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BCHEM 253 – METABOLISM IN HEALTH AND DISEASES 1
Lecture 6
The Energetics of the Citric Acid Cycle
Christopher Larbie, PhD
Introduction
The 2 ATP’s produced during glycolysis are only a small fraction of the potential energy
available from glucose. Under anaerobic conditions, animals convert glucose into 2
molecules of lactate. Much of the potential energy of the glucose molecule remains
untapped. Under Aerobic conditions a much more dynamic pyruvate metabolism
occurs. The 2 moles of NADH produced by glyceraldehyde-3-phosphate
dehydrogenase are oxidized in the electron transport chain back to NAD +. The electron
transport chain generates a proton gradient that drives the synthesis of 5 ATP molecules
from ADP and Pi. Furthermore, the pyruvate formed by glycolysis is converted to
acetyl-CoA by pyruvate dehydrogenase (generating another 2 moles of NADH per
glucose and another 5 ATPs by oxidative phosphorylation). The acetyl -CoA formed
enters into the citric acid cycle where it is completely oxidized into CO 2 . The electrons
liberated by oxidation are captured by NAD+ or FAD which are then transferred via the
electron transport chain ultimately to O 2 , the final electron acceptor. The electron
transport chain is coupled to generating a proton gradient which produces a proton
motive force that drives the synthesis of ATP. This allows the net production of 32
molecules of ATP to be formed per glucose molecule. The function of the citric acid
cycle is to harvest high energy electrons from carbon fuels.
The citric acid cycle is the central metabolic hub of the cell, the gateway of aerobic
metabolism. The citric acid cycle produces intermediates which are precursors for fatty
acids, amino acids, nucleotide bases, cholesterol and porphyrins. The citric acid cycle is
shown below. The citric acid cycle occurs in the mitochondria of eukaryotes.
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New carbons enter the citric acid cycle through acetyl-CoA. The acetyl group may come
from pyruvate, fatty acids, ketone bodies, ethanol or alanine. The two carbons of acetylCoA are transferred to oxaloacetate to yield the first tricarboxylic acid citrate in a
reaction catalyzed by citrate synthase. A dehydration followed by a rehydration
rearranges citrate into isocitrate. Two successive decarboxylations coupled to the
generation of 2 NADH produce succinyl-CoA. Four steps later, oxaloacetate is
regenerated along with a GTP, FADH2 and NADH.
The citric acid cycle may seem like an elaborate way to oxidize acetate into carbon
dioxide, but there is chemical logic to the cycle. In order to directly oxidize acetate into
two molecules of CO 2 , a C—C bond must be broken. Under the mild conditions found
in cells, there is insufficient energy to break the bond. Biological systems often break
C—C bonds between carbon atoms α and β to a carbonyl group. Examples are aldolase
and transaldolase.
Another common type of C—C cleavage is α-cleavage of an α-hydroxy-ketone which
we have seen before in transketolase and pyruvate decarboxylase, two TPP dependent
enzymes.
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Neither of these common strategies for cleavage of C—C bonds is available to acetate.
Instead the acetate is activated in the form acetyl-CoA, condensed with oxaloacetate to
form citrate and then carrying out a β-cleavage in subsequent steps.
The Net reaction of the citric acid cycle is:
3NAD+ + FAD + GDP + Pi + Acetyl-CoA + 2H2 O → 3NADH + FADH2 + GTP + CoA +
2CO2 + 3H+
The Aerobic Fate of Pyruvate
In eukaryotes, the reactions of the citric acid cycle occur in the mitochondria. The
mitochondrion is enclosed by a double membrane. All of the glycolytic enzymes are
found in the cytosol of the cell. Charged molecules such as pyruvate must be
transported in and out of the mitochondria. Small charged molecules with molecular
weights of less than 10,000 can freely diffuse through the outer mitochondrial
membrane through aqueous channels called porins. A transport protein called pyruvate
translocase specifically transports pyruvate through the inner mitochondrial membrane
into the matrix of the mitochondrion.
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Once in the mitochondria, pyruvate dehydrogenase converts pyruvate into acetyl-CoA and
CO2 .
