2 NADH

1
Acetyl-CoA
Score Per glucose
A
2 NADH 2 NADH
2 ATP
2 CO2
Handout 8-1
2
Acetyl-CoA

• O
• CH3 - C OH Co-enzyme A ?
  Acetyl CoA
• Acetic acid, acetate

Acetate group
3
Acetyl-CoA
Per glucose
B
2 oxaloacetate
2 ATP
2 NADH2 NADH 2 NADH 2 NADH
2 CO2 2 CO2 2 CO2
6 CO2
4
GTP is energetically equivalent to ATP

• GTP ADP ? GDP ATP
• ?Go 0
• G guanine (instead of adenine in ATP)

5
Acetyl-CoA
Per glucose
C
2 oxaloacetate
2 NADH 2 NADH 2 NADH 2 NADH
2 ATP 2 ATP
2 CO2 2 CO2 2 CO2
2
Succinic dehydrogenase
6
FAD flavin adenine dinucleotide
Business end (flavin)
ribose
adenine
ribose
FAD 2H. ? FADH2
7
Acetyl-CoA
D
Per glucose
oxaloacetate
2 NADH 2 NADH 2 NADH 2 NADH 2 FADH2 2 NADH
2 ATP 2 ATP
2 CO2 2 CO2 2 CO2
Note label is in OA after one turn of
cycle, half the time on top, half on bottom. So
no CO2 from Ac-CoA after just one turn. (CO2 in
first turn from OA).
Succinic dehydrogenase
8
E
Per glucose
2 NADH 2 NADH 2 NADH 2 NADH 2 FADH2 2 NADH
2 ATP 2 ATP
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 Paltry increase in ATP so far
2 CO2 2 CO2 2 CO2
9
Oxidation of NAD by O2

• NADH2 1/2 O2   --gt  NAD H2O
• ?Go -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)

10
Iron-sulfur protein
10
NADH2
Free energy
heme
50 Cs long
Cytochromes are proteins
Ubiquinone Coenzyme Q
Handout 8-3
11
Handout 8-4
12
(outside)
Nelson and Cox, Principles of Biochemistry
13
Schematic idea of H being pumped out
Handout 8-4
14
FoF1 Complex Oxidative phosphorylation (ATP
formation))
Handout 8-4
15
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 Is
pH drops
pH rises
NADH
NADH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
16
ADP Pi
Artificially produced mitochondrial membrane
vesicle
ATP is formed from ADP Pi
17
Dinitrophenol (DNP) an uncoupler of oxidative
phosphorylation
?
-
H
DNPs -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.
18
Chemiosmotic theory Proton motive force
(pmf) Chemical gradient Electrical gradient
Electrochemical gradient Peter Mitchell 1961
(without knowing mechanism) Water-pump-dam
analogy Some evidence
19
What about E. coli? Cell membrane houses all
components
the inside
FoF1 Complex Oxidative phosphorylation (ATP
formation)
Handout 8-4
the outside
20
The mechanism of ATP formation The ATP
synthetase (or ATP synthase) The F0F1 complex
the inside
the outside
Gamma subunit is inserted inside
ATP synthetase
21
ATP synthetase
inside
outside
22
ADP
Pi
Three conformational states of the a-b subunit
L, T, and O
Handout 8-5
23
Motor experiment
(MW 42K)
24
Actin labeled by tagging it with fluorescent
molecules
Attached to the gamma subunit
Actin is a muscle protein polymer
Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida
Kazuhiko Kinosita Jr. (1997) Direct observation
of the rotation of F1-ATPase. Nature, 386, 299 -
302.
Testing the ATP synthetase motor model by
running it in reverse (no H gradient, add ATP)
25
Run reaction in reverse add ATP, drive
counter-clockwise rotation of cam
1
2
3
4
5
Here the cam has no driving motor (E) attached
any more
Start here
ATP
ATP hydrolysis
?
?
?
?
x
counter-clockwise
Note modified slightly 10/25/09 for clarity all
large arrows are now just the reverse of the the
ATP synthesis directions.
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Outside
Inside
Chloroplasts
Mitochondria
Outside
Inside
29
View of the c-subunits making up the F0 subunit
using atomic force microscopy
Animation of the Fo rotation driven by the
influx of H ions (wheels within wheels).M.E.
Girvin
Norbert Dencher and Andreas Engel
30
Handout 8-5
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ATP 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)

34
Handout 8-1
35
X
Fumarate
More favorable ?GO with FAD
X
ATP generated by the ATP synthetase is called is
oxidative phosphorylation, or oxphos
36
Nelson and Cox, Principles of Biochemistry
37
Handout 8-6
Grand total (E. coli) 17 2 19 per ½
glucose or 38 per 1 glucose
Handout labeled 8-6
38
Cellular location (eukaryotes)
CYTOPLASM
MITOCHONDRIA
Handout labeled 8-6
39
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.

