The document discusses cellular respiration, which occurs in three stages: 1) acetyl-CoA production from organic fuels like glucose and fatty acids, 2) acetyl-CoA oxidation in the citric acid cycle (CAC) to produce NADH, FADH2, and GTP, and 3) oxidative phosphorylation to generate large amounts of ATP. The citric acid cycle involves a series of chemical reactions that generate energy in the form of ATP, NADH, and FADH2. These stages capture energy from nutrients and facilitate the production of ATP to fuel cellular work.

1. 16|
The
Citric
Acid
Cycle
© 2013 W. H. Freeman and Company

2. Only
a
small
amount
of
energy
available
in
glucose
is
captured
in
glycolysis
2
ΔG′° = –146 kJ/mol
Glycolysis
Full oxidation (+ 6 O2)
ΔG′° = –2,840 kJ/mol
6 CO2 + 6 H2O
Glucose
Only the 1st stage of
Glucose oxidation

3. Cellular
Respira>on
•
Process
in
which
cells
consume
O2
and
produce
CO2
•
Provides
more
energy
(ATP)
from
glucose
than
glycolysis
•
Also
captures
energy
stored
in
lipids
and
amino
acids
•
Used
by
animals,
plants,
and
many
microorganisms
•
Occurs
in
three
major
stages:
-­‐
acetyl
CoA
producEon
(from
organic
fuel
molecules)
-­‐
acetyl
CoA
oxidaEon
(in
the
CAC
to
produce
CO2)
-­‐
electron
transfer
and
oxidaEve
phosphorylaEon
(reduced
coenzymes
from
CAC
give
their
e–’s
to
O2
forming
ATP
in
the
process)

4. Respira>on:
Stage
1
Acetyl-­‐CoA
Produc>on
• AcEvated
form
of
acetate
• C-­‐skeleton
of
sugars
and
faKy
acids
are
converted
to
acetyl-­‐
CoA
before
entering
the
CAC
some
a.a.
enter
CAC
via
other
intermediates
• Pyruvate
dehydrogenase
complex
(PDH)
• MulEple
copies
of
3
enzymes
• 5
reacEons
by
3
enzymes,
whereby
the
intermediates
remain
bound
to
the
enzyme
molecule
unEl
forming
the
final
product
• 5
cofactors
(4
derived
from
vitamins)

5. Respira>on:
Stage
2
Acetyl-­‐CoA
oxida>on
Generates
NADH,
FADH2,
and
one
GTP

6. Respira>on:
Stage
3
Oxida>ve
Phosphoryla>on
Generates
a
lot
of
ATP

7. In
eukaryotes,
citric
acid
cycle
occurs
in
mitochondria
• Glycolysis
occurs
in
the
cytoplasm
• Citric
acid
cycle
occurs
in
the
mitochondrial
matrix†
• OxidaEve
phosphorylaEon
occurs
on
and
in
the
inner
membrane
†Except
succinate
dehydrogenase,
which
is
located
in
the
mitochondrial
inner
membrane

8. Conversion
of
Pyruvate
to
Acetyl-­‐CoA
• Net
ReacEon:
– Oxida*ve
decarboxyla*on
of
pyruvate
– First
carbons
of
glucose
to
be
fully
oxidized
(remember:
2
pyr/glc)
• Catalyzed
by
PDH
– Requires
5
coenzymes
– TPP,
lipoate,
and
FAD
are
prostheEc
groups
– NAD+
and
CoA-­‐SH
are
co-­‐substrates
TPP
–
thiamine
(B1)
FAD
–
riboflavin
(B2)
NAD
–
niacin
(B3)
CoA
–
panthothenic
acid
(B5)
Derived
from:

9. Structure
of
Coenzyme
A
• Coenzymes
are
not
a
permanent
part
of
the
enzymes’
structure.
– They
associate,
fulfill
a
funcEon,
and
dissociate
• The
funcEon
of
CoA
is
to
accept
and
carry
acetyl
groups
• Thioesters
have
a
high
acyl
group
transfer
potenEal
(donate
their
acyl
groups
to
different
groups)