This reaction may look straight forward but it is complex. Pyruvate dehydrogenase is a
humongous enzyme that can be visualized by electron micrographs. Pyruvate
dehydrogenase is a multienzyme complex held together by noncovalent interactions.
There are three different enzymes and five coenzymes. The three individual enzymes
are E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihyrolipoyl
dehydrogenase. The multienzyme complex consists of 24 subunits of E1, 24 subunits of
E2 and 12 subunits of E3. In E1 we find a thiamine pyrophosphate coenzyme. In E2
there is a lipoic acid coenzyme. In E3 there is a FAD coenzyme. The molecular weight of
pyruvate dehydrogenase isolated from E. coli is 4.6 million Daltons, slightly larger than
a ribosome. In mammals, pyruvate dehydrogenase is twice that size and has two
additional regulatory subunits. One is a protein kinase which phosphorylates three
serine residues in E1; the other is a phosphatase which hydrolytically removes the same
phosphoryl groups from the serines of E1.
The structure of the pyruvate dehydrogenase complex is shown below. The E2,
dihydrolipoyl transacetylase are shown as red balls. These 24 subunits create the core
upon which the pyruvate dehydrogenase complex is built. The E1, pyruvate
dehydrogenase enzyme is an α2 β2 dimer shown as the orange and yellow balls. There
are 24 of the αβ dimers in the complex. There are 12 subunits of the E3, dihyrolipoyl
dehydrogenase complex shown in green. The E3 subunits associate into dimers, so there
are six E3 dimers in the complex.
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Lets look at the chemistry going on in this complex.
In E1, pyruvate dehydrogenase enzyme, we have a tightly bound thiamine
pyrophosphate coenzyme or prosthetic group. We have encountered this cofactor
before, pyruvate decarboxylase and transketolase. The reaction catalyzed in this subunit
is similar to that of pyruvate decarboxylase.
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The next step in the enzyme catalyzed reaction involves a new cofactor we haven’t
encountered yet. It is lipoic acid. Lipoic acid is not found free in nature. Instead lipoic
acid is covalently attached to a lysine residue through an amide linkage. The enzyme
that catalyzes the formation of the lipoamide linkage uses ATP to generate AMP and
pyrophosphate. Lipoic acid is an important cofactor because it couples acyl -group
transfers with electron transfers during the oxidation and decarboxylation of α-keto
acids. Because the ring strain inherent in the disulphide cyclic structure is relieved upon
reduction, lipoic acid has a strong negative reduction potential, E o’ = − 0.30 V.
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To summarize the chemistry up to this point, this enzyme has taken pyruvate, used TPP
to catalyze the decarboxylation of an α-ketoacid to form the hydroxyethyl-TPP
intermediate which is stable enough to isolate. The hydroxyethyl-TPP was then
oxidized by and concomitantly transferred to lipoamide to form an acetyllipoamide.
The acetyllipoamide is the final product of E1. The lipoamide prosthetic group is part of
the E2 Dihydrolipoyl transacetylase enzyme. The next step in the course of the reaction
mechanism occurs in the active site of the E2, Dihydrolipoyl transacetyl ase enzyme,
which catalyzes the transfer of the acetyl group from lipoamide to coenzyme A.
If you look back to the structure of lipoamide, you will see it has a long fl exible tether
linking the amide to the E2 enzyme. The length of this tether is about 45 Å. This is long
enough to reach the active site of E1 to form the acetyllipoamide and then carry this
intermediate to the active site of the E2 enzyme to form acetyl-CoA and the
dihydrolipoamide which is then carried to yet the third active site, the E3,
dihyrolipoamide dehydrogenase enzyme.
The E3, dihyrolipoamide dehydrogenase enzyme regenerates lipoamide for another
round of catalysis. This enzyme is a flavoprotein, meaning, it has a tightly bound FAD
prosthetic group. Flavins undergo 2 electron reductions, but can accept electrons one at
a time, unlike NAD+ which can only accept two electrons at a time in the form of a
hydride. This gives flavoproteins more catalytic diversity than NAD coenzymes.
