Download Glycolysis

Biochemistry of Metabolism
Glycolysis
Copyright © 1998-2006 by Joyce J. Diwan.
All rights reserved.
6 CH OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
H
1
OH
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 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
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++.
NH2
ATP
N
N
adenosine triphosphate
O

O
O
P
O
O

P
O
N
O
O

P
O
O
adenine
CH2

N
O
H
H
OH
H
OH
H
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 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
1
H
2
OH
Hexokinase H
OH
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.
6 CH2OH
Induced fit:
5
H
4
Glucose binding
to Hexokinase
stabilizes a
conformation
in which:
OH
O
H
OH
H
2
3
H
OH
glucose
 the C6 hydroxyl of the
bound glucose is close to
the terminal phosphate of
ATP, promoting catalysis.
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
4
1
OH
Mg
2+
OH
H
OH
3
H
1
H
2
OH
Hexokinase H
OH
glucose-6-phosphate
glucose
Hexokinase
 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 OPO 2
2
3
5
O
H
4
OH
H
OH
3
H
H
2
OH
H
1
OH
6 CH OPO 2
2
3
1CH2OH
O
5
H
H
4
OH
HO
2
3 OH
H
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.
Phosphofructokinase
6 CH OPO 2
2
3
O
5
H
H
4
OH
6 CH OPO 2
2
3
1CH2OH
O
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
5
Mg2+
1CH2OPO32
H
H
4
OH
HO
2
3 OH
H
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.
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
2
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
dihydroxyacetone
phosphate
5
C
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
glyceraldehyde-3phosphate
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.
1CH2OPO3
2C
O
HO 3C
H 4C
H
H
2
H
Aldolase
2
CH
OPO
2
3
3
OH
2C
OH
1CH2OH
2
CH
OPO
2
3
6
dihydroxyacetone
phosphate
5
C
fructose-1,6bisphosphate
O
+
O
1C
H 2C OH
2
CH
OPO
3
2
3
glyceraldehyde-3phosphate
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.
lysine
2
1CH2OPO3
H
+
H3N
C
CH2
CH2
CH2
CH2

NH3
COO
2C

HO
H
H
NH (CH2)4
+
Enzyme
CH
3
C
OH
C
OH
4
5
2
CH
OPO
2
3
6
Schiff base intermediate of
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.
Triosephosphate Isomerase
H
H
C
OH
C
O
+
H H
CH2OPO32
dihydroxyacetone
phosphate
+
H
OH
H H
C
C
+
OH
CH2OPO32
enediol
intermediate
+
H
O
C
H
C
OH
CH2OPO32
glyceraldehyde3-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.
OH
O
HC
O
O
C
C
CH2OPO32
CH2OPO32
proposed
enediolate
intermediate
phosphoglycolate
transition state
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.
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
b-strands & a-helices.
TIM
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
these to a-helices.
TIM
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
O
HC
TIM
O
O
C
C
CH2OPO32
CH2OPO32
proposed
enediolate
intermediate
phosphoglycolate
transition state
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.
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
NAD+
1C
H
2
C
OH
+ Pi
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
C
2
OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
6. Glyceraldehyde-3-phosphate Dehydrogenase
catalyzes:
glyceraldehyde-3-P + NAD+ + Pi 
1,3-bisphosphoglycerate + NADH + H+
Glyceraldehyde-3-phosphate
Dehydrogenase
H
O
NAD+
1C
H
2
C
OH
+ Pi
2
CH
OPO
2
3
3
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
C
2
OH
2
CH
OPO
2
3
3
1,3-bisphosphoglycerate
Exergonic oxidation of the aldehyde in glyceraldehyde3-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
H
H3N+
C
COO
CH2
SH
cysteine
O
1C
H 2 C OH
2
3 CH2OPO3
glyceraldehyde-3phosphate
A cysteine thiol at the active site of Glyceraldehyde3-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
Oxidation to a
carboxylic acid
(in a ~ thioester)
occurs, as NAD+
is reduced to
NADH.
Enz-Cys
O
OH
HC
CH
SH
S
OH
OH
CH
CH
CH2OPO32
glyceraldehyde-3phosphate
CH2OPO32
thiohemiacetal
intermediate
NAD +
NADH
Enz-Cys
S
O
OH
C
CH
CH2OPO32
acyl-thioester
intermediate
Pi
Enz-Cys
SH
2
O3PO
O
OH
C
CH
CH2OPO32
1,3-bisphosphoglycerate
The “high energy” acyl thioester is attacked by Pi to
yield the acyl phosphate (~P) product.
H
O
H
H
C
C
NH2
+
N
O

