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Flow of glucose in E. coli
Macromolecules
Polysaccharides
Lipids
Nucleic Acids
Proteins
yn
th
e
tic
pa
t
hw
ay
monomers
bi
os
intermediates
glucose
Each arrow = a specific chemical reaction
2
3
-2 ATP
+ 4 ATP
= + 2 ATP
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•
•
•
•
•
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.
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Overall reaction of glycolysis to pyruvate INCLUDING the generation of ATP
1 glucose + 2 ADP + 2 Pi + 2 NAD
Δ Go = -18 kcal/mole
2 pyruvate + 2 ATP + 2 NADH2
So overall reaction goes essentially completely to the right.
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Handout 7-4b
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The 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])
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Handout 7-4b
• 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.
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Oxidizing agents around for NAD:
1) Oxygen
Defer
2) Pyruvate
In E. coli, humans:
Pyruvate  lactate, NADH2  NAD, coupled
In Yeast:
Pyruvate  ethanol + CO2
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Glucose
GAL-3-P
Glucose
NAD
1,3-Di-PGA
NADH2
Biosynthetic pathway to NAD
Lactate
Pyruvate
excreted
Handout 7-1b
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Fermentation: anaerobiosis (no oxygen)
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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)
The efficiency of fermentation
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glucose--> 2 lactates,
without considering the couplings for the formation of
ATP's (no energy harnessing):
Δ Go = -45 kcal/mole
So 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.
Fermentation goes all the way to the right
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glucose--> 2 lactates,
without considering the couplings for the formation of ATP's (no energy
harnessing):
Δ Go = -45 kcal/mole kcal/mole
Out of this comes 2 ATPs, worth 14 kcal/mol.
So the efficiency is about 14/45 = ~30%
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.
That’s fermentation, for now.
Energy yield
But all this spewing turns out to be wasteful.
Glucose could be completely oxidized, to: … CO2
That 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)
Complete oxidation of glucose,
Much more ATP
But nature’s solution is a bit complicated.
The fate of pyruvate is now different
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Acetyl-CoA
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Score:
Per glucose
2 NADH
2 NADH
2 ATP
2 CO2
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Acetyl-CoA
O
||
CH3 - C –OH
+ Co-enzyme A
Acetic acid, acetate

Acetyl ~CoA
Acetate group
Acetyl-CoA
Per glucose20
2 oxaloacetate
2 NADH
2 NADH
2 NADH
2 NADH
2 ATP
2 CO2
2 CO2
2 CO2
6 CO2
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GTP is energetically equivalent to ATP
GTP + ADP  GDP + ATP
ΔGo = ~0
G= guanine (instead of adenine in ATP)
Acetyl-CoA
Per glucose
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2 oxaloacetate
2 NADH
2 NADH
2 NADH
2 NADH
2 ATP
2 ATP
2 CO2
2 CO
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2 CO2
Succinic dehydrogenase
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FAD = flavin adenine dinucleotide
FAD + 2H.  FADH2
Acetyl-CoA
Per glucose24
oxaloacetate
2 NADH
2 NADH
2 NADH
2 NADH
2 FADH2
2 NADH
2 ATP
2 ATP
2 CO2
2 CO2
2 CO2
Succinic dehydrogenase
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Per glucose
2 NADH
2 NADH
2 NADH
2 NADH
2 FADH2
2 NADH
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
2 CO2
2 CO2
2 CO2
2 ATP
2 ATP
Oxidation of NAD by O2
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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)
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NADH2
H
H
Handout 8-3
Ubiquinone; Coenzyme Q
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Handout 8-4
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Schematic idea of H+ being pumped out
nal
Handout 8-4
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FoF1
complex
Handout 8-4
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Chemiosmotic theory
Proton motive force (pmf)
Chemical gradient
Electrical gradient
Electrochemical gradient
Peter Mitchell 1961
Water-pump-dam analogy
Some evidence:
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Artificial phospholipid membrane
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
pH drops
NADH
NADH
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
pH rises
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+ H+
H+ H+
H+
H+ H+
H+
H+
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H+ H+
H+
H+ H+
H+
H+
H+
H+
H+
H+
ADP + Pi
H+ ATP
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
Artificially produced mitochondrial membrane vesicle
ATP is formed from ADP + Pi
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Dinitrophenol (DNP): an uncoupler of oxidative phosphorylation

-
+ H+
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.
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The mechanism of ATP formation:
The ATP synthetase
(or ATP synthase)
The F0F1 complex
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outside
inside
Gamma subunit: cam
ATP synthetase
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inside
outside
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Alpha+beta
Gamma
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Motor experiment
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Actin labeled
By tagging it with
fluorescent molecules
Actin is a muscle
protein polymer
Testing the ATP synthetase motor model
by running it in reverse (no H+ gradient, add ATP)
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}
ATP synthetase
Run reaction in reverse, add ATP, drive counter-clockwise rotation of cam
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3
2
1
5
ATP hydrolysis



This is oxidative phosphorylation of ADP

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Actin labeled
By tagging it with
fluorescent molecules
Actin is a muscle
protein polymer
Testing the ATP synthetase motor model
by running it in reverse
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desktop
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Synthase.mov movie
ATP accounting
•
•
•
•
•
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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)
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ATP
ATP
ATP
Similar to handout 8-2
Handout 8-6
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OXPHOS:
1 NADH from
glycolysis
Substrate
level
phosphorylation
(SLP): 2 ATP
1ATP from
Glycolysis
1 NADH from
Krebs entry
3NADH from
Krebs
1 FADH2 from
Krebs
Total: 17 ATP
1 ATP (GTP)
from Krebs
5 NADH = 15 ATP
1 FADH2 = 2 ATP
Grand total (E.
coli):
17 + 2 = 19
per ½ glucose
or 38 per 1
glucose
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ATP 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.
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Efficiency
• 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
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Cellular location
(eukaryotes):
CYTOPLASM
MITOCHONDRIA
Handout 8-6
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Nucleic acids
Prof. Mowshowitz, next time
But wait:
I will be back for one more lecture (#11) on energy metabolism and intermediary metabolism