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Lecture 4
Glycolysis
(Embden-Meyerhof Pathway)
Cleavage of glucose and
Substrate-level phosphorylation
of ADP to ATP
The “Powertrain” of Human Metabolism (Overview)
CARBOHYDRATES
Glucose
PROTEINS
LIPIDS
Amino acids
Fatty acids
Oxaloacetate
O2
Glycogen
Glucose-6-P
Pyruvate
Acetyl-CoA
CO2
Glycolysis
Lactate
Ribose-5-P
NADPH
NADH
ATP
H2O
Ketone
bodies
Cholesterol
NADH
p. 21
There are two fundamentally different ways
to generate ATP:
1. Substrate-level phosphorylation of ADP to ATP
2. Proton gradient-dependent ATP synthesis
Substrate-level Phosphorylation (ATP)
Sred
Sox + Pi
–∆G
P~Sox
+∆G
P~Sox + ADP
Sox + ADP~P (ATP)
Example
i) P~Sox + H2O
Sox + Pi
∆Go’= –49.6
ii) ADP + Pi
ATP + H2O
∆Go’= +30.5
Pi = HPO42-
Σ ∆Go’= –19.1
First Reaction in Glycolysis
Glucose
(Glc)
Glucose-6-phosphate
?
(Glucose-6-P)
(Glc-6-P)
(G6P)
∆G Calculations on 1. Reaction in Glycolysis (Phosphorylation of Glucose)
Summary of Chalk Board Calculations
∆Go’ or ∆G (kJmol-1)
Glc + Pi Î Glc-6-P +
5 mM 1 mM 0.083 mM
H2O
+13.9
+21.1
Keq
3.7x10-3
2.8x10-4
(intracellular concentrations)
∆Go’ = –RTlnKeq
Keq = e^(-∆Go’/RT) = e^(-13,900 Jmol-1/8.315 Jmol-1K-1 x 298 K) = 0.0037
∆G = ∆Go’ + RTln[P]/[S]
∆G = +13.9 kJmol-1 + (8.315 Jmol-1K-1 x 310 K) x ln[83 x10-6]/[5 x 10-3][1 x 10-3]
∆G = +13.9 kJmol-1 + (2.578 kJmol-1) x 2.81
∆G = +13.9 + 7.2 = +21.1 kJmol-1
(intracellular conditions are even more unfavorable than standard conditions
for the reaction to proceed as desired)
Q: How to drive glucose phosphorylation forward despite large positive ∆G?
A: Couple to much more favorable reaction (larger negative ∆G) such as to
the hydrolysis of ATP!!
∆Go’ or ∆G (kJmol-1)
ATP
+
H2O
Î
ADP
+
Pi
-30.5
-45.9
Keq
2.2x105
5.4x107
Intracellular [ATP]/[ADP][Pi] = 500 or higher (“phosphorylation potential” )
∆G = ∆Go’ + RTln[P]/[S]
∆G = - 30.5 + RTln 1/500
∆G = - 30.5 + (- 15.4) = - 45.9 kJmol-1
Combination (coupling) of both reactions via an enzyme (hexokinase):
∆Go’ or ∆G (kJmol-1)
Keq
Glc + Pi Î Glc-6-P
Intracellular conditions
+
H2O
+13.9
+21.1
3.7x10-3
2.8x10-4
ATP + H2O Î ADP
Intracellular conditions
+
Pi
-30.5
-45.9
2.2x105
5.4x107
-16.6
-24.8
8.1x102
1.5x104
Glc + ATP Î Glc-6-P
Intracellular conditions
+
ADP
Note:
Coupling of a reaction to ATP hydrolysis can shift its Keq up to 108 –fold !!