NH2
+
2e + H
N
R
R
NAD+
NADH
Recall that NAD+ accepts 2 e plus one H+ (a hydride)
in going to its reduced form.
Phosphoglycerate Kinase
O
OPO32 ADP ATP O
O
1C
H 2C OH
2
3 CH2OPO3
1,3-bisphosphoglycerate
C
1
Mg
2+
H 2C OH
2
3 CH2OPO3
3-phosphoglycerate
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.
Phosphoglycerate Mutase
O
O
C
1
O
O
C
1
H 2C OH
2
3 CH2OPO3
H 2C OPO32
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.
Phosphoglycerate Mutase
O
O
C
1
H 2C OH
2
3 CH2OPO3
3-phosphoglycerate
histidine
O
O
H
C
1
H 2C OPO3
3 CH2OH
2
2-phosphoglycerate
An active site histidine side-chain
participates in Pi transfer, by
donating & accepting phosphate.
The process involves a
2,3-bisphosphate intermediate.
View an animation of the
Phosphoglycerate Mutase reaction.
H3N+
COO
C
CH2
C
HN
HC
CH
NH

O
O
C
1
H 2C OPO32
2
3 CH2OPO3
2,3-bisphosphoglycerate
Enolase
O
O

C
1
H 2 C OPO32
3 CH2OH
H

O
O

C
C
OH
O
O
1
OPO32
CH2OH
C
2C
OPO32
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.
Pyruvate Kinase
O
O
C
1
C
2
ADP ATP
O
O
C
1
OPO32
3 CH2
phosphoenolpyruvate
C
2
O
3 CH3
pyruvate
10. Pyruvate Kinase catalyzes:
phosphoenolpyruvate + ADP  pyruvate + ATP
Pyruvate Kinase
O
O
ADP ATP
C
1
C
2
O
O
C
C
1
OPO32
3 CH2
phosphoenolpyruvate
C
2
O
O
1
OH
3 CH2
enolpyruvate
C
2
O
3 CH3
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.
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
Dehydrogenase
NADH + H+
Glycolysis
continued.
Recall that
there are 2
GAP per
glucose.
1,3-bisphosphoglycerate
ADP
Phosphoglycerate Kinase
ATP
3-phosphoglycerate
Phosphoglycerate Mutase
2-phosphoglycerate
Enolase
H2O
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.
Glyceraldehyde-3-phosphate
Dehydrogenase
H
Fermentation:
Anaerobic
organisms lack a
respiratory chain.
O
NAD+
1C
H
2
C
OH
3 CH2OPO3
+ Pi
2
glyceraldehyde3-phosphate
OPO32
+ H+ O
NADH
1C
H
C
2
OH
3 CH2OPO3
2
1,3-bisphosphoglycerate
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.
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
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.
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
HC
OH
CH3
CH3
pyruvate
lactate
Skeletal muscles ferment glucose to lactate during
exercise.
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
Lactate Dehydrogenase
O
O
C
C
NADH + H+ NAD+
O
O
O
C
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.
Pyruvate
Decarboxylase
Alcohol
Dehydrogenase
CO2
NADH + H+ NAD+
O
O
C
C
O
CH3
pyruvate
H
O
C
CH3
acetaldehyde
H
H
C
OH
CH3
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.
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.
Glycolysis Enzyme/Reaction
DGo'
DG
kJ/mol kJ/mol
Hexokinase
Phosphoglucose Isomerase
Phosphofructokinase
Aldolase
Triosephosphate Isomerase
Glyceraldehyde-3-P Dehydrogenase
& Phosphoglycerate Kinase
-20.9 -27.2
+2.2
-1.4
-17.2 -25.9
+22.8
-5.9
+7.9 negative
-16.7
-1.1
Phosphoglycerate Mutase
Enolase
Pyruvate Kinase
+4.7
-3.2
-23.0
-0.6
-2.4
-13.9
*Values in this table from D. Voet & J. G. Voet (2004) Biochemistry, 3rd Edition, John
Wiley & Sons, New York, p. 613.
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.
6 CH2OH
5
H
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
2
H
1
OH
Hexokinase H
OH
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.
6 CH2OH
5
H
Glucokinase
is a variant of
Hexokinase
found in liver.
4
OH
O
H
OH
H
2
3
H
OH
glucose
6 CH OPO 2
2
3
5
O
ATP ADP
H
H
1
OH
4
Mg2+
OH
H
OH
3
H
1
H
2
OH
Hexokinase H
OH
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.
 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.
Glycogen
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
Glucose-6-P
Glucose + Pi
Glycolysis
Pathway
Glucokinase,
with high KM
Glucose-1-P
for glucose,
allows liver to
store glucose
Pyruvate
as glycogen in
Glucose metabolism in liver.
the fed state
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].
Pyruvate Kinase
O
O
Pyruvate Kinase, the
ADP
C
last step Glycolysis, is
1
2
C
OPO
controlled in liver partly
3
2
by modulation of the
3 CH2
amount of enzyme.
phosphoenolpyruvate
ATP
O
O
C
1
C
2
O
3 CH3
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.
Phosphofructokinase
6 CH OPO 2
2
3
O
5
H
H
4
OH
6 CH OPO 2
2
3
1CH2OH
O
ATP ADP
HO
2
3 OH
H
fructose-6-phosphate
5
Mg2+
1CH2OPO32
H
H
4
OH
HO
2
3 OH
H
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.
60
low [ATP]
PFK Activity
50
40
30
high [ATP]
20
10
0
0
0.5
1
1.5
[Fructose-6-phosphate] mM
2
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-1-P
Glucose
Hexokinase or Glucokinase
Glucose-6-Pase
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.