10. Structure
of
Lipoyllysine
• ProstheEc
groups
are
strongly
bound
to
the
protein
– The
lipoic
acid
is
covalently
linked
to
the
enzyme
via
a
lysine
residue
(lipoyllysine)
– Undergo
reversible
redox
reacEons
between
thiols
and
disulfides;
hence
can
serve
as
an
electron
(Hydrogen)
carrier
and
an
acyl
carrier

11. Pyruvate
Dehydrogenase
Complex
(PDH)
•
Large
(up
to
10
MDa)
mulEenzyme
complex
-­‐
large
enough
to
be
seen
with
cryoEM
-­‐
pyruvate
dehydrogenase
(E1)
-­‐
dihydrolipoyl
transacetylase
(E2)
-­‐
dihydrolipoyl
dehydrogenase
(E3)
-­‐
each
present
in
mul3ple
copies
• Advantages
of
mulEenzyme
complexes:
‒ short
distance
between
catalyEc
sites
allows
channeling
of
substrates
from
one
catalyEc
site
to
another
‒ channeling
minimizes
side
reacEons
‒
regulaEon
of
acEvity
of
one
subunit
affects
the
enEre
complex

12. 3D
Reconstruc>on
from
Cryo-­‐EM
data
The
bovine
enzyme
Core:
60
idenEcal
copies
of
E2)
E1
acEve
site
has
bound
TPP
E3
acEve
site
has
bound
FAD
Protein
kinase
and
phosphoprotein
phosphatase
are
part
of
the
complex

13. Overall
Reac>on
of
PDH
E1
• Step
1:
DecarboxylaEon
of
pyruvate
to
an
aldehyde
• Step
2:
OxidaEon
of
aldehyde
to
a
carboxylic
acid
‒ Electrons
reduce
lipoamide
and
form
a
thioester
E2
•
Step
3:
FormaEon
of
acetyl-­‐CoA
(product
1)
E3
• Step
4:
ReoxidaEon
of
the
lipoamide
cofactor
• Step
5:
RegeneraEon
of
the
oxidized
FAD
cofactor
‒ Forming
NADH
(product
2)

15. The
Citric
Acid
Cycle
(CAC)
C-­‐C
bond
formaEon
to
make
citrate
IsomerizaEon
via
dehyd./rehydraEon
OxidaEve
decarboxy-­‐
laEons
to
give
2
NADH
Substrate-­‐level
phosphorylaEon
to
give
GTP
DehydrogenaEon
to
give
reduced
FADH2
HydraEon
DehydrogenaEon
to
give
NADH

16. The
Citric
Acid
Cycle
• Per
each
turn
of
the
cycle:
‒ One
acetyl
group
enters
(2
C)
and
2
CO2
leave
‒ One
molecule
of
oxaloacetate
is
used
to
make
citrate
and
one
molecule
is
regenerated
(no
net
change
in
OA
concentraEon;
which
is
very
low)
‒ 4
of
the
8
steps
are
oxidaEons
(the
energy
of
oxidaEon
is
conserved
in
NADH
and
FADH2)
• Not
limited
to
energy
produc>on
‒ 4-­‐
and
5-­‐C
intermediates
serve
as
precursors
for
different
products
‒ To
replace
these
intermediates,
cells
use
anaplero*c
(replenishing)
reacEons

17. C-­‐C
Bond
Forma>on
by
Condensa>on
of
Acetyl-­‐CoA
and
Oxaloacetate
(step
1)
• CondensaEon
of
acetyl-­‐CoA
and
oxaloacetate
• The
only
reacEon
with
C-­‐C
bond
formaEon
• Uses
Acid/Base
Catalysis
– Carbonyl of oxaloacetate
is a good electrophile (stabilization of carbanions)
– Methyl group has been converted to methylene
• Rate-­‐limi*ng
step
of
CAC
• AcEvity
largely
depends
on
[oxaloacetate]
• Highly
thermodynamically
favorable/irreversible
– Regulated
by
substrate
availability
and
product
inhibiEon

18. Induced
Fit
in
the
Citrate
Synthase
• oxaloacetate
binds
first
à
creaEng
a
binding
site
for
acetyl-­‐CoA
• Avoids
unnecessary
hydrolysis
of
thioester
in
acetyl-­‐
CoA
b)
Closed
conforma>on:
Binding
of
OAA
creates
binding
for
acetyl-­‐CoA
ReacEve
carbanion
is
protected
a) Open
conforma>on:
Free
enzyme
does
not
have
a
binding
site
for
acetyl-­‐CoA

