Loknayak Jaiprakash Institute of Technology

(Dept. Of Science & Technology,Govt. Of

Bihar)

Biology
Module-8
Biochemistry of metabolism
Lecture -1-Glycolysis

Dr. Kumari Seema

Assistant Professor
Department: Applied Sciences & Humanities
1
 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.
2
 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

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++.
3
 NH2

ATP N
N
adenosine triphosphate
O O O N N

O P O P O P O CH2 adenine
O
O O O H H
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.
4
 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 DG of hydrolysis.
5
 6 CH2OH 6 CH OPO 2
2 3
ATP ADP
Induced fit: H
5 O H H
5 O H
H H
Glucose binding 4
OH H 1
Mg 2+
4
OH H 1

OH OH OH OH
to Hexokinase 3 2 3 2
H OH Hexokinase H OH
stabilizes a
glucose glucose-6-phosphate
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.
6
 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.
7
 6 CH OPO 2
2 3
5 6 CH OPO 2 1CH2OH
H O H 2 3
O
H
4 H 1 5 H HO 2
OH
OH OH H 4 3 OH
3 2
OH H
H OH
Phosphoglucose Isomerase
glucose-6-phosphate fructose-6-phosphate

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.
8
 Phosphofructokinase
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH Mg2+ H 4 3 OH
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate

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.
9
 2
1CH2OPO3

2C O
H O
2
HO 3C H Aldolase 3
CH 2 OPO3 1C

H 4C OH 2C O + H 2C OH
2
H C OH 1CH2OH 3 CH2OPO3
5
2
6 CH 2 OPO3 dihydroxyacetone glyceraldehyde-3-
phosphate phosphate
fructose-1,6-
bisphosphate
Triosephosphate Isomerase

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.
10
 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 2OPO3
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 e-amino group of the active site lysine, to form a
protonated Schiff base intermediate.
Cleavage of the bond between C3 & C4 follows.
11
 2
1CH2OPO3

2C O
H O
2
HO 3C H Aldolase 3
CH 2 OPO3 1C

H 4C OH 2C O + H 2C OH
2
H C OH 1CH2OH 3 CH2OPO3
5
2
6 CH 2 OPO3 dihydroxyacetone glyceraldehyde-3-
phosphate phosphate
fructose-1,6-
bisphosphate
Triosephosphate Isomerase

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.
12
 Triosephosphate Isomerase
H H OH H O
+ + + +
H C OH H H C H H C
C O C OH H C OH
CH2OPO32 CH2OPO32 CH2OPO32

dihydroxyacetone enediol glyceraldehyde-

phosphate intermediate 3-phosphate

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.
13
 OH
HC O O O
C C
CH 2OPO32 CH 2OPO32
proposed phosphoglycolate
enediolate transition state
intermediate analog

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.
14
Triosephosphate Isomerase
structure is an ab barrel, or
TIM barrel.
In an ab barrel there are
8 parallel b-strands surrounded
by 8 a-helices.
Short loops connect alternating TIM
b-strands & a-helices.

15
TIM barrels serve as scaffolds
for active site residues in a
diverse array of enzymes.
Residues of the active site are
always at the same end of the
barrel, on C-terminal ends of
b-strands & loops connecting
TIM
these to a-helices.
There is debate whether the many different enzymes with
TIM barrel structures are evolutionarily related.
In spite of the structural similarities there is tremendous
diversity in catalytic functions of these enzymes and
little sequence homology.
16
 OH
HC O O O
C C
CH 2OPO32 CH 2OPO32
proposed phosphoglycolate
enediolate transition state
TIM intermediate analog

Explore the structure of the Triosephosphate Isomerase

(TIM) homodimer, with the transition state inhibitor
2-phosphoglycolate bound to one of the TIM monomers.
Note the structure of the TIM barrel, and the loop that
forms a lid that closes over the active site after binding
of the substrate.
17
 Glyceraldehyde-3-phosphate
Dehydrogenase
H O + H+ O OPO32
1C NAD+ NADH 1C
+ Pi
H C OH H C OH
2 2
2 2
3 CH 2 OPO3 3 CH 2 OPO3

glyceraldehyde- 1,3-bisphospho-
3-phosphate glycerate

6. Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 
1,3-bisphosphoglycerate + NADH + H+
18
 Glyceraldehyde-3-phosphate
Dehydrogenase
H O + H+ O OPO32
1C NAD+ NADH 1C
+ Pi
H C OH H C OH
2 2
2 2
3 CH 2 OPO3 3 CH 2 OPO3

glyceraldehyde- 1,3-bisphospho-
3-phosphate glycerate

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.
19
 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.
20
 Enz-Cys SH O OH

