1 - 1 Lec #2
Carbohydrate Catabolism for ATP Generation
lactate
anaerobic
Glycolysis
2x pyruvate
Glucose
Gluconeogenesis
ATP
Oxidative
phosphorylation
NADH
FADH2
ethanol
acetylCoA
aerobic
TCA Cycle
The course can be divided roughly into two sections: degradation (usually coupled to
conversion of released energy into ATP) and biosynthesis.
We will begin with a review of the core of metabolism that was touched on at the end of
2360: glycolysis, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation
involving the electron transport chain (ETC).
As will become evident as we progress through the various sections, virtually all of
metabolism is linked back to this core pathway and can easily be thought of as branches
leading from or to the core.
It is important to realize therefore that, while we often asign the role of ATP generation to
this section, it is equally important for producing many of the intermediates required in
biosynthetic pathways and also for metabolizing products from other degradative
pathways.
Of course, remember: ΔG'o = -RTlnK'eq and ΔG'o = -nFΔE'o
1. Glycolysis and Gluconeogenesis
The term "glycolysis" literally means the breakdown of sugar, but has come to be used
to refer specifically to the breakdown of glucose to pyruvate.
The term "gluconeogenesis" means literally the birth or generation of glucose and has
come to refer to the reversal of glycolysis involving a few specific enzymes in addition to
those in the glycolysis pathway.
As with many pathways, the first step of glycolysis catalyzed by hexokinase is
irreversible, and commits the carbohydrate to the degradative pathway. This also
requires a separate enzyme to reverse the process, glucose-6-phosphatase.
1-2
1
ATP
ADP + H+
2
HOH2C
Hexokinase
G'o=-16.7 kJ/mol
O
HO
O3POH2C
HO
OH
O
HO
HO
OH
OH
Glucose
OH
Glucose-6-phosphate
(Glc-6-P)
Pi Glucose-6-phosphatase H2O
G'o=-13.8 kJ/mol
2
2
2
O3POH2C
O
HO
CH2OPO3
Phosphoglucose isomerase
G'o=+1.7 kJ/mol
CH2OH
OH
O
HO
OH
OH
Fructose-6-phosphate
(Frc-6-P)
Glc-6-P
3
ATP
ADP + H+
2
2
CH2OPO3
CH2OH
OH
O
OH
OH
OH
OH
CH2OPO3
Phosphofructokinase
G'o=-16.7 kJ/mol
Fructose-1,6-bisphosphatase
G'o=-13.8 kJ/mol
2
CH2OPO3
OH
O
OH
OH
Fructose-1,6-bisphosphate
(Frc-1,6-bisP)
Frc-6-P
Pi
H2O
Phosphofructokinase is also an irreversible reaction in vivo necessitating the need for a
separate enzyme to reverse the process for gluconeogenesis.
These two enzymes make up the site at which the glycolysis pathway is regulated, and
the key concept underlying control is energy levels. The reaction progressing to the
right (energy release) occurs under conditions of low energy, while the reaction to the
left (glucose synthesis for energy storage) occurs under conditions of high energy.
1-3
The presence of high concentrations of ATP and citrate in the cell signal a high energy situation
where more energy is not needed and the breakdown of glucose to make more can be stopped.
At the same time that energy generation is stopped, excess energy can be stored in the form of
glucose and glycogen. This is accomplished in part by ATP and citrate inhibiting
phosphofructokinase and activating fructose-1,6-bisphosphatase.
The presence of high concentrations of AMP and ADP in the cell signal a low energy situation
where more energy is needed and where there is no excess energy to store as glucose or
glycogen. This is accomplished in part by AMP and ADP activating phosphofrutokinase and
inhibiting fructose-1,6-bisphosphatase.
feedback
energy
storage
Glycogen
Phosphofructokinase
Glucose-6-P
Frc-6-P
ATP
citrate
AMP
ADP
Frc-1,6-P
pyruvate
ATP
citrate
Fructose-1,6-bisphosphatase
energy
release
AMP/ADP
This is accomplished by both enzymes being allosteric and capable of responding to both
activators and inhibitors. The example of phosphofructokinase responding to [fructose-6phosphate] illustrates this.
