Download The Citric Acid Cycle

SECTION
8
The Citric Acid Cycle
Learning Objectives
✓ Why is the reaction catalyzed by the pyruvate dehydrogenase complex a
crucial juncture in metabolism?
✓ How is the pyruvate dehydrogenase complex regulated?
✓ What is the advantage of oxidizing acetyl CoA in the citric acid cycle?
✓ How is the citric acid cycle regulated?
Y
ou learned in Chapter 15 that glucose can be metabolized in glycolysis to
pyruvate, yielding some ATP. However, the process of glycolysis is inefficient,
capturing only a fraction of the energy inherent in a glucose molecule as ATP.
More of the energy can be accessed if the pyruvate is completely oxidized to carbon dioxide and water. The combustion of fuels to carbon dioxide and water to
generate ATP is called cellular respiration and is the source of more than 90% of
the ATP required by human beings. Cellular respiration, unlike glycolysis, is an aerobic process, requiring molecular oxygen—O2. In eukaryotes, cellular respiration
takes place inside the double-membrane bounded mitochondria, whereas glycolysis is cytoplasmic.
Cellular respiration can be divided into two parts. First, carbon fuels are completely oxidized with a concomitant generation of high-transfer-potential electrons
in a series of reactions variously called the citric acid cycle (CAC), the tricarboxylic
acid (TCA) cycle, or the Krebs cycle. In the second part of cellular respiration,
referred to as oxidative phosphorylation, the high-transfer-potential electrons are
transferred to oxygen to form water in a series of oxidation–reduction reactions.
This transfer is highly exergonic, and the released energy is used to synthesize ATP.
We will focus on the citric acid cycle in this section, leaving oxidative phosphorylation until Section 9.
The citric acid cycle is the central metabolic hub of the cell. It is the gateway
to the aerobic metabolism of all fuel molecules. The cycle is also an important
source of precursors for the building blocks of many other molecules such as
amino acids, nucleotide bases, and porphyrin (the organic component of heme).
The citric acid cycle component oxaloacetate also is an important precursor to
glucose (p. 254).
We begin this section by examining one of the most important reactions in
living systems: the conversion of glucose-derived pyruvate into acetyl CoA, an activated acetyl unit and the actual substrate for the citric acid cycle. This reaction
links glycolysis and cellular respiration, thus allowing for the complete combustion of glucose, a fundamental fuel in all living systems. We will then study the
citric acid cycle itself, the final common pathway for the oxidation of all fuel molecules, carbohydrates, fats, and amino acids.
Chapter 17: Preparation for
the Cycle
Chapter 18: Harvesting
Electrons from the Cycle
CHAPTER
17
Preparation
for the Cycle
17.1 Pyruvate Dehydrogenase Forms
Acetyl Coenzyme A from
Pyruvate
17.2 The Pyruvate Dehydrogenase
Complex Is Regulated by Two
Mechanisms
17.3 The Disruption of Pyruvate
Metabolism Is the Cause of
Beriberi
The majestic Brooklyn Bridge links Brooklyn with Manhattan in New York City. Pyruvate
dehydrogenase links glycolysis with cellular respiration by converting pyruvate into acetyl
CoA. Unlike the Brooklyn Bridge, however, molecular traffic flows in only one direction. [Ron
Chapple Stock/Alamy.]
A
s you learned in Chapter 15, the pyruvate produced by glycolysis can have
many fates. In the absence of oxygen (anaerobic conditions), the pyruvate is
converted into lactic acid or ethanol, depending on the organism. In the presence
of oxygen (aerobic conditions), it is converted into a molecule, called acetyl coenzyme A (acetyl CoA; Figure 17.1), that is able to enter the citric acid cycle. The
path that pyruvate takes depends on the energy needs of the cell and the oxygen
availability. In most tissues, pyruvate is processed aerobically because oxygen is
readily available. For instance, in resting human muscle, most pyruvate is
processed aerobically by first being converted into acetyl CoA. In very active
muscle, however, much of the pyruvate is processed to lactate because the oxygen supply cannot meet the oxygen demand.
