Oxidation of NAD by O2

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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)

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Iron-sulfur protein
NADH2
H
heme
Ubiquinone Coenzyme Q
H
Handout 8-3
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Handout 8-4
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Nelson and Cox, Principles of Biochemistry
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Schematic idea of H being pumped out
nal
Handout 8-4
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FoF1 Complex Oxidative phosphorylation (ATP
formation)
Handout 8-4
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Chemiosmotic theory (Mitchell hypothesis) Proton
motive force (pmf) Chemical gradient Electrical
gradient Electrochemical gradient Peter
Mitchell 1961 (without knowing mechanism) Water-p
ump-dam analogy 3 exampels of evidence
supporting themitchell hypothesis
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Experiment 1
Artificial phospholipid membrane
H
H
H
H
H
H
H
H
H
H
H
H
ETC Complex Is
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Experiment 2
Experiment 2
ADP Pi
Artificially produced mitochondrial membrane
vesicle
ATP is formed from ADP Pi
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Dinitrophenol (DNP) an uncoupler of oxidative
phosphorylation
Experiment 3
- H
-
H
DNPs -OH is weakly acidic in this environment
(pH7) 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
ATP synthetase
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ATP synthetase
inside
outside
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Three conformational states of the a-b subunit
L, T, and O
Handout 8-5
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Motor experiment
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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)
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Run reaction in reverse, add ATP, drive
counter-clockwise rotation of cam
1
2
3
4
5
ATP
ATP hydrolysis
?
?
?
?
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desktop
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View of the c-subunits making up the F0 subunit
using atomic force microscopy
Norbert Dencher and Andreas Engel
Animation of the Fo rotation driven by the
influx of H ions (wheels within wheels).M.E.
Girvin
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Handout 8-5
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Hongyun Wang and George Oster
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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)

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

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Cellular location (eukaryotes)
CYTOPLASM
MITOCHONDRIA
Handout labeled 8-6
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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

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

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Alternative sources of carbon and energy
French fries fat (oil) triglyceride
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(Triglyceride)
Lipases (hydrolysis)
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Handout 9-1 left
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ATP
DHAP (dihydroxy acetone phosphate)
glycerol
???
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D
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ATP
glycerol
- O2
Glycerol cannot be fermented. E. coli CANNOT grow
on glycerol in the absence of air These pathways
are real, and they set the rules. No magic is
involved
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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
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Handout 9-2
Deamination and transamination of amino acids
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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
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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
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Biosynthesis of monomers

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

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

1. Lipids
2. Polysaccharides
3. Proteins

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NADPH
Phospholipid
Handout 9-3
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Biosynthesis of the polysaccharide hyaluronic acid
UTP
Activated form of the sugar activated for
condensation
Glucuronic acid-1-phosphate (Glucu)
Growing hyaluronic acid chain
Hyaluronic acid Glucu-NAcGA-Glucu-NAcGA-Glucu-N
AcGA-

Hyaluronate 1 Next add an acetyl-glucosamine
monomer (NAcGA) Then repeat the addition of a
glucuronic acid monomer Then another
acetyl-glucosamine monomer Etc. So 2 enzymes
to form this alternating polymer from monomers
PPi
Handout 9-4
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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.

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• 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?

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Nucleic acids

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