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Biochemistry I
Citric Acid Cycle
CO2
CO2
Oxidative
Decarboxylations
and Energy Output
Chapter 17 – part 2
Isocitrate Dehydrogenase
http://www.wiley.com/college/fob/quiz/quiz16/16-2.html
Dr. Ray
Key Concept Map For
Citric Acid Cycle (TCA)
Is TCA cycle an aerobic or anaerobic pathway?
Strictly
aerobic
Cellular Respiration:
1. TCA is first stage, with removal of high
energy electrons from carbon fuels.
2. Electrons reduce O2 to generate a proton
gradient across Inner Mitochondrial Membrane
3. H+ gradient is used to synthesize ATP.
• Oxidative Phosphorylation involves
steps (2) and (3).
Ref: Lippincott's Illustrated Reviews: Biochemistry, 3rd Ed., Fig 9.10
Step 1: Citrate Synthase Mechanism – Summary
• Brings substrates into _____________________________________
close proximity, orienting them, & polarizing bonds
• Uses general _________________________
acid-base catalysis
• The hydrolysis of the _____________
powers the synthesis of a
thioester
new molecule from 2 precursors (C-C bond formation)
1. How is the wasteful hydrolysis of acetyl CoA prevented (since this also
has a high-energy thioester linkage)?
Citrate synthase is well suited to hydrolyze citryl CoA, not acetyl CoA.
Induced fit conformational changes prevent undesirable side reactions
Oxaloacetate + Acetyl CoA + H2O  Citrate + CoASH
CO2CH2
HO
OH2
C
CO2-
CH2
CO2-
+
CoASH
Step 2: Aconitase - Isomerization
1) Can a tertiary ROH be oxidized?
2) Can a secondary ROH be oxidized?
• The tertiary hydroxyl group is not properly located in the citrate molecule
for the oxidative decarboxylations that follow, so it is moved in this step.
• Isomerization of citrate to isocitrate:
1. moves 3o alcohol on C3 up to form a 2o alcohol on C2
2. alcohol oxygen is now beta to carboxylate (COO-) attached to C3,
which is needed to facilitate oxidative b-decarboxylation in next step
Dehydration
Rehydration
3o
at C3
Isomerization by ACONITASE always moves the
central alcohol AWAY from acetyl-CoA side
• Aconitase is a metalloenzyme with a
[4Fe-4S] iron-sulfur cluster at the active site
2o
at C2
The Citric Acid Cycle Problems:
2. What statement is NOT correct about the citrate synthase reaction in
the citric acid cycle?
A) its products include coenzyme A and citrate
B) it forms a tricarboxylic acid
C) its substrates include acetyl-CoA and oxaloacetate
D) it is coupled to the hydrolysis of ATP
E) acetyl CoA is the nucleophile in this reaction
3. The isomerization of citrate to isocitrate:
A) is the only unnecessary step of the citric acid cycle.
B) protects cells from the toxic effects of carbon monoxide (CO).
C) converts a tertiary alcohol, which cannot easily be oxidized, to a
secondary alcohol that can be oxidized.
D) is a reduction reaction.
E) is an oxidation reaction.
- Alcohol oxidation will occur in next step
Step 3: Isocitrate Dehydrogenase – oxidative decarboxylation
6C Isocitrate  5C a-Ketoglutarate + CO2
The oxidative b-decarboxylation of isocitrate is catalyzed by:
Isocitrate Dehydrogenase:
C1
C2
C3
C4
b-keto acid
C1
b
b
oxidation
a
C2
C3
b-decarboxy
lation
• The rate of formation of a-ketoglutarate is important in determining the
overall rate of the citric acid cycle.
• This oxidation generates the first high energy electron carrier (NADH)
in the cycle (energy extracted via oxidation).
 The CO2 that is removed began the cycle as part of oxaloacetate
(labeled C1, C2, C3, C4 above), not as part of acetyl CoA.
