1. 2 3 -2 ATP + 4 ATP = + 2 ATP 4 So does this solve the direction problem? Only for a second … Where does this ATP come from, if we are E. coli growing.

1

g l u c o s e

monomers

MacromoleculesPolysaccharides LipidsNucleic AcidsProteins

biosy

nthet

ic p

athw

ay

intermediates

F l o w o f g l u c o s e i n E . c o l i

E ac h a rro w = a sp e c ific c h em ica l re ac tio n

2

3

-2 ATP+ 4 ATP= + 2 ATP

4

• So does this solve the direction problem? Only for a second …

• Where does this ATP come from, if we are E. coli growing in minimal medium…

• Glucose is the only carbon source.• Need to make ATP from glucose, and

this TAKES energy.• Need only to regenerate ATP from ADP:

Via GLYCOLYSIS, e.g.

5

Overall reaction of glycolysis to pyruvate INCLUDING the generation of ATP

1 glucose + 2 ADP + 2 Pi + 2 NAD 2 pyruvate + 2 ATP + 2 NADH2

Δ Go = -18 kcal/mole

So overall reaction goes essentially completely to the right.

6

Handout 7-4b

7The second way the cell gets a reaction to go in the desired direction:

1) A coupled reaction.

One of two ways the cell solves the problem of getting a reaction to go in the desired direction Glucose + ATP glucose-6-P04 + ADP, Δ Go = -3.4 kcal/mole

2) The second way:• Removal of the product of an energetically unfavorable

reaction• Uses a favorable downstream reaction• “Pulls” the unfavorable reaction• Operates on the second term of the Δ G equation.• Δ G = Δ Go + RTln([products]/[reactants])

8

Handout 7-4b

9• So glucose pyruvic acid

• ADP ATP, as long as we have plenty of glucose• Are we all set?

• No…. What about the NAD.. We left it burdened with those electrons.

• Soon all of the NAD will be in the form of NADH2

• Glycolysis will screech to a halt !!• Need an oxidizing agent in plentiful supply to keep taking

those electron off the NADH2, to regenerate NAD so we can continue to run glucose through the glycolytic pathway.

10Oxidizing agents around for NAD:

1) Oxygen

Defer

2) Pyruvate

In E. coli, humans:

Pyruvate lactate, NADH2 NAD, coupled

In Yeast:

Pyruvate ethanol + CO2

11

12

Glucose NADH2NAD

Lactate Pyruvate

GAL-3-P 1,3-Di-PGA

Biosynthetic pathway to NAD

Handout 7-1b

excreted

Glucose

13

14Fermentation: anaerobiosis (no oxygen)

Lactate fermentation

Ethanolic fermentation

Mutually exclusive, depends on organism

Other types, less common fermentations, exist– (e.g., propionic acid fermentation, going on in Swiss cheese)

15The efficiency of fermentation

glucose--> 2 lactates,

without considering the couplings for the formation of

ATP's (no energy harnessing): 

Δ Go = -45 kcal/moleSo 45 kcal/mole to work with.

Out of this comes 2 ATPs, worth 14 kcal/mol.

So the efficiency is about 14/45 = ~30%

Where did the other 31/45 kcal/mole go?

Wasted as HEAT.

16Fermentation goes all the way to the right

Since 2 ATPs ARE produced, taking them into account, for the reaction:

Glucose + 2 ADP + 2 Pi 2 lactate + 2 ATP

ΔGo = -31 kcal/mole (45-14)

Very favorable.All the way to the right. Keep bringing in glucose, keep spewing out lactate,Make all the ATP you want.

glucose--> 2 lactates, without considering the couplings for the formation of ATP's (no energy harnessing):  Δ Go = -45 kcal/mole kcal/moleOut of this comes 2 ATPs, worth 14 kcal/mol. So the efficiency is about 14/45 = ~30%

That’s fermentation, for now.

17Energy yield

Complete oxidation of glucose,

Much more ATP

But nature’s solution is a bit complicated.

The fate of pyruvate is now different

But all this spewing turns out to be wasteful.Glucose could be completely oxidized, to: … CO2That is, burned.

How much energy released then?Glucose + 6 O2 6 CO2 + 6 H2O ΔGo = -686 kcal/mole !Compared to -45 to lactate (both w/o/ ATP considered)

18

2 NADH2 NADH

2 ATP

Acetyl-CoA

2 CO2

Score:Per glucose

19Acetyl-CoA

O

||

CH3 - C –OH + Co-enzyme A Acetyl ~CoA

Acetic acid, acetate

Acetate group

20

2 ATP

Acetyl-CoA

2 CO2

2 CO2

2 CO2

2 oxaloacetatePer glucose

2 NADH2 NADH2 NADH2 NADH

6 CO2

21

GTP is energetically equivalent to ATP

GTP + ADP GDP + ATP

ΔGo = ~0

G= guanine (instead of adenine in ATP)

22

2 NADH2 NADH2 NADH2 NADH

2 ATP

2 ATP

Acetyl-CoA

2 CO2

2 CO2

2 CO2

2

Succinic dehydrogenase

2 oxaloacetate

Per glucose

23FAD = flavin adenine dinucleotide

FAD + 2H. FADH2

24

2 NADH2 NADH2 NADH2 NADH2 FADH22 NADH

2 ATP

2 ATP

Acetyl-CoA

2 CO2

2 CO2

2 CO2

Succinic dehydrogenase

oxaloacetatePer glucose

25

2 NADH2 NADH2 NADH2 NADH2 FADH22 NADH

2 ATP

2 ATP

2 CO2

2 CO2

2 CO2

Glucose + 6 O2 6 CO2 + 6 H2O :

By glycolysis plus one turn of the Krebs Cycle:

1 glucose (6C) 2 pyruvate (3C) 6 CO2

2 X 5 NADH2 and 2 X 1 FADH2 produced per glucose

4 ATPs per glucose

NADH2 and FADH2 still must be reoxidized ….