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The final step of the reaction catalyzed by this E3 enzyme is the transfer of the 2
electrons from the tightly bound FADH2 to the transiently bound NAD+ to generate
NADH + H+.
This is a usual electron transfer. The common role of FAD is to accept electrons from
NADH. One thing about flavoproteins is that the protein bound flavins have a great
variety of reduction potentials. In this enzyme the reduction potential is shifted such
that FADH2 is the electron donor and NAD+ is the acceptor.
Typical flavoprotein
FAD + 2e- + 2H+ → FADH2
NAD + 2e- + H+ → NADH
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Eo’≈ 0 V
Eo’ = -0.315 V
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THE CYCLE
I. Citrate Synthase
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The first reaction of the citric acid cycle is the condensation of acetyl-CoA and
oxaloacetate to form citrate and CoA-SH. The enzyme that catalyzes this reaction is
called citrate synthase. (ΔGo’ = -32.2 kJ/mol)
The change in free energy based on the steady state concentrations of oxaloacetate,
acetyl-CoA and citrate in the mitochondria isolated from pig hearts is: ΔG = -53.9
kJ/mol (a very exergonic reaction and irreversible).
The mechanism of citrate synthase is shown below. In the active site of the enzyme
there are two histidines and an aspartate which function as general acids and bases
during catalysis.
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The first step is to generate an enol of acetyl-CoA. Asp-375 functions as a general base
abstracting a proton from the methyl group of Acetyl-CoA. His-274 concertedly
functions as a general acid donating a proton to the carbonyl to form the enol. The enol
generated in the first step is converted into a nucleophile by the abstraction of the enol
hydrogen by His- 274 now functioning as a general base.
The electrons of the double bond attack the electrophilic center of the ketone of
oxaloactete. His-320 functions in concert as a general acid donating its proton to the
carbonyl oxygen of oxaloacetate to form citryl-CoA. Citryl-CoA spontaneously
hydrolyses while it is still bound to the active site to generate coenzyme A and citrate.
Citrate synthase is a homodimer with symmetry as shown below. It has a sequential
order kinetic mechanism. First the enzyme binds oxaloacetate which induces the large
conformational change shown (b). This is yet another example of induced fit. The
conformation change induced by oxaloacetate binding creates the acetyl-CoA binding
site and seals oxaloacetate form the aqueous solvent.
Citrate synthase is allosterically regulated by NADH and Succinyl- CoA.
• It is the first step of a metabolic pathway.
• It catalyzes an irreversible step in the pathway.
• It is a homodimer with symmetry.
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II. Aconitase
Citrate is a tertiary alcohol. It is difficult to oxidize tertiary alcohols because forming the
ketone would involve breaking a carbon-carbon bond. To get around this problem,
citrate is isomerized into isocitrate. Isocitrate is a secondary alcohol which can be easily
oxidized to the ketone by NAD+. The enzyme that catalyzes this migration of a
hydroxyl group is aconitase. This enzyme catalyzes the dehydration of citrate of form
cis-aconitate and then rehydrating the double bond to form isocitrate. (ΔGo’ = +6.7
kJ/mol)
The change in free energy based on the steady state concentrations of citrate and
isocitrate in the mitochondria isolated from pig hearts is: ΔG = +0.8 kJ/mol (near
equilibrium).
Aconitase is an iron sulphur protein (shown above). It contains four iron atoms
complexed to four inorganic sulphides and three cysteine sulphur atoms called a 4Fe-4S
iron- sulphur cluster. One of the iron atoms has an open coordination site that
complexes with the carboxylate group of C3 and the hydroxyl group of citrate. This iron
residue facilitates the dehydration and rehydration reaction and accounts for the
stereospecifity of the reaction.
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III. Isocitrate Dehydrogenase
This is the first oxidation step of the pathway.
Isocitrate dehydrogenase catalyzes the oxidation of isocitrate to oxalosuccinate which is
a β−keto acid. This β−keto acid spontaneously decarboxylates to form α−ketoglutarate.
ΔGo’ = -8.4 kJ/mol. The change in free energy based on the steady state concentrations
of isocitrate and α−ketoglutarate in the mitochondria isolated from pig hearts is: ΔG = 17.5 kJ/mol (A very exergonic, irreversible reaction).
