carbohydrate catabolism for atp generationphosphofructokinase is also an irreversible reaction in...
TRANSCRIPT
Glucose 2x pyruvateGlycolysis
Gluconeogenesis
lactate
ethanol
anaerobic
acetylCoA
TCA Cycle
NADHFADH2
OxidativephosphorylationATP aerobic
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
Carbohydrate Catabolism for ATP Generation
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 - 1 Lec #2
OHOH2C
HO
OH OH
OO3POH2C
HO
OH OH
ATP ADP + H+
HexokinaseG'o=-16.7 kJ/mol
H2OPi Glucose-6-phosphataseG'o=-13.8 kJ/mol
Glucose Glucose-6-phosphate (Glc-6-P)
2
OO3POH2C
HO
OH OH
Glc-6-P
2
O
OH
OHCH2OPO3
2
OH
CH2OH
Fructose-6-phosphate (Frc-6-P)
Phosphoglucose isomeraseG'o=+1.7 kJ/mol
O
OH
OHCH2OPO3
2
OH
CH2OH
Frc-6-P
O
OH
OHCH2OPO3
2
OH
CH2OPO3
Fructose-1,6-bisphosphate (Frc-1,6-bisP)
ATP ADP + H+
PhosphofructokinaseG'o=-16.7 kJ/mol
H2OPi
Fructose-1,6-bisphosphataseG'o=-13.8 kJ/mol
2
1
2
3
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 - 2
HOHO
HO
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.
Frc-6-P Frc-1,6-P
Phosphofructokinase
Fructose-1,6-bisphosphatase
ATPcitrate
AMPADP
Glycogen
Glucose-6-P
citrateATPpyruvate
energystorage
energyrelease
This is accomplished by both enzymes being allosteric and capable of responding to both activators and inhibitors. The example of phosphofructokinase responding to [fructose-6-phosphate] illustrates this.
1 - 3
AMP/ADP
feedback
I = inhibitorA = activator
I A
T-state R-state
Velo
city
[Fructose-6-phosphate]
+ Activator(AMP / ADP)
+ Inhibitor(ATP / citrate)
O
OH
OHCH2OPO3
2
OH
CH2OPO3
Frc-1,6-bisP
2 CH2OPO3
CO
C HHO
C OHH
C OHH
CH2OPO3
CH2OPO3
CO
CH2OH
C
O
H
C OHH
CH2OPO3
DHA-P
Glyceraldehyde-3-phosphate (Ga-3-P)
Aldolase∆G'o=+23.8 kJ/mol
4
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.
5
CH2OPO3
CO
CH2OH
Dihydroxyacetone phosphate (DHA-P)
C
O
H
C OHH
CH2OPO3
Triose phosphate isomerase
Ga-3-P∆G'o=+7.5 kJ/mol
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.
Glucose 2 Glyceraldehyde-3-P 2 Pyruvate
2ATP 4ATP2NADH
preparative energy producing
2
2
2
2
2
2
1 - 4
C
O
H
C OHH
CH2OPO3
Ga-3-P
2
6
C
O
OPO3
C OHH
CH2OPO32
Glyceraldehyde-3-phosphate dehydrogenase
∆G'o=+6.2 kJ/mol K'eq=0.08
Pi NAD+ NADH + H+
1,3-bisphosphoglycerate (1,3-bisPGA)
7
C
O
OPO3
C OHH
CH2OPO32
1,3-bisPGA
C
O
O
C OHH
CH2OPO32
3-phosphoglycerate (3-PGA)
3-Phosphoglycerate kinase
∆G'o=-18.8 kJ/mol K'eq=2 x 103
ADP ATP
2
CO2
C OHH
CH2OPO32
3-PGA
8
CO2
C OPO3H
CH2OH
2
2-phosphoglycerate (2-PGA)
Phosphoglycerate mutase
∆G'o=+4.2 kJ/mol K'eq=0.2
CO2
C OPO3H
CH2OH
2
2-PGA
CO2
C OPO3
CH2
2
phosphoenolpyruvate (PEP)
9
Enolase
∆G'o=+1.8 kJ/mol K'eq=0.3
H2O
1 - 5
2
CO2
C O
CH3
PEP
CO2
C OPO3
CH2
2
Pyruvate
10
Pyruvate kinaseΔG'o=-31.4 kJ/mol
ADP + H+ ATP
The pyruvate kinase reaction is irreversible in vivo and to reverse the reaction for gluconeogenesis requires two enzymatic steps.