19. Isomeriza>on
by
Dehydra>on/
Rehydra>on
(step
2)

20. Aconitase
• EliminaEon
of
H2O
from
citrate
gives
a
cis
C=C
bond
– Lyase
• Citrate,
a
terEary
alcohol,
is
a
poor
substrate
for
oxidaEon
• Isocitrate,
a
secondary
alcohol,
is
a
good
substrate
for
oxidaEon
• AddiEon
of
H2O
to
cis-­‐aconitate
is
stereospecific
(either
to
form
isocitrate
or
citrate)
• Cytosolic
isozyme
uses
NADP+
as
a
cofactor
• Thermodynamically
unfavorable/reversible
– Product
is
consumed
rapidly
by
the
next
step
(concentraEon
kept
low)
to
pull
reacEon
forward

21. Iron-­‐Sulfur
Center
in
Aconitase
• Water
removal
from
citrate
and
subsequent
addiEon
to
cis-­‐
aconitate
are
catalyzed
by
the
iron-­‐sulfur
center:
sensiEve
to
oxidaEve
stress.
• The
iron-­‐sulfur
center
acts
in
both
substrate
binding
and
catalysis.

22. Aconitase
is
a
“moonligh>ng”
enzyme
• When
Fe
is
deficient,
aconitase
loses
its
Fe-­‐S
center
and
acquires
a
new
role
in
Fe
homeostasis
• Cytosolic
Aconitase
is
an
enzyme
(with
Fe-­‐S)
and
a
regulator
of
protein
synthesis
(
–
Fe)
• In
humans
Fe
levels
must
be
regulated:
too
liKle
à
anemia;
too
much
à
liver
damage
• Transferrin:
carries
Fe
in
the
blood
• Transferrin
receptor:
receives
and
endocytoses
Fe
• Ferri*n:
stores
excess
Fe
inside
the
cells
• Apoaconitase
(
–
Fe)
regulates
protein
levels
by
stabilizing
or
destabilizing
the
mRNA
of
transferrin
receptor
or
ferriEn
• Apoaconitase
à
êferriEn
and
é
TfR
synthesis
(the
results
would
be
an
increase
in
cellular
[Fe])

23. Oxida>ve
Decarboxyla>on
#2
(step
3)
• Carbon
is
lost
as
CO2
and
NADH
is
generated
• Carbon
lost
as
CO2
did
NOT
come
from
acetyl-­‐CoA
• OxidaEon
of
the
alcohol
to
a
ketone
• Transfers
a
hydride
to
NAD+
• Cytosolic
isozyme
uses
NADP+
as
a
cofactor
• Highly
thermodynamically
favorable/irreversible
• Regulated
by
product
inhibiEon
and
ATP

24. Final
Oxida>ve
Decarboxyla>on
(step
4)
• Last
oxidaEve
decarboxylaEon
– Net
full
oxidaEon
of
all
carbons
of
glucose
• Succinyl-­‐CoA
is
another
higher-­‐energy
thioester
bond
• Highly
thermodynamically
favorable/irreversible
– Regulated
by
product
inhibiEon

25. Origin
of
C-­‐atoms
in
CO2
Both
CO2
carbon
atoms
are
derived
from
oxaloacetate
We have lost 2 CO2 already, so we have a net complete oxidation of
glucose after two pyruvates go through the CAC.
But its not the actual carbons from pyruvate (in red) in each cycle.
H2C
C
H2C
HO
COOH
COOH
COOH
Citrate Isocitrate
H2C
HC
C
H
COOH
COOH
COOH
HO
α-ketoglutarate
H2C
CH2
C
COOH
COOHO
Succinyl-CoA
H2C
CH2
C
COOH
SCoAO

26. α-­‐Ketoglutarate
Dehydrogenase
• Complex
similar
to
pyruvate
dehydrogenase
– Same
coenzymes,
idenEcal
mechanisms
– AcEve
sites
different
to
accommodate
different-­‐sized
substrates
– E1
aa
sequences
differ
(and
specificity)
E2
are
very
similar
E3
are
idenEcal