This irreversible reaction is allosterically regulated. NADH and ATP are allosteric
inhibitors. ADP is an allosteric activator which lowers the Km for isocitrate by a factor
of 10. When the concentration of ADP is low, the Km for citrate is well above the
physiological concentration making the enzyme essentially active. With ADP bound in
the allosteric binding site, the Km is lowered by a factor of ten making the enzyme
active at physiological concentrations.
IV. α−Ketoglutarate Dehydrogenase
The next step in the TCA cycle is the oxidative decarboxylation of α−ketoglutarate to
form succinyl-CoA and NADH. ΔGo’ = -30.0 kJ/mol
The change in free energy based on the steady state concentrations of α−ketoglutarate
and succinyl-CoA in the mitochondria isolated from pig hearts is: ΔG = -43.9 kJ/mol (a
very exergonic, irreversible reaction).
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If you look at the chemical transformation involved, you will note the similarity
between this enzymatic reaction and the one catalyzed by pyruvate dehydrogenase.
Pryruvate dehydrogenase:
Pyruvate + CoA + NAD+ →acetyl-CoA + CO 2 + NADH
α−Ketoglutarate dehydrogenase:
α−ketoglutarate + CoA + NAD+ → succinyl-CoA + CO 2 + NADH
Both of these reactions include the decarboxylation of an α-keto acid and the
subsequent formation of an high energy thioester linkage with coenzyme A.
α−Ketoglutarate dehydrogenase is a humongous multienzyme complex that is very
similar to the pyruvate dehydrogenase multienzyme complex.
The mechanism for α−Ketoglutarate dehydrogenase is identical to that of pyruvate
dehydrogenase.
V. Succinyl-CoA Synthetase
So far we have generated 2 molecules of CO 2 and 2 molecules of NADH for each
molecule of pyruvate that enters the TCA cycle. The electrons of NADH will be routed
through the electron transport chain and ultimately generated 2.5 equivalents of ATP by
oxidative phosphorylation. The succinyl-CoA contains a high energy bond which is
going to be utilized in this step of the cycle.
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The enzyme succinyl-CoA synthetase couples the conversion of succinyl-CoA into
succinate with the synthesis of GTP from GDP and Pi. The standard free energy change
for hydrolyzing succinyl-CoA into succinate and coenzyme A is -33.8 kJ/mole. The
standard free energy required to synthesize GTP from GDP and Pi is + 30.5 kJ/mole. If
we couple these two reactions together than the standard free energy change is -3.3
kJ/mole. This enzyme catalyzes a substrate level phosphorylation to generate the only
NTP produced directly in the citric acid cycle. The GTP produced is converted into ATP
by the enzymatic activity of nucleoside diphosphate kinase:
ADP + GTP →ATP + GDP
For succinyl-CoA synthetase: ΔGo’ = -3.3 kJ/mol. The change in free energy based on
the steady state concentrations of all the reactants and products in the mitochondria
isolated from pig hearts is: ΔG ≈ 0 (a near equilibrium reaction).
The mechanism for this enzyme is shown below.
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In the first step an enzyme bound
inorganic phosphate group attacks the
thioester to convert the high energy
thioester bond into a high energy
acylphosphate bond.
This enzyme bound succinyl-phosphate
then undergoes nucleophilic attack by an
active site histidine residue to form
succinate and a phosphorylated histidine
intermediate.
In the last step of this mechanism, GDP is
bound and the phosphate group is
transferred to GDP forming GTP. The
potential energy of the thioester bond has
been conserved first by the formation of
succinyl-phosphate, then by the formation
of the phosphorylated histidine residue
and finally by the substrate level
phosphorylation of GDP to form GTP.
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VI. Succinate Dehydrogenase
In the next step succinate dehydrogenase is going to oxidize succinate into fumarate.
The oxidation of an alkane is not sufficiently exergonic to reduce NAD + Eo’ = - 0.315.