CO2
C O
CH3
PEP
CO2
C OPO3
CH2
2
Pyruvate
Pyruvate carboxylase biotin
ATP + H2O ADP + Pi
PEP carboxy kinase
GTP GDPCO2 CO2
C O
H2C
CO2
CO2
Glc Glc-6-P Frc-6-P Frc-1,6bisP PEP Pyruvate
OAA
Oxaloacetate (OAA)
Summary
Glycolysis to release energy
Gluconeogenesis to store energy
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 - 6
Lec #3
NADHNAD+
CO2
C O
CH3
Pyruvate
Anaerobic in muscle
CO2
HC OH
CH3
Lactate
Lactate dehydrogenase
G'o=-25.1 kJ/mol
NADH +H+ NAD+ Lactate will accumulate in muscle when insufficient oxygen is transported to the tissue.
A return to normal levels of oxygen allows the lactate to be reconverted to pyruvate for further metabolism.
Anaerobic in yeast
CO2
C O
CH3
Pyruvate
C
O
CH3
Acetaldehyde
Pyruvate decarboxylase
NADH +H+ NAD+
Thiamine pyrophosphate (TPP)
CO2
H
CH2
OH
CH3
Ethanol
Alcohol dehydrogenase
1 - 7
The decarboxylation in the first step is irreversible and gives rise to CO2 evolution (bubbling) during fermentation to produce alcohol.
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.
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.
CO2
C O
CH3
Pyruvate
C
O
CH3
Acetyl CoA
Pyruvate dehydrogenase complex
TPPLipoic acidFAD
CO2
S-CoA
NAD+ NADH + H+CoASH + H+
G'o=-33.4 kJ/mol Keq'=7.6 x 105
an oxidative decarboxylation
1 - 8Regulation of pyruvate dehydrogenase
Pyr deH2ase (active)
Pyr deH2ase-P (inactive)
ATP
ADP
Pi
Proteinkinase
Proteinphosphatase
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.H2O
Mechanism of pyruvate dehydrogenase
N
N
NH2
H3C
H2C
N
C S
CH3
H
CH2
H2C O
P O
O
OP
O
O
O
Cl
Active portion that you are responsible for
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
N
C S
HCl
Can form a stable carbanion.Lipoic acid (lipoate)
S
H2CCH2
CH
S
CH2
CH2
Lipoate Is covalently attached to the enzyme through an amide bond with a lysine.
Active portion that you are responsible for
H2C
CH2
CO2
1 - 9Pyruvate 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.
EN
C SCO2
CO
CH3
Pyruvate
TPP carbanion
H+
EN
C SC
CH3
C
HO
O
O
EN
C SC
CH3
HO
EN
C SC
CH3HOAcetol-TPP complex -a 2-carbon fragment
The pathway followed from this stage is enzyme specific.
N
C SC
CH3HO
N
C SC
H3C
HO
H+
S
H2CCH2
CH
S
H+
PdC
PdH
PdH
CO2
1 - 10
N
CS
C
CH3O
N
CS
C
H3C
O S
H2CCH2
CH
HS
H
H
N
CS
CCH3
O
H
Acetaldehyde
H
N
CSC
CH3
O S
H2CCH2
CH
HS
Acetyl-dihydrolipoyl-PdH
CoASH
Acetyl-CoA
C
CH3
OS-CoA
SH
H2CCH2
CH
HSS
H2CCH2
CH
S
Dihydrolipoyl-PdHLipoyl-PdH
FADFADH2
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.
H+
H+
PdH
PdH
PdH
PdH
PdHPdH
PdC
PdC
CO2
CO
CH2
CO2
SCoAC
O
H2C
Acetyl CoA
OAA
H
H+
CO2CHO
CH2
CO2
CH2
CO2
Citrate
Citrate synthaseG'o=-32.2 kJ/mol K'eq=3 x 105
CoASH + H+
* **
*1
2
1 - 11
CO2CHO
CH2
CO2
CH2
CO2
Citrate
*
*
CO2CH
HC
CO2
CH2
CO2
Isocitrate
*
*
CO2C
CH
CO2
CH2
CO2
cis-Aconitate
*
*
OH
Aconitase
G'o=+6.3 kJ/mol K'eq=0.08
H2O
CO2CH
HC
CO2
CH2
CO2
Isocitrate
*
*
OH
CH2
C
CO2
CH2
CO2
-ketoglutarate
*
*
O
CO2NAD+ + H+ NADH+H+
Isocitrate dehydrogenase
G'o=-20.9 kJ/mol K'eq=4.8 x 103
3
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.
