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To understand how the pyruvate dehydrogenase
complex acts as a mediator of flow of carbon
compounds between the glycolytic pathway and the
citric acid cycle.
To understand the central role of the citric acid
cycle in mitochondrial energy metabolism.
The structures of the intermediates in the cycle.
The names of the enzymes that catalyze each step.
The cofactors and products involved in each step.
The citric acid cycle (Krebs cycle, tricarboxylic acid
cycle) is a series of reactions in mitochondria that
bring about the catabolism of acetyl residues.
The acetyl groups are fed into the citric acid cycle,
which enzymatically oxidizes them to CO2. The
energy released by oxidation is conserved in the
reduced electron carriers NADH and FADH2.
The reduced coenzymes are then oxidized
themselves, giving up protons and electrons. The
electrons are transferred, eventually, to O2, via a
chain of electron-carrying molecules known as the
In the course of electron transfer, the large amount
of energy released is conserved in the form of ATP
Catabolism ofCatabolism of
proteins, fats,proteins, fats,
in the threein the three
steps ofsteps of
Biomedical ImportanceBiomedical Importance
The citric acid cycle acts as a final common pathway for the
oxidation of carbohydrate, lipids, and protein, because glucose, fatty
acids, and many amino acids can all be metabolized to acetyl-CoA or
intermediates in the cycle.
The citric acid cycle also plays a major role in gluconeogenesis,
transamination, deamination, and lipogenesis. The liver is the only
tissue where all of these occur to a significant extent.
When large numbers of liver cells are damaged or destroyed, in
acute hepatitis or cirrhosis, this can have major repercussions on
Very few genetic abnormalities exist for enzymes of the citric acid
cycle, suggesting such abnormalities are incompatible with normal
development and highlighting the vital nature of the process.
Pyruvate hydrogenase is a key enzyme needed to convert pyruvate
to acetyl-CoA. A variety of disorders in pyruvate metabolism are due
to defects in this enzyme.
Pyruvate transport into mitochondriaPyruvate transport into mitochondria
Pyruvate generated in the cytoplasm by glycolysis
must be transported across the inner mitochondrial
membrane via a pyruvate/H+
This transport uses some of the energy stored in
the mitochondrial inner membrane electrical
Conversion of pyruvate to acetyl-CoA byConversion of pyruvate to acetyl-CoA by
pyruvate dehydrogenasepyruvate dehydrogenase
Within the mitochondria, pyruvate is oxidatively
decarboxylated to acetyl-CoA.
This reaction is catalyzed by several different enzymes
working sequentially in a multi-enzyme complex collectively
designated as the pyruvate dehydrogenase complex.
Pyruvate + NAD+
+ CoA→Acetyl-CoA + NADH + H+
Coenzyme A (CoA)Coenzyme A (CoA)
Coenzyme A (CoA). A hydroxyl
group of pantothenic acid is
joined to a modified ADP moiety
by a phosphate ester bond, and
its carboxyl group is attached to
β-mercaptoethylamine in amide
linkage. The hydroxyl group at
the 3´position of the ADP moiety
has a phosphoryl group not
present in ADP itself. The -SH
group of the mercaptoethylamine
moiety forms a thioester with
acetate in acetyl-coenzyme A
Pyruvate Dehydrogenase ComplexPyruvate Dehydrogenase Complex
Contains three enzymes each present in multiple copies:
1. pyruvate dehydrogenase (E1)
2. dihydrolipoyl transacetylase (E2)
3. dihydrolipoyl dehydrogenase (E3)
The complex from mammals has 60 copies of E2, which contains
lipoate, and constitutes the core of the complex.
In addition, five different coenzymes or prosthetic groups are
1. thiamine pyrophosphate (TPP)
2. flavin adenine dinucleotide (FAD)
3. coenzyme A (CoA)
4. nicotinamide adenine dinucleotide (NAD)
All are clustered for efficient handling of intermediates
Lipoic acid (lipoate) in
amide linkage with the side
chain of a Lys residue. The
lipoyllysyl moiety is the
prosthetic group of
transacetylase (E2 of the
complex). The lipoyl group
occurs in oxidized
(disulfide) and reduced
(dithiol) forms and acts as a
carrier of both hydrogen
and an acetyl (or other acyl)
Oxidation decarboxylation of pyruvate toOxidation decarboxylation of pyruvate to
acetyl-CoA by the pyruvate dehydrogenaseacetyl-CoA by the pyruvate dehydrogenase
Pyruvate reacts with thiamine pyrophosphate bound to
E1. Pyruvate undergoes decarboxylation to the
hydroxylethyl derivative, with a loss of CO2.
The acetyl group and 2 electrons from TPP are
transferred to the oxidized form of the lipoyllysyl group of
the core enzyme (E2).
A transestenification occurs where the -SH group of CoA
replaces the -SH group of E2 to yield acetyl-CoA and the
reduced form of the lipoyl group.
E3 transfers two hydrogens from the reduced lipoyl
groups to E2 to FAD.
The reduced FADH2 of E3 transfers a hydride ion to
, forming NADH.
Biomedical ImplicationsBiomedical Implications
Mutations in the genes for the subunits of the pyruvate
dehydrogenase complex, or a dietary thiamine deficiency,
can have severe consequences.
Thiamine-deficient animals are unable to oxidize pyruvate
normally. Particularly important in the brain, which
usually obtains energy from the aerobic oxidation of
glucose. e.g. Beriberi is a disease that results from
thiamine deficiency and is characterized by loss of neural
Alcoholics can also develop thiamine deficiency because
much of their dietary intake of calories is vitamin-free.
