Berg • Tymoczko • Stryer

Biochemistry
Sixth Edition

Chapter 17:
The Citric Acid Cycle

Copyright © 2007 by W. H. Freeman and Company

Outline
1. Pyruvate Dehydrogenase Links Glycolysis to the Citric
Acid Cycle
2. The Citric Acid cycle Oxidizes Two-Carbon Units
3. Entry to the Citric Acid Cycle and Metabolism through It
Are Controlled.
4. The Citric Acid Cycle Is a Source of Biosynthetic
Precursors
5. The Glyoxylate Cycle Enables Plants and Bacteria to Grow
on Acetate.
• The complete oxidation of glucose derivatives to carbon dioxide for
harvesting high-energy electrons from carbon fuel.
• The citric acid cycle (or tricaboxylic acid cycle)
• The final common pathways for the oxidation of fuel molecules such as
amino acids, fatty acids, and carbohydrates
• Most fuel molecules enter the cycle as acetyl coenzyme A
• Also source of most energy and precursor for building blocks of many
other molecules (amino acids, nucleotide bases, cholesterol, and
porphyrin (heme)
 Crestas mitocondriales -> plegamientos d membrana
interna. Membrana interna muy gruesa por gran cantidad
de proteínas.
Beta oxidación de acidos grasos también se da en ella

Figure 17.1 Mitochondrion. The double membrane of the mitochondrion is

evident in this electron micrograph. The numerous invaginations of the inner
mitochondrial membrane are called cristae. The oxidative decarboxylation of
pyruvate and the sequence of reactions in the citric acid cycle take place within
the matrix.
 The Citric Acid Cycle Harvests High-Energy
Electrons
acetyl CoA

isocitrate via citrate

Oxalacetate
(regenerated)
Se liberan dos e-

Malato
-ketoglutarate
Subcinato

succinate
Se liberan dos e-
Se pega coenzima A subsinil

Figure 17.2 Overview of the citric acid cycle. The citric acid cycle oxidizes
two- carbon units, producing two molecules of CO2, one molecule of GTP, and
high- energy electrons in the form of NADH and FADH2.
Oxigeno recibe electrones provenientes de ciclo de Krebs y se convierte
en agua. Por eso, en condiciones anaeróbicas no se da.
Membrana interna
mitocondrial ->
contienen proteínas
Quienes portan acetil CoA para transferencia de
e-

Figure 17.3 Cellular respiration. The citric acid cycle constitutes the first stage in
cellular respiration, the removal of high-energy electrons from carbon fuels
(left). These electrons reduce O2 to generate a proton gradient (red pathway),
which is used to synthesize ATP (green pathway). The reduction of O2 and the
synthesis of ATP constitute oxidative phosphorylation.
 17.1 Pyruvate Dehydrogenase Links Glycolysis to
the Citric Acid Cycle

Acetyl CoA, the fuel for the

citric acid cycle from
breakdown of glycogen, fatty
acid, and amino acids.
In aerobic condition, pyruvate is
transported into mitochondria in
exchange for OH- by the pyruvate
carrier, an antiporter and then
oxidatively decarboxylated to
Acetyl CoA (irreversible reaction).

Figure 17.4 The link between glycolysis and the citric acid cycle. Pyruvate
produced by glycolysis is converted into acetyl CoA, the fuel of the citric
acid cycle.
Proteinas transportadoras permiten paso de sustancias
por membrana.
Piruvato -> Cotransporte con hidroxilos
Luego es convertido a acetilCoA. En reacción -> salen 2
e- (NADH) y dióxido de carbono por complejO
enzimatico piruvato deshidrogenasa (sustratos:
piruvato NAD Y COA. Alfa ceto glutarato
deshidrogenasa (gemela de a anterior y hace parte del
ciclo. Alfa cetoglutarato en vez de piruvato.
 Pyruvate dehydrogenase complex