H2O
2. TCA Cycle
reaction intermediate
4
CH2
C
CO2
CH2
CO2
-ketoglutarate
*
*
O
CH2
C
S-COA
CH2
CO2
Succinyl CoA
*
*
O
CO2NAD+ NADH+H+
-Ketoglutarate dehydrogenase
G'o=-33.4 kJ/mol K'eq=7.6 x 105
1 - 12
TPPLipoic acidFAD
Same mechanism as described for pyruvate dehydrogenase
CoASH + H+
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.
5CoASH
GDP + Pi GTP
SuccinylCoA synthetase
G'o=-2.9 kJ/mol K'eq=3.7
CH2
C
S-COA
CH2
CO2
Succinyl CoA
*
*
OCH2
CO2
CH2
CO2
Succinate
At this stage, it is no longer possible to differentiate the two carbons that entered the TCA cycle in this round.
6
CH2
CO2
CH2
CO2
Succinate
C
O2C
C
CO2
Fumarate
H
HSuccinate dehydrogenase (Complex 2 of ETC)
G'o= 0 kJ/mol K'eq=1
FADH2FAD
C
O2C
C
CO2
Fumarate
H
HFumaraseΔG'o= 0 kJ/mol K'eq=1
H2O1 - 13
CH2
CO2
C
CO2
H OH
Malate
7
8
CH2
CO2
C
CO2
H OH
Malate
CH2
CO2
C
CO2
O
OAA
Malate dehydrogenaseΔG'o=+29.7 kJ/mol K'eq=1.3 x 10-5
NAD+ NADH + H+ This is obviously not a favourable reaction but "product pull" from citrate synthase pulls the reaction to completion by displacing the equilibrium towards OAA.
Summary(including the Electron Transport Chain, not yet covered in detail)
Glycolysis : Glucose + O2 2 Pyruvate + 2 H2O +2 H+
TCA cycle: 2 Pyruvate + 2 H+ + 5O2 6 CO2 + 4 H2O
______________________________________________________
Overall: Glucose + 6 O2 6 CO2 + 6 H2O
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.
Lec #4
ΔGo' = -2868 kJ/mol!! NO ATP or NADH produced !!
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 - 14
Glycolysis breakdown
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+
or Pyr + 2.5 O2 + H+ 3 CO2 +2 H2O 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, andb) 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).
CO2
C O
CH3
PEP
CO2
C OPO3
CH2
2
Pyruvate
Pyruvate carboxylase
ATP +H2O ADP +Pi
PEP carboxy kinase
GTP GDP
CO2 CO2
C O
H2C
CO2
CO2
CO2
C O
H2C
CO2
1
OAA
This is both anaplerotic and gluconeogenetic.
Activated by AcCoA.
This is both anaplerotic and gluconeogenetic.
2
OAA
∆G'o=+2.0 kJ/mol biotin
∆G'o=-2.8 kJ/mol
PEP
CO2
C OPO3
CH2
2
PEP carboxylase
HCO3-
CO2
C O
H2C
CO2
This is anaplerotic and has a role in C4 plants.
3
1 - 17
Pi
OAA
∆G'o=-28.6 kJ/mol
Pyruvate
CO2
C O
CH3Malic enzyme
CO2
CO2
HC OH
H2C
CO2
This is anaplerotic and has a role in C4 plants.
4
Malate
∆G'o=-1.7 kJ/mol
NADPH + H+ NADP+
Glucose
PEP
PyruvateCitrate
Malate
Fum
Succ SuccCoA
OAA
AcCoA
Isocitrate
2 CO2
4
12
3
Summary
CO2
1 - 184. 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 5 C6 12 NADPH 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
OO3POH2C
HO
OH O
NADP+NADPH + H+
Glucose-6-phosphate dehydrogenase
∆G'o=-0.4 kJ/mol Gluconolactone-6-phosphate
2
1
OO3POH2C
HO
OH OH
Glucose-6-phosphate (Glc-6-P)
2
HO HO
1 - 19
OO3POH2C
HO
OH OLactonase
∆G'o=-20.5 kJ/mol
Gluconolactone-6-phosphate
2
2
OHO3POH2C
HO
OH O
2
O
H2O
3
OHO3POH2C
HO
OH O
2
O
CO2
HC OH
CHHO
HC OH
HC OH
CH2OPO32
NADP+NADPH + H+
Gluconate-6-phosphate dehydrogenase
H2C OH
C O
HC OH
HC OH
CH2OPO32
Gluconate-6-phosphate
Gluconate-6-phosphate
CO2
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.