I
I = inhibitor
A = activator
A
Velocity
T-state
R-state
+ Activator
(AMP / ADP)
+ Inhibitor
(ATP / citrate)
[Fructose-6-phosphate]
2
4
CH2OPO3
2
2
CH2OPO3
1-4
CH2OPO3
2
CH2OPO3
OH
O
O
O
C
C
CH2OH
OH
HO
OH
Frc-1,6-bisP
C
H
H
C
OH
H
C
OH
Aldolase
o
∆G' =+23.8 kJ/mol
Dihydroxyacetone phosphate
(DHA-P)
O
2
H
C
H
C
CH2OPO3
OH
2
CH2OPO3
With a Keq = 9 x 10-5, this is not a "favourable" reaction and it
goes to completion in the direction of glycolysis only because
subsequent reactions remove the products and displace the
equilibrium. This is referred to as "product pull". The
reaction is obviously favorable for gluconeogenesis.
Glyceraldehyde-3-phosphate
(Ga-3-P)
O
2
CH2OPO3
5
O
H
C
H
C
C
CH2OH
DHA-P
Triose phosphate
isomerase
∆G'o=+7.5 kJ/mol
OH
2
CH2OPO3
Ga-3-P
At this point in the pathway, the "preparative phase" is finished. Glucose has been broken
down into two glyceraldehyde-3-phosphate molecules at the expense of two ATPs.
The "energy producing phase" follows in which the glyceraldehyde-3-phosphate is
converted to pyruvate with the production of both ATP and NADH.
preparative
Glucose
2ATP
energy producing
2 Pyruvate
2 Glyceraldehyde-3-P
4ATP
2NADH
1-5
6
O
H
C
H
C
Pi NAD+
OH
2
CH2OPO3
Ga-3-P
7
O
NADH + H+
Glyceraldehyde-3-phosphate
dehydrogenase
∆G'o=+6.2 kJ/mol
K'eq=0.08
2
H
OPO3
C
OH
2
CH2OPO3
1,3-bisphosphoglycerate
(1,3-bisPGA)
O
O
2
H
C
C
OPO3
C
OH
ADP
ATP
3-Phosphoglycerate kinase
2
CH2OPO3
∆G'o=-18.8 kJ/mol
K'eq=2 x 103
1,3-bisPGA
H
C
O
C
OH
2
CH2OPO3
3-phosphoglycerate
(3-PGA)
8
CO2
H
C
CO2
2
OH
Phosphoglycerate mutase
2
CH2OPO3
∆G'o=+4.2 kJ/mol
K'eq=0.2
3-PGA
H
C
OPO3
CH2OH
2-phosphoglycerate
(2-PGA)
9
H2O
CO2
CO2
2
H
C
OPO3
CH2OH
2-PGA
2
Enolase
∆G'o=+1.8 kJ/mol
K'eq=0.3
C
OPO3
CH2
phosphoenolpyruvate
(PEP)
1-6
10
ADP + H+
ATP
CO2
C
CO2
2
OPO3
C
Pyruvate kinase
ΔG'o=-31.4 kJ/mol
CH2
O
CH3
Pyruvate
PEP
The pyruvate kinase reaction is irreversible in vivo and to reverse the reaction for
gluconeogenesis requires two enzymatic steps.
CO2 ATP + H2O
CO2
C
ADP + Pi
O
C
Pyruvate carboxylase
biotin
CH3
GTP
CO2
O
GDP CO2
PEP carboxy kinase
H2C
Pyruvate
CO2
C
2
OPO3
CH2
PEP
CO2
Oxaloacetate
(OAA)
Lec #3
Summary
Glycolysis to release energy
Glc
Glc-6-P
Frc-6-P
Frc-1,6bisP
Gluconeogenesis to store energy
NAD+
Pyruvate
PEP
OAA
NADH
What happens next depends on whether or not oxygen is
present and also the organism, but in all cases NADH has
to be converted back to NAD+ so that the breakdown of
glucose can continue.
1-7
Anaerobic (low O2)
When oxygen levels are low, oxidative phosphorylation cannot take place and it is necessary
to oxidize NADH back to NAD+ enzymatically in order to keep glycolysis going. This can be
accomplished in a number of ways and the two that are most familiar occur in muscle tissue
and yeast.
Anaerobic in muscle
NADH +H+
CO2
C
NAD+
Lactate will accumulate in
muscle when insufficient
oxygen is transported to the
tissue.
CO2
O
HC
OH
Lactate dehydrogenase
CH3
G'o=-25.1 kJ/mol
A return to normal levels of
oxygen allows the lactate to be
reconverted to pyruvate for
further metabolism.
