Biochemistry I, Spring Term
Lecture 29
April 3, 2013
Lecture 29: Glycolysis
Key Terms:
 Glycolysis:
1. Location: cytosol
2. Input: glucose
3. Output: pyruvate
4. Net energy prod.: 2 ATP, 2
NADH
5. Key controlling step: PFK



Kinase (X + ATP → X-P + ADP)
Dehydrogenase (Redox)
Substrate-level phosphorylation
CH2OH
OH
OH
Glucose
Glycolysis
OH
ADP
O P-O-CH
O
Glucose-6-P
HO
[isomerase]
HO
Fructose-6-P
ATP
[phosphofructokinase]
3
OPO3
H2C
Glucose
Glucose
ATP
[hexokinase]
O
OH
ADP
Fructose-1-6-P
H
HO
CH2-OH
2
O3P-O-CH2
O
HO
OH
CH2-O-PO3
OH
[aldolase]
HO
O
glyceraldehyde-3-P [triose phos. isomerase] dihydroxyacetone
phosphate
+ Pi [glyceraldehyde-3-P
+
OPO3 NAD
dehydrogenase]
H2C
NADH 1,3 bisphosphoglycerate
O-PO3
ADP
HO
OPO3
[phosphoglycerate kinase]
O
H2C
ATP
O
3-phosphoglycerate
Glucose-6-P
Fructose-6-P
Fructose-1,6-P
Pyruvate
HO
Pyruvate
Fatty Acid Oxidation
Fatty Acids
Acetyl-CoA
NADH
O
OH
H2C
H2O
O3P
CH2
FAD Electron Transport
& Oxidative Phosphorylation
Citric Acid
Cycle
O
O
O2
O3P
O
O
O
H2O
[phosphoglycerate mutase]
2-phosphoglycerate
O
[enolase]
phosphoenol pyruvate
ADP
[pyruvate kinase]
ATP
Pyruvate CH3
O
O
O
Coupling and Energy Capture
DHAP
What you should note:
 Reactions on the left with unfavorable
energy changes (red arrows) become
favorable in glycolysis due to coupling.
 The energy released (yellow highlight) from
oxidation and dephosphorylations on the
left is efficiently captured as NADH or ATP
in glycolysis.
Glyc_ald-3-P
[aldolase]
F-1,6-P
Pi
G-6-P
oxidation
F-6-P
Pi
Glucose
Glucose
DC
Glyc_ate-1,3-P
Pi
Glyc_ate-2-P
PEP
ATP
ADP
F-6-P
G-6-P
DC
DC - Direct coupling
IC - Indirect coupling
EC - Energy capture
ATP
NADH
DHAP EC
Gly_aldF-1,6-P
IC 3-P
[aldolase]
ADP
Glyc_ate-3-P
Pi
Glyc_ate1,3-P
EC
ATP
Glyc_ate3-P
IC
Glyc_ate2-P
PEP
EC
ATP
Pyr
Hypothetical Reactionsno coupling or energy capture
1
Pyr
Actual Gibbs energy change during glycolysis - showing coupling and energy capture
Biochemistry I, Spring Term
Lecture 29
April 3, 2013
1. Hexose kinase Reaction: Glucose + ATP  Glucose-6-P + ADP
Glucose
G-6-P
Group transfer reaction: Phosphate is transferred from ATP to glucose.
O
O
+
OH
OH
OH OH
O
O
O P O P O P O
O
O
O
O
N
N
ATP

N
N
O
H2C


Pi
Glucose
NH2
H
O
ATP
DC
ADP
G-6-P
O
P O
O
CH2
O
+
ADP
OH
OH
OH OH
OH OH
Traps glucose in the cell as G-6-P
Favorable hydrolysis of ATP directly coupled
to phosphorylation of glucose.
Transfer of the phosphate group on ATP to water
is negligible because water is excluded from the
active site by a conformational change of the
enzyme. Binding of the substrates causes a large
change in the structure of the enzyme to produce
the catalytically competent conformation.
Glucose accumulation in cell – An example of indirect coupling:
Hexose kinase keeps the concentration of glucose inside the cell below its equilibrium value, making the flow of
glucose into the cell spontaneous (G<0). Calculate the sign of the Gibbs Free energy for the transport of glucose
across the cell membrane in the absence (left) and presence (right) of hexose kinase activity to see that this is the
case.
[G] IN
[G] IN
G  G o  RT ln
 ([oIN ]  [oOUT ] )  RT ln
[G]OUT
[G]OUT
3. Phosphofructokinase (PFK):
Fructose -6-P + ATP→ Frucose-1,6-bis phosphate (F16P)

