Biochemistry of Metabolism

Glycolysis

Copyright © 1998-2004 by Joyce J.

Diwan.
All rights reserved.
 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.
 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 okin ase H OH
gluco se 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++.
 NH2

ATP N
N
adenosine triphosphate
N
O O O 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.
 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 okinase H OH
glu cose glu cose -6-ph osphate

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.
 glucose

Hexokinase
Induced fit:
Binding of glucose to Hexokinase promotes a large
conformational change by stabilizing an alternative
conformation in which:
 the C6 hydroxyl of the bound glucose is close to the
terminal phosphate of 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. 
 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.
 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.
 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.
 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.
 lysine 1CH 2OPO3
2
H
 2C NH (CH2)4 Enzyme
H3N+
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 -amino group of the active site lysine, to form a
protonated Schiff base intermediate.
Cleavage of the bond between C3 & C4 follows.
 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.
 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
CH 2 O PO 3 2  CH 2 O PO 3 2  CH 2 O PO 3 2 

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.
 OH
HC 
O O O 
C C
2 2
CH 2 O PO 3 CH 2 O PO 3
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.
Triosephosphate Isomerase
structure is an  barrel, or
TIM barrel.
In an  barrel there are
8 parallel -strands
surrounded by 8 -helices.
Short loops connect alternating
TIM
-strands & -helices.
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
-strands & loops connecting TIM
these to -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.
 OH
HC O O O
C C
CH 2 O PO 3 2  CH 2 O PO 3 2 
p ro p o sed 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
TIM in te rm e d ia te a n a lo g

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
 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 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

6. Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 
1,3-bisphosphoglycerate + NADH + H+
 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 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.
 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.
 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

NAD + thiohemiacetal
carboxylic acid intermediate
NADH
(in a ~ thioester)
O OH
occurs, as NAD+
Enz-Cys S C CH CH 2OPO 3 2
is reduced to acyl-thioester
Pi
NADH. 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.
 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.
 P h o s p h o g ly c e ra te K in a s e
O O PO 3 2  A D P A T P 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.
 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 l y 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.
 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
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 C
HN CH

An active site histidine HC NH



side-chain participates in Pi
O O
transfer, by donating & accepting
C
the phosphate. 1
H 2C OPO32
The process involves a
CH2OPO32
2,3-bisphosphate intermediate. 3
2,3-bisphosphoglycerate
View an animation of the
Phosphoglycerate Mutase reaction.
 E n o la s e
O O O O
C 1
C
1
H C O PO 3 2  2
C O PO 3 2  + H 2 O
2
3 CH 2 O H 3 CH 2
2 -p h o s p h o g ly c e ra te p h o s p h o e n o lp y ru v a te

9. Enolase catalyzes
2-phosphoglycerate  phosphoenolpyruvate + H2O
This Mg++-dependent dehydration reaction is inhibited
by fluoride.
Fluorophosphate forms a complex with Mg++ at the active
site.
 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
C O PO 3 2  2
C OH 2
C O

3 CH 2 3 CH 2 3 CH 3
p h o sp h o e n o lp y ru v a te e n o l p y r u v a te p y r u v a te

10. Pyruvate Kinase catalyzes:

phosphoenolpyruvate + ADP  pyruvate + ATP
 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 l p y r u 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.
 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
 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
 Glycolysis

Balance sheet for ~P bonds of ATP:

2
 How many ATP ~P bonds expended? ________

 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
________
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. 
 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
Fermentation: + P i
H C OH H C OH
2 2
Anaerobic 3 CH 2 O PO 3
2
3 CH 2 O PO 3
2

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

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, that may be excreted.
The complete pathway, including Glycolysis and the
reoxidation of NADH, is called fermentation.
E.g., Lactate L a c ta te D e h y d ro g e n a s e
Dehydrogenase O O O O
catalyzes reduction of C NADH + H+ NAD+ C
the keto in pyruvate to C O HC OH
a hydroxyl, yielding CH 3 CH 3
lactate, as NADH is p y ru v a te la c ta te
oxidized to NAD+.
Skeletal muscles ferment glucose to lactate during
exercise, when aerobic metabolism cannot keep up with
energy needs.
Lactate released to the blood may be taken up by other
tissues, or by muscle after exercise, and converted via the
reversible Lactate Dehydrogenase back to pyruvate, e.g.,
for entry into Krebs Cycle.
 L a c ta te D e h y d ro g e n a s e
O O O O
C NADH + H+ NAD+ C
C O HC OH
CH 3 CH 3
p y ru v a te la c ta te

Lactate is also a significant energy source for neurons

in the brain.
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.
 P y ru v a te A lc o h o l
D e c a rb o x y la s e D e h y d ro g e n a se
O O
CO 2 H NADH + H+ NAD+ H
C O
C H C OH
C O
CH 3 CH 3
CH 3
p y ru v a te a c e ta ld e h y d e e th a n o l

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.
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.
 Go' G
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, 3 rd Edition, John
Wiley & Sons, New York, p. 613.
Three Glycolysis enzymes catalyze spontaneous reactions:
Hexokinase, Phosphofructokinase & Pyruvate Kinase.
Control of these enzymes determines the rate of the
Glycolysis pathway.
 Local control involves dependence of enzyme-
catalyzed reactions on concentrations of pathway
substrates or intermediates within a cell.
 Global control involves hormone-activated production
of second messengers that regulate cellular reactions for
the benefit of the organism as a whole.
Local control of Hexokinase and Phosphofructokinase will
be discussed here. Regulation by hormone-activated cAMP
signal cascade will be discussed later.
 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 okinase H OH
glu cose glu cose -6-ph osphate

Hexokinase is inhibited by its product glucose-6-

phosphate.
Glucose-6-phosphate inhibits by competition at the
active site, as well as by allosteric interactions at a
separate site on the enzyme.
 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 okinase H OH
glu cose glu cose -6-ph osphate

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.
Glucokinase, a variant of Hexokinase found in liver, has
a high KM for glucose. It is active only at high [glucose].
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.
Liver Glucokinase is subject to inhibition by glucokinase
regulatory protein (GKRP).
The ratio of Glucokinase to GKRP changes in different
metabolic states, providing a mechanism for modulating
glucose phosphorylation.
 Glycogen Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.

Glucokinase, with its high KM for glucose, allows the liver

to store glucose as glycogen, in the fed state when blood
[glucose] is high.
 Glycogen Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-1-P Glucose-6-P Glucose + Pi
Glycolysis
Pathway
Pyruvate
Glucose metabolism in liver.

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].
 P h o s p h o fru c to k in a s e
6 CH
2O PO 3 2  1 CH 2 O H 6 CH
2O PO 3 2  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

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.
 60

50 low [A T P ]

40

PFK Activity
30

20
high [A T P]

10

0
0 0.5 1 1.5 2
[F ru ctose-6-p h osp h ate] m M

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.
 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.