CH3
Pyruvate
Lactate
Anaerobic in yeast
CO2
CO2
O
NADH +H+
NAD+
OH
H
C
C
O
CH3
Pyruvate
Pyruvate decarboxylase
Thiamine pyrophosphate
(TPP)
CH2
Alcohol dehydrogenase
CH3
CH3
Acetaldehyde
Ethanol
The decarboxylation in the first step is irreversible and gives rise to CO2
evolution (bubbling) during fermentation to produce alcohol.
Aerobic (normal O2)
When oxygen levels are normal, oxidative phosphorylation can occur to regenerate NAD+ and
pyruvate can be metabolized more completely generating more NADH.
an oxidative decarboxylation
CoASH + H+ NAD+
CO2
C
NADH + H+ CO2
O
CH3
Pyruvate
O
C
Pyruvate dehydrogenase
complex
G'o=-33.4 kJ/mol
Keq'=7.6 x 105
TPP
Lipoic acid
FAD
S-CoA
CH3
Acetyl CoA
1-8
Regulation of pyruvate dehydrogenase
Pyr deH2ase
(active)
Pi
1. High energy signals: NADH, ATP
and AcCoA inhibit directly.
2. Low energy signals: NAD+, CoA
and AMP activate.
3. NADH also activates protein kinase
leading to inactivation.
ATP
Protein
phosphatase
Protein
kinase
H2O
ADP
Pyr deH2ase-P
(inactive)
Mechanism of pyruvate dehydrogenase
Pyruvate dehydogenase requires four cofactors (thiamine pyrophosphate, lipoic acid, FAD and
NAD+) in addition to coenzyme A. We will first look at the structures of the coenzymes and
then outline the mechanism. Similarities to the mechanism of pyruvate decarboxylase, which
also uses TPP, will be highlighted. (**In the future, check out α-ketoglutarate dehydrogenase,
α-ketoacyl dehydrogenase and α-ketoisovalerate dehydrogenase.**)
Thiamine pyrophosphate
NH2
CH3
Active portion that you are responsible for
H2
C
N
N
CH2
Cl
C
N
H3C
H
O
O
H2C
S
O
O
P
P
O
O
O
N
HCl
C
S
Can form a stable carbanion.
Lipoic acid (lipoate)
S
S
CH
H2C
C
H2
H2
C
CH2
C
H2
Active portion that you are responsible for
CO2
C
H2
Lipoate Is covalently attached to the
enzyme through an amide bond with a
lysine.
1-9
Pyruvate dehydrogenase is actually a multimeric complex of as many as 12 subunits some of
which have a discrete enzymatic activity. However, we will not delineate the various activities and
instead focus on the overall "pyruvate dehydrogenase" reaction.
At the same time we will be looking at the pyruvate decarboxylase reaction mechanism.
PdH = pyruvate dehydrogenase
PdC = pyruvate decarboxylase
And in the first stages of the mechanism, both PdH and PdC will be designated as E where both
utilize TPP in a similar reaction that decarboxylates pyruvate.
E
E
CO2
O
O
N
H+
C
N
C
S
O
S
C
C
TPP carbanion
C
HO
CH3
CH3
Pyruvate
CO2
E
N
The pathway followed from
this stage is enzyme specific.
C
N
C
S
C
HO
PdC
C
C
HO
C
HO
CH3
Acetol-TPP complex
-a 2-carbon fragment
CH3
N
S
H3C
C
S
H+
C
CH3
S
PdH
N
H+
E
HO
S
S
CH
H2C
C
H2
PdH
1 - 10
PdH
PdC
N
N
H 3C
C
H
C
S
C
O
HS
O
CH3
S
CH
H
H
PdC
H
+
H
S
C
H 2C
C
H2
PdH
N
+
C
C
CH3
PdH
N
CH3
H
S
C
C
O
O
Acetaldehyde
S
HS
S
PdH
CH
H 2C
C
H2
Acetyl-dihydrolipoyl-PdH
CoASH
CH3
C
S-CoA
O
Acetyl-CoA
S
HS
S
SH
CH
H 2C
C
H2
PdH
CH
H 2C
FADH2
Lipoyl-PdH
FAD
PdH
C
H2
Dihydrolipoyl-PdH
NAD+
NADH + H+
At a minimum,therefore, the pyruvate dehydrogenase complex harbours a dihydrolipoate
transacetylase, a dihydrolipoate dehydrogenase, NADH-FADH2 oxidoreductase, and pyruvate
decarboxylase activities all under the name pyruvate dehydrogenase.
This leads directly to the Tricarboxylic Acid (TCA) Cycle.