F-1,6-P
Favorable hydrolysis of ATP directly coupled to phosphorylation of
fructose-6-P.
NH2
O
O
H
P
O
O
O
O
CH2
O
H2C
+
HO
N
N
O
O
O P O P O P O
O
O
O
O
N
O
P
O
O
CH2
O
2
H2C
+
HO
OH
OH
OH OH
O
O
OH
ATP
O
P
N
OH
DC
ADP
ATP
ADP
F-6-P
F-1,6-P
O
O
Pi
F-6-P
Biochemistry I, Spring Term
4. Aldolase ReactionIndirect Coupling. The
large free energy change
in the last step of
glycolysis
(PEP
to
pyruvate)
keeps
the
concentration
of
all
previous
intermediates
low, allowing the aldolase
reaction
to
proceed
spontaneously.
Lecture 29
April 3, 2013
fructose 1,6
bis phosphate
CH2OPO3
O
CH2OH
O
OH
O
CH2OPO3
HO
OH
OH
Glucose
C-O-PO3
O CH2OH
ATP
DC
Aldolase
OH
OH
OH
Go = +24 kJ/mol
CH2OPO3
ADP
OH
F-6-P
ATP
DC
G = - 6 kJ/mol
OH
NADH
DHAP EC
Gly_aldF-1,6-P
C-O-PO3
IC 3-P
C-O-PO
Glyc_ate1,3-P
ATP
EC
Glyc_ate3-P
IC
Glyc_ate2-P
3
HO
PEP
OH
5. Glyceraldehyde-3-P Dehydrogenase: This reaction
proceeds in two steps. The first step is the oxidation of
the aldehyde to the carboxylic acid using NAD+ as the
electron acceptor. This results in the formation of a
covalent enzyme intermediate. The second step is the
phosphorylation of the carboxylic acid by inorganic
phosphate. Both reactions are catalyzed by a single
enzyme.
The Reaction Steps are (see diagram to right)
I. ES complex, active site Cys is deprotonated.
II. Thio group is a nucleophil, attacks aldehyde, proton
is transferred to NAD+ as a hydride (H-), net transfer
of 2 electrons and one proton.
III. NADH is released. 3-P-G remains bound to the
enzyme as a stable thioester intermediate.
IV. Attach of Pi, producing 1,3-bisphosphoglycerate
I
II
S
S
H
O
H
H2 N
O
O
CHOH
Glyceraldehyde-3-P
(Gly_ald-3-P)
NADH
EC
H
1,3 phospho
glycerate
(Glyc_ate-1,3-P)
Pi
Glyc_ate1,3-P
CH2OPO3
+
N
III
IV
S
O
H
O
O
H
H2 N
S
O
OH
CH2OPO3
glyceraldehyde-3
phosphate
CHOH
P
CH2OPO3
O
CHOH
CH2OPO3
O
+
OPO3
OH
CH2OPO3
1,3 bis
phospho
glycerate
O
OH
CH2OPO3
ADP
H O
ATP
NH2
NH2
N
R
NAD+
(Oxidized)
3
Glyc_ate3-P
H
O
O
O
ATP
HO
O
H
3-phosphoglycerate
(Glyc_ate-3-P)
EC
O
CHOH
H2 N
CH2OPO3
+
N
H
H
N
oxidation
O
Pyr H3C
OH
DHAP
Gly_aldF-1,6-P
IC 3-P
[aldolase]
O
EC
ATP
[aldolase]
OH
3-phospho
glycerate
OH
CH2OPO3
ADP
O
O
H
HO
G-6-P
dihydroxy
acetone phosphate
CH2OH
N
R
NADH
(Reduced)
HO
O
OH
CH2OPO3
3-phospho
glycerate