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Citrate Cycle
Lecture 28
Key Concepts
• The Citrate Cycle captures energy using redox reactions
• Eight reactions of the Citrate Cycle
• Key control points in the Citrate Cycle regulate metabolic
flux
• 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?
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.
Overview of the Citrate Cycle
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The citrate cycle captures energy using redox reactions
Acetyl-CoA, derived from pyruvate (from glycolysis), is the
“normal” entry substrate into the cycle
Pyruvate can also be derived from amino acid catabolism,
and acetyl-CoA can be derived from both amino a acids
and fatty acids.
Large enzyme complexes are involved
As name states, it is a cyclical pathway
“Products” of citrate cycle are CO2, NADH, FADH2 and
GTP
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.
Function of the Citrate Cycle
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Primary function : oxidize acetyl-CoA
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
It 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 is acetyl-CoA
– The exhaust is two molecules of CO2
– The energy output is eight electrons (redox energy) and one GTP
that is converted to ATP (phosphoryl transfer energy).
Redox energy is used to generate ATP
• "currency exchange" for redox energy and ATP
synthesis in the mitochondria
– for every 2 NADH molecules that are reoxidized in the electron
transport chain, ~5 molecules of ATP are produced by oxidative
phosphorylation (~2.5 ATP/ NADH).
– For every 2 FADH2 molecules that are reoxidized by the electron
transport chain, only ~3 molecules of ATP are produced (~1.5
ATP/FADH2)
• Why the difference?
– differences in where these two coenzymes enter the electron
transport chain.
• Based on 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.
Pathway overview
Other names for citrate cycle
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) or the
citric acid cycle, although we will refer to it as the citrate
cycle.
Considering that the pKa values of the three carboxylate groups in citric
acid are 3.1, 4.7 and 6.4, why is "citrate cycle" a better term than
"citric acid cycle" or "tricarboxylic acid cycle"?
Pathway Questions
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.
Pathway Questions
What is the overall net reaction of citrate cycle?
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O →
CoA + 2 CO2 + 3 NADH + 2 H+ + FADH2 + GTP
∆Gº’ = -57.3 kJ/mol
Pathway Questions
What are the key regulated enzymes in citrate 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.
Pathway Questions
What are the key regulated enzymes in citrate cycle?
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 - 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, succinylCoA and ATP.
Pathway Questions
What are examples of 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.
The complete oxidation of glucose to CO2 and H2O
is summarized by the reaction:
Glucose (C6H12O6) + 6O2 → 6CO2 + 6H2O
∆Gº’ = -2,840 kJ/mol
∆G = -2,937 kJ/mol
Citrate cycle is where most of this CO2 is produced
Where does the rest of it come from?
8 Reactions of the
Citrate Cycle
In the first half of the cycle,
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 succinylCoA by four successive
reactions that lead to the
formation of one GTP (ATP),
one FADH2 and one NADH.
8 Reactions of the
Citrate Cycle
Reaction 1: Condensation of oxaloacetate and acetyl-CoA
by citrate synthase to form citrate
Purpose: commit the acetate unit of acetyl-CoA to oxidative
decarboxylation
Reaction follows an ordered mechanism:
1. oxaloacetate binds, inducing a conformational change in the enzyme
that facilitates:
2. acetyl-CoA binding
3. formation of the transient intermediate, citryl-CoA
4. rapid hydrolysis that releases CoA-SH and citrate
Reaction 2: Isomerization of citrate by aconitase to form
isocitrate
reversible two step isomerization reaction
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
water is added back to convert the double bond in cis-aconitate to a
single bond with a hydroxyl group on the terminal carbon
Aconitase is one of the targets of fluorocitrate
Fluorocitrate is derived from
fluoroacetate. Fluoroacetatecontaining plants, such as acacia
found in parts of Australia and Africa,
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 α-ketoglutarate, CO2 and
NADH
First of two decarboxylation steps in the citrate cycle
First reaction to generate NADH used for energy conversion reactions
in the electron transport system
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
Reaction 4: Oxidative decarboxylation of by α-ketoglutarate
dehydrogenase to form succinyl-CoA, CO2 and NADH
Second oxidative decarboxylation reaction (produces NADH)
α-Ketoglutarate dehydrogenase complex utilizes essentially the same
catalytic mechanism we have already described for the pyruvate
dehydrogenase reaction
Includes the binding of substrate to an E1 subunit (α-ketoglutarate
dehydrogenase), followed by decarboxylation and formation of a
TPP-linked intermediate
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 (∆Gº' =
-32.6 kJ/mol) is used in the succinyl-CoA synthetase reaction to carry
out a phosphoryl transfer reaction (∆Gº' = +30.5 kJ/mol) that leads to
the production of GTP (or ATP) and succinate.
Nucleoside diphosphate kinase interconverts GTP and ATP by a readily reversible
phosphoryl transfer reaction: GTP + ADP ↔ GDP + ATP (∆Gº' = 0 kJ/mol).
Reaction 6: Oxidation of succinate by succinate
dehydrogenase to form fumarate
This coupled redox reaction directly links the 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. 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.
Reaction 7: Hydration of fumarate by fumarase to form
malate
Fumarase is a highly stereo-specific enzyme
Catalyzes the reversible hydration of the C=C double bond in fumarate
to generate the L-isomer of malate.
Stereospecificity of enzymes is an important feature that we will see again
and again in metabolism.
Enantiomers are Mirror Images
Also called Stereoisomers
COO-
COO-
HO
H
H
OH
H
H
H
H
COO-
COO-
L-malate
L-Dogbert
D-malate
Plane of
Reflection
D-Dogbert
Reaction 8: Oxidation of malate by malate dehydrogenase
to form oxaloacetate
Final reaction in the citrate cycle (Yeah!)
Oxidation in the presence of NAD+ of the hydroxyl group of malate to
form oxaloacetate
Change in standard free energy for this reaction is unfavorable (∆Gº' =
+29.7 kJ/mol)
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, ∆G).
Bioenergetics of the citrate cycle
Glycolysis + pyruvate dehydrogenase reaction + citrate cycle = net reaction:
Glucose + 2 H2O + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi →
6 CO2 + 10 NADH + 6 H+ + 2 FADH2 + 4 ATP
Reducing power of 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.
The complete oxidation of
glucose by the pyruvate
dehydrogenase complex
and the citrate cycle leads
to the production of 6 CO2
molecules as “waste”.
Use of radioactively-labeled
metabolic intermediates such as
14C-acetyl CoA
What happens to the two carbon atoms
that enter the citrate cycle as acetylCoA?
In the first turn of the cycle, both carbons
are incorporated into oxaloacetate.
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.
→ Intermediates must be continually
regenerated in a cyclic pathway to
maintain metabolic flux.
We now do this with 13C labeled
compounds as well.
Regulation Of The
Citrate Cycle
The three main control points:
1. citrate synthase
2. isocitrate dehydrogenase
3. α-ketoglutarate dehydrogenase
Pyruvate dehydrogenase and
pyruvate carboxylase control the
rate of citrate cycle flux by
providing acetyl CoA and
oxaloacetate, respectively.
These enzymes are also
regulated.