HC CH CH2OPO32
glyceraldehyde-3-
OH OH phosphate

Oxidation to a Enz-Cys S CH CH CH2OPO32

carboxylic acid NAD + thiohemiacetal
NADH intermediate
(in a ~ thioester)
O OH
occurs, as NAD+
Enz-Cys S C CH CH2OPO32
is reduced to acyl-thioester
Pi
NADH. intermediate
O OH
2
Enz-Cys SH O3PO C CH CH2OPO32
1,3-bisphosphoglycerate

The “high energy” acyl thioester is attacked by Pi to

yield the acyl phosphate (~P) product.
21
 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.

22
 Phosphoglycerate Kinase
O OPO32 ADP ATP O O
1C 1
C
H 2C OH H 2C OH
2 Mg2+ 2
3 CH 2 OPO3 3 CH 2 OPO3

1,3-bisphospho- 3-phosphoglycerate
glycerate

7. Phosphoglycerate Kinase catalyzes:

1,3-bisphosphoglycerate + ADP 
3-phosphoglycerate + ATP
This phosphate transfer is reversible (low DG), since
one ~P bond is cleaved & another synthesized.
The enzyme undergoes substrate-induced conformational
change similar to that of Hexokinase.
23
 Phosphoglycerate Mutase
O O O O
C
1
C
1
H 2C OH H 2C OPO32
2
3 CH2OPO3 3 CH2OH
3-phosphoglycerate 2-phosphoglycerate

8. Phosphoglycerate Mutase catalyzes:

3-phosphoglycerate  2-phosphoglycerate

Phosphate is shifted from the OH on C3 to the

OH on C2.

24
 Phosphoglycerate Mutase
O O
histidine
O O
H
1
C 1
C
2 H3N+ C COO
H 2C OH H 2C OPO3
2 CH2
3 CH2OPO3 3 CH2OH
C
3-phosphoglycerate 2-phosphoglycerate
HN CH

HC NH

An active site histidine side-chain
participates in Pi transfer, by
donating & accepting phosphate. O O
1
C
The process involves a H 2C OPO32
2,3-bisphosphate intermediate. 2
3 CH2OPO3
2,3-bisphosphoglycerate
View an animation of the
Phosphoglycerate Mutase reaction.
25
 Enolase

O H  
O OH O
O O O
C C 1
C
1
H 2 C OPO32 C OPO32 2C OPO32

3 CH2OH CH2OH 3 CH2

2-phosphoglycerate enolate intermediate phosphoenolpyruvate

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.
26
 Pyruvate Kinase
O O O O
ADP ATP
1
C 1
C

2
C OPO32 2
C O

3 CH2 3 CH3
phosphoenolpyruvate pyruvate

10. Pyruvate Kinase catalyzes:

phosphoenolpyruvate + ADP  pyruvate + ATP

27
 Pyruvate Kinase

O O O O O O
C ADP ATP C C
1 1 1

2
C OPO32 C
2
OH 2
C O

3 CH2 3 CH2 3 CH3

phosphoenolpyruvate enolpyruvate pyruvate

This phosphate transfer from PEP to ADP is spontaneous.

 PEP has a larger DG 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.
28
 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 29
 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 30
 Glycolysis

Balance sheet for ~P bonds of ATP:

 How many ATP ~P bonds expended? ________

2

 How many ~P bonds of ATP produced? (Remember

4
there are two 3C fragments from glucose.) ________

 Net production of ~P bonds of ATP per glucose:

