Download Citrate Cycle Supplemental Reading Key Concepts

Bioc 460 - Dr. Miesfeld Spring 2008
Citrate Cycle Supplemental Reading
Key Concepts
- The Citrate Cycle captures energy using redox reactions
- Eight enzymatic reactions of the Citrate Cycle
- Key control points in the citrate cycle regulate metabolic flux
KEY CONCEPT QUESTIONS IN THE CITRATE CYCLE:
What role does NADH and FADH2 have in connecting the citrate cycle to ATP synthesis?
Why is the citrate cycle considered the hub of metabolism?
Biochemical Applications of the Citrate Cycle:
Fluoroacetate is found in poisonous plants and
it is the active ingredient in "compound 1080"
which is used by ranchers to kill coyotes and
foxes. Cells convert fluoracetate to fluorocitrate,
a potent inhibitor of the citrate cycle enzyme
mitochondrial aconitase.
The Citrate Cycle Captures Energy Using Redox Reactions
The citrate cycle is central to aerobic metabolism and ATP production because it contains four
dehydrogenase reactions that generate NADH and FADH2 that are reoxidized by the electron
transport chain to generate large amounts of ATP by
Figure 1.
oxidative phosphorylation (lecture 30). The citrate cycle is
considered the "hub" of cellular metabolism because it not
only links the oxidation of metabolic fuels (carbohydrate,
fatty acids and proteins) to ATP synthesis, but it also
provides shared metabolites for numerous other metabolic
pathways as shown in figure 1.
Unlike glycolysis which occurs in the cytosol, all of
the enzymes in the citrate cycle, electron transport chain
and oxidative phosphorylation reside in the mitochondrial
matrix where pyruvate is converted to acetyl-CoA by the
enzyme pyruvate dehydrogenase. As illustrated in figure
2, pyruvate can also be derived from amino acid
catabolism, and acetyl-CoA can be derived from both
amino a acids and fatty acids. The primary function of the
citrate cycle in terms of energy conversion reactions is to
oxidize acetyl-CoA, and in the process, transfer four pairs
of electrons from citrate cycle intermediates to 3 moles of
NADH and 1 mole of FADH2 during each turn of the cycle.
As shown in figure 3, the citrate cycle is a "metabolic
engine" in which all eight of the cycle intermediates are continually replenished to maintain a
smooth-running energy conversion process. The fuel for this metabolic engine is acetyl-CoA, the
exhaust is two molecules of CO2, and the energy output is eight electrons (redox energy) and
one GTP that is converted to ATP (phosphoryl transfer energy). The electron transport system
and oxidative phosphorylation convert the available redox energy into ATP through a series of
reactions that reoxidize NADH and FADH2 in the presence of O2.
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Figure 2,
Figure 3.
The "currency exchange" for redox energy and ATP synthesis in the mitochondria is such that for
every 2 NADH molecules that are reoxidized in the electron transport chain, there is a production
of ~5 molecules of ATP by oxidative phosphorylation (~2.5 ATP/ NADH). Oxidation of 2 FADH2
molecules by the electron transport chain results in only ~3 molecules of ATP (~1.5 ATP/FADH2)
because of differences in where these two coenzymes enter the electron transport chain. Based
on this ATP currency exchange ratio, and the one substrate level phosphorylation reaction, each
turn of the cycle produces ~10 ATP for every acetyl-CoA that is oxidized. Since regeneration of
NAD+ and FAD inside the mitochondrial matrix is required to maintain flux through the citrate cycle
(four of the enzymes require NAD+ or FAD as coenzymes), this metabolic engine is dependent on
a constant supply of O2 just like a combustion engine. The reliance on redox reactions in the
electron transport chain to maintain metabolic flux in the citrate cycle is similar to the way that the
glyceraldehyde-3-P dehydrogenase reaction in glycolysis requires the regeneration of NAD+ by
lactate dehydrogenase under anaerobic conditions.
Hans Krebs, a biochemist who fled Nazi Germany for England in 1933, first described the citrate
cycle in 1937. The citrate cycle is sometimes called the Krebs cycle or the tricarboxylic acid
cycle (citrate is a tricarboxylate), although we will refer to it as the citrate cycle because citrate is
the first product of the pathway and the unprotonated form of citric acid is the predominant species
at physiological pH (the pKa values of the three carboxylate groups are 3.1, 4.7 and 6.4).
Figure 4 shows the eight citrate cycle reactions. The citrate cycle is distinguished from linear
metabolic pathways in that oxaloacetate is both the substrate for the first reaction (citrate
synthase) and the product of the last reaction (malate dehydrogenase), which means that it must
be regenerated from citrate after each turn of the cycle.