Electrostatic Catalysis:
b-Decarboxylation Reactions Require an Electron Sink
Mechanism of Covalent catalysis via Schiff base (imine) formation:
1. Uncatalyzed Rxn
mechanism:
• breaking Ca-Cb
a b
bond requires
nearby carbonyl as
an electron sink
2. Chemically
1) How can energy
catalyzed
of anionic enolate
mechanism:
be lowered?
a primary amine
that forms a:
positively (+)
charged imine
ELECTRON SINK
2) Why is lower reaction
faster than upper reaction?
Many decarboxylation reactions occur in the Citric Acid Cycle, and most
have an electron sink like a positively charged Schiff Base !
Step 3: Isocitrate Dehydrogenase
Loss of CO2 (Mechanism in Chapter 15)
b-keto carboxylic acid
carbanion + carbon dioxide
ketone
O
H2C
b-carbon
A
--H2C
O
Oxidative
b-decarboxylation
ketone + CO2
O
B
H3C
O
-
O
O C O
O C O
A. Loss of CO2 from a beta-keto acid or equivalent; Only the Ca-Cb bond
can by cleaved because the resulting carbanion is resonance stabilized
as an enolate; carbanion only stable adjacent (alpha) to a carbonyl
• Since carbanions alpha to carbonyl are stabilized by resonance, the
b-keto acid can cleave to give this carbanion.
Thus only Ca-Cb bonds are cleaved !
B. Reprotonation of the carbanion C by an acid at the active site
• When the CO2 is lost, it leaves as a GAS; this reaction is “irreversible”
for all practical purposes
• Since this reaction is “irreversible,” Nature has had to design another mechanism
for adding CO2 to molecules. In almost all cases, addition of CO2 uses the
biotin cofactor and requires energy in the form of ATP.
Step 3: Isocitrate Dehydrogenase
Mechanism of Oxidative b-decarboxylation
Know
Produces first CO2 and NADH of citric acid cycle via two steps: mechanism
1. Oxidation of 2o alcohol at C2 to a ketone to form oxalosuccinate (betaketo acid), and reduction of NAD+ to NADH
2. Next undergoes b-decarboxylation of carboxylate (beta) to ketone at C2
3. Mn2+ stabilizes enolate O-, which protonates to enol then tautomerizes to
more stable ketone forming a-ketoglutarate
C5
C4
C3
C2
C1
Earlier isomerization by aconitase always moves
the central alcohol AWAY from acetyl-CoA side
Came from
Acetyl CoA
C6
1
2
3
- flow)
1)
_________
double
bond
(encourage
e
Mn2+ function? 2) _________ intermediate O(-) charge
C5
C4
C3
C2
C1
Mechanism of
Isocitrate Dehydrogenase
oxidative b-decarboxylation
(A)
(D)
(B)
(E)
Isocitrate + NAD+
 a-Ketoglutarate + NADH + CO2
Q: In what order do these steps occur?
(C)
(F)
Lehninger – Principles of Biochemistry, 5th Ed,
animations (chapter 16), by Nelson and Cox,
2008 W. H. Freeman & Company
Step 4: a-Ketoglutarate Dehydrogenase
Oxidative a-decarboxylation
5C a-Ketoglutarate  4C Succinyl-CoA + CO2
• Produces second CO2 and NADH of citric acid cycle, by another oxidative
decarboxylation step but this time removing the carboxy group a- to C=O
• The substantial energy released upon oxidation of C1 carbon from
ketone to a carboxylic acid derivative, is stored as:
(1) a reduced NADH
(2) a ‘high energy’ thioester (a carboxylic acid derivative)
• a-decarboxylation of terminal carboxyl is much more complx than
b-decarboxylation, so catalyzed by a multienzyme complex homologous to
pyruvate dehydrogenase PDHC (do NOT need to know mechanism)
1) Which step in glycolysis does this most resemble, in terms of
energy extraction and recovery?
where aldehyde
oxidation forms high
energy acyl phosphate
(1,3-BPG), a molecule
that allows substrate
level phosphorylation
Step 5: Succinyl-CoA Synthetase:
Substrate-level Phosphorylation
Succinyl-CoA + GDP + Pi  Succinate + GTP + CoASH
• Succinyl CoA is an energy-rich thioester compound:
(1) DG° for the hydrolysis of
succinyl CoA is about - 33.5 kJ/mol
(2) DGo’ for hydrolysis of ATP
to from ADP + Pi is - 30.5 kJ/mol
1) Can succinyl CoA be used for
substrate level phosphorylation?