No oxygen yet to be consumed

No water produced yet

Per glucose

26Oxidation of NAD by O2

NADH2 + 1/2 O2   -->  NAD + H2O

ΔGo = -53 kcal/mole

If coupled directly to ADP ATP (7 kcal cost),46 kcal/mole waste, and heat

So the electrons on NADH (and FADH2) are not passed directly to oxygen, but to intermediate carriers,

Each transfer step involves a smaller packet of free negative energy change (release)

27

Handout 8-3

NADH2

Ubiquinone; Coenzyme Q

H

H

28

Handout 8-4

29

30

nal

Schematic idea of H+ being pumped out

Handout 8-4

31

FoF1 complex

Handout 8-4

32

Chemiosmotic theory

Proton motive force (pmf)Chemical gradientElectrical gradient Electrochemical gradient

Peter Mitchell 1961

Water-pump-dam analogy

Some evidence:

33

H+H+

H+H+H+H+

H+H+

H+H+

H+H+

H+

H+H+

H+

H+

H+

H+

H+H+

H+

H+

H+

H+

H+H+

H+

H+

H+

H+

H+H+

H+

H+

H+

ETC Complex I’s

NADH

H+

H+H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

NADH

Artificial phospholipid membrane

pH drops pH rises

34

H+

H+

H+

H+

H+

H+

H+

H+ H+

H+

H+

H+

H+

H+

H+H+H+

H+

H+

H+

H+

H+

H+

H+H+

H+H+

H+

H+

H+

H+

H+

H+

H+

H+H+

H+

H+

H+H+

ADP + Pi

ATP

Artificially produced mitochondrial membrane vesicle

ATP is formed from ADP + Pi

35Dinitrophenol (DNP): an uncoupler of oxidative phosphorylation

DNP’s -OH is weakly acidic in this environment

DNP can easily permeate the mitochondrial inner membrane

Outside the mitochondrion, where the H+ concentration is high, DNP picks up a proton

After diffusing inside, where the H+ concentration low, it gives up the proton.

So it ferries protons from regions of high concentration to regions of low concentration, thus destroying the proton gradient.

Electron transport chain goes merrily on and on, but no gradient is formed and no ATP is produced.

- + H+

36

The mechanism of ATP formation:

The ATP synthetase (or ATP synthase)

The F0F1 complex

37

ATP synthetase

outside

insideGamma subunit: cam

38

outside

inside

39

Alpha+beta

Gamma

40

Motor experiment

41

Testing the ATP synthetase motor model by running it in reverse (no H+ gradient, add ATP)

Actin labeledBy tagging it with fluorescent molecules

Actin is a muscle protein polymer

42

ATP synthetase

}

43

1234 5

Run reaction in reverse, add ATP, drive counter-clockwise rotation of cam

ATP hydrolysis

This is oxidative phosphorylation of ADP

44

Testing the ATP synthetase motor model by running it in reverse

Actin labeledBy tagging it with fluorescent molecules

Actin is a muscle protein polymer

45

desktop

46

Synthase.mov movie

47ATP accounting

• Each of the 3 ETC complex (I, III, IV) pumps enough H+ ions to allow the formation of 1 ATP.

• So 3 ATPs per pair of electrons passing through the full ETC.

• So 3 ATPs per 1/2 O2

• So 3 ATPs per NADH2

• But only 2 ATPs per FADH2 (skips complex 1)

48

49

ATP

ATP

ATP

Similar to handout 8-2

50

Substrate level phosphorylation (SLP): 2 ATP

1ATP from Glycolysis

1 ATP (GTP) from Krebs

OXPHOS:

1 NADH from glycolysis

1 NADH from Krebs entry

3NADH from Krebs

1 FADH2 from Krebs

Total: 17 ATP

5 NADH = 15 ATP 1 FADH2 = 2 ATP

Grand total (E. coli):17 + 2 = 19 per ½ glucoseor 38 per 1 glucose

Handout 8-6

51ATP accounting

• 38 ATP/ glucose in E. coli

• 36 ATP/glucose in eukaryotes– Cost of bringing in the electrons from NADH from glycolysis into the

mitochondrion = 1 ATP per electron pair

– So costs 2 ATPs per glucose, subtract from 38 to get 36 net.

52Efficiency

• 36 ATP/ glucose, worth 7 X 36 = 252 kcal/mole of glucose

• ΔGo for the overall reaction glucose + 6 O2→ 6CO2 + 6 H2O:-686 kcal/ mole

• Efficiency = 252/686 = 37%

• Once again, better than most gasoline engines.

• and Energy yield:36 ATP/ glucose vs. 2 ATP/glucose in fermentation(yet fermentation works)

• So with or without oxygen, get energy from glucose

53Cellular location

(eukaryotes):

CYTOPLASM

MITOCHONDRIA

Handout 8-6

54Nucleic acids

Prof. Mowshowitz, next time

But wait:

I will be back for one more lecture (#11) on energy metabolism and intermediary metabolism