268
269
NH2
N
O
O
–
–
O
O
P
O
O
O
2
H
O
O
O
P
–
O
OH
O
H
N
HN
N
O
CH3
C
N
O
P
CH3
HO
17.1 Pyruvate Dehydrogenase
N
Figure 17.1 Coenzyme A. Coenzyme A is
the activated carrier of acyl groups. Acetyl
CoA, the fuel for the citric acid cycle, is
formed by the pyruvate dehydrogenase
complex.
C
S
CH3
O
Acetyl coenzyme A (Acetyl CoA)
A schematic portrayal of the citric acid cycle is shown in Figure 17.2. The
citric acid cycle accepts two-carbon acetyl units in the form of acetyl CoA. These
two-carbon acetyl units are introduced into the cycle by binding to a four-carbon
acceptor molecule. The two-carbon units are oxidized to CO2, and the resulting
high-transfer-potential electrons are captured. The acceptor molecule is regenerated, capable of processing another two-carbon unit. The cyclic nature of these
reactions enhances their efficiency.
In this chapter, we examine the enzyme complex that catalyzes the formation
of acetyl CoA from pyruvate, how this enzyme is regulated, and some pathologies
that result if the function of the enzyme complex is impaired.
17.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A
from Pyruvate
Glycolysis takes place in the cytoplasm of the cell, but the citric acid cycle takes
place in the mitochondria (Figure 17.3). Pyruvate must therefore be transported into the mitochondria to be aerobically metabolized. This transport is
facilitated by a special transporter (p. 324). In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to
form acetyl CoA.
Pyruvate + CoA + NAD + ¡ acetyl CoA + CO2 + NADH + H +
Matrix
Inner
mitochondrial
membrane
Outer
mitochondrial
membrane
Figure 17.3 Mitochondrion. The double membrane of the mitochondrion
is evident in this electron micrograph. The oxidative decarboxylation of
pyruvate and the sequence of reactions in the citric acid cycle take place
within the matrix. [(Left) Omikron/Photo Researchers.]
O
H3C
C
Acetyl unit
Four-carbon
acceptor
Six-carbon
molecule
GTP
or
ATP
2 CO2
High-transfer-potential electrons
Figure 17.2 An overview of the citric acid
cycle. The citric acid cycle oxidizes twocarbon units, producing two molecules of
CO2, one molecule of GTP or ATP, and
high-transfer-potential electrons.
Table 17.1 Pyruvate dehydrogenase complex of E. coli
Glucose
Enzyme
Abbreviation
Number
of chains
Pyruvate
dehydrogenase
component
E1
24
TPP
Oxidative
decarboxylation
of pyruvate
Dihydrolipoyl
transacetylase
E2
24
Lipoamide
Transfer of
acetyl group
to CoA
Dihydrolipoyl
dehydrogenase
E3
12
FAD
Regeneration of
the oxidized form
of lipoamide
Glycolysis
Pyruvate
CO2
Prosthetic
group
2 e−
Acetyl CoA
Reaction
cataiyzed
Abbreviations: TPP, thiamine pyrophosphate; FAD, flavin adenine dinucleotide.
Citric
acid
cycle
2 CO2
GTP
This irreversible reaction is the link between glycolysis and the citric acid cycle
(Figure 17.4). This reaction is a decisive reaction in metabolism: it commits
the carbon atoms of carbohydrates to oxidation by the citric acid cycle or to
the synthesis of lipids. Note that the pyruvate dehydrogenase complex produces CO 2 and captures high-transfer-potential electrons in the form of
NADH, thus foreshadowing the key features of the reactions of the citric acid
cycle.
The pyruvate dehydrogenase complex is a large, highly integrated complex
of three distinct enzymes (Table 17.1), each with its own active site. The pyruvate dehydrogenase complex is a member of a family of extremely large similar complexes with molecular masses ranging from 4 million to 10 million
daltons (Figure 17.5). As we will see, their elaborate structures allow substrates
to travel efficiently from one active site to another, connected by tethers to the
core of the complex.