C6
6 ribulose (C5)
xylulose C5
xylulose C5
ribose C5 ribose C5
xylulose C5
xylulose C5
C7 +C3 C6 + C4 C6 + C3
C7 +C3 C6 + C4 C6 + C3
1
2
2
3
3
4
4
5
Basically, there are 5 "steps" some of which involve more than one enzymatic reaction.
HO HO
HO
H2C OH
C O
HC OH
HC OH
CH2OPO32
Ribulose-5-phosphate
11 - 20 Lec #5
CH2OH
C O
CHHO
HC OH
CH2OPO32
Xylulose-5-phosphate
HC O
HC OH
HC OH
HC OH
CH2OPO32
Ribose-5-phosphate
Ribose phosphate isomerase
Ribulose phosphate 3-epimerase
K'eq = 0.8 K'eq = 3
2
CH2OH
C O
CHHO
HC OH
CH2OPO32
Xylulose-5-phosphate
HC O
HC OH
HC OH
HC OH
CH2OPO32
Ribose-5-phosphate
HC O
HC OH
CH2OPO32
Glyceraldehyde-3-phosphate
CHHO
HC OH
HC OH
HC OH
CH2OPO32
Sedoheptulose-7-phosphate
C
CH2OH
O
+ +
Transketolase
TPP
C5 + C5 C7 + C3
HC O
HC OH
CH2OPO32
Glyceraldehyde-3-phosphate
CHHO
HC OH
HC OH
HC OH
CH2OPO32
Sedoheptulose-7-phosphate
C
CH2OH
O
+
3 C7 + C3 C4 + C6
C O
CHHO
HC OH
HC OH
CH2OPO32
Fructose-6-phosphate
CH2OH
HC OH
HC OH
CH2OPO3
2
Erythrose-4-phosphate
HC O
+Transaldolase
1 - 214 C4 + C5 C6 + C3
HC OH
HC OH
CH2OPO3
2
Erythrose-4-phosphate
HC O
+
CH2OH
C O
CHHO
HC OH
CH2OPO32
Xylulose-5-phosphate
C O
CHHO
HC OH
HC OH
CH2OPO32
Fructose-6-phosphate
CH2OH
HC O
HC OH
CH2OPO32
Glyceraldehyde-3-phosphate
+Transketolase
TPP
5 C3 + C3 C6
HC O
HC OH
CH2OPO32
Glyceraldehyde-3-phosphate
CH2OH
C O
CH2OPO32
Dihydroxyacetone phosphate
C O
CHHO
HC OH
HC OH
CH2OPO32
Fructose-6-phosphate
CH2OH
Triose phosphate isomerase
C O
CHHO
HC OH
HC OH
CH2OPO32
Fructose-1,6-bisphosphate
CH2OPO3
2
Aldolase
Fructose-1,6-bisphosphatase
H2O Pi
In summary:
6 C6 6 C5 5 C6
12 NADPH (energy yield)Glc-6P
** All Frc-6-P converted by:
Phosphogluco isomerase so cycle can continue.**
6 CO2
1 - 22
EN
CS
CH2OH
CO
CH
TPP carbanion
H+
EN
CS
C
CH
HOH2C
HO
EN
CS
CHOH2C
OH
EN
CS
C
HOH2C
HO
a 2-carbon fragment bound to TPP
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.
R1
HO R1
O
H
C
R1
OH
C
R2
OH
H+
EN
CS
C
CH
HOH2C
OR2
O
H
H
EN
CS
CH2OH
CO
CH
R2
HO H+
C
R1
OH
C
R2
OH
and can be C3, C4 or C5 aldoses
CH2OH
CO
CH
R1
HO
CH2OH
CO
CH
R2
HOand can be C5, C6 or C7 ketoses
1 - 235. Electron Transport Chain and Oxidative Phosphorylation
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.