1 - 11
2. TCA Cycle
* CO2
1
CO2
O
H
C
O
*
H 2C
CH2
C
CoASH + H+
H2O
+
*
SCoA
H
Acetyl CoA
CO2
Citrate synthase
G'o=-32.2 kJ/mol
K'eq=3 x 105
* CH2
HO
C
CO2
CH2
CO2
OAA
Citrate
2
HO
* CO2
* CO2
* CO2
* CH2
* CH2
* CH2
C
CO2
CH2
Aconitase
G'o=+6.3 kJ/mol
K'eq=0.08
C
H
HC
CO2
C
OH
CH
CO2
CO2
H 2O
CO2
Isocitrate
Citrate
CO2
cis-Aconitate
reaction intermediate
3
* CO2
* CO2
NAD+ + H+ NADH+H+ CO2
* CH2
H
C
HC
CO2
OH
CO2
Isocitrate
Isocitrate dehydrogenase
G'o=-20.9 kJ/mol
K'eq=4.8 x 103
* CH2
CH2
C
O
CO2
-ketoglutarate
In some organisms, this is considered to be the slow or rate determining step in the TCA
cycle. As such, its turn over rate determines the overall rate of the TCA Cycle.
Significantly, isocitrate dehydrogenase is an allosteric enzyme that is activated by ADP
(low energy signal) and inhibited by ATP and NADH (high energy signals).
Regulation at this site also influences the glycolysis pathway because inhibition results in
a build up of citrate which affects the phosphofructokinase / fructose-1,6-bisphosphatase
control site.
1 - 12
*
* CO2
4
+
CoASH + H
NAD+
* CH2
CH2
C
NADH+H+
* CH2
-Ketoglutarate dehydrogenase
O
CO2
-ketoglutarate
CO2
CO2
G'o=-33.4 kJ/mol
K'eq=7.6 x 105
TPP
Lipoic acid
FAD
Same mechanism as
described for pyruvate
dehydrogenase
CH2
C
O
S-COA
Succinyl CoA
Both of the decarboxylation steps are irreversible because of the evolution of CO2 and
lack of a system for adding it back (biotin + ATP).
Also note that while two carbons have been released as CO2, they are not the same two
carbons that entered as acetylCoA in this particular round of the TCA cycle.
5
* CO2
CoASH
GDP + Pi
* CH2
CO2
GTP
At this stage, it is no
longer possible to
differentiate the two
carbons that entered the
TCA cycle in this round.
CH2
CH2
C
SuccinylCoA synthetase
CH2
G'o=-2.9 kJ/mol
K'eq=3.7
O
CO2
S-COA
Succinyl CoA
Succinate
6
CO2
FAD
FADH2
H
C
CH2
CH2
CO2
Succinate
CO2
Succinate dehydrogenase
(Complex 2 of ETC)
G'o= 0 kJ/mol
K'eq=1
C
O 2C
H
Fumarate
1 - 13
7
H
CO2
H2O
CO2
C
H
C
H
O2C
Fumarate
8
C
CH2
CO2
OH
CH2
CO2
Malate
CO2
H
Fumarase
ΔG'o= 0 kJ/mol
K'eq=1
C
NAD+
NADH + H+ CO2
C
OH
Malate dehydrogenase
ΔG'o=+29.7 kJ/mol
K'eq=1.3 x 10-5
CH2
CO2
This is obviously not a
favourable reaction but
O "product pull" from citrate
synthase pulls the reaction
to completion by displacing
the equilibrium towards
OAA.
OAA
Malate
Lec #4
Summary
(including the Electron Transport Chain, not yet covered in detail)
Glycolysis : Glucose + O2
TCA cycle: 2 Pyruvate + 2 H+ + 5O2
2 Pyruvate + 2 H2O +2 H+
6 CO2 + 4 H2O
______________________________________________________
Overall:
Glucose + 6 O2
6 CO2 + 6 H2O
ΔGo' = -2868 kJ/mol
!! NO ATP or NADH
produced !!
The object of the following sections is to demonstrate how the overall reactions can be
derived from the individual reactions of the pathways.
The key to generating the overall reaction is the final steps (11 and 12 in the glycolysis
scheme and 10, 11 and 12 in the TCA cycle scheme) that are not actually part of the
pathways.
The reason they are included is to return the ATP and NADH/FADH2 which do not
appear in the overall process to ADP and NAD+/FAD.