• The cleavage of the thioester bond of succinyl CoA is coupled to the
phosphorylation of a purine nucleoside diphosphate GDP, in a reaction
catalyzed by succinyl CoA synthetase (succinate thiokinase)
• Subsequently, the g-phosphoryl group can be readily transferred
from GTP to ADP to form ATP, in a reaction catalyzed by
nucleoside diphosphokinase (an energy neutral reaction):
DGo’ = 0
Step 5: Succinyl-CoA Synthetase Mechanism
1.
2.
3.
4.
Exergonic thioester hydrolysis is coupled to endergonic substratelevel phosphorylation of GDP to GTP by formation of several ‘highenergy’ compounds which conserve the initial thioester energy.
Phosphorolysis (Pi is nucleophile) of high-energy thioester to form highenergy succinyl-phosphate
Transfer phosphate to His to form high energy covalent phospho-His
intermediate and succinate
Substrate-Level
Movement of phosphorylated His near to GDP
Phosphorylation
Transfer phosphate to GDP to form GTP
Phosphorolysis
Know Mechanism
and Energy
Transformations
Regeneration of Oxaloacetate
Introduce oxygen as -OH, which is then transformed into a C=O
6
7
Oxidation
Hydration
8
Oxidation
Remaining three reactions of the citric acid cycle (steps 6, 7 & 8),
all involve 4C compounds and constitute the final stage of the
citric acid cycle - the regeneration of oxaloacetate.
1. Oxaloacetate is regenerated allowing another round of the citric
acid cycle to occur
2. More energy is extracted by two more oxidations, in the form of
one FADH2 and one NADH
Many Metabolic Enzymes Require Cofactors
Holoenzyme = Cofactor + Apoenzyme
Holoenzyme only has biological activity when cofactor is bound.
Cofactor (or coenzyme) = small non-amino acid molecule required for the
catalytic activity of an enzyme. Often derived from dietary vitamins and
minerals. Cofactor can be an organic molecule, metal ion, or
organometallic complex. Cofactors can be either:
• Cosubstrate = small organic molecule that associates only transiently
with an enzyme. Later associates with another enzyme.
• Prosthetic Group = a molecule that is permanently and tightly bound
to an enzyme (often, but not always covalently).
 NADH and FADH2 are MOBILE electron carriers essential for metabolism!
• NAD+ and NADP+ shuttle reducing equivalents
throughout metabolism, are derived from niacin.
• They are cosubstrates for dehydrogenase enzymes.
• Once reduced, they transport electrons released by
fuel oxidation (glucose metabolism) into the mitochondrial electron transport chain for ATP synthesis.
NADH: Activated Carrier of Electrons for Fuel Oxidation
NADH is an activated carriers of electrons during fuel oxidation:
Reactive site
• Oxidation of ROH to RCHO or RCOR’
• Reduction of NAD+ to NADH
NADH – is used in catabolic reactions
uses free energy of metabolite oxidation
to generate ATP, the cell’s “energy
currency”
NADPH – in anabolic reactions is used
as “second energy currency = reducing
power” for driving biosynthesis reactions
In the oxidation of a substrate, the nicotinamide ring of NAD+ accepts a
hydrogen ion and two electrons, which are equivalent to a hydride ion (H:-)
NAD+ + 2e- + H+  NADH
NADH Oxidation and Reduction: H2 Removal & Addition
R
H
A
R
O
OH
R
R
Oxido-reductase
Overall reaction:
H
H
CONH2
+
N
R
•
•
•
•
NAD+
H
H
R C R'
OHH
Base :
CONH2
+
N
R
NADH
+
R C R' + H
O
Dehydrogenases
TRENDS:
(H:-) hydride ion (H+ + 2e-) can be transferred to NAD+ (NADP+) or flavin
Driving force in the “forward” direction is electrostatic (charge
neutralization), attraction of hydride (negative charge) and pyridinium moiety
of cofactor (which has a positive charge)
Driving force in “reverse” direction is the resonance gained when the
pyridinium becomes aromatic
These two driving forces balance one another so that the reaction can be
“pushed” one way or the other by the protein structure:
NAD+ ⇆ NADH reaction can occur in both directions !