0.0
-0.3
-0.6
+0.3
+0.6
+0.9
E'o
NADH
Cyt c
SuccCoQ
O2
I
III
IV
II
∆E'o = 0.36 v∆G'o = -69.5 kJ/mol
∆E'o = 0.19 v∆G'o = -36.7 kJ/mol
∆E'o = 0.58 v∆G'o = -111.6 kJ/mol
∆E'o ~ 0.0 v∆G'o ~ 0 kJ/mol
remember : ∆G'o = -n ∆E'o = 96.5 kJ/v.mol (Faraday's const.)∆G'o = -RTlnK'eq R = 8.3 J/mol.K (gas constant)
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 mainlyon the overall picture and less on the individual components.
1 - 24
NADH + H+ NAD+ Succ Fum 1/2 O2 + 2H+ H2O
4 H+ 4 H+ 2 H+
I
II
III IV
CoQ
CoQH2
CoQ
Cytcox
Cytcred OUTSIDE
INSIDE
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.
ADP + Pi ATP + H2O
4 H+
The transfer of 4 protons through the ATPaseis coupled to the phosphorylation of one ADP to ATP (ie. 4 H+ = 1 ATP).
This is a summary of the chemiosmotic theory of oxidative phosphorylation as intially proposed by Peter Mitchell.
ATPase
INSIDE
OUTSIDE
FAD
+ + + + +
- - - - - - -
1 - 25In summary:
from Glycolysis/TCA in ETC ATPase
1 NADH 10 H+ pumped 2.5 ATP
1 FADH2 (succinate) 6 H+ pumped 1.5 ATP
NADH + H+
1/2 O2 H2O
2.5 ADP + 2.5 Pi + 2.5 H+ 2.5 ATP + 2.5 H2O
Succinate (FADH2) Fumarate (FAD)
1/2 O2 H2O
1.5 ADP + 1.5 Pi + 1.5 H+ 1.5 ATP + 1.5 H2O
Glucose
2 Pyruvate
2 AcCoA 2 Citrate
2 OAA
2 CO2
2 CO2
2 CO2
2 NADH 4 ATP (2 ATP net) 2 NADH
2 NADH
2 NADH
2 NADH
2 FADH2
2 GTP
Therefore:1 Glucose 6 CO2
2 ATP 2 ATP (net)2 GTP 2 ATP10 NADH 25 ATP2 FADH2 3 ATP__________________Yield 32 ATP/glucose (x 30.5 kJ/mol ATP = 976 kJ/mol)
Lec #62 ATP
NAD+
976/2868 x 100 = 34.0% efficient)
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:
Ga3P
Pyruvate
AcCoA Citrate
OAA
CO2
CO2
CO2
NADH 2 ATP
NADH
NADH
NADH
NADH
FADH2
GTP
Therefore:1 Ga-3-P 3 CO2
2 ATP 2 ATP1 GTP 1 ATP5 NADH 12.5 ATP1 FADH2 1.5 ATP_________________Yield 17 ATP
From TCA cycle intermediate:
SuccMal
AcCoA Citrate
OAA
CO2
CO2
CO2
FADH2
NADH
NADH
NADH
NADH
FADH2
GTP
Therefore:1 Succ 4 CO2
1 ATP 1 ATP1 GTP 1 ATP5 NADH 12.5 ATP2 FADH2 3 ATP_________________Yield 17.5 ATP
OAAPyr
NADHATP
CO2
If Malic enzyme (Mal to Pyr) is used the energy yield would be one ATP less.
*
*
NADH + CO2
start/finish
start/finish
Common to all energy calculations
fatty acids
amino acids
1 - 27
Glc
Frc-6-P
AcCoA
Citrate
Frc-1,6-bisP
ATPCitrate
ATPCitrate
AMPADP
AMPADP
+
+
-
-Pyruvate
AcCoAATPNADH
CoAAMPNAD+
SuccCoAATPNADH
-
-
+
OAA
AcCoA +
Isocitrate
-KG
ADP +
ADP +ATP -
SuccCoASuccCoANADH -
Summary of Regulation
High energy molecules signal a slow down of glycolysis and the TCA cycle and turn on gluconeogenesis.
Low energy molecules signal an increase in glycolysis and the TCA cycle
6. Summary of Carbohydrate Catabolism
1. Glycolysis and gluconeogenesis
2. TCA Cycle
3. Anaplerotic reactions
4. Pentose phospate pathway
5. Electron transport chain / oxidative phosphorylation
6. Energy calculations
7. Regulation
NADH activatesprotein kinase
-