1 - 14
Glycolysis breakdown
1. Glucose + ATP
Glc-6-P + ADP + H+
2. Glc-6-P
Frc-6-P
3. Frc-6-P + ATP
Frc-1,6-bisP +ADP + H+
4. Frc-1,6-bisP
Ga-3-P + DHA-P
5. DHA-P
Ga-3-P
6. 2 Ga-3-P + 2 Pi + 2 NAD+
2 1,3-bisPGA + 2 NADH + 2 H+
7. 2 1,3-bisPGA + 2 ADP
2 3-PGA + 2 ATP
8. 2 3-PGA
2 2-PGA
9. 2 2-PGA
2 PEP + 2 H2O
10. 2 PEP + 2 ADP +2 H+
2 Pyruvate + 2 ATP
11. 2ATP + 2 H2O
2 ADP + 2 Pi + 2H+
12. 2 NADH +2 H+ + O2
2 NAD+ + 2H2O
__________________________________________________________
Glucose + O2
2 Pyruvate + 2 H2O + 2H+
1 - 15
TCA cycle breakdown
(for 1 pyruvate)
1. Pyr + H+ + CoASH + NAD+
AcCoA + CO2 +NADH + H+
2. AcCoA + OAA + H2O
Citrate + CoASH + H+
3. Citrate
Isocitrate
4. Isocitrate + NAD+ + H+
-KG + CO2 + NADH + H+
5. -KG + NAD+ + CoASH + H+
Succ-CoA + CO2 + NADH + H+
6. Succ-CoA + GDP + Pi
Succ + CoASH + GTP
7. Succ + FAD
Fum + FADH2
8. Fum + H2O
Mal
9. Mal + NAD+
OAA + NADH + H+
10 4 NADH + 4 H+ + 2 O2
4 NAD+ + 4 H2O
11. FADH2 + 0.5 O2
FAD + H2O
12. GTP + H2O
GDP + Pi + H+
_____________________________________________________________
Pyr + 2.5 O2 + 3 H2O + 3 H+
3 CO2 + 5 H2O + 2 H+
Pyr + 2.5 O2 + H+
3 CO2 +2 H2O
or
or for 2 pyruvate (from 1 glucose)
2 Pyr + 5 O2 + 2 H+
6 CO2 + 4 H2O
1 - 16
3. Balancing or Anaplerotic Reactions
Many intermediates in both the glycolysis pathway and the TCA cycle are used in other
pathways as starting materials or are generated in other pathways as degradation products.
In order to keep the pool sizes of the intermediates in these two core pathways in
synchrony, a number of balancing or anaplerotic reactions have evolved.
If one focuses just on the basic reactions of the two pathways, reflection on the following
questions will illustrate why it is important to have reactions to link them and allow the
interconversion of intermediates.
1. If a cell is growing on a TCA cycle intermediate such as succinate as the sole carbon
source:
a) how are glucose and other carbohydrates needed for cell wall and membrane synthesis
generated; and
b) how is AcCoA generated such that energy can be produced from the TCA cycle?
2. If a cell is growing on pyruvate or lactate as the sole carbon source:
a) how are TCA cycle intermediates produced, and
b) how is glucose produced (the answer to this is obviously gluconeogenesis)?
3. Finally, if a cell is growing on glucose as the sole carbon source, how are TCA cycle
intermediates generated?
The answers lie in four reactions, two of which we have already dealt with in
gluconeogenesis (the reversal of glycolysis).
1
CO2 ATP +H2O
CO2
C
ADP +Pi
CO2
O
This is both anaplerotic
and gluconeogenetic.
C
O
Pyruvate carboxylase
∆G' =+2.0 kJ/mol
biotin
o
CH3
Pyruvate
Activated by AcCoA.
H 2C
CO2
OAA
2
CO2
C
O
GTP
GDP CO2
CO2
2
C
H 2C
CO2
OAA
PEP carboxy kinase
∆G'o=-2.8 kJ/mol
CH2
PEP
OPO3
This is both anaplerotic
and gluconeogenetic.
1 - 17
3
CO2
CO2
HCO3-
2
C
Pi
C
O
This is anaplerotic and
has a role in C4 plants.
OPO3
H 2C
PEP carboxylase
CH2
∆G'o=-28.6 kJ/mol
PEP
CO2
OAA
4
NADPH + H+
NADP+
CO2
CO2
CO2
HC
C
OH
O
H 2C
Malic enzyme
CH3
∆G'o=-1.7 kJ/mol
This is anaplerotic and
has a role in C4 plants.