Pyruvate + NAD+ + CoA Acetyl CoA + CO2 + NADH+H +

High-energy-potential electrons in the form of NADH
are captured; many of the key features of the
reactions of the citric acid cycle itself.
Componentes de la
piruvato deshidogenasa

TPP ->Tiamina (grupo

prostético}

Otras coenzimas:
Conezima A
NAD
A large, highly integrated complex of three kinds
of enzymes (4-10 million daltons) that carry out
a series of remarkably complicated reactions.
(a member of a family protein that includes
- ketoglutarate dehydrogenase)

Figure 17.5 Electron micrograph of the pyruvate dehydrogenase complex from E.

coli.
 Mechanism: The Synthesis of Acetyl Coenzyme A from
Pyruvate Requires Three Enzymes and Five
Coenzymes
The reaction requires the participation of the three enzymes of the pyruvate
dehydrogenase complex, each composed of several polypeptide chains, and
five coenzymes: thiamine pyrophosphate (TPP), lipoic acid, and FAD serve
as catalytic cofactors, and CoA and NAD+ . (At least two additional enzymes
required for regulation of activity of the complex)

vitamin B1

thiazole ring
Three steps of the conversion of pyruvate into acetyl CoA that are coupled
to preserve the free energy derived the decarboxylation step to drive
the formation of NADH and acetyl CoA
 Step 1. Decarboxylation of pyruvate to form Hydroxyethyl-
TPP

TPP -> Tiamina pirofosfatp

Highly acidic than most (=C-) groups, with a pK a value near

10 Ready to nucleophilic attack to carbonyl group of
 The positively charged thiazole ring
of TPP acts as electron sink

decarboxylation

protonation

Figure 17.6 Mechanism of the E1 decarboxylation reaction. E1 is the pyruvate dehydrogenase

component of the pyruvate dehydrogenese complex. A key feature of the prosthetic group, TPP, is that
the carbon atom between the nitrogen and sulfur atoms in the thiazole ring is much more acidic
than most =CH- groups, with a p K a value near 10. (1) This carbon center ionizes to form a carbanion .
(2) The carbanion readily adds to the carbonyl group of pyruvate. (3) This addition is followed by the
decarboxylation of pyruvate. The positively charged ring of TPP acts as an electron sink that
stabilizes the negative charge that is transferred to the ring as part of the decarboxylation. (4)
Protonation yields hydroxyethyl-TPP.
Step 2 . Oxidation of hydroxyethyl-TPP to form acetyl group and
its transfer to lipoamide

Pyruvate dehydrogenase
E1 component

E2 E2

E1 Y E2 -> complejos de la enzima

Step 3 . Formation of Acetyl CoA (in
E2)

Energy-rich thioester
bond is preserved
Step 4 . Regeneration of oxidized form of lipoamide by dihydrolipoyl
dehydrogenase (E3)

Unusual, because the common role for FAD is to receive electrons from
NADH. The electron transfer potential of FAD is altered by its association
with the enzyme and enables it to transfer electrons to NAD+ .
Flavoproteins
 Flexible Linkages Allow Lipoamide to Move Between
Different Active Sites

Amarillo

An atomic model of the

complex reconstituted from
all of the components known
from different complexes
and species

Figure 17.7 Schematic representation of the pyruvate dehydrogenase complex.

The transacetylase core (E2) is shown in red, the pyruvate dehydrogenase
component (E1) in yellow, and the dihydrolipoyl dehydrogenase (E3) in green.
 Homologous to biotin-
brazo binding domains such
as pyruvate
carboxylase

Figure 17.8 Structure of the transacetylase (E2) core. Each red ball represents a
trimer of three E2 subunits. Notice that each subunits consists of three
domains: a lipoamide-binding domain, a small domain for interaction with E3,
and a large transacetylase catalytic domain. The transacetylase domain has
three subunits, with one depicted in red and the others in white in the ribbon
 {Por fuera del
NAD conezima quita los e- al FAD y queda todo como antes = NADH
ciclo de Krebs}
hidroxieltil
FAD reducido

Brazo lipoamida oxidado: asi debe estar

Brazo lipoamida reducido

acetil
 The structural integration of three kinds of enzymes makes the coordinated
catalysis of a complex reaction possible.
The proximity of one enzyme to another increases the overall reaction rate and
minimizes side reactions.