27. Genera>on
of
GTP
through
Thioester
(step
5)
• Substrate
level
phosphorylaEon
• Energy
of
thioester
allows
for
incorporaEon
of
inorganic
phosphate
into
ADP
or
GDP
to
make
ATP
or
GTP
• Goes
through
a
phospho-­‐enzyme
intermediate
• Produces
GTP,
which
can
be
converted
to
ATP,
or
ATP
directly
(2
isozymes
in
animal
cells,
specific
for
GDP
or
ADP)
• Slightly
thermodynamically
favorable/reversible
– Product
concentraEon
kept
low
to
pull
forward

28. Oxida>on
of
an
Alkane
to
Alkene
(step
6)
• Bound
to
mitochondrial
inner
membrane
– Complex
II
in
the
ETC
• OxidaEon
of
the
alkane
to
alkene
requires
FAD
• FAD
is
covalently
bound
• 3
Fe-­‐S
clusters
• Near
equilibrium/reversible
– Product
concentraEon
kept
low
to
pull
forward

29. Hydra>on
Across
a
Double
Bond
(step
7)
• Highly
stereospecific
– AddiEon
of
water
is
always
trans
and
forms
L-­‐malate
– Cannot
work
on
maleate
(cis
isomer
of
fumarate)
– OH-­‐
adds
to
fumarate…
then
H+
adds
to
the
carbanion
– Cannot
disEnguish
between
inner
carbons,
so
either
can
gain
–OH
• Slightly
thermodynamically
favorable/reversible
– Product
concentraEon
kept
low
to
pull
reacEon
forward

30. Oxida>on
of
Alcohol
to
a
Ketone
(step
8)
• Final
step
of
the
cycle
• Regenerates
oxaloacetate
for
citrate
synthase
• Highly
thermodynamically
UNfavorable/reversible
– Oxaloacetate
concentraEon
kept
VERY
low
by
citrate
synthase
(
<
10–6
M)
• Pulls
the
reacEon
forward

31. One
Turn
of
the
Citric
Acid
Cycle

32. Net
Result
of
the
Citric
Acid
Cycle
• Net
oxidaEon
of
two
carbons
to
CO2
– Equivalent
to
two
carbons
of
acetyl-­‐CoA
– but
NOT
the
exact
same
carbons
• Energy
captured
by
electron
transfer
to
NADH
and
FADH2
• Generates
1
GTP,
which
can
be
converted
to
ATP
Acetyl-­‐CoA
+
3NAD+
+
FAD
+
GDP
+
Pi
+
2
H2O
à
2CO2
+
3NADH
+
FADH2
+
GTP
+
CoA
+
3H+

33. Direct
and
Indirect
ATP
Yield

35. CAC
is
an
amphibolic
pathway
• Amphibolic-­‐
serves
in
both
catabolism
and
anabolism
• Precursors
for
many
molecules
• Needs
to
be
replenished
(by
anapleroEc
reacEons)
Red
arrows

36. Anaplero>c
Reac>ons
• Must
replenish
the
intermediates
in
order
for
the
cycle
and
central
metabolic
pathway
to
conEnue
• 4-­‐carbon
intermediates
are
formed
by
carboxylaEon
of
3-­‐carbon
precursors
• The
replenishing
and
consuming
reacEons
are
in
dynamic
balance
([CAC
intermediates]
is
~
constant)

37. Anaplero>c
Reac>ons
• Must
replenish
the
intermediates
in
order
for
the
cycle
and
central
metabolic
pathway
to
conEnue
• 4-­‐carbon
intermediates
are
formed
by
carboxylaEon
of
3-­‐carbon
precursors
• The
replenishing
and
consuming
reacEons
are
in
dynamic
balance
([CAC
intermediates]
is
~
constant)
• Regulatory
enzyme
–
inac*ve
in
the
absence
of
acetyl-­‐CoA
• More
acetyl-­‐CoA,
more
acEvity
è
more
OAA
to
react
with
acetyl-­‐CoA
to
start
the
cycle