CO2
Pyruvate
Malate
Summary
CO2
Glucose
AcCoA
Pyruvate
PEP
Citrate
1
2
Isocitrate
3
OAA
4
Malate
2 CO2
Fum
Succ
SuccCoA
1 - 18
4. Pentose Phosphate Pathway
(or hexose monophosphate shunt or phosphogluconate pathway)
Basically this is an alternate pathway for glucose degradation found particularly in animal cells
where NADPH is required. Fat cells are a prime example.
As with many degradative pathways, it can be broken down into:
(a) an energy producing phase:
C6
C5 + CO2
2NADPH
(b) a rearrangement phase:
C5
C6
And to provide enough carbons for the rearrangement phase to take place, it is necessary to work
with multiple molecules with the lowest common denominator being 6 C5 and 5 C6 which results in:
6 C6
6 C5
12 NADPH
5 C6
6 CO2
This is roughly equivalent to 30 ATP (2.5 ATP / NADPH) suggesting that the efficiency is similar to
that of glycolysis / TCA cycle (which isn't that surprising since most ATP is derived from the ETC.
Energy Producing Phase
1
2
O3POH2C
NADP+
O
HO
NADPH + H+
2
O3POH2C
O
HO
HO
OH
OH
Glucose-6-phosphate
(Glc-6-P)
Glucose-6-phosphate
dehydrogenase
∆G'o=-0.4 kJ/mol
HO
OH
O
Gluconolactone-6-phosphate
1 - 19
2
2
2
H2O
O3POH2C
O3POH2C
O
HO
Lactonase
HO
OH
O
HO
OH
∆G' =-20.5 kJ/mol
o
O
Gluconate-6-phosphate
Gluconolactone-6-phosphate
3
2
O
OH
HO
CO2
H 2C
O3POH2C
HC
OH
HO
OH
O
HO
NADP+
NADPH + H+ CO2
O
Gluconate-6-phosphate
HC
HC
OH
Gluconate-6-phosphate
dehydrogenase
HC
OH
HC
OH
OH
2
CH2OPO3
2
CH2OPO3
Ribulose-5-phosphate
Rearrangement Phase
It is easiest to follow the rearrangement phase by first considering a
summary of the organization which converts 6 C5 into 5 C6.
6 ribulose (C5)
1
xylulose C5
xylulose C5
ribose
C5
ribose
C5
xylulose C5
2
C7 +C3
3
4
C6 + C4
C6 + C3
5
2
C7 +C3
O
CH
HO
OH
C
OH
3
C6 + C4
4
C6
C6 + C3
xylulose C5
Basically, there are 5 "steps" some of which involve more than one enzymatic reaction.
H 2C
OH
1 - 20
Lec #5
1
C
Ribulose phosphate
3-epimerase
O
HC
OH
HC
OH
Ribose phosphate
isomerase
CH2OH
C
HO
O
K'eq = 0.8
CH
HC
K'eq = 3
2
CH2OPO3
Ribulose-5-phosphate
OH
HC
O
HC
OH
HC
OH
HC
OH
2
CH2OPO3
2
CH2OPO3
Xylulose-5-phosphate
2
C5 + C5
Ribose-5-phosphate
C7 + C3
CH2OH
C
CH2OH
HC
O
C
HC
OH
HC
OH
HO
HO
O
+
CH
HC
OH
HC
2
O
HC
OH
HC
OH
HC
OH
CH2OPO3
HC
OH
Glyceraldehyde3-phosphate
TPP
+
2
OH
Ribose5-phosphate
C7 + C3
HC
Transketolase
CH2OPO3
Xylulose5-phosphate
3
CH
2
CH2OPO3
O
2
CH2OPO3
Sedoheptulose7-phosphate
C4 + C6
CH2OH
C
HO
O
CH
HC
HC
HC
HC
OH
+
OH
OH
2
CH2OPO3
Sedoheptulose7-phosphate
HC
CH2OH
HC
O
HC
OH
HC
OH
C
O
OH
Transaldolase
2
CH2OPO3
Glyceraldehyde3-phosphate
HO
2
+
O
CH
HC
OH
HC
OH
CH2OPO3
Erythrose-4phosphate
2
CH2OPO3
Fructose-6phosphate
1 - 21
4
C4 + C5
C6 + C3
CH2OH
CH2OH
HC
C
HC
O
OH
Transketolase
+
HC
HO
CH
OH
HC
2
HC
OH
+
HC
OH
HC
OH
HC
O
HC
OH
2
CH2OPO3
Glyceraldehyde3-phosphate
2
CH2OPO3
Xylulose5-phosphate
C3 + C3
O
CH
2
CH2OPO3
Erythrose-4phosphate
HC
HO
O
TPP
OH
CH2OPO3
5
C
O
Fructose-6phosphate
C6
CH2OH
Triose phosphate
isomerase
C
2
O
2
CH2OPO3
CH2OPO3
Glyceraldehyde3-phosphate
Dihydroxyacetone
phosphate
2
CH2OH
CH2OPO3
Aldolase
C
HO
H2O
O
C
Pi
HO
CH
HC
OH
HC
OH
Fructose-1,6bisphosphatase
CH
HC
OH
HC
OH
2
2
CH2OPO3
CH2OPO3
Fructose-1,6bisphosphate
In summary:
6 C6
6 CO2
6 C5
12 NADPH (energy yield)
5 C6
O
Fructose-6** All Frc-6-P phosphate
converted by:
Phosphogluco
isomerase
so cycle can
continue.**
Glc-6P
1 - 22
Mechanism of Transketolase
Transketolase requires thiamine pyrophosphate (like pyruvate dehydrogenase) and the following
mechanism should be compared to what happens in that enzymatic process.