8 e−
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.
The Synthesis of Acetyl Coenzyme A from Pyruvate Requires Three
Enzymes and Five Coenzymes
We will examine the mechanism of action of the pyruvate dehydrogenase
complex in some detail because it catalyzes a key juncture in metabolism—the
link between glycolysis and the citric acid cycle that allows the complete oxidation of glucose. The mechanism of the pyruvate dehydrogenase reaction is
wonderfully complex, more so than is suggested by its simple stoichiometry.
The reaction requires the participation of the three enzymes of the pyruvate
dehydrogenase complex as well as five coenzymes. The coenzymes thiamine
pyrophosphate (TPP), lipoic acid, and FAD serve as catalytic coenzymes, and
CoA and NAD ⫹ are stoichiometric coenzymes. Catalytic coenzymes, like
enzymes, are not permanently altered by participation in the reaction. Stoichiometric coenzymes function as substrates.
Figure 17.5 Electron micrograph of the
pyruvate dehydrogenase complex from
E. coli. [Courtesy of Dr. Lester Reed.]
NH2
H
+
N
N
S
S
O
H2N
N
O
H3C
O
Thiamine pyrophosphate (TPP)
270
O
P
–
P
O
O
2–
S
OH
H
O
O
Lipoic acid
The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation, oxidation, and the transfer of the resultant acetyl group to CoA.
O
C
H3C
O
CO2
C
O
C
Decarboxylation
–
–
H3C
O
2 e–
C
Oxidation
17.1 Pyruvate Dehydrogenase
O
CoA
+
H3C
271
C
H3C
Transfer to CoA
S
CoA
O
Pyruvate
Acetyl CoA
These steps must be coupled to preserve the free energy derived from the decarboxylation step to drive the formation of NADH and acetyl CoA.
1. Decarboxylation. Pyruvate combines with the ionized (carbanion) form of TPP
and is then decarboxylated to yield hydroxyethyl-TPP.
R⬘
H3C
N+
H
R⬘
H3C
N+
– +
C
+ CO2
C
CH3
S
R
TPP
OH
H
O
H3C
Carbanion
of TPP
N+
Hydroxyethyl-TPP
Pyruvate
S
R
R⬘
H3C
2 H+
C
O
S
R
O
–
R⬘
H3C
N+
–
This reaction is catalyzed by the pyruvate dehydrogenase component (E1) of the
multienzyme complex. TPP is the coenzyme of the pyruvate dehydrogenase component.
S
R
Carbanion of TPP
2. Oxidation. The hydroxyethyl group attached to TPP is oxidized to form an
acetyl group while being simultaneously transferred to lipoamide, a derivative of
lipoic acid. Note that this transfer results in the formation of an energy-rich
thioester bond.
R⬘
H3C
N+
OH
–
C
S
R
–
+
CH3
S
H
Hydroxyethyl-TPP
(ionized form)
HS
N+
S
+ H3C
S
R
Lysine
side chain
S
C
H
R⬙
O
R⬙
Lipoamide
O
H
N
H
R⬘
H3C
+ H+
Carbanion of TPP
HN
Acetyllipoamide
O
The disulfide group of lipoamide is reduced to its disulfhydryl form in this reaction. The reaction, also catalyzed by the pyruvate dehydrogenase component E1,
yields acetyllipoamide.
3. Formation of Acetyl CoA. The acetyl group is transferred from acetyllipoamide
to CoA to form acetyl CoA. Dihydrolipoyl transacetylase (E2) catalyzes this reaction. The energy-rich thioester bond is preserved as the acetyl group is transferred
to CoA. Acetyl CoA, the fuel for the citric acid cycle, has now been generated from
pyruvate.