An elevated level of blood pyruvate is often an indicator of
defects in pyruvate oxidation.
Reactions of the citric acid cycleReactions of the citric acid cycle
Overview of citric acid cycleOverview of citric acid cycle
Eight successive reaction steps.
The six carbon citrate is formed from two carbon
acetyl-CoA and four carbon oxaloacetate.
Oxidation of citrate yields CO2 and regenerates
oxaloacetate, which plays essentially a catalytic
The energy released is captured in the reduced
coenzymes NADH and FADH2.
Step 1: Formation of citrateStep 1: Formation of citrate
Citrate synthase catalyzes the condensation of
acetyl-CoA with oxaloacetate to form citrate
Oxaloacetate binds first and induces a
conformational change, creating a binding site for
CoA is liberated and recycled
Step 2: Formation of isocitrate via cis-Step 2: Formation of isocitrate via cis-
The enzyme aconitase catalyzes the reversible
transformation of citrate to isocitrate, through the
intermediary formation of the tricarboxylic acid cis-
The reaction proceeds to the right because
isocitrate is rapidly consumed in the next step of the
Step 3: Oxidation of isocitrate toStep 3: Oxidation of isocitrate to αα--
ketoglutarate and COketoglutarate and CO22
Isocitrate dehydrogenase catalyzes oxidative
decarboxylation of isocitrate to form α-ketoglutarate
Step 4: Oxidation ofStep 4: Oxidation of αα-ketoglutarate to-ketoglutarate to
succinyl-CoA and COsuccinyl-CoA and CO22
This step is another oxidative decarboxylation, in which α-
ketoglutarate is converted to succinyl-CoA and CO2 by the α-
ketoglutarate dehydrogenase complex. The energy of oxidation of
the substrate is conserved in the formation of the thioester bond of
This reaction is virtually identical to the pyruvate dehydrogenase
reaction, except that E1 of the pyruvate dehydrogenase complex
binds pyruvate, and E2 of the α-ketoglutarate dehydrogenase
complex binds α-ketoglutarate
Arsenite inhibits the reaction causing α-ketoglutarate to accumulate.
Step 5: Conversion of succinyl-CoA toStep 5: Conversion of succinyl-CoA to
In this step, the thioester bond of the substrate is broken and
used to drive the synthesis of a phosphoanhydride bond in
either GTP or ATP, forming succinate.
The enzyme becomes phosphorylated at a His residue as an
intermediate in this reaction, and this is transferred to ADP or
GDP to form ATP or GTP. Animal cells have two isozymes, one
specific for ADP and one for GDP. If GTP is formed, ATP can
be generated by nucleoside diphosphate kinase:
GTP + ADP GDP + ATP
Step 6: Oxidation of succinate to fumarateStep 6: Oxidation of succinate to fumarate
The flavoprotein succinate dehydrogenase oxidizes
succinate to fumarate
In eukaryotes, succinate dehydrogenase is tightly bound
to the inner mitochondrial membrane, and is the only
enzyme of the citric acid cycle that is membrane-bound.
Malonate, an analog of succinate, is a strong competitive
inhibitor of succinate dehydrogenase and therefore blocks
the citric acid cycle.
Step 7: Hydration of fumarate to malateStep 7: Hydration of fumarate to malate
The reversible hydration of fumarate to L-malate is
catalyzed by fumarase (fumarate hydratase)
Step 8: Oxidation of malate to oxaloacetateStep 8: Oxidation of malate to oxaloacetate
In the last reaction of the citric acid cycle, NAD-
linked L-malate dehydrogenase catalyzes the
oxidation of L-malate to oxaloacetate
Under standard thermodynamic conditions, the
equilibrium of this reaction lies far to the left, but in
cells, oxaloacetate is continually removed by the
highly exergonic citrate synthase reaction in step 1.
This keeps the concentration of oxaloacetate in the
cells low (10-6
Products of one turn of the
citric acid cycle. Three
NADH, one FADH2, one GTP
(or ATP), and two CO2 are
released in oxidative
All cycle reactions are
shown in one direction
only, but keep in mind that
most of the reactions are
The two carbons appearing as CO2 are not the same two carbons that
entered the cycle as the acetyl group of acetyl-CoA (they are in
oxaloacetate, and will be released in subsequent cycles)
The cycle generates the equivalent of 12 ATP’s from one acetyl-CoA
(3 NADH = 9 ATPs, 1 FADH2 = 2 ATPs, 1 ATP (GTP) directly)
Vitamins play key roles in the citric acidVitamins play key roles in the citric acid
Four of the soluble vitamins of the B complex have
precise roles in the functioning of the citric acid cycle:
1. Riboflavin - in the form of flavin adenine dinucleotide
(FAD), a cofactor in the dehydrogenase complexes.
2. Niacin - in the form of nicotinamide adenine
dinucleotide (NAD), a cofactor for three dehydrogenases.
3. Thiamine/vitamin B - as thiamine dephosphate, the
coenzyme for the α-ketoglutarate dehydrogenase
4. Pantothenic acid - a part of coenzyme A, the cofactor
attached in acetyl-CoA and succinyl-CoA
Mar. 3, 2020
Dec. 5, 2019
Mar. 21, 2019
Sep. 13, 2018
Reading: Harper's Biochemistry Chapter 18, Lehninger Principles of Biochemistry 3rd Ed. pp. 567-583.