Figure 17.9 Reactions of the pyruvate dehydrogenase complex. At the top

(center), the enzyme (represented by a yellow, a green, and two red spheres) is
unmodified and ready for a catalytic cycle. (1) Pyruvate is decarboxylated to form
hydroxyethyl-TPP. (2) The lipoamide arm of E2 moves into the active site of E1,
(3) E1 catalyzes the transfer of the two-carbon group to the lipoamide group to
form the acetyl-lipoamide complex. (4) E2 catalyzes the transfer of the acetyl
moiety to CoA to form the product acetyl CoA. The dihydrolipoamide arm then
swings to the active site of E3. E3 catalyzes (5) the oxidation of the
dihydrolipoamide acid and (6) the transfer of the protons and electrons to NAD+
to complete the reaction cycle.
17.2 The Citric Acid Cycle Oxidizes Two-Carbon
Units Synthase Forms Citrate from Oxaloacetate and
Citrate
Acetyl Coenzyme A
Por 1 glucosa, 2 acetil CoA
Aldol condensation reaction
Hydrolysis of a high-energy thioester intermediate drives overall reaction.
Very important to minimize the side reaction such as hydrolysis of acetyl CoA

Citrate synthase

Estado de transición

Synthase, an enzyme catalyzing a synthetic reaction in which two units are

joined without the direct participation of ATP or another nucleoside
triphosphate.
 Mechanism: The Mechanism of Citrate Synthase Prevents
Undesirable Reactions
Homodimer of 49-kd subunit (mammalian)
binds sequential, ordered kinetics; oxaloacetate first (induces 19 o rotation, 15Å
movement, creating a binding site for acetyl CoA), followed by acetyl CoA.
(analogous to hexokinase).
Close proximity, orienting, and polarizing certain bonds (two histidines and
one aspartate residues in the active site)

Figure 17.10 Conformational changes in citrate synthase on binding oxaloacetate.

The small domain of each subunit of the homodimer is shown in yellow; the
yellow; the large domains are shown in blue. (Left) Open form of enzyme alone.
(Right) Closed form of the liganded enzyme.
 The catalytic residues crucial for hydrolysis of the thioester linkage are
not appropriately positioned until citryl CoA is formed ( c.f ., hexokinase,
triose phosphate isomerase)
Figure 17.11 Mechanism of synthesis of citryl CoA by citrate synthase. (1) In the substrate complex
(left). His 274 donates a proton to the carbonyl oxygen of acetyl CoA to promote the removal of a
methyl proton by Asp 375 to form the enol intermediate (center). (2) Oxaloacetate is activated by the
transfer of a proton from His 320 to its carbonyl carbon atom. (3) Simultaneously, the enol of acetyl CoA
attacks the carbonyl carbon of oxaloacetate to form a carbon-carbon bond linking acetyl CoA and
oxaloacetate. His 274 is reprotonated. Citryl CoA is formed. His 274 participates again as a proton
donor to hydrolyze the thioester (not shown), yielding citrate and CoA.
Citrate Is Isomerized into
Isocitrate
Se mueve hidroxilo de c3 a c2
Quita h y oh, forma agua. Toma agua otra vez y los vuelve a poner pero en distinto orden

Catalyzed by Aconitase

Estado de transición

Tertiary hydroxyl group

unsuitable for
oxidative
decarboxylation
 Centro hierro-azufre -> hacen transferencias

Bind citrate and then isocitrate through their

carboxylate and hydroxyl group, facilitating
dehydration and rehydration.