HS
CoA
SH + H3C
C
S
C
CoA
H
S
R⬙
Acetyllipoamide
CH3 +
HS
H
O
Coenzyme A
HS
O
Acetyl CoA
R⬙
Dihydrolipoamide
H
S
S
Reactive disulfide bond
Lipoamide
The pyruvate dehydrogenase complex must “reset” lipoamide so that the
complex can catalyze another set of reactions. The complex cannot complete
another catalytic cycle until the dihydrolipoamide is oxidized to lipoamide. In a
fourth step, the oxidized form of lipoamide is regenerated by dihydrolipoyl dehydrogenase (E3). Two electrons are transferred to an FAD prosthetic group of the
enzyme and then to NAD⫹.
272
17 Preparation for the Cycle
NAD+
HS
S
+ FAD
H
H
R⬙
Dihydrolipoamide
E1(␣2␤2)
FAD + NADH + H+
S
HS
E3(␣␤)
+ FADH2
R⬙
Lipoamide
This electron transfer from FAD to NAD⫹ is unusual, because the common
role for FAD is to receive electrons from NADH. The electron-transfer potential of FAD is increased by its association with the enzyme, enabling it to transfer electrons to NAD ⫹ . Proteins tightly associated with FAD are called
flavoproteins.
Flexible Linkages Allow Lipoamide to Move
Between Different Active Sites
E2(␣3)
Figure 17.6 A 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. The number
and type of subunits of each enzyme is
given parenthetically.
The structures of all of the component enzymes of the pyruvate dehydrogenase
complex are known, albeit from different complexes and species. Thus, it is now
possible to construct an atomic model of the complex to understand its activity
(Figure 17.6).
The core of the complex is formed by the transacetylase component E2.
Transacetylase consists of eight catalytic trimers assembled to form a hollow
cube. Each of the three subunits forming a trimer has three major domains
(Figure 17.7). At the amino terminus is a small domain that contains a flexible
lipoamide cofactor. The lipoamide domain is followed by a small domain that
Lipoamide
domain
Lipoamide
Domain
interacting with
E3 component
Figure 17.7 Structure of the transacetylase
(E2) core. Each red ball represents a trimer
of three E2 subunits. Notice that each
subunit 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 subunit depicted in red and the
other two in white in the ribbon
representation.
A
trimer
Transacetylase
domain
NAD+
NADH + H+
FADH2
TPP
Pyruvate CO2
FAD
TPP
H
TPP
6
S
1
E1
S
CH3
E3
S
S
E2
5
TPP
S
FAD
TPP
FAD
OH
C
S
2
H
FAD
TPP
SH HS
S
OH
FAD
C
S
CH3
O
3
4
S
CH3
SH
Acetyl CoA CoA
Figure 17.8 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.
interacts with E3 within the complex. A larger transacetylase domain completes
an E2 trimer. The eight E2 trimers are surrounded by twenty-four copies of E1
(an ␣2␤2 tetramer ) and 12 copies of E3 (an ␣␤ dimer) surround the E2 core.
How do the three distinct active sites work in concert? The key is the long, flexible lipoamide arm of the E2 subunit, which carries substrate from active site
to active site (Figure 17.8).
1. Pyruvate is decarboxylated at the active site of E1, forming the hydroxyethylTPP intermediate, and CO2 leaves as the first product. This active site lies deep
within the E1 complex, connected to the enzyme surface by a 20-Å-long
hydrophobic channel.
2. E2 inserts the lipoamide arm of the lipoamide domain into the deep channel
in E1 leading to the active site.
3. E1 catalyzes the transfer of the acetyl group to the lipoamide. The acetylated
arm then leaves E1 and enters the E2 cube to visit the active site of E2, located
deep in the cube at the subunit interface.
4. The acetyl moiety is then transferred to CoA, and the second product, acetyl
CoA, leaves the cube. The reduced lipoamide arm then swings to the active site
of the E3 flavoprotein.
273
274
17 Preparation for the Cycle
5. At the E3 active site, the lipoamide is oxidized by coenzyme FAD. The reactivated lipoamide is ready to begin another reaction cycle.