Roles of two redox cofactors NAD+ & FAD in metabolism
• Both NAD+/NADH and FAD/FADH2 must undergo reversible redox
reactions as they function in electron transfer by accepting electrons, then
passing them on to other electron carriers. Thus they are regenerated and
reused in additional metabolic cycles.
1. When is FAD used instead of NAD+ as an oxidizing agent in
metabolic reactions?
• In catabolism, NAD+ functions biochemically in quite exergonic oxidation
of alcohols to aldehydes or ketones, oxidation of these to carboxylic acids,
and reactions involving decarboxylations (both a and b ).
• In catabolism, FAD functions biochemically to oxidize alkanes (such as
succinate) to alkenes (such as fumarate), a simple dehydrogenation.
2. When is NADP+ used instead of NAD+ in metabolic reactions?
• NADPH/NADP+ are used in anabolic (reductive) pathways (biosynthesis)
Step 6: Sucinnate Dehydrogenase: oxidation
Succinate + FAD  Fumarate + FADH2
• Stereospecific dehydrogenation of 4C succinate to 4C fumarate
• Metabolite oxidation, since loss of H2
OUT
IN
• The FADH2 produced by the oxidation of succinate does not dissociate
from the enzyme, but electrons are transferred directly from enzyme-bound
FADH2 into the electron transport chain.
• Succinate dehydrogenase (complex II) is an integral membrane protein
containing an iron-sulfur prosthetic group. The protein is attached to the
inner mitochondrial membrane (IMM) and feeds electrons directly into
the electron-transport chain, the link between the citric acid cycle and
ATP formation (Chapter 18 – Oxidative Phosphorylation).
Step 6: Sucinnate Dehydrogenase:
Succinate + FAD  Fumarate + FADH2
• FAD is reduced as succinate is oxidized.
• This is the only membrane-bound enzyme of the citric acid cycle, with
a covalently linked prosthetic group (which is unusual).
• This is also the only step in oxidative glucose catabolism to use FAD.
1. FAD is a dinucleotide linked to Adenine
by two phsophate groups.
2. Note FAD, NAD+, CoA and ATP all
contain a adenosine monophosphate unit
(AMP, in red)
• Humans must obtain riboflavin in their
diets as vitamin B2
• The oxidative decarboxylation
of pyruvate (PDHC) and the
sequence of reactions in the
citric acid cycle all take place
within the matrix (inner
compartment) of mitochondria.
Step 7: Fumarase
hydration
4C Fumarate  4C Malate
• Fumarase catalyzes hydration of fumarate to malate
• Reaction involves addition of H2O (OH-/H+) across a double bond,
via a carbanion transition state
• Note this reaction is stereospecific as a new chiral center is
produced in malate
 forms only L-malate (not D-malate)
Step 8: Malate Dehydrogenase oxidation
Malate + NAD+  Oxaloacetate + NADH
• oxidize 2o alcohol at C2, to a ketone to regenerate oxaloacetate
• produce third NADH in one turn of citric acid cycle
C2
1. How many turns of the citric acid cycle will result from the
complete oxidative catabolism of one glucose molecule?
1 glucose  2 pyruvate  2 Acetyl CoA (+ 2CO2)
Regeneration of Oxaloacetate
• The last 3 reactions of the citric acid cycle constitute a metabolic motif
that is also used in fatty acid synthesis and degradation as well as in the
degradation of some amino acids (see end of Chapter 15).