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Figure 4.
1. What does the citrate cycle accomplish for the cell?
• Transfers 8 electrons from acetyl-CoA to the coenzymes NAD+ and FAD to form 3 NADH and 1
FADH2 which are then re-oxidized by the electron transport chain to produce ATP by the
process of oxidative phosphorylation.
• Generates 2 CO2 as waste products and uses substrate level phosphorylation to generate 1 GTP
which is converted to ATP by nucleoside diphosphate kinase.
• Supplies metabolic intermediates for amino acid and porphyrin biosynthesis.
2. What is the overall net reaction of the citrate cycle?
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O 
CoA + 2 CO2 + 3 NADH + 2 H+ + FADH2 + GTP
ΔGº’ = -57.3 kJ/mol
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3. What are the key enzymes in the citrate acid cycle?
Pyruvate dehydrogenase – not a citrate cycle enzyme but it is critical to flux of acetyl-CoA through
the cycle; this multisubunit enzyme complex requires five coenzymes, is activated by NAD+, CoA
and Ca2+ (in muscle cells), and inhibited by acetyl-CoA, ATP and NADH.
Citrate synthase – catalyzes the first reaction in the pathway and can be inhibited by citrate,
succinyl-CoA, NADH and ATP; inhibition by ATP is reversed by ADP.
Isocitrate dehydrogenase - catalyzes the oxidative decarboxylation of isocitrate by transferring two
electrons to NAD+ to form NADH, and in the process, releasing CO2, it is activated by ADP and
Ca2+ and inhibited by NADH and ATP.
α-ketoglutarate dehydrogenase - is functionally similar to pyruvate dehydrogenase in that it is a
multisubunit complex, requires the same five coenzymes and catalyzes an oxidative
decarboxylation reaction that produces CO2, NADH and succinyl-CoA; it is activated by Ca2+ and
AMP and it is inhibited by NADH, succinyl-CoA and ATP.
4. What are examples of the citrate cycle in real life?
Citrate is produced commercially by fermentation methods using the microorganism Aspergillus
niger. Every year almost a half of million tonnes (5 x 108 kg) of citrate are produced worldwide by
exploiting the citrate synthase reaction. Citrate is used as a flavor enhancer and food preservative.
Eight Reactions Of The Citrate Cycle
In the first four reactions, the two carbon acetate group of acetyl-CoA is linked to the four carbon
oxaloacetate substrate to form a six carbon citrate molecule. Citrate is then transformed to
isocitrate to set up two decarboxylation reactions yielding two NADH and the high energy four
carbon cycle intermediate succinyl-CoA. In the second half of the cycle, oxaloacetate is
regenerated from succinyl-CoA by four successive reactions that lead to the formation of one GTP
(ATP), one FADH2 and one NADH.
• Reaction 1: Condensation of oxaloacetate and acetyl-CoA by citrate synthase to form citrate
The purpose of the first reaction in the citrate cycle is to commit the acetate unit of acetyl-CoA to
oxidative decarboxylation. The citrate synthase reaction follows an ordered mechanism in which
oxaloacetate binds first, inducing a conformational change in the enzyme that facilitates acetylCoA binding. Formation of the transient intermediate, citryl-CoA, is followed by a rapid hydrolysis
reaction that releases CoA-SH and citrate as shown in figure 5.
Figure 5.
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• Reaction 2: Isomerization of citrate by aconitase to form isocitrate
This reversible isomerization reaction involves a two step mechanism in which the intermediate,
cis-aconitate, is formed by a dehydration reaction that requires the participation of an iron-sulfur
cluster (4Fe-4S) in the enzyme active site. Next, water is added back to convert the double bond
in cis-aconitate to a single bond with a hydroxyl group on the terminal carbon (figure 6).
Figure 6.
Aconitase is one of the targets of the toxic compound fluorocitrate which is derived from
fluoroacetate, a naturally occurring poison found in native Australian and African plants (and
compound 1080). Fluoroacetate-containing plants are so deadly that Australian sheep herders
have reported finding sheep with their heads still in the bush they were feeding on when they died.
• Reaction 3: Oxidative decarboxylation of isocitrate by isocitrate dehydrogenase to form αketoglutarate, CO2 and NADH
Reaction 3 is the first of two decarboxylation steps in the citrate cycle, and also the first reaction to
generate NADH which is used for energy conversion reactions in the electron transport system.
Eukaryotic cells actually contain two isocitrate dehydrogenases, the mitochondrial NAD+dependent enzyme that functions in the citrate cycle, and a second NADP+-dependent enzyme
that is present in both the cytosol and mitochondria. As shown in figure 7, NAD+ catalyzes an
oxidation reaction that generates the transient intermediate oxalosuccinate. In the presence of the
divalent cations Mg2+ or Mn2+, oxalosuccinate is decarboxylated to form α-ketoglutarate.
