Summary Citric Acid Cycle

Chapter
16
-­‐
Citric
Acid
Cycle


TCA
(tricarboxylic
acid
cycle)
Citric
acid
cycle
and
Krebs
cycle.

Named
after
Sir
Hans
Krebs,
Nobel
Laureate.

He
worked
as
an

assistant
professor
for
Otto
Warburg
(Nobel
Prize
1931)
and
his
position
terminated
1933
and
at,
Sir
Fredrick
Gowland

Hopkin's
(Nobel
prize
1929)
request
he
left
Germany
to
hold
a
Rockefeller
Studentship
at
the
School
of
Biochemistry,

Cambridge.
In
1953
he
earned
the
Nobel
Laureate
in
Medicine
for
his
discovery
of
the
citric
acid
cycle

Discovery
of
the
cycle

•   Discovered
by
observing
the
reduction
of
compounds
in
muscle
tissue.

Certain
key
molecules
(succinate,
oxaloacetate)

served
as
catalysts
in
O2
consumption
and
oxidative
metabolism
of
glucose
and
pyruvate.

•   Szent-­‐Gyorgyi
determined
the
catalytic
affect
of
small
amounts
of
future
TCA
intermediates

•   Knoop
(also
key
in
fatty
acid
metabolism)
the
formation
of
citrate
form
OAA
and
Pyruvate

•   Krebs
found
a
cycle
of
reforming
catalytic
amount
of
oxaloacetate



The
Krebs
cycle
is
a
central
pathway
for
recovering
energy
from
three
major
metabolites:
carbohydrates,
fatty
acids,
and
amino

acids.



Most
enter
the
cycle
through
Acetyl~CoA.

The
two
carbons
entered
at
this
step
are
lost
as
CO2
(the
reason
you
breath
out
CO2).

The
carbon
atoms
that
enter
by
A-­‐CoA
leave
after
the
second
turn
of
the
cycle.




Synthesis
of
the
TCA

•   Reactions
take
place
in
mitochondria
-­‐
thus
transport
of
reactants
and
products
are
important

•   Overall
reaction
involves
the
entry
of
a
2
carbon
compound
(acetyl
CoA)
into
the
cycle
with
the
loss
of
2
CO2
and

formation
of
3
NADH,
FADH2
and
GTP
or
ATP.





Acetyl
CoA
+
2
O2
+12
ADP
+
12
Pi
-­‐>
2
CO2

+CoASH
+12
ATP
+
H2O

•   No
net
change
in
the
concentration
of
the
4
carbon
compound
oxaloacetate.

•   The
carbons
lost
as
CO2
are
from
previous
A-­‐CoAs
not
from
the
reactant
A-­‐Co



Think
of
why
this
is
a
cycle
vs.
pathway
-­‐
not
because
it
is
written
that
way.

-   Oxaloacetate
-­‐
only
a
small
amount
is
needed
-­‐
catalytic
role

-   Anapleurotic
-­‐
“filling
up”
cycle
can
be
used
as
entry
and
exit
for
production
of
other
essential
metabolites

The
TCA

•   Takes
place
in
mitochondria
in
the
matrix

•   Like
glycolysis
pathway,
the

TCA
is
highly
regulated

•   Sources
of
Acetyl
CoA
(another
cross
road
metabolite)

-   glycolysis
-­‐
via
PDH

•   ß
oxidation
of
fatty
acids

•   selected
amino
acids



Getting
there
-­‐
don’t
forget
PC!

Pyruvate
Dehydrogenase
(PDH)
-­‐
Entry
of
glucose
metabolites
into
cycle
is
through
formation
of
acetyl-­‐CoA
by
oxidative

decarboxylation
of
pyruvate

-   In
eukaryotes,
all
of
the
TCA
enzymes
and
the
PDH
are
found
in
the
mitochondria.

Either
in
the
inner
compartment
or

the
matrix
of
the
mitochondrion.



-   Pyruvate
is
made
in
the
cytosol
and
transported
by
a
H+
/
pyruvate
symporter.











PDH
Exists
as
large
multiunit
complex


§   Coenzymes
-­‐
Vitamin
B1-­‐
thiamine
pyrophosphate
(TPP),
panthanoic

+
acid
(CoA),
riboflavin
(FAD),
Niacin
(NAD )
and
lipoamide

•

(3
different
subunits)

E1
Pyruvate
Dehydrogenase
-­‐
24
copies
/
E2
Dihydrolipoyl
Translacetylase
-­‐
24

copies
/
E3
Dihydrolipoyl
Dehydrogenase
-­‐
12
copies

-­‐
increases
local
concentration
of
substrate
for
each
subunit


-­‐
multi-­‐enz
complexes
allows
little
chance
for
diffusion
and
side
reactions
and


direct
transfer
of
substrate
from
E1
to
E2
to
E3


The
three
enzymes
of
the
PDH


,






















5
catalytic
steps,
each
with
different

coenzymes.




1
E1-­‐Decarboxylation
of
pyruvate
-­‐
(condensation
with
TPP)

•   TPP
adds
to
carbonyl
carbon

•   carbanion
intermediate-­‐
necessary
to
attack
negative
charged
C=O
carbon

resonance-­‐stabilized
by
ring
of
thiamine
pyrophophate

2
E2

transfer
from
TPP
to
lipoamide
(amide
linkage)

•   2
lipoamides
involved

•   act
as
transfer/carrier
arm
for
acetyl
group
***
active
site
studies
show

distortion
for
disulfide
bond
to
form…

•   disulfide
(oxidized
form
is
converted
to
the
mercapto
(reduced)
form

3

E2

transfer
of
acetyl
group
to
CoA

4

E3
Dihydrolipoyl
dehydrogenase


•   oxidizes
the
amide
group
of
E2
by
the
reduction
of
the
Cys-­‐Cys
disulfide
bond
of
E3

5
E
3
is
reoxidized
by
NAD+
in
a
transient
reduction
involving
FAD
(bound
to
the
enzyme).

This
prepares
the
enzyme
for

another
round,
and
produces
reduced
NADH







Reaction
of
the
cycle

Citrate
Synthase
(CS)
-­‐
catalyzes
the
condensation
of
acetyl-­‐CoA
and
OAA
in
a
highly
exergonic
fashion.

There
is
a
substantial

conformational
change
in
the
enzyme
when
substrate
binds.
“hides”
water
from
the
active
site
and
then
forms
A-­‐CoA

binding
site.
-­‐
ordered
sequential
reaction



Citrate
Synthase
(CS)

-­‐aldol
condensation
of
Acetyl
CoA
and
oxaloacetate

-­‐involves
two
His
and
one
Asp

-­‐
ordered
reaction
leading
to
tertiary
changes

-­‐
induced
fit
caused
by
OAA
binding
forms
reactive
site















-­‐
order
of
binding
helps
stop
Acetyl
CoA
hydrolysis


-­‐
two
neutral
His
involved
in
catalysis

-­‐
His
donates
H
to
C=O
oxygen
of
Acetyl
CoA
and
OAA

-­‐
Asp
is
the
proton
acceptor

-­‐
loss
of
acetyl
CoA
CH3
hydrogen
to
Asp

-­‐
condensation
forms
CitrylCoA





Aconitase
-­‐
catalyzes
the
isomeration
of
citrate
to
isocitrate
via
steriospecific
dehydration
and
rehydration.

(a
two
step

reaction).