• A methylene group (CH2) is converted into a carbonyl group (C = O) in
three steps: an oxidation, a hydration, and a second oxidation reaction
Oxidation
forming FADH2
Hydration
Oxidation
forming NADH
The Citric Acid Cycle Problems:
Wkbk SelfTest #8,
(Wkbk Pbm # 5)
1. Given the biochemical intermediates of the pyruvate dehydrogenase
reaction and the citric acid cycle shown, answer the following questions.
(a) Name the intermediates A and B
A=
B=
(b) In isocitrate, which atoms come
1
2
from acetyl CoA?
8
B
2
7
3
6
5
4
A
(c) Which reaction is catalyzed by
a-ketoglutarate dehydrogenase?
(d) Which enzyme catalyzes step 1?
(e) Which reactions are oxidations?
Name the enzyme that catalyzes
each of them.
The Citric Acid Cycle Problems:
Wkbk SelfTest #8,
(Wkbk Pbm # 5)
1. Given the biochemical intermediates of the pyruvate dehydrogenase
reaction and the citric acid cycle shown, answer the following questions.
(f) At which reaction does substratelevel phosphorylation occur?
Name the enzyme and the
products of this reaction.
1
8
2
B
2
(g) Which of the reactions require an
FAD cofactor? Name the enzyme
(h) Indicate the decarboxylation
reactions and name the enzymes.
7
3
6
5
4
A
http://www.wiley.com/college/fob/quiz/quiz16/16-2.html
The Citric Acid Cycle Problems:
1. Which of the following best describes the net organic products
formed during the oxidation of ONE molecule of glucose to six
molecules of CO2 during glucose catabolism (including glycolysis,
pyruvate dehydrogenase complex, and citric acid cycle reactions)?
A)
B)
C)
D)
E)
4 ATP + 8 NADHmatrix + 2 NADHcytosol + 2 FADH2
6 ATP + 10 NADH
4 ATP + 8 NADH + 2 FADH2
2 FADH2 + 8 NADHmatrix + 2 NADHcytosol
4 ATP + 10 NADHmatrix + 2 FADH2
TCA Provides Intermediates for Biosynthesis
As a major metabolic hub of the cell, the citric acid cycle
is central pathway in catabolism in oxygenated environment (aerobic),
also provides intermediates for biosynthesis  Anabolism
Examples: Most of the carbon atoms in porphyrins come from succinyl CoA.
• Many of the amino acids are derived from a-ketoglutarate & oxaloacetate.
• Citrate is a starting material for fatty acid and cholesterol biosynthesis.
• The important point now is that
citric acid cycle intermediates must
be replenished if any are drawn off
for biosynthesis.
• Suppose that a lot of oxaloacetate
is converted into amino acids for
protein synthesis and, subsequently,
the energy needs of the cell rise.
• Citric acid cycle will operate to a reduced extent unless new oxaloacetate is formed,
because acetyl CoA cannot enter the cycle unless it condenses with oxaloacetate.
Even though oxaloacetate is recycled in TCA,
a minimal level must be maintained to allow the
citric acid cycle to function.
Amphibolic Functions of the Citric Acid Cycle
1. Do anabolic reactions
require free energy to
occur?
2. For catabolism of Acetyl CoA, are
the cellular concentrations of OAA
and Acetyl CoA the same?
• ________________ amounts of
TCA intermediates are needed to
maintain the degradative
(catabolic) function of the cycle,
with release and conservation of
free energy.
3. Is there NET production of OAA
during the Citric Acid cycle?
TCA is both catabolic and anabolic
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fob/quiz/quiz16/16-15.html
• In a reaction outside TCA, pyruvate (3C)
and CO2 (1C) can combine to replenish
4C oxaloacetate. This rxn is catalyzed
by pyruvate carboxylase.
Regulation of the Citric Acid Cycle
High Energy Charge:
large [ATP]/[AMP] ratio
Control of the Citric Acid Cycle:
• Regulated primarily by the
concentrations of ATP and NADH
(final products of pathway)
• Key control points in TCA are
the two reactions with the
largest driving force (DG’):
isocitrate dehydrogenase
a-ketoglutarate dehydrogenase
- If cell’s energy charge
[ATP]/[AMP] is high (because
excess ATP is present) then these
two enzymes and the entire TCA
pathway is downregulated via
_______________________.
Regulation of the Citric Acid Cycle
Low Energy Charge:
small [ATP]/[AMP] ratio
During exercise use a lot of ATP, so
entire TCA pathway is up-regulated.