________
2

31
Balance sheet for ~P bonds of ATP:
 2 ATP expended
 4 ATP produced (2 from each of two 3C fragments
from glucose)
 Net production of 2 ~P bonds of ATP per glucose.
Glycolysis - total pathway, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
In aerobic organisms:
 pyruvate produced in Glycolysis is oxidized to CO2
via Krebs Cycle
 NADH produced in Glycolysis & Krebs Cycle is
reoxidized via the respiratory chain, with production
of much additional ATP.
32
 Glyceraldehyde-3-phosphate
Dehydrogenase
H O + H+ O OPO32
1C NAD+ NADH 1 C
Fermentation: + Pi
H C OH H C OH
2 2
2 2
Anaerobic 3 CH2OPO3 3CH2OPO3
organisms lack a glyceraldehyde- 1,3-bisphospho-
respiratory chain. 3-phosphate glycerate

They must reoxidize NADH produced in Glycolysis

through some other reaction, because NAD+ is needed for
the Glyceraldehyde-3-phosphate Dehydrogenase reaction.
Usually NADH is reoxidized as pyruvate is converted to
a more reduced compound.
The complete pathway, including Glycolysis and the
reoxidation of NADH, is called fermentation.
33
 Lactate Dehydrogenase
O O O O
C NADH + H+ NAD+ C
C O HC OH
CH3 CH3
pyruvate lactate

E.g., Lactate Dehydrogenase catalyzes reduction of

the keto in pyruvate to a hydroxyl, yielding lactate, as
NADH is oxidized to NAD+.
Lactate, in addition to being an end-product of
fermentation, serves as a mobile form of nutrient energy,
& possibly as a signal molecule in mammalian organisms.
Cell membranes contain carrier proteins that facilitate
transport of lactate.
34
 Lactate Dehydrogenase
O O O O
C NADH + H+ NAD+ C
C O HC OH
CH3 CH3
pyruvate lactate

Skeletal muscles ferment glucose to lactate during

exercise, when the exertion is brief and intense.
Lactate released to the blood may be taken up by other
tissues, or by skeletal muscle after exercise, and converted
via Lactate Dehydrogenase back to pyruvate, which may
be oxidized in Krebs Cycle or (in liver) converted to back
to glucose via gluconeogenesis
35
 Lactate Dehydrogenase
O O O O
C NADH + H+ NAD+ C
C O HC OH
CH3 CH3
pyruvate lactate

Lactate serves as a fuel source for cardiac muscle as

well as brain neurons.
Astrocytes, which surround and protect neurons in the
brain, ferment glucose to lactate and release it.
Lactate taken up by adjacent neurons is converted to
pyruvate that is oxidized via Krebs Cycle.
36
 Pyruvate Alcohol
Decarboxylase Dehydrogenase
O O
CO2 H NADH + H+ NAD+ H
C O
C H C OH
C O
CH3 CH3
CH3
pyruvate acetaldehyde ethanol

Some anaerobic organisms metabolize pyruvate to

ethanol, which is excreted as a waste product.
NADH is converted to NAD+ in the reaction
catalyzed by Alcohol Dehydrogenase.
37
Glycolysis, omitting H+:
glucose + 2 NAD+ + 2 ADP + 2 Pi 
2 pyruvate + 2 NADH + 2 ATP
Fermentation, from glucose to lactate:
glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP

Anaerobic catabolism of glucose yields only 2 “high

energy” bonds of ATP.

38
 DGo' DG
Glycolysis Enzyme/Reaction kJ/mol kJ/mol
Hexokinase -20.9 -27.2
Phosphoglucose Isomerase +2.2 -1.4
Phosphofructokinase -17.2 -25.9
Aldolase +22.8 -5.9
Triosephosphate Isomerase +7.9 negative
Glyceraldehyde-3-P Dehydrogenase -16.7 -1.1
& Phosphoglycerate Kinase
Phosphoglycerate Mutase +4.7 -0.6
Enolase -3.2 -2.4
Pyruvate Kinase -23.0 -13.9
*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John
Wiley & Sons, New York, p. 613.
39
Flux through the Glycolysis pathway is regulated by
control of 3 enzymes that catalyze spontaneous reactions:
Hexokinase, Phosphofructokinase & Pyruvate Kinase.
 Local control of metabolism involves regulatory effects
of varied concentrations of pathway substrates or
intermediates, to benefit the cell.
 Global control is for the benefit of the whole organism,
& often involves hormone-activated signal cascades.
Liver cells have major roles in metabolism, including
maintaining blood levels various of nutrients such as
glucose. Thus global control especially involves liver.
Some aspects of global control by hormone-activated
signal cascades will be discussed later.
40
 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