Figure 7.
• Reaction 4: Oxidative decarboxylation of α-ketoglutarate by α-ketoglutarate dehydrogenase to
form succinyl-CoA, CO2 and NADH
In this second oxidative decarboxylation reaction, the α-ketoglutarate dehydrogenase complex
utilizes essentially the same catalytic mechanism we have already described for the pyruvate
dehydrogenase reaction. This includes the binding of substrate to an E1 subunit (α-ketoglutarate
dehydrogenase), followed by decarboxylation and formation of a TPP-linked intermediate. Figure 8
on the next page summarizes the α-ketoglutarate dehydrogenase reaction, which not surprisingly,
is similar to pyruvate dehydrogenase in that it has a large change in free energy (ΔGº' = -33.5
kJ/mol) and is allosterically-regulated by changes in the energy charge of the cell.
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Figure 8.
• Reaction 5: Conversion of
succinyl-CoA to succinate by
succinyl-CoA synthetase in a
substrate level phosphorylation
reaction that generates GTP
The available free energy in the
thioester bond of succinyl-CoA
(ΔGº' = -32.6 kJ/mol) is used in the
succinyl-CoA synthetase reaction to
carry out a phosphoryl transfer reaction
(ΔGº' = +30.5 kJ/mol) that leads to the
production of GTP (or ATP) and
succinate (figure 9). The enzyme
nucleoside diphosphate kinase
interconverts GTP and ATP by a readily
reversible phosphoryl transfer reaction:
GTP + ADP <===> GDP + ATP (ΔGº' =
0 kJ/mol).
Figure 9.
• Reaction 6: Oxidation of succinate by succinate dehydrogenase to form fumarate
This coupled redox reaction directly links the
Figure 10.
citrate cycle to the electron transport system
through the redox conjugate pair FAD/FADH2
which is covalently linked to the enzyme
succinate dehydrogenase, an inner
mitochondrial membrane protein. As shown in
figure 10, oxidation of succinate results in the
transfer of an electron pair to the FAD moiety,
which in turn, passes the two electrons to the
electron carrier coenzyme Q in complex II of the
electron transport system.
Figure 11.
• Reaction 7: Hydration of fumarate by fumarase to
form malate
Fumarase is a highly stereo-specific enzyme that
catalyzes the hydration of the C=C double bond in
fumarate to generate the L-isomer of malate. This
reversible reaction is shown in figure 11.
• Reaction 8: Oxidation of malate by malate
dehydrogenase to form oxaloacetate
The final reaction in the citrate cycle involves
the oxidation of the hydroxyl group of malate to
form oxaloacetate in the presence of NAD+ as
shown in figure 12. This leads to the production
of the last of 3 NADH molecules generated by
the citrate cycle. The two carbon atoms
Figure 12.
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originating from acetyl- Figure 13.
CoA in reaction 1 are
incorporated into
oxaloacetate and not
lost as CO2 until
subsequent turns.
While the change in
standard free energy
for this reaction is
unfavorable (ΔGº' =
+29.7 kJ/mol), the
extremely low
concentration of
oxaloacetate inside the
mitochondrial matrix
ensures that the
reaction is pulled to
the right under
physiological
conditions (as a result
of actual free energy ΔG).
Now that we have looked at all eight citrate cycle reactions individually, let's review the
bioenergetics of the pathway by summing up the ΔGº' values as shown above in figure 13. By
combining the glycolytic pathway, the pyruvate dehydrogenase reaction and the citrate cycle into
one complete circuit we obtain the following net reaction:
Glucose + 2 H2O + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi --->
6 CO2 + 10 NADH + 6 H+ + 2 FADH2 + 4 ATP
The reducing power of the 10 NADH and 2 FADH2 can
be converted to ATP equivalents using the currency
exchange ratios of ~2.5 ATP/NADH and ~1.5
ATP/FADH2 to yield ~28 ATP, which when combined
with the 4 ATP synthesized by substrate
phosphorylation, generates a maximum of ~32 ATP as
shown in figure 14 (~30 ATP in muscle cells). The
complete oxidation of glucose by the pyruvate
dehydrogenase complex and the citrate cycle leads to
the production of 6 CO2 molecules as waste.
When Krebs first described the citrate cycle in
1937, it wasn't possible to follow the fate of individual
carbon atoms throughout the cycle, and therefore it
wasn't until techniques were developed using
radioactively-labeled metabolic intermediates that it
became clear exactly how each enzyme reaction
functioned. Based on the results of these 14C-labeling
experiments, figure 15 shows how a game of "follow
the bouncing carbon" can be played to see what
Figure 14.