6. The final product, NADH, is produced with the reoxidation of FADH2 to FAD.
The structural integration of three kinds of enzymes and the long flexible
lipoamide arm make the coordinated catalysis of a complex reaction possible.
The proximity of one enzyme to another increases the overall reaction rate and
minimizes side reactions. All the intermediates in the oxidative decarboxylation of pyruvate remain bound to the complex throughout the reaction
sequence and are readily transferred as the flexible arm of E 2 calls on each
active site in turn.
17.2 The Pyruvate Dehydrogenase Complex
Is Regulated by Two Mechanisms
Glucose
Pyruvate
Pyruvate
dehydrogenase
complex
Acetyl CoA
CO2
Lipids
Figure 17.9 From glucose to acetyl CoA.
The synthesis of acetyl CoA by the
pyruvate dehydrogenase complex is a key
irreversible step in the metabolism of
glucose.
The pyruvate dehydrogenase complex is stringently regulated by multiple
allosteric interactions and covalent modifications. As stated earlier, glucose can be
formed from pyruvate through the gluconeogenic pathway (p. 251). However, the
formation of acetyl CoA from pyruvate is an irreversible step in animals and thus
they are unable to convert acetyl CoA back into glucose. The oxidative decarboxylation of pyruvate to acetyl CoA commits the carbon atoms of glucose to either of
two principal fates: (1) oxidation to CO2 by the citric acid cycle with the concomitant generation of energy or (2) incorporation into lipid, inasmuch as acetyl CoA
is a key precursor for lipid synthesis (Chapter 28 and Figure 17.9). High concentrations of reaction products inhibit the reaction: acetyl CoA inhibits the
transacetylase component (E2) by directly binding to it, whereas NADH inhibits
the dihydrolipoyl dehydrogenase (E3). High concentrations of NADH and acetyl
CoA inform the enzyme that the energy needs of the cell have been met or that
enough acetyl CoA and NADH have been produced from fatty acid degradation
(p. 407). In either case, there is no need to metabolize pyruvate to acetyl CoA. This
inhibition has the effect of sparing glucose, because most pyruvate is derived from
glucose by glycolysis.
The key means of regulation of the complex in eukaryotes is covalent
modification—in this case, phosphorylation (Figure 17.10). Phosphorylation of
the pyruvate dehydrogenase component (E1) by a specific kinase switches off the
activity of the complex. Deactivation is reversed by the action of a specific phosphatase. Both the kinase and the phosphatase are physically associated with the
transacetylase component (E2), again highlighting the structural and mechanistic importance of this core. Moreover, both the kinase and the phosphatase are
themselves regulated.
To see how this regulation works under biological conditions, consider muscle that is becoming active after a period of rest (Figure 17.11). At rest, the muscle will not have significant energy demands. Consequently, the NADH/NAD⫹,
ATP
ADP
P
Figure 17.10 The regulation of the
pyruvate dehydrogenase complex. A
specific kinase phosphorylates and
inactivates pyruvate dehydrogenase (PDH),
and a phosphatase activates the
dehydrogenase by removing the
phosphoryl group. The kinase and the
phosphatase also are highly regulated
enzymes.
Kinase
Active
PDH
Inactive
PDH
Phosphatase
Pi
H2O
(A) HIGH ENERGY CHARGE
Pyruvate
(B) LOW ENERGY CHARGE
NAD+
PDH
+
PDH
−
−
−
+
NADH
NADH
Acetyl CoA
CAC
17.3 Disruption of Pyruvate Metabolism
Pyruvate
NAD+
Acetyl CoA
ADP
e
−
ATP
CAC
275
ADP
ATP
−
e
Figure 17.11 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
pyruvate and ADP, which inhibit the kinase
that phosphorylates PDH.
acetyl CoA/CoA, and ATP/ADP ratios will be high. These high ratios stimulate the
kinase, promoting phosphorylation and, hence, deactivation of the pyruvate
dehydrogenase complex. In other words, high concentrations of immediate
(acetyl CoA and NADH) and ultimate (ATP) products of the pyruvate dehydrogenase complex inhibit its activity. Thus, pyruvate dehydrogenase is switched off
when the energy charge is high.