The oxidation of an alkane is sufficiently exergonic to reduce FAD. E o’ = 0.0308
Fumarate + 2e - +2H+→ Succinate
Eo’ = 0.031
ΔEo’ = - 0.0002
V= ΔGo’ = + 0.04 kJ/mol
The change in free energy based on the steady state concentrations of all the reactants
and products in the mitochondria isolated from pig hearts is: ΔG ≈ 0; (a near
equilibrium reaction).
Succinate dehydrogenase is an integral membrane bound enzyme that is part of the
electron transport chain. All of the other enzymes of the citric acid cycle are soluble
proteins found in the mitochondrial matrix. Succinate dehydrogenase also contains
three iron sulphur clusters as part of its electron transport chain. The electrons captured
by the FAD are passed to the iron sulphur clusters to coenzyme Q (quinine) to produce
the reduced form QH2 (dihydroquinone).
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VII. Fumarase
Fumarase catalyzes the trans addition of water to the double bond of fumarate to
produce malate. The reaction catalyzed by fumarase is stereospecific. (ΔGo’ = -3.8
kJ/mol). The change in free energy based on the steady state concentrations of all the
reactants and products in the mitochondria isolated from pig hearts is: ΔG ≈ 0 (A near
equilibrium reaction).
VIII. Malate Dehydrogenase
The last step is catalyzed by malate dehydrogenase. Malate dehydrogenase catalyzes
the oxidation of the hydroxyl group of malate into a ketone to generate oxaloacetate
and complete the journey around the cycle. ΔGo’ = +29.7 kJ/mol. The change in free
energy based on the steady state concentrations of malate and oxaloacetate in the
mitochondria isolated from pig hearts is: ΔG ≈ 0 (a near equilibrium reaction). Note that
the standard change in free energy is large and endergonic. In order to drive this
reaction the concentration of oxaloacetate must be maintained at a very low
concentration (less than 10-6 M) in the mitochondrial matrix.
IX The Net reaction of the citric acid cycle is:
3NAD+ + FAD + GDP + Pi + Acetyl-CoA + 2H2 O
→ 3NADH + FADH2 + GTP + CoA + 2CO 2 + 3H+ ΔGo’ = - 40 kJ/mol
The change in free energy based on the steady state concentrations of all the reactants
and products in the mitochondria isolated from pig hearts is: ΔG = -115 kJ/mol
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REGULATION OF THE CITRIC ACID CYCLE
I. Changes in Free Energy
3 Molecules of NADH and 1 molecule of FADH 2 are generated each turn of the Citric
acid cycle. The eight electrons captured are transported by electron carriers to O 2
generating a proton gradient that drives the oxidative phosphorylation of ADP to
generate ATP. The stoichiometry of electron transport and oxidative phosphorylation is
2.5 ATP per NADH and 1.5 ATP per FADH2 . As a result 9 ATP are generated by
electron transport-oxidative phosphorylation per turn of the cycle.
Molecular oxygen does not participate in the citric acid cycle. However, the cycle
operates only under aerobic conditions because NAD + and FAD cannot be regenerated
in the absence of oxygen. Glycolysis has an aerobic and anaerobic mode, but the citric
acid cycle only operates under aerobic conditions.
II. Regulation of Pyruvate Dehydrogenase
Pyruvate is an important metabolite. It is the product of glycolysis and a substrate for
gluconeogenesis. Under anaerobic conditions, pyruvate is fermented into lactate or
alcohol to regenerate NAD+. Under aerobic conditions pyruvate is converted into
acetyl-CoA by pyruvate dehydrogenase. This is an irreversible step in animals. Animals
are unable to directly convert acetyl-CoA back to pyruvate. The oxidative
decarboxylation of pyruvate to form acetyl-CoA commits the acetyl group to either
complete oxidation by the citric acid cycle into CO 2 or incorporation in fatty acids and
lipids. This irreversible branch point is regulated by several means. This enzyme is
allosterically regulated. High concentrations of acetyl-CoA inhibit the transacetylase
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component of E2. High concentrations of NADH inhibits the dihydrolipoyl
dehydrogenase component of E3.