38. Bio>n
is
a
CO2
carrier
• Vitamin
B7
(bioEn)
is
required
in
our
food
• It
is
a
cofactor
(prostheEc
group)
in
carboxylases
• BioEn
is
a
specialized
carrier
of
1-­‐C
groups
in
their
most
OXIDIZED
state
(CO2)
• Pyruvate
carboxylase
has
4
idenEcal
subunits
each
carrying
a
molecule
of
bioEn
• It
is
present
in
many
foods
and
intesEnal
bacteria
are
able
to
synthesize
it,
hence
bioEn
deficiency
is
rare
• ConsumpEon
of
raw
eggs
in
large
quanEEes
leads
to
bioEn
deficiency
since
egg
white
have
the
protein
avidin
which
binds
bioEn
very
Eghtly
and
prevents
its
absorpEon
in
the
intesEne

39. Biological
tethers
allow
flexibility
• All
enter
the
cells
on
the
same
transporter
• All
are
covalently
aKached
to
proteins
• All
provide
flexible
arms
on
the
enzymes
to
which
they
are
covalently
bound
• All
act
as
tethers
that
move
intermediates
from
one
acEve
site
to
the
next

40. Regula>on
of
the
Citric
Acid
Cycle
Cell
has
high
energy
demands!
Cell
is
supplied
with
enough
energy
[NADH]/[NAD+]
and
[ATP]/[ADP]
are
high

41. Regula>on
of
the
Citric
Acid
Cycle
• Regulated
at
highly
thermodynamically
favorable
and
irreversible
steps
– PDH,
citrate
synthase,
IDH,
and
αKDH
• General
regulatory
mechanism
– AcEvated
by
substrate
availability
– Inhibited
by
product
accumulaEon
– Overall
products
of
the
pathway
are
NADH
and
ATP
• Affect
all
regulated
enzymes
in
the
cycle
• Inhibitors:
NADH
and
ATP
• AcEvators:
NAD+
and
AMP
• Ca2+
in
muscles
acEvates
the
cycle
(Ca2+
signals
muscle
contracEon
è
need
for
energy)

42. Regula>on
of
Pyruvate
Dehydrogenase
• Also
regulated
by
reversible
phosphorylaEon
of
E1
– PhosphorylaEon:
inacEve
– DephosphorylaEon:
acEve
• PDH
kinase
and
PDH
phosphatase
are
part
of
mammalian
PDH
complex
– Kinase
is
acEvated
by
ATP
• High
ATP
à
phosphorylated
PDH
à
less
acetyl-­‐CoA
• Low
ATP
à
kinase
is
less
acEve
and
phosphatase
removes
phosphate
from
PDH
à
more
acetyl-­‐CoA
– Phosphatase
is
acEvated
by
insulin,
PEP,
and
AMP,
and
inhibited
by
ATP,
NADH,
and
Acetyl-­‐CoA

43. Addi>onal
Regulatory
Mechanisms
• Citrate
synthase
is
also
inhibited
by
succinyl-­‐CoA
– α-­‐ketoglutarate
is
an
important
branch
point
for
amino
acid
metabolism
– Succinyl-­‐CoA
communicates
flow
at
this
branch
point
to
the
start
of
the
cycle
• RegulaEon
of
isocitrate
dehydrogenase
controls
citrate
levels
– Aconitase
is
reversible
– InhibiEon
of
IDH
leads
to
accumulaEon
of
isocitrate
and
reverses
aconitase
– Accumulated
citrate
leaves
mitochondria
and
inhibits
PFK-­‐1
in
glycolysis

44. CAC
muta>ons
lead
to
cancer
• MutaEons
in
CAC
enzymes
are
very
rare
in
man
• GeneEc
defects
in
fumarase
à
smooth
muscle
and
kidney
cancer
• MutaEons
in
succinate
DH
à
tumors
of
the
adrenal
glands
• Both
enzymes
are
defined
as
tumor
suppressor
genes
• IDH
mutaEon
leads
to
a
new
funcEon
of
the
enzyme
the
net
result
of
which
is
the
development
of
glial
cell
tumors
in
the
brain

45. Ques>on
5
(Take
home
exam)
Due:
NEXT
WEEK
(js>ban@birzeit.edu)
• Please
solve
ques>ons:
1. 5
(NAD
redox
carriers)
2. 10
(OAA
in
mito)
3. 18
(14C-­‐glucose)
4. 19
(Beriberi)
For
wri9en
answers,
I
prefer
to
have
them
typed
in
Word.
I
can
accept
the
assignment
in
one
file
sent
to
my
email.
For
answers
that
require
solving
mathema3cally,
you
can
either
type
them
or
write
them
down
and
scan
them.