(2.8x10-4 Î 1.5x104)
Hexokinase (induced fit)
Binding of glucose (red)
The 1. Reaction of Glycolysis
Example of a phosphoryl transfer or kinase reaction
CHO
H
HO
OH
Glucose (Glc)
NH2
ATP
H
H
OH
H
OH
N
N
O
-O P
O
-
O
Enzyme
N
O
O
P O
P O
-
O
O
O
-
H
CH2O H
N
OH
H
OH
Electrophile
(P)
Nucleophile
(-OH)
H+
p. 26
CHO
NH2
H
OH
HO
N
N
H
O
H
OH
H
-O P
O
O
P O
P O
-
-
N
N
O
O-
O
O
OH
O
H
CH2O-
OH
H
OH
CHO
NH2
H
HO
Glc-6-P
OH
ADP
H
O-
H
H
OH
OH
CH2O
O
P
O-
OP
N
O
O
P O
N
O
OH
O
N
N
OH
H
OH
O-
p. 26
(Liver)
Aerobic Glycolysis (Overview)
Fischer projection-open chain
CHO
H
CHO
OH
H
1
HO
CH2OH
2
H
HO
H
OH
H
OH
ATP
ADP
3
HO
H
H
OH
H
OH
H
OH
H
OH
ATP
ADP
5
H
6
CH2OPO32-
CH2OPO32-
4
OH
+
OH
4 HC
CH2OPO32-
H
Haworth
projection
GLC
Ring
form
GLC-6-P
CH2OPO32-
CH2OH
OH
H
OH
OH
H
2
H
OH
ATP
H
H
OH
ADP
ATP
HPO42NAD+
6
OH
OH
G-3-P
OH
OH
H
3
OH
O
CH2OPO32-
O
H
OH
H
OH
O3POH2C
5
5
6 CH OPO 22
3
F-1,6-bisP
CH2OH
O
OH
H
OH
1
OH
O3POH2C
O
H
O
H
F-6-P
DHAP
3 CH2OH
H
4
H
O
O
3
HO
H
CH2OH
2
2
O
OH
1 CH2OPO32-
1 CH2OPO32-
H
ADP
NADH
+ H+
2 Molecules
-O
-
C
O
-O
O
C
O
10
CH3
PYR
C
OPO32-
ATP
O
ADP
CH2
PEP
H
H2O
C
O
8
9
O
OPO32-
-O
OPO32-
CH2OH
2-PGA
7
H
H
OH
CH2OPO32-
3-PGA
ATP
ADP
3,4
2,5
1,6
C
O
OH
CH2OPO32-
1,3 bisPGA
p. 25
Fischer projection-open chain
CHO
H
OH
H
1
HO
CH2OH
CHO
2
H
H
OH
H
OH
CH2OH
HO
ATP
O
OH
ADP
HO
H
H
H
OH
H
OH
H
OH
H
OH
CH2OPO32-
CH2OPO32-
Haworth
projection
Ring
form
GLC
GLC-6-P
Isomerization
F-6-P
Reaction 2:
Phosphogluco isomerase or Glucose-6-P ketolisomerase (see p 24)
p. 25
CH2OH
1 CH2OPO322
O
3
HO
1 CH2OPO32-
H
H
OH
H
OH
HO
ATP
ADP
H
H
3
4
5
6
CH2OPO32-
2
O
O
4
3 CH2OH
H
OH
+
OH
4 HC
CH2OPO32-
DHAP
H
5
5
O
OH
G-3-P
Phosphorylation
F-6-P
F-1,6-bisP
6 CH OPO 22
3
Reaction 3:
Phosphofructokinase-1 (PFK-1) or
ATP:Fructose-6-P 1-phosphotransferase
p. 25
CH2OH
1 CH2OPO322
O
3
HO
H
H
OH
H
OH
CH2OPO32-
F-6-P
p. 25
1 CH2OPO32-
HO
ATP
ADP
H
H
3
4
5
6
2
O
O
4
3 CH2OH
H
OH
Cleavage
F-1,6-bisP
H
5
+
4 HC
OH
CH2OPO32-
DHAP
5
O
OH
G-3-P
6 CH OPO 22
3
Reaction 4:
Aldolase or
Fructose-1,6-BisP glyceraldehyde-3-P lyase
Aldol Cleavage in Glycolysis
Requirements for cleavage: C-OH must be β to carbonyl carbon
R1
R1
H
C
O
C
R5
O
+
R2
C
R5
C
R3
O
R2
C
R3
C
O
R4
H
H
R4
R1
R2
C
O
C
R3
H
p. 27
Aldol Cleavage in Glycolysis
(Reaction No. 4)
CH2OP
βC
HO
H
α
O
C
H
C
O
Rest
HO
CH2OP
C
O
C
H
H
H
H
C
Rest
O
Reverse Reaction (Aldol Condensation)
Condensation
Requirements for condensation: H on C that is α to carbonyl carbon on substrate 1 (C-H acidic)
and need for carbonyl group on substrate 2
Resonance stabilized
Substrate 1
R1
R2
H
C
O
C
R3
R1
R1
R2
C
O
C
R3
R2
C
O
C
R3
H
R1
H
R5
C
C
O
R2
C
R3
R5
C
OH
O
R4
Substrate 2
R4
p. 27
CH2OH
1 CH2OPO322
O
3
HO
H
H
OH
H
OH
CH2OPO32-
F-6-P
1 CH2OPO32-
HO
ATP
ADP
H
H
3
4
5
6
2
O
O
4
3 CH2OH
H
OH
+
OH
4 HC
CH2OPO32-
F-1,6-bisP
DHAP
H
5
5
O
OH
G-3-P
6 CH OPO 22