If high rate of TCA then need more:
• Oxaloacetate - produced from pyruvate
(by pyruvate carboxylase)
• Acetyl CoA - produced from either
- pyruvate (by PDHC) or
- fatty acids (by b-oxidation pathway)
Stereochemistry and the Citric Acid Cycle
Oxaloacetate  Citrate  a-Ketoglutarate
• Citrate is prochiral, and aconitase (in step 2) can
interact asymmetrically with the substrate and
distinguish between its two carboxymethyl groups:
C1 of citrate is different from C5
• Enzymes are chiral molecules made up of L-amino acids,
so they can catalyze asymmetric reactions!
• Only one carboxy methyl will fit in enzyme active site
once the COO- and OH groups are bound to the enzyme.
1. If C4 of oxaloacetate is radioactively labeled with 14C, at top C1 of aKG
where will the label end up in a-ketoglutarate?
Note 2 carbons that go in as acetyl CoA (green) are NOT
the two that come out in one turn of TCA.
C1
C2
C3
C4*
C1*
C1*
C2
C2
C3
2
C3
C4
C4
C5
C5
at top C1 of aKG
Citric Acid Cycle :
Isotope Labeling Problems
Oxaloacetate
CH2
-OOC
C O
+ CH3
1
H2O
COO- 1
CH2
2=
-OOC C OH 3 -OOC
CH2
4
C
C
O
b-decarboxylation
CoASH
COO-
O
S CoA
COOCH2
C OH
5
COOH C OH
2
3
-OOC C H
CH2
CH2
COO-
COO- [3a]
OCitrate
substrate level
phosphorylation
GTP +
CoASH
CoA
CO2
S
C O
4
CH2
CH2
COOC O
CH2
[3b]
COO-
a-Ketoglutarate
CH2
CoASH
5
CH2
=
Succinyl CoA
COO- 1
2
CH2
CH2
COO-
2nd CO2
1. Start with
label at C4 of
Oxaloacetate.
Q: Where is
label in a-keto
glutarate?
3
COO- 4
CH2
GDP + Pi
COO-
a-Ketoglutarate
Oxalosuccinate
OC O
CH2
COO-
Succinate
rotate OAA
H2O
COOCH2
6
H
Succinate
COOC
CH2
COO-
1
2
3
4
5
COO-
a-decarboxylation
COOC O
O
H C H
C C H
-O CH2
Isocitrate
Acetyl CoA
1
2
3
4
5
COOC O
CO2
7
HO C H
CH2
C
-OOC
COO-
H
Fumarate
COOMalate
8
1
COO2 O C
3
CH2
4
COO-
=
COO- 4
CH2 3
-OOC
C O 2
1
Oxaloacetate
Stereochemistry and the Citric Acid Cycle
Succinyl CoA  Succinate  Fumarate
Scrambling of 14C at C4 of succinate, occurs
during the succinate dehydrogenase reaction (step 6)
• Succinate and Fumarate have 2-fold rotational symmetry (plane of
symmetry), so they are NOT prochiral, and terminal atoms are scrambled.
Substrate can enter enzyme active site with either terminal atom at bottom.
Thus a 14C label on C4 of succinate will be scrambled, and appear at
both C1 and C4 of fumarate and malate (for half the molecules of each)
Label EITHER at C1* or C4*, NOT both
C1
C2
C3
C4*
=
C1*
C2
C3
C4*
C1*
C2
C3
C4*
2. If C1 (carbonyl) of Acetyl CoA is labeled with 14C, where will the label
end up in fumarate & malate after 1 turn of TCA? How many positions
in a particular molecule of malate will be labeled?.