The starting point is the stable carbanion of TPP which carries out a nucleophilic attack on the
carbonyl carbon of C5, C6 and C7 ketoses.
E
E
N
H+
CH2OH
N
C
HOH2C
S
TPP carbanion
HO
S
C
C
O
C
HO
CH
CH
R1
O
R1
H
O
E
H
N
C
HOH2C
R1
E
N
C
S
C
C
HO
O
H+
HOH2C
H
S
C
a 2-carbon fragment
OH bound to TPP
C
R2
E
N
HOH2C
C
E
S
N
C
H
O
CH2OH
CH
H
CH
O
HO
C
H+
can be C5, C6 or C7
CH
ketoses
O
H
C
R2
O
H
and
R1
R1
CH
CH2OH
and
HO
HO
R2
CH2OH
C
S
C
O
O
O
C
R2
C
R2
can be C3, C4 or C5 aldoses
5. Electron Transport Chain and Oxidative Phosphorylation
1 - 23
Glycolysis and the TCA cycle generate reduced electron carrier, NADH and FADH2 that must be
oxidized back to NAD+ and FAD in order for the pathways to continue to function.
Under aerobic conditions, this is achieved in the electron transport chain and the energy
released in the oxidation reactions is coupled to the phosphorylation of ADP to form ATP. This is
the process of oxidative phosphorylation.
Basically there is a series of oxidation-reduction reactions as the electrons are passed through a
number of intermediates in the cell membrane. In the following graph, the key intermediates that
shuttle electrons among the four complexes in the mitochondrial membrane that comprise the
electron transport chain are shown and the free energy change (calculated from the change in
standard reduction potentials) are indicated.
remember :
∆G'o = -n ∆E'o
= 96.5 kJ/v.mol (Faraday's const.)
-0.6
∆G'o = -RTlnK'eq
R = 8.3 J/mol.K (gas constant)
NADH
o
∆E'
=
0.36
v
-0.3
I
∆G'o = -69.5 kJ/mol
II
Succ
0.0
CoQ
∆E'o = 0.19 v
o
o
III
∆E' ~ 0.0 v
E'
∆G'o = -36.7 kJ/mol
∆G'o ~ 0 kJ/mol
Cyt c
+0.3
IV
+0.6
+0.9
∆E'o = 0.58 v
∆G'o = -111.6 kJ/mol
O2
The mitochondrial membrane can be broken down and fractionated into four complexes, labelled
I, II, III and IV, each made up of a large number of proteins, pigments and lipids. As shown in the
diagram each is capable carrying out a specific part of the electron transfer process. Complex I
transfers electrons from NADH to Coenzyme Q; complex III transfers electrons from CoQ to
cytochrome c; and complex IV transfers electrons from cytochrome c to molecular oxygen.
Complex II contains the succinate dehydrogenase activity with FAD and transfers electrons from
succinate to CoQ.
Some of the components of the complexes are known but not all. Therefore, we will focus mainly
on the overall picture and less on the individual components.
4 H+
4 H+
1 - 24
OUTSIDE
2 H+
Cytcred
Cytcox
CoQH2
I
IV
III
CoQ
CoQ
II
FAD
NADH + H+
+
NAD
INSIDE
Succ
+
Fum
1/2 O2 + 2H
H2O
Complex I contains FMN, Fe-S protein and 42 other proteins.
Complex II contains FAD and succinate dehydrogenase.
Complex III contains cytochromes b and c1 and 11 other proteins.