Figure 17.12 Binding of citrate to the iron-sulfur complex of aconitase. A 4Fe-4S

iron-sulfur cluster is a component of the active site of aconitase. Notice that one
of the iron atoms of the cluster binds to a COO- group and an OH group of
Isocitrate Is Oxidized and Decarboxylated to α-
Ketoglutarate

Isocitrate + NAD+ -ketoglutarate + CO2 + NADH+H+

Isocitrate dehydrogenase Decarboxilaciones oxidativas:

(important regulation step)
Se quitan electrones y carboxilo

Unstable -ketoacid
Succinyl Coenzyme A Is Formed by the
Oxidative Decarboxylation of α-Ketoglutarate

Enlace tiolester con energia

-ketoglutarate

dehydrogenase
complex
Una de las gemelas

The reaction mechanism is entirely analogous to the pyruvate dehydrogenase

complex. The -ketoglutarate dehydrogenase component (E1) and transsuccinylase
(E2) are different but homologous to the corresponding enzymes in the pyruvate
dehydrogenase complex, whereas dihydrolipoyl dehydrogenase components (E3)
are identical.
A Compound with High Phosphoryl-Transfer Potential Is
Generated from Succinyl Coenzyme A

Energy-rich thioester
compound (ΔGo’=-33.5kJ/mol)

Succinyl CoA
synthetase (succinate
thiokinase)
Mechanism: Succinyl Coenzyme A Synthetase Transforms
Types of Biochemical Energy
Succinyl phosphate phosphohistidine
(energy-rich) (energy-rich)

 subunit

Energy-rich thioester compound

(ΔGo ’=-33.5 kJ/mol)

Swing over to a
 subunit
bound nucleoside
diphosphate

The only step in the TCA cycle of a substrate level

phosphorylation GTP + ADP ™ GDP + ATP (by nucleoside
Figure 17.13 Reaction mechanism of succinyl CoA synthetase. The reaction
proceeds through a phosphorylated enzyme intermediate. (1)
Orthophosphate displaces coenzyme A, which generates another energy-
rich compound, succinyl phosphate. (2) A histidine residue removes the
phosphoryl group with the concomitant generation of succinate and
phosphohistidine. (3) The phosphohistidine residue then swings over to a
bound nucleoside diphosphate, and (4) the phosphoryl group is transferred
to form the nucleoside triphosphate.
 22 heterodimer
One  pair is
the functional
unit

Figure 17.14 Structure of succinyl CoA synthetase. The enzyme is composed of

two subunits. The α subunit contains a Rossmann fold that binds the ADP
component of CoA, and the β subunit contains a nucleotide-activating region
called the ATP-grasp domain. The ATP-grasp domain is shown here binding a
molecule of ADP. Notice the histidine residue is between the CoA and the ADP.
This histidine residue picks up the phosphoryl group from near the CoA and
swings over to transfer it to the nucleotide bound in the ATP-grasp domain.
 Oxaloacetate Is Regenerated by the Oxidation of
Succinate
Activacion del succinato

Se quitan 2 e-
pierden 2 e-
Succinate
dehydrogenase Fumarase

Esta en
membrana
interna
mitocondrial

Malate
dehydrogenase

ΔGo ’=+29.7 kJ/mol

E-FAD + succinate E-FADH2 + fumarate

Succinate dehydrogenase
FAD is covalently attached to a histidine; Iron-sulfur
cluster Embedded in inner mitochondrial membrane
Directly associated with the electron-transport chain
Fumarase Solo hace el isómero L

stereospecific trans addition of

a hydrogen atom and a
hydroxyl group (only L-malate
is formed)
 The Citric Acid Cycle Produces High-Transfer-Potential
Electrons, GTP, and CO2
Acido citrico

Se pierden electrones y
carbonos que provenían OH de C3 A C2
de acetilCoA Cetoacido
de 4 c
Energías equivalentes:
NADH = 2,5 atp
FAD= 1,5 ATP
GTP = ATP
Per acetyl CoA
Pierde COOH Se rompe
2.5 ATP/NADH x 3 7,5 ATP
1.5 ATP/FADH2 x 1
en GTP

NAD+ and FAD are

En total, se ganan
regenerated in the
n 10 ATP por cada
mitochondrion only
e by the transfer acetil CoA Se rompe
electron to O2 .