Hexokinase is inhibited by product glucose-6-phosphate:

 by competition at the active site
 by allosteric interaction at a separate enzyme site.
Cells trap glucose by phosphorylating it, preventing exit
on glucose carriers.
Product inhibition of Hexokinase ensures that cells will
not continue to accumulate glucose from the blood, if
[glucose-6-phosphate] within the cell is ample.
41
 6 CH2OH 6 CH OPO 2
2 3
ATP ADP
5 O 5 O
H H H H
H H
4 1 4 1
Glucokinase OH H
Mg2+
OH H

is a variant of OH OH OH OH
3 2 3 2
Hexokinase H OH Hexokinase H OH
found in liver. glucose glucose-6-phosphate

 Glucokinase has a high KM for glucose.

It is active only at high [glucose].
 One effect of insulin, a hormone produced when blood
glucose is high, is activation in liver of transcription of
the gene that encodes the Glucokinase enzyme.
 Glucokinase is not subject to product inhibition by
glucose-6-phosphate. Liver will take up & phosphorylate
glucose even when liver [glucose-6-phosphate] is high.
42
 Glucokinase is subject to inhibition by glucokinase
regulatory protein (GKRP).
The ratio of Glucokinase to GKRP in liver changes in
different metabolic states, providing a mechanism for
modulating glucose phosphorylation.

43
 Glycogen Glucose
Glucokinase, Hexokinase or Glucokinase
with high KM Glucose-6-Pase
for glucose, Glucose-1-P Glucose-6-P Glucose + Pi
allows liver to Glycolysis
Pathway
store glucose
as glycogen in Pyruvate
the fed state Glucose metabolism in liver.
when blood [glucose] is high.
Glucose-6-phosphatase catalyzes hydrolytic release of Pi
from glucose-6-P. Thus glucose is released from the liver
to the blood as needed to maintain blood [glucose].
The enzymes Glucokinase & Glucose-6-phosphatase, both
found in liver but not in most other body cells, allow the
liver to control blood [glucose].
44
 Pyruvate Kinase
Pyruvate Kinase, the O O O O
ADP ATP
last step Glycolysis, is 1
C 1
C
2
controlled in liver partly 2
C OPO3 2
C O
by modulation of the 3 CH2 3 CH3
amount of enzyme. phosphoenolpyruvate pyruvate

High [glucose] within liver cells causes a transcription

factor carbohydrate responsive element binding protein
(ChREBP) to be transferred into the nucleus, where it
activates transcription of the gene for Pyruvate Kinase.
This facilitates converting excess glucose to pyruvate,
which is metabolized to acetyl-CoA, the main precursor
for synthesis of fatty acids, for long term energy storage.
45
 Phosphofructokinase
6 CH OPO 2 1CH2OH 6 CH OPO 2 1CH2OPO32
2 3 2 3
O ATP ADP O
5 H HO 2 5 H HO 2

H 4 3 OH Mg2+ H 4 3 OH
OH H OH H
fructose-6-phosphate fructose-1,6-bisphosphate

Phosphofructokinase is usually the rate-limiting step of

the Glycolysis pathway.
Phosphofructokinase is allosterically inhibited by ATP.
 At low concentration, the substrate ATP binds only at
the active site.
 At high concentration, ATP binds also at a low-affinity
regulatory site, promoting the tense conformation.
46
 60

50 low [ATP]

PFK Activity
40

30

20
high [ATP]

10

0
0 0.5 1 1.5 2
[Fructose-6-phosphate] mM

The tense conformation of PFK, at high [ATP], has lower

affinity for the other substrate, fructose-6-P. Sigmoidal
dependence of reaction rate on [fructose-6-P] is seen.
AMP, present at significant levels only when there is
extensive ATP hydrolysis, antagonizes effects of high ATP.
47
 Glycogen Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.

Inhibition of the Glycolysis enzyme Phosphofructokinase

when [ATP] is high prevents breakdown of glucose in a
pathway whose main role is to make ATP.
It is more useful to the cell to store glucose as glycogen
when ATP is plentiful.
48
Thank You
49