In Mammals, pyruvate dehydrogenase is regulated by phosphorylation by pyruvate
dehydrogenase kinase a regulatory enzyme that is part of the mammalian pyruvate
dehydrogenase complex. Pyruvate dehydrogenase kinase is allosterically activated by
NADH and acetyl-CoA such that when the mitochondrial matrix concentrations of
these two effectors rises, they stimulate the phosphorylation of a serine residue on the
pyruvate dehydrogenase E1 subunit, which blocks the first step of catalysis, the
decarboxylation of pyruvate. Pyruvate allosterically inhibits the kinase’s activity.
Eventually the mitochondrial matrix concentrations of NADH and acetyl -CoA will
come down and the enzyme will need to be reactivated. The reactivation of this enzyme
is accomplished by another enzyme, pyruvate dehydrogenase phosphatase, a Ca 2+
activated enzyme that binds to the dehydrogenase complex, hydrolyses the
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phosphorylated serine, reactivating the enzyme. This phosphatase will remain
associated with the dehydrogenase complex as long as the NADH/NAD + and the
acetyl-CoA/CoA ratios remain low. High concentrations of NADH or acetyl-CoA
inactivate the phosphatase and cause it to dissociate from the pyruvate dehyrdrogenase
complex. Insulin and Ca2+ ions activate the phosphatase.
Important to note that the cAMP dependent phosphorylation cascade has no effect on
the phosphorylation of pyruvate kinase. The cascade produced by insulin however does
activate the phosphatase.
III. Regulation of the Citric Acid Cycle
The citric acid cycle must be carefully regulated by the cell. If the citric acid cycle were
permitted to run unchecked, large amounts of metabolic energy would be wasted in the
over production of reduced coenzymes and ATP. Conversely if the citric acid cycle ran
too slowly, ATP would not be generated fast enough to sustain the cell.
By looking at the changes in free energy of the reactions of the citric acid cycle, it is cle ar
that there are three irreversible steps. These three reactions of the cycle, citrate synthase,
isocitrate dehydrogenase and α-ketoglutarate dehydrogenase operate with large
negative free energy changes under the concentrations of products and reactants in the
matrix of the mitochondria.
Because the citric acid cycle is linked to oxygen consumption to regenerate NAD +, the
citric acid cycle is regulated primarily by product feedback inhibition. Glycolysis and
glycogen metabolism are under complex systems of allosteric and hormonal control.
The citric acid cycle in contrast is regulated by three simple mechanisms.
1. Substrate availability
2. Product inhibition
3. Competitive feedback inhibition.
The allosteric effectors that control the flux of metabolites through the citric acid cycle
are shown on the next page. The principle signals are acetyl-CoA, succinyl-CoA, ATP,
ADP, AMP, NAD+ and NADH.
All of the regulatory enzymes of the citric acid cycle including pyruvate dehyrogenase
are allosterically inhibited by NADH. The combination of the electron transport chain
and oxidative phosphorylation produce ATP form NADH, consequently ATP is an
allosteric inhibitor of pyruvate dehydrogenase and isocitrate dehydrogenase. The TCA
cycle is turned on however by high ratios of either ADP/ATP or NAD+/NADH which
indicate that the cell has run low of NADH or ATP.
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IV. Citric Acid Cycle Intermediates Are Precursors for Biosynthetic Reactions
It is easy to think of the citric acid cycle as a catabolic pathway oxidizing acetate into
CO2 and generating ATP. Many of the intermediates, α-ketoglutarate, succinyl-CoA,
fumarate and oxaloacetate produced in the citric acid cycle, are precursors for
important biomolecules.
1. A Transamination reaction converts glutamate into α-ketoglutarate or vice versa.
Glutamate is a precursor for the synthesis of other amino acids and purine
nucleotides.
2. Succinyl-CoA is a precursor for porphyrins.
3. Oxaloacetate can be transaminated to form aspartate. Aspartate itself is a
precursor for other amino acids and pyrimidine nucleotides. Oxaloacetate is a
substrate for gluconeogenesis.