3
Aldolase Reaction
p. 25
R
P
Aldolase
∆Go’= + 23.9 kJ/mol
∆G = - 0.23 kJ/mol
∆Go’= - 14.2 kJ/mol
∆G = - 18.8 kJ/mol
PFK-1
R
Common
In termediate
P
Aldolase
∆Go’= + 23.9 kJ/mol
∆G = - 0.23 kJ/mol
∆Go’= - 14.2 kJ/mol
∆G = - 18.8 kJ/mol
Phosphogluco isomerase
∆Go’= + 1.7 kJ/mol
∆G = - 2.9 kJ/mol
Hexokinase
PFK-1
R
Common
In termediate
∆Go’= - 16.6 kJ/mol
∆G = - 24.8 kJ/mol
P
Aldolase
∆Go’= + 23.9 kJ/mol
∆G = - 0.23 kJ/mol
∆Go’= - 16.6 kJ/mol
∆G = - 24.8 kJ/mol
∆Go’= - 14.2 kJ/mol
∆G = - 18.8 kJ/mol
Hexokinase
PFK-1
R
Common
In termediate
P
Aldolase
∆Go’= + 23.9 kJ/mol
∆G = - 0.23 kJ/mol
“ripple effect”
Pyruvate
Kinase
∆Go’= - 31.7 kJ/mol
∆G = - 23.0 kJ/mol
CH2OH
1 CH2OPO322
O
3
HO
H
H
OH
H
OH
CH2OPO32-
F-6-P
1 CH2OPO32-
HO
ATP
ADP
H
H
3
4
5
6
2
O
O
4
3 CH2OH
H
OH
+
OH
4 HC
CH2OPO32-
F-1,6-bisP
DHAP
H
5
5
O
OH
G-3-P
6 CH OPO 22
3
Reaction 5:
Triose phosphate isomerase (ketolisomerase)
p. 25
4 HC
H
5
O
OH
G-3-P
Reaction 6: The Big Deal!
6 CH OPO 22
3
23
HPO42+
NAD
6
Glyceraldehyde-3-P dehydrogenase
or
NADH
+ H+
Glyceraldehyde-3-P NAD+ oxidoreductase
(phosphorylating)
OPO32-
H
3,4
2,5
1,6
C
O
OH
CH2OPO32-
1,3 bisPGA
p. 25
NAD(P)H: Safe and Soluble Carrier of “Hydrogen”
H
2H
O
C
H+
Hydride acceptor/donor
N
H O
C
NH2
(Nicotinamide)
+
H
NH2
..
N
R
O
-O
P
O
O
NADH or NADPH
H
H
OH
H
OH
H
NH2
N
N
O
N
O
P
O
N
O
H
O-
H
H
H
OH
OX
NAD+ or NADP+
p. 28
∆G/∆E Calculations on 6. Reaction in Glycolysis
Summary of Chalk Board Calculations
Glyceraldehyde-3-P + Pi + NAD+ Î 1,3-Bisphoshoglycerate + NADH + H+
Can be formally written as two reactions (coupled by enzyme):
I. Glyceraldehyde-3-P + H2O + NAD+ Î 3-Phoshoglycerate + NADH + H+
II. 3-Phoshoglycerate + Pi Î 1,3-Bisphoshoglycerate + H2O
Compare ∆Go’ of both reactions
I. Glyceraldehyde-3-P + H2O + NAD+ Î 3-Phoshoglycerate + NADH + H+
Similar to oxidation of acetaldehyde to acetate (see Table 3, reactions 3 and 12)
Acetaldehyde + H2O + NAD+ Î Acetate + NADH + H+
∆Go’ = -nF∆Eo
∆Eo = EoOxidant – EoReductant
∆Eo = EoNAD+ – EoAcetaldehyde
∆Eo = - 0.32V – (-0.58V)
∆Eo = + 0.26V
∆Go’ = - 2 (electrons) x 96.5 kJmol-1V-1 x 0.26V
∆Go’ = - 50.2 kJmol-1
II. 3-Phoshoglycerate + Pi Î 1,3-Bisphoshoglycerate + H2O
See Table 4 for ∆Go’ of
Hydrolysis of 1,3-Bisphosphoglycerate (∆Go’ = - 49.6 kJmol-1)
or
Formation of 1,3-Bisphosphoglycerate (∆Go’ = + 49.6 kJmol-1)
Conclusion:
In a first approximation (because we are only looking at ∆Go’ values),
the oxidation of glyceraldehyde-3-P to 3-phosphoglycerate yields about
the same amount of energy (-50.2 kJmol-1) as is required to produce
1,3-bisphosphoglycerate from 3-phosphoglycerate and Pi (+49.6 kJmol-1).
Again, these two reactions do not occur in isolation but are coupled or
combined by the enzyme Glyceraldehyde-3-P Dehydrogenase.
Therefore, the ∆Go’ (and in fact ∆G) of the overall reaction is close to zero.