As exercise begins, the concentrations of ADP and pyruvate will increase as
muscle contraction consumes ATP and glucose is converted into pyruvate to meet
the energy demands. Both ADP and pyruvate activate the dehydrogenase by inhibiting the kinase. Moreover, the phosphatase is stimulated by Ca2⫹, a signal that also
initiates muscle contraction. A rise in the cytoplasmic Ca2⫹ level to stimulate muscle contraction elevates the mitochondrial Ca2⫹ level. The rise in mitochondrial
Ca2⫹ activates the phosphatase, enhancing pyruvate dehydrogenase activity.
In some tissues, the phosphatase is regulated by hormones. In liver,
epinephrine binds to the ␣-adrenergic receptor to initiate the phosphatidylinositol pathway (p. 180), causing an increase in Ca2⫹ concentration that activates
the phosphatase. In tissues capable of fatty acid synthesis (such as the liver and
adipose tissue), insulin (the hormone that signifies the fed state) stimulates the
phosphatase, increasing the conversion of pyruvate into acetyl CoA. In these tissues, the pyruvate dehydrogenase complex is activated to funnel glucose to
pyruvate and then to acetyl CoA and ultimately to fatty acids.
Clinical Insight
Defective Regulation of Pyruvate Dehydrogenase Results in a
Pathological Condition
In people with a phosphatase deficiency, pyruvate dehydrogenase is always phosphorylated and thus inactive. Consequently, glucose always has to take the anaerobic path and is processed to lactate rather than acetyl CoA. This condition results in
unremitting lactic acidosis—high blood levels of lactic acid. In such an acidic environment, many tissues malfunction, most notably the central nervous system. ■
17.3 The Disruption of Pyruvate Metabolism
Is the Cause of Beriberi
The importance of the coordinated activity of the pyruvate dehydrogenase
complex is illustrated by disorders that result from the absence of a key coenzyme. Recall that thiamine pyrophosphate is a coenzyme for the pyruvate dehydrogenase activity of the pyruvate dehydrogenase complex. Beriberi, a
neurological and cardiovascular disorder, is caused by a dietary deficiency of
QUICK QUIZ List some of the
advantages of organizing the
enzymes that catalyze the formation of
acetyl CoA from pyruvate into a single
large complex.
276
17 Preparation for the Cycle
“A certain very troublesome affliction,
which attacks men, is called by the
inhabitants Beriberi (which means
sheep). I believe those, whom this same
disease attacks, with their knees
shaking and the legs raised up, walk
like sheep. It is a kind of paralysis, or
rather Tremor: for it penetrates the
motion and sensation of the hands and
feet indeed sometimes of the whole
body.”
—Jacob Bonitus, a physician
working in Java in 1630
Figure 17.12 Milled and polished rice.
Brown rice is milled to remove only the
outer husk. Further milling (polishing)
removes the inner husk also, resulting in
white rice. [Image Source/Age Fotostock.]
thiamine (also called vitamin B1). Thiamine deficiency results in insufficient
pyruvate dehydrogenase activity because thiamine pyrophosphate cannot be
formed. The disease has been and continues to be a serious health problem in
the Far East because rice, the major food, has a rather low content of thiamine.
This deficiency is partly ameliorated if the whole rice grain is soaked in water
before milling; some of the thiamine in the husk then leaches into the rice kernel (Figure 17.12). The problem is exacerbated if the rice is polished, because
only the outer layer contains significant amounts of thiamine. Beriberi is also
occasionally seen in alcoholics who are severely malnourished and thus thiamine deficient. The disease is characterized by neurological and cardiac symptoms. Damage to the peripheral nervous system is expressed as pain in the
limbs, weakness of the musculature, and distorted skin sensation. The heart
may be enlarged and the cardiac output inadequate.