4. Citrate can be exported out of the mitochondria into the cytostol where is broken
down by ATP citrate lyase to yield oxaloacetate and acetyl-CoA. (Citrate
Oxaloacetate shuttle shown below)
5. The acetyl-CoA produced is a precursor for fatty acids. The oxaloacetate
produced in this reaction is reduced to malate which can be either be transported
back into the matrix of the mitochondria where it is reoxidized into oxaloacetate
or malate can be oxidatively decarboxylated to pyruvate by malic enzyme which
can then be transported back to the mitochondria.
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V. Anaplerotic Reactions
Reciprocal arrangements replenish the intermediates removed from the citric acid cycle
for biosynthesis. The reactions that replenish the citric acid cycle are called anaplerotic
reactions which means filling up reactions. Anaplerotic reactions include PEP
carboxylase and pyruvate carboxylase both of which synthesize oxaloacetate from
pyruvate.
Pyruvate carboxylase is one of the most important anaplerotic reactions. This enzyme
catalysis the first step of gluconeogenesis (synthesis of glucose from non-carbohydrate
precursors) from pyruvate and is found exclusively in the mitochondria. The enzyme
has a covalently bound biotin cofactor. Since this enzyme functions in gluconeogenesis,
it is allosterically regulated. This enzyme requires acetyl-CoA to be bound at an
allosteric binding site in order to activate bicarbonate with ATP.
PEP carboxylase is found in yeast, bacteria and plants but not in animals.
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Malic enzyme catalyses the reaction shown above. The malic enzyme is found in the
cytosol or mitochondria of many plants and animals and is a NADP + dependent
enzyme.
Other metabolites fed into the citric acid cycle are succinyl-CoA from the degradation of
odd-chain fatty acids, α-ketoglutarate and oxaloacetate from the transamination
reactions of glutamate and aspartate.
VI. The Glyoxylate Cycle of Plants, Yeast and Bacteria
Plants, fungi, algae, protozoans and bacteria can thrive on two carbon compounds such
as acetate, ethanol and acetyl-CoA, as their sole carbon source. In the citric acid cycle,
we have seen how acetyl-CoA is oxidized into 2 molecules of CO 2 to generate ATP.
There is no net synthesis in the TCA cycle since we end up with oxaloacetate, the exact
same compound we began with. The TCA cycle has evolved to produce ATP from
acetyl-CoA. This cycle cannot produce massive amounts of biosynthetic precursors
needed for growth on acetate unless alternative reactions are available. In order to grow
and thrive on acetate, the two CO 2 producing reactions of the citric acid cycle need to be
bypassed. The living organisms listed above can grow on acetate by employing a
modification of the citric acid cycle called the glyoxylate cycle which takes two carbon
compounds and converts them into four carbon compounds.
This cycle bypasses the oxidative decarboxylations of the citric acid cycle by using two
alternative enzymes, Isocitrate lyase and malate synthase. Isocitrate lyase cleaves
isocitrate into glyoxylate and succinate. Malate synthase takes glyoxylate and
condenses it with another acetyl-CoA to form Malate and CoA. The net effect is the
preservation of carbon units using two molecules of acetyl-CoA to generate malate
which is then converted by malate dehydrogenase into oxaloacetate which can be then
converted into PEP and on through gluconeogenesis. The enzymes of the glyoxylate
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cycle are contained in glyoxysomes which are organelles devoted to this cycle. There
are enzymes common to both the TCA and glyoxylate cycles as well as isozymes and
functionally distinct enzymes allowing these two organelles to operate independently
in these two cycles.
The glyoxylate cycle allows seeds to grow in the dark where photosynthesis is
impossible. Many seeds are rich in lipids allowing these organisms to degrade fatty
acids to generate acetyl-CoA. The glyoxylate cycle then can take two of these acetylCoA’s to generate oxaloacetate and from there other intermediates.
The glyoxysomes lack succinate dehydrogenase, fumarase and malate dehydrogenase.
Consequently intermediates must be shuttle back and forth between the glyoxysome
and the mitochondria. Succinate is transported to the mitochondria where it is
converted into oxaloacetate. The transamination of aspartate is necessary because
oxaloacetate cannot be transported out of the mitochondria. Aspartate then moves from
the mitochondria back to the glyoxysome where it is transaminated back to oxaloacetate
and completes the shuttle.
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