1. 2 3 -2 atp + 4 atp = + 2 atp 4 so does this solve the direction problem? only for a second …...
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g l u c o s e
monomers
MacromoleculesPolysaccharides LipidsNucleic AcidsProteins
biosy
nthet
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athw
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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
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-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 2 pyruvate + 2 ATP + 2 NADH2
Δ Go = -18 kcal/mole
So overall reaction goes essentially completely to the right.
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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])
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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
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Glucose NADH2NAD
Lactate Pyruvate
GAL-3-P 1,3-Di-PGA
Biosynthetic pathway to NAD
Handout 7-1b
excreted
Glucose
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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)
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2 NADH2 NADH
2 ATP
Acetyl-CoA
2 CO2
Score:Per glucose
19Acetyl-CoA
O
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CH3 - C –OH + Co-enzyme A Acetyl ~CoA
Acetic acid, acetate
Acetate group
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2 ATP
Acetyl-CoA
2 CO2
2 CO2
2 CO2
2 oxaloacetatePer glucose
2 NADH2 NADH2 NADH2 NADH
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)
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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
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2 NADH2 NADH2 NADH2 NADH2 FADH22 NADH
2 ATP
2 ATP
Acetyl-CoA
2 CO2
2 CO2
2 CO2
Succinic dehydrogenase
oxaloacetatePer glucose
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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)
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Handout 8-3
NADH2
Ubiquinone; Coenzyme Q
H
H
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Handout 8-4
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nal
Schematic idea of H+ being pumped out
Handout 8-4
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FoF1 complex
Handout 8-4
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Chemiosmotic theory
Proton motive force (pmf)Chemical gradientElectrical gradient Electrochemical gradient
Peter Mitchell 1961
Water-pump-dam analogy
Some evidence:
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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+
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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
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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+
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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+
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The mechanism of ATP formation:
The ATP synthetase (or ATP synthase)
The F0F1 complex
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ATP synthetase
outside
insideGamma subunit: cam
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outside
inside
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Alpha+beta
Gamma
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Motor experiment
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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
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ATP synthetase
}
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1234 5
Run reaction in reverse, add ATP, drive counter-clockwise rotation of cam
ATP hydrolysis
This is oxidative phosphorylation of ADP
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Testing the ATP synthetase motor model by running it in reverse
Actin labeledBy tagging it with fluorescent molecules
Actin is a muscle protein polymer
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desktop
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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)
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ATP
ATP
ATP
Similar to handout 8-2
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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