Thiamine pyrophosphate is not just crucial to the conversion of pyruvate
to acetyl CoA. In fact, this coenzyme is the prosthetic group of three important
enzymes: pyruvate dehydrogenase, a-ketoglutarate dehydrogenase (a citric acid
cycle enzyme, p. 283), and transketolase. Transketolase functions in the pentose
phosphate pathway, which will be considered in Chapter 25. The common feature of enzymatic reactions utilizing TPP is the transfer of an activated aldehyde
unit. As expected in a body in which TPP is deficient, the levels of pyruvate and
␣-ketoglutarate in the blood of patients with beriberi are higher than normal.
The increase in the level of pyruvate in the blood is especially pronounced after
the ingestion of glucose. A related finding is that the activities of the pyruvate
dehydrogenase complex and the ␣-ketoglutarate dehydrogenase complex in
vivo are abnormally low. The low transketolase activity of red blood cells in
beriberi is an easily measured and reliable diagnostic indicator of the disease.
Why does TPP deficiency lead primarily to neurological disorders? The nervous system relies essentially on glucose as its only fuel. The product of glycolysis—
pyruvate—can enter the citric acid cycle only through the pyruvate dehydrogenase
complex. With that enzyme deactivated, the nervous system has no source of fuel.
In contrast, most other tissues can use fats as a source of fuel for the citric acid cycle.
Symptoms similar to those of beriberi appear in organisms exposed to mercury or arsenite (AsO33-). Both substances have a high affinity for sulfhydryls in
close proximity to one another, such as those in the reduced dihydrolipoyl groups
of the E3 component of the pyruvate dehydrogenase complex (Figure 17.13).
The binding of mercury or arsenite to the dihydrolipoyl groups inhibits the complex and leads to central nervous system pathologies. The proverbial phrase “mad
2,3-Dimercaptopropanol
(BAL)
HS
S
–
O
Excreted
As
HS
S
SH
R
H
Dihydrolipoamide
from pyruvate
dehydrogenase
component E3
As
+
Arsenite
HO
SH
As
O–
HO
HO
S
2 H2O
HO
SH
O
–
SH
S
R
H
Arsenite chelate
on enzyme
R
H
Restored enzyme
Figure 17.13 Arsenite poisoning. Arsenite inhibits the pyruvate dehydrogenase complex by
inactivating the dihydrolipoamide component of the transacetylase. Some sulfhydryl
reagents, such as 2,3-dimercaptoethanol, relieve the inhibition by forming a complex with
the arsenite that can be excreted.
Figure 17.14 Mad Hatter. The Mad Hatter
is one of the characters that Alice meets at
a tea party in her journey through
Wonderland. Real hatters worked with
mercury, which inhibited an enzyme
responsible for providing the brain with
energy. The lack of energy would lead to
peculiar behavior, often described as “mad.”
[The Granger Collection.]
as a hatter” refers to the strange behavior of poisoned hat makers who used mercury nitrate to soften and shape animal furs (Figure 17.14). This form of mercury
is absorbed through the skin. Similar symptoms afflicted the early photographers,
who used vaporized mercury to create daguerreotypes.
Treatment for these poisons is the administration of sulfhydryl reagents with
adjacent sulfhydryl groups to compete with the dihydrolipoyl residues for binding with the metal ion. The reagent–metal complex is then excreted in the urine.
Indeed, 2,3-dimercaptopropanol (see Figure 17.13) was developed after World
War I as an antidote to lewisite, an arsenic-based chemical weapon. This compound was initially called BAL, for British anti-lewisite.