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Figure 15.
happens to the two carbon atoms that enter the
citrate cycle as acetyl-CoA. In the first turn of the
cycle, both carbons are incorporated into
oxaloacetate, and it isn't until the second cycle that
the first carbon is released as CO2 by the isocitrate
dehydrogenase reaction. The second carbon is not
lost as CO2 until the α-ketoglutarate reaction in the
fourth turn of the cycle. This is a good way to
demonstrate that intermediates must be continually
regenerated in a cyclic pathway to maintain
metabolic flux.
REGULATION OF THE CITRATE CYCLE
The citrate cycle is quite different than glycolysis in
that the end product of the pathway, oxaloacetate,
is the substrate for the first reaction, meaning that
flux through the pathway is continuously monitored
by resetting the level of available substrate after
each turn of the cycle. In addition to substrate
availability, the two other regulatory mechanisms
at play in the citrate cycle are product inhibition
and feedback control of key enzymes. Figure 16
illustrates that the three main control points within
the citrate cycle are regulation of citrate synthase,
isocitrate dehydrogenase, and α-ketoglutarate
dehydrogenase activities. Figure 16 also shows the
factors that regulate pyruvate dehydrogenase and
pyruvate carboxylase activity as these two
enzymes control the rate of citrate cycle
Figure 16.
flux by providing acetyl CoA and
oxaloacetate, respectively. Importantly,
pyruvate carboxylase is allosterically
activated by acetyl-CoA to increase
production of oxaloacetate and thereby
maintain flux through the citrate cycle.
Pyruvate carboxylase plays a key role
in providing intermediates for the citrate
cycle by being the "tag team partner"
for pyruvate dehydrogenase by
balancing the input of oxaloacetate
with that of acetyl-CoA. For example,
when flux through the citrate cycle is
decreased due to loss of cycle
intermediates to anabolic pathways,
acetyl-CoA accumulates in the
mitochondrial matrix and allosterically
activates pyruvate carboxylase.
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ANSWERS TO KEY CONCEPT QUESTIONS IN THE CITRATE CYCLE:
The coenzymes NADH and FADH2 function as carrier molecules that transport electron pairs from
redox reactions in the citrate cycle to the electron transport system where they are reoxidized to
provide redox energy that can be harnessed for ATP synthesis. The primary role of the citrate
cycle is to strip 4 electron pairs (8 e-) from the acetate group of acetyl-CoA and transfer them to 3
NAD+ and 1 FAD molecules to form 3 NADH and 1 FADH2, respectively. Reoxidation of NADH
and FADH2 by the electron transport system generates a proton gradient across the inner
mitochondrial membrane that drives ATP synthesis inside the mitochondrial matrix through a
process called oxidative phosphorylation. The ATP currency exchange for reoxidation of NADH by
the coupled electron transport system/oxidative phosphorylation reactions is ~2.5 ATP/NADH and
~1.5 ATP/FADH2. Based on the yield of NADH and FADH2 from the citrate cycle, and the ATP
currency exchange, each turn of the citrate cycle produces ~10 ATP for every acetyl-CoA that
enters. Moreover, complete oxidation of glucose to 6 CO2 and 6 H2O by the combined reactions of
glycolysis, the pyruvate dehydrogenase complex, and the citrate cycle, results in the maximum
production of ~32 ATP/glucose (only ~30 ATP are produced per glucose in muscle cells because
of the glycerol-3P shuttle as described in lecture 30).
The citrate cycle is considered the hub of metabolism because it provides the crucial link between
oxidation of acetyl-CoA and ATP synthesis, and in addition, provides metabolic precursors for the
biosynthesis of amino acids, fatty acids, cholesterol, heme and glucose. The citrate cycle
intermediates α-ketoglutarate and oxaloacetate provide carbon skeletons for amino acid
biosynthesis, whereas, succinyl-CoA combines with glycine to from δ-aminolevulinic acid, a
precursor for heme biosynthesis. Citrate is transported out of the mitochondria and converted
back to acetyl-CoA and oxaloacetate in the cytosol by the enzyme citrate lyase. Cytosolic acetylCoA is used for fatty acid and cholesterol biosynthesis, and oxaloacetate can be converted to
phosphoenolpyruvate by the enzyme phosphoenolpyruvate carboxykinase (PEPCK) for use in
glucose synthesis. Another citrate cycle intermediate, malate, can also be used for
gluconeogenesis following export to the cytosol where it is converted first to oxaloacetate by
cytosolic malate dehydrogenase, and converted to phosphoenolypyruvate by the enzyme PEPCK.
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