Figure 17.15 The citric acid cycle. Notice that since succinate is a symmetric
molecule, the identity of the carbons from the acetyl unit is lost.
Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2H 2 O €
2 CO2 + 3 NADH + FADH2 + GTP + 2 H+ + CoA
(The carbon atoms that enter each cycle are not the ones that
leave)
 17.3 Entry to the Citric Acid Cycle and Metabolism Through
It Are Controlled
The Pyruvate Dehydrogenase Complex Is
Regulated Allosterically and by Reversible
Phosphorylation

Irreversible commitment step

mitocondria Acetyl CoA inhibits the transacetylase (E2),

while NADH inhibits the dihydrolipoyl
dehydrogenase (E3)
Llave que regula la entrada de acetilCoA

Two fates of acetyl CoA; CO2 or fatty acids

The net effect of inhibition of PDH;

Para guardar energia. Ciclo intermediario en síntesis de lípidos
sparing
Figure 17.16 From glucose to acetyl CoA. The synthesis of acetyl CoA by the
glucose
pyruvate dehydrogenase complex is a key irreversible step in the metabolism of
glucose.
 Regulacion por modificación covalente
In eukaryotes,

The kinase is associated with the transacetylase component (E2) of PDH.

Both the kinase and phosphatase are regulated.

Estado pospandrial

Figure 17.17 Regulation of the pyruvate dehydrogenase complex. A specific

kinase phosphorylates and inactivates pyruvate dehydrogenase (PDH), and a
phosphatase activates the dehydrogenase y removing the phosphoryl group. The
kinase and the phosphatase also are highly regulated enzymes.
 High NADH/NAD + , Acetyl CoA/CoA, and ATP/ADP promote phosphorylation
(inhibition of PDH)

In muscle, Ca+2 stimulates the phosphatase.

In liver, epinephrine-initiated phosphatidyl inositol pathway through -adrenergic
receptor increases Ca2+ , which stimulates phosphatase
In the liver and adipose tissue, insulin stimulates the phosphatase

Regulacion por alosterismo

No hay AMP en
mitocondria

Los tres son

inhibidores
alostéricos en buen Nad activador y
estado de energía sustrato
ADP activador

Figure 17.18 Response of the pyruvate dehydrogenase complex to the energy charge.
The pyruvate dehydrogenase complex is regulated to respond to the energy charge of
the cell.
(A) The complex is inhibited by its immediate products, NADH and acetyl CoA, as well as
by the ultimate product of cellular respiration, ATP. (B) The complex is activated by
In people with a phosphatase deficiency, pyruvate
dehydrogenase is always inactive; lactic acidosis, low pH,
many tissues malfunction, most notably central nervous
system
 The Citric Acid Cycle Is Controlled at Several Regulación del ciclo
Points
Isocitrate dehydrogenase is stimulated by ADP,
which increases the enzyme’s affinity for
substrates; the binding of isocitrate, NAD+ , Mg 2+ ,
and ADP is mutually cooperative.

-ketoglutarte dehydrogenase is inhibited by

NADH and succinyl CoA and high energy
charge.

Accumulation of citrate in cytosol; allosteric

inhibitor of phosphofructokinase, source of
acetyl CoA for fatty acid synthesis

Accumulation of -ketoglutarate; precursor of

several amino acids

In many bacteria, the synthesis of citrate from

oxaloacetate and acetyl CoA carbon units is
an important control point; ATP increase the
value of KM for acetyl CoA.