Complex IV contains cytochromes a and a3 and 13 other proteins.
The energy released in the oxidation-reduction reactions is used to "pump" protons across the
membrane from inside to outside creating a region of high proton concentration (low pH) and
positive charge on the outside and low proton concentration (high pH) and negative charge on
the inside.
An energized (entropically unfavorable or "unrandomized") state is created involving a proton
gradient and an electrical gradient across the membrane utilizing the oxidation energy.
It is the proton and electrical gradients of the energized state that are used to produce ATP and the
ATPase (also ATP synthase) effects the coupling of the energized state to ATP production. In
simple terms, the ATPase couples the flow of protons through the membrane to the phosphorylation
of ADP.
4 H+
OUTSIDE
+++++
The transfer of 4 protons through the ATPase
is coupled to the phosphorylation of one ADP
to ATP (ie. 4 H+ = 1 ATP).
ATPase
-------
ADP + Pi
INSIDE
ATP + H2O
This is a summary of the chemiosmotic
theory of oxidative phosphorylation as intially
proposed by Peter Mitchell.
1 - 25
In summary:
from Glycolysis/TCA
in ETC
1 NADH
1 FADH2 (succinate)
ATPase
10 H+ pumped
2.5 ATP
6 H+ pumped
1.5 ATP
NADH + H+
NAD+
2.5 ADP + 2.5 Pi + 2.5 H+
2.5 ATP + 2.5 H2O
H2O
1/2 O2
Succinate (FADH2)
Fumarate (FAD)
1.5 ADP + 1.5 Pi + 1.5 H+
1.5 ATP + 1.5 H2O
H2O
1/2 O2
Glucose
Lec #6
2 ATP
2 CO2
2 Pyruvate
2 NADH
4 ATP
(2 ATP net)
2 AcCoA
2 Citrate
2 NADH
2 NADH
2 OAA
2 NADH
Therefore:
1 Glucose
2 CO2
2 CO2
6 CO2
2 ATP
2 ATP (net)
2 GTP
2 ATP
2 FADH2
10 NADH
25 ATP
2 GTP
2 FADH2
3 ATP
__________________
Yield
32 ATP/glucose (x 30.5 kJ/mol ATP = 976 kJ/mol)
976/2868 x 100 = 34.0% efficient)
2 NADH
1 - 26
This general procedure can be used to determine the ATP yield realized from the breakdown of any
glycolysis or TCA cycle intermediate completely to CO2.
From a glycolysis intermediate:
fatty acids
amino acids
Ga3P
CO2
Pyruvate
NADH
2 ATP
AcCoA
Citrate
NADH
Therefore:
1 Ga-3-P
NADH
3 CO2
OAA start/finish
CO2
NADH
2 ATP
2 ATP
1 GTP
1 ATP
5 NADH
12.5 ATP
1 FADH2
1.5 ATP
_________________
Yield
17 ATP
CO2
NADH
FADH2
GTP
From TCA cycle intermediate:
*
Succ
Mal
FADH2
OAA
NADH + CO2
CO2
Common to all energy calculations
CO2
Pyr
NADH
AcCoA
ATP
Citrate
NADH
Therefore:
1 Succ
NADH
4 CO2
1 ATP
1 ATP
1 GTP
1 ATP
5 NADH
12.5 ATP
3 ATP
2 FADH2
_________________
Yield
17.5 ATP
* If Malic enzyme (Mal to Pyr) is used
the energy yield would be one ATP
less.
OAA start/finish
CO2
NADH
CO2
FADH2
GTP
NADH
1 - 27
Summary of Regulation
ATP
Citrate
-
AMP
ADP
+
NADH
activates
protein kinase
Glc
Frc-6-P
-
Frc-1,6-bisP
ATP
Citrate
+
AMP
ADP
-
AcCoA
Pyruvate ATP
NADH
CoA
AMP
AcCoA +
NAD+
AcCoA
High energy
molecules signal a
slow down of
glycolysis and the TCA
cycle and turn on
gluconeogenesis.
OAA
2. TCA Cycle
3. Anaplerotic reactions
4. Pentose phospate pathway
5. Electron transport chain / oxidative phosphorylation
6. Energy calculations
7. Regulation
+
Isocitrate
ATP
ADP
-KG
SuccCoA
1. Glycolysis and gluconeogenesis
SuccCoA
ATP
NADH
ADP
Citrate
Low energy
molecules signal an
increase in glycolysis
and the TCA cycle
6. Summary of Carbohydrate Catabolism
+
SuccCoA
NADH
-
+