40
Efficiency

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

41
Alternative sources of carbon and energy

• Shake milk
• milk sugar lactose disaccharide
• glucose galactose
• beta-galactosidase
• HOH ? glucose
  galactose
• glucose ? glycolysis, etc.
• galactose
• ?
• ?(3 enzymatic steps)
• ?
• glucose

42
Alternative sources of carbon and energy
Bun starch poly-alpha-glucose
G-1-P ? G-6-P glycolysis
43
Alternative sources of carbon and energy
Lettuce cellulose polysaccharide Poly-beta
glucose ? (stays as the polysaccharide) We
have no enzyme for catabolizing cellulose

44
Alternative sources of carbon and energy
French fries fat (oil) triglyceride
45
(Triglyceride)
Lipases (hydrolysis)
Glycolysis (at DHAP)
46
Handout 9-1 left
47
Alternative sources of carbon and energy
Hamburger protein Proteases (e.g., trypsin) ? ?
20 AAs Stomach acid (pH1) also helps by
denaturing protein making it accessable to
proteolytic attack Each of the 20 AAs has its
own catabolic pathway, and ends up in the
glycolytic or Krebs cycle pathways But first,
the N must be removed
48
Handout 9-2
Deamination and transamination of amino acids
49
E.g., degradation of phenylalanine (6 steps)
Phe builds up and gets metabolized to an
injurious product (phenyl pyruvate)
transaminase
Products Fumaric acid ? Krebs and
Acetoacetate ? 2 Acetyl-CoA
? Krebs
50
You are what you eat
Catabolism
Anabolism
Anabolism
ATP
STARCH
GLYCOGEN
glucose
GLYCOLYSIS
ATP
pyruvate
ATP
ATP
ATP
ATP
FATS
FATS
FATS
FATS
FATS
acetyl-CoA
KREBS
O.A.
-K.G.
-K.G.
-K.G.
ATP
ATP
ATP
ATP
AMINO ACIDS
ATP
AMINO ACIDS
AMINO ACIDS
AMINO ACIDS
AMINO ACIDS
E.T.C.
NAD
NAD
NAD
PROTEINS
PROTEINS
PROTEINS
PROTEINS
PROTEINS
NADH
2
ATP
O
H
O
2
2
Handout 9-2
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Handout 9-2
Handout 9-2
53
Biosynthesis of monomers

• E.g.,
• Fatty acids (acetyl CoA from Krebs cycle)
• Amino acids (Serine 3-phospho-glyceric acid
  from glycolysis)

54
Handout 9-1 Start at bottom
P
P
Handout 9-1 right
55
Handout 9-3
Phosphoester group
(Glycolytic intermediate)
Glutamate is the amino donor
hydrolysis
Handout 9-3
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Biosynthesis of macromolecules
58
Biosynthesis of macromolecules

• Lipids
• Polysaccharides
• Proteins

59
NADPH
Phospholipid
Handout 9-3
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Polypaccharide Hyaluronic acid
n
62
Polysaccharide synthesis Hyaluronic acid (joint
lubricant)
Glucuronic acid
N-acetyl-glucosamine
COO-
Enz.1
Enz. 2
Enz.1

Enz. 2
Enz.1
Enz. 2
Enz.1
Enz. 2
Enz.1
Enz. 2
Enz.1
Hyaluronic acid (polysaccharide)
via 2 enzymes
63
Biosynthesis of proteins

• e.g., an enzyme like hexokinase
  met-val-his-leu-gly ..
• If this done like lipids and polysaccharides, we
  need an enzyme for each linkage
• First an enzyme that will condense val to met to
  make met-val.
• Then an enzyme with a different substrate
  specificity, which adds his to met-val to make
  met-val-his.
• Since there are 500 AAs in hexokinase, we need
  500 enzymes to do the job.
• If there are 3000 proteins in E. coli, then we
  need 500 X 3000 1.5 million enzymes to make
  all the different primary structure of all the
  proteins.
• But even then, it wont work, as each of these
  million enzymes is also a protein that needs to
  be synthesized.
• We need a better plan to polymerize the amino
  acids in the right order.

64
Problem 1

• Getting specific reaction rates to go in real
  time
• Enzymes
• Getting the reactions to go in the desired
  direction
• Coupled reactions favorable metabolic paths
  (also enzymes)
• 
  
• Getting the information to make the specific
  3-dimensional enzymes
• Just need to specify the primary structure ..
  How?

Problem 2
Problem 3
65
Nucleic acids

• Prof. Mowshowitz will continue with this next
  chapter in the story,
• leading to the biosynthesis of the all-important
  proteins.

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