SUMMARY
17.1 Pyruvate Dehydrogenase Forms Acetyl Coenzyme A from Pyruvate
Most fuel molecules enter the citric acid cycle as acetyl CoA. The link
between glycolysis and the citric acid cycle is the oxidative decarboxylation
of pyruvate to form acetyl CoA. In eukaryotes, this reaction and those of
the cycle take place inside mitochondria, in contrast with glycolysis, which
takes place in the cytoplasm. The enzyme complex catalyzing this reaction,
the pyruvate dehydrogenase complex, consists of three distinct enzyme
activities. Pyruvate dehydrogenase catalyzes the decarboxylation of pyruvate and the formation of acetyllipoamide. Dihydrolipoyl transacetylase
forms acetyl CoA, and dihydrolipoyl dehydrogenase regenerates the active
transacetylase. The complex requires five cofactors: thiamine pyrophosphate, lipoic acid, coenzyme A, NAD⫹, and FAD.
17.2 The Pyruvate Dehydrogenase Complex Is Regulated
by Two Mechanisms
The irreversible formation of acetyl CoA from pyruvate is an important
regulatory point for the entry of glucose-derived pyruvate into the citric
acid cycle. The pyruvate dehydrogenase complex is regulated by feedback
inhibition by acetyl CoA and NADH. The activity of the pyruvate dehydrogenase complex is stringently controlled by reversible phosphorylation by
an associated kinase and phosphatase. High concentrations of ATP and
NADH stimulate the kinase, which phosphorylates and inactivates the complex. ADP and pyruvate inhibit the kinase, whereas Ca2⫹ stimulates the
phosphatase, which dephosphorylates and thereby activates the complex.
17.3 The Disruption of Pyruvate Metabolism Is the Cause of Beriberi
The importance of the pyruvate dehydrogenase complex to metabolism,
especially to catabolism in the central nervous system, is illustrated by
beriberi. Beriberi is a neurological condition that results from a deficiency
of thiamine, the vitamin precursor of thiamine pyrophosphate. The lack of
TPP impairs the activity of the pyruvate dehydrogenase component of the
pyruvate dehydrogenase complex. Arsenite and mercury are toxic because of
their effects on the complex. These chemicals bind to the lipoic acid coenzyme of dihydrolipoyl dehydrogenase, inhibiting the activity of this enzyme.
277
Summary
278
17 Preparation for the Cycle
Key Terms
lipoic acid (p. 270)
pyruvate dehydrogenase (E1) (p. 271)
acetyllipoamide (p. 271)
dihydrolipoyl transacetylase (E2)
(p. 271)
acetyl CoA (p. 268)
citric acid cycle (p. 268)
pyruvate dehydrogenase complex
(p. 269)
thiamine pyrophosphate (TPP)
(p. 270)
dihydrolipoyl dehydrogenase (E3)
(p. 272)
flavoproteins (p. 272)
beriberi (p. 275)
Answer to QUICK QUIZ
The advantages are as follows:
1. The reaction is facilitated by having the active sites in
proximity.
2. The reactants do not leave the enzyme until the final
product is formed. Constraining the reactants minimizes
loss due to diffusion and minimizes side reactions.
3. All of the enzymes are present in the correct amounts.
4. Regulation is more efficient because the regulatory
enzymes—the kinase and phosphatase—are part of the
complex.
Problems
1. Naming names. What are the five enzymes (including
regulatory enzymes) that constitute the pyruvate dehydrogenase complex? Which reactions do they catalyze?
2. Coenzymes. What coenzymes are required by the pyruvate dehydrogenase complex and what are their roles?
3. More coenzymes. Distinguish between catalytic coenzymes and stoichiometric coenzymes in the pyruvate dehydrogenase complex.
4. A potent inhibitor. Thiamine thiazolone pyrophosphate
binds to pyruvate dehydrogenase about 20,000 times as
strongly as does thiamine pyrophosphate, and it competitively inhibits the enzyme. Why?
R⬘
H3C
H3C
N+
R⬘
N
H
R
S
TPP
O
R
S
Thiazolone analog
of TPP
5. Lactic acidosis. Patients in shock often suffer from lactic acidosis owing to a deficiency of O2. Why does a lack of
O2 lead to lactic acid accumulation? One treatment for
shock is to administer dichloroacetate, which inhibits the
kinