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

-

+