Figure 17.19 Control of the citric acid cycle. The citric acid cycle is regulated
primarily by the concentration of ATP and NADH. The key control points are the
 17.4 The Citric Acid Cycle Is a Source of Biosynthetic
Precursors Intermediarios para anabolismo
The citric acid cycle as a major metabolic hub of the
cell Acetil coa sale en
forma de citrato
hacia el citolasma
para hacer acidos
grasos
+ grupo amino

+ grupo amino

+ hierro= grupo hemo

Figure 17.20 Biosynthetic role of the citric acid cycle. Intermediates are drawn
off for biosyntheses (shown by red arrows) when the energy needs of the cell
are met. Intermediates are replenished by the formation of oxaloacetate from
pyruvate.
 The Citric Acid Cycle Must Be Capable of Being Rapidly
Replenished
Pyruvate + CO2 + ATP + H2 O oxaloacetate + ADP + Pi + 2
H+

Pyruvate carboxylase
active only in the presence of acetyl CoA
If energy charge is high, oxaloacetate
is converted into glucose

If energy charge is low, oxaloacetate

replenishes the citric acid cycle (a cycle any
of the intermediates can be replenished this
way)

anaplerotic (“fill up”) reaction

Reacciones anapleroticas:
para reponer
intermediarios del ciclo
que se van gastando

Figure 17.21 PATHWAY INTEGRATION: pathways active during exercise after a night’s rest.
The rate of the citric acid cycle increases during exercise, requiring the replenishment
of oxaloacetate and acetyl CoA. Oxaloacetate is replenished by its formation from
pyruvate.
 The Disruption of Pyruvate Metabolism Is the Cause of
Beriberi and Poisoning by Mercury and Arsenic
Beriberi (TPP, vitamin B1 deficiency disease) Reacción
Higher levels of pyruvate and -ketoglutarate in blood
anaplerotica?
TPP (thiamine pyrophosphate) is the prosthetic group
of three important enzyme
Pyruvate dehydrogenase
-ketoglutarate dehydrogenase
Transketolase (pentose phosphate pathway)

Nerve system relies essentially on glucose as its only fuel

(TPP deficiency leads primarily to neurological
disorders)

Arsenite causes symptoms similar to those of beriberi

mo y
n el

Figure 17.22 Arsenite poisoning. Arsenite inhibits the pyruvate dehydrogenase

complex by inactivating the dihydrolipoamide component of the transacetylase.
Some sulfhydryl regents, such as 2,3-dimercaptoethanol, relieve the inhibition by
forming a complex with the arsenite that can be excreted.
The Citric Acid Cycle May Have Evolved From Preexisting
Pathways

Assembly of preexisting reaction pathways

17.5 The Glyoxylate Cycle Enables Plants and Bacteria to
Grow on Acetate

Acetate + CoA-SH + ATP Acetyl CoA + AMP + PPi

Acetyl CoA PPi is further hydrolyzed to 2Pi
synthetase

In plants and bacteria

2 Acetyl CoA + NAD+ + H2 O succinate + 2 CoA + NADH + 2 H+

Glyoxylate cycle in bacteria and plant

1. converts two carbon acetyl units into four-carbon units
(succinate) for energy production and biosynthesis that can be
converted into carbohydrates by a combination of the citric acid
cycle and gluconeogenesis
2. bypasses the two decarboxylation steps of the citric acid cycle.
3. Two molecules of acetyl CoA enter per turn of the glyoxylate cycle
4. In plant, the cycle takes place in glyoxysomes.
5. In bacteria and plant, acetyl CoA can be synthesized from acetate
by an ATP driven reaction catalyzed by acetyl CoA synthetase
 17.5 The Glyoxylate Cycle Enables Plants and
Bacteria to Grow on Acetate

Ciclo del glioxilato

Sólo en plantas.
Se hace glucosa a partir de lípidos

No tenemo

Figure 17.22 The glyoxylate pathway. The glyoxylate cycle allows plants and some
microorganisms to grow on acetate because the cycle bypasses the decarboxylation
steps of the citric acid cycle. The reactions of this cycle are the same as those of the
citric acid cycle except for the ones catalyzed by isocitrate lyase and malate synthase,
which are boxed in blue.