One C labeled: half molecules labeld at C1 and half labeled at C4
Citric Acid Cycle : Isotope Labeling Problem
Oxaloacetate
CH2
1
C O
-OOC
+ CH3
#C
O
b-decarboxylation
CoASH
COO-
H2O
COO- 1
CH2
2=
-OOC C OH 3 -OOC
CH2
4
#C
O
S CoA
COOCH2
C OH
COOH C OH
2
CH2
CH2
# COO- [3a]
Citrate
COO-
substrate level
phosphorylation
a-decarboxylation
S
C O
4
CH2
CH2
CoASH
# COO-
# COO-
a-Ketoglutarate
5
CH2
1
CH2 2
CH2 3
COO- 4
Succinate
6
C
#
2
3
COO- 4
CH2
Succinate
H2O
# COO-
7
HO C H
CH2
C
-OOC
CH2
symmetric molecule so label
# 50% at C1, and 50% at C4
#COO-
H
#COO- 1
# COO-
Succinyl CoA
#COO-
=
CH2
GDP + Pi
# either at C1 or
at C4, not at both
#
OC O
CH2
CH2
OR
Oxalosuccinate
GTP +
CoASH
CoA
CO2
COOC O
COOC O
C C H
-O CH2
Isocitrate
Acetyl CoA
1
2
3
4
5
O
COOC O
1
H C H 2
3
CH2
[3b] # COO- 4
5
a-Ketoglutarate
3
-OOC C H
# COO-
5
O-
CO2
H
Fumarate
8
2. Start with label
# (C=O) of
at C1
AcetylCoA.
Where is label in
oxaloacetate?
One C labeled:
half molecules at C1
and half at C4
1 # COO2 O C
3
CH2
4
COO-
#
=
COO- 4
CH2 3
-OOC
C O 2
1
#
#
# COOMalate
Second round of TCAOxaloacetate
starts with OAA
labelled on ½ molecules at C1 and ½ at C4
(a)
COO
#C
(d)
-
O
S-CoA
#
CH3
C
O
CH3
Indicates new
C-C bond
#
=
+ CO2
1
#
#
2
1
Pyruvate Dehydrogenase Complex
2
#
#
OAA labeled
either at
C1* or C4*
NOT both
2. Start with
label # at C1
(C=O) of
AcetylCoA.
Half molecules
of OAA labeled
at C1 and half
at C4
8
#
#
#
Q: Which atoms
(and to what percent)
is OAA labeled?
7
4
#
#
#
#
14C
labeling in the
Citric Acid Cycle
problems
3
The two CO2 that come
off during one round of
the cycle are NOT the
two that go in as Acetyl
CoA in that round
6
#
5
Metabolic Fate of Pyruvate
3. If Pyruvate is isotopically labeled at its C3 (methyl) position with
14C, where will the radioactive label appear after one round of
the citric acid cycle?
C2 & C3 of OAA labeled,
50% label at each C
•
•
•
Will any labeled CO2 be released? No, CO2 comes from OAA
Answer by drawing out the product of the pyruvate dehydrogenase step,
and the key steps in the citric acid cycle from acetyl CoA to
oxaloacetate.
Indicate the labeled carbon in glucose with an asterisk (*) and trace this
label through every metabolite till oxaloacetate.
Citric Acid Cycle:
Oxaloacetate (4C)  Citrate (6C)
 a-Ketoglutarate (5C)  Succinyl-CoA (4C)  Succinate (4C)
 Fumarate (4C)  Malate (4C)  Oxaloacetate (4C)
*
(a)
(d)
COO-
S-CoA
C
C
O
O
=
1
*
CH3
3. What if we
start with
label at C2
(methyl) of
AcetylCoA.
*
Half molecules
of OAA labeled
at C2 and half
at C3
*
+ CO2
2
*
Pyruvate Dehydrogenase Complex
CH3
Isotope Labeling
Problem:
*
*
*
3
The two CO2 that come
off during one round of
the cycle are NOT the
two that go in as Acetyl
CoA in that round
*
*
*
Q: Which atoms
(and to what percent)
is OAA labeled?
7
4
*
*
*
14C
labeling in the
Citric Acid Cycle
problem
2
1
8
OAA labeled
either at C2*
or C3*
new
C-C bond
6
*
*
5