculty of Pharmacy
of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 P h o s p h o g ly c e r a te M u ta s e
O O O O
1
C 1
C
2
H 2C O H H 2 C O PO 3
2
3 CH 2 O PO 3 3 CH 2 O H
3 -p h o s p h o g ly c e r a te 2 -p h o s p h o g ly c e r a te

8. Phosphoglycerate Mutase catalyzes:

3-phosphoglycerate  2-phosphoglycerate

Phosphate is shifted from the OH on C3 to the

OH on C2.

Faculty of Pharmacy
23
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 P h o s p h o g ly c e r a te M u ta s e
O O
histidine
O O
H
1
C 1
C
2 H3N+ C COO
H 2C O H H 2 C O PO 3
2 CH2
3 CH 2 O PO 3 3 CH 2 O H
C
3 -p h o s p h o g l y c e r a t e 2 -p h o s p h o g ly c e r a te
HN CH

HC NH
An active site histidine side-chain 

participates in Pi transfer, by
O O
donating & accepting phosphate. C
1
The process involves a H 2C OPO32
2,3-bisphosphate intermediate. 3CH2OPO3
2

2,3-bisphosphoglycerate
View an animation of the
Phosphoglycerate Mutase reaction.
Faculty of Pharmacy
24
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 E n o la s e
O  H 
O  OH O
O O O
C C 1
C
1
H 2 C O P O 32 C O P O 32 2C O P O 32

3 C H 2O H C H 2O H 3CH2
2 -p h o s p h o g ly c e ra te e n o la te in te r m e d ia te p h o s p h o e n o lp y r u v a te

9. Enolase catalyzes:
2-phosphoglycerate  phosphoenolpyruvate + H2O
This dehydration reaction is Mg++-dependent.
2 Mg++ ions interact with oxygen atoms of the substrate
carboxyl group at the active site.
The Mg++ ions help to stabilize the enolate anion
intermediate that forms when a Lys extracts H+ from C #2.
25
Faculty
Faculty of Pharmacy
of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 P y r u v a te K in a s e
O O O O
ADP ATP
1
C 1
C

2
C O PO 3 2  2
C O

3 CH 2 3 CH 3
p h o s p h o e n o lp y r u v a te p y r u v a te

10. Pyruvate Kinase catalyzes:

phosphoenolpyruvate + ADP  pyruvate + ATP

Faculty of Pharmacy
26
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 P y r u v a te K in a s e

O O O O O O
C ADP ATP C C
1 1 1
2
2
C O PO 3 2
C OH 2
C O

3 CH 2 3 CH 2 3 CH 3
p h o s p h o e n o lp y ru v a te e n o lp y ru v a te p y ru v a te

This phosphate transfer from PEP to ADP is spontaneous.

 PEP has a larger G of phosphate hydrolysis than ATP.
 Removal of Pi from PEP yields an unstable enol, which
spontaneously converts to the keto form of pyruvate.
Required inorganic cations K+ and Mg++ bind to anionic
residues at the active site of Pyruvate Kinase.
Faculty of Pharmacy
27
Faculty of Pharmacy ©M. S.©M. S. Ramaiah
Ramaiah University
University of Applied
of Applied Sciences
Sciences
 glucose Glycolysis
ATP
Hexokinase
ADP
glucose-6-phosphate
Phosphoglucose Isomerase
fructose-6-phosphate
ATP
Phosphofructokinase
ADP
fructose-1,6-bisphosphate
Aldolase

glyceraldehyde-3-phosphate + dihydroxyacetone-phosphate
Triosephosphate
Isomerase
Glycolysis continued
Faculty of Pharmacy
28
©M. S. Ramaiah University of Applied Sciences
 glyceraldehyde-3-phosphate
NAD+ + Pi Glyceraldehyde-3-phosphate
NADH + H+ Dehydrogenase
1,3-bisphosphoglycerate
Glycolysis ADP
continued. Phosphoglycerate Kinase
ATP
Recall that 3-phosphoglycerate
there are 2 Phosphoglycerate Mutase
GAP per 2-phosphoglycerate
glucose.
H2O Enolase
phosphoenolpyruvate
ADP
Pyruvate Kinase
ATP
pyruvate 29
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences
 Summary

• Glycolysis is a determined sequence of ten enzyme-

catalyzed reactions
• Glycolysis occurs, with variations, in nearly all
organisms, both aerobic and anaerobic
• The second half of glycolysis is known as the pay-
off phase
• Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2
NADH + 2 H+ + 2 ATP + 2 H2O

30
Faculty of Pharmacy ©M. S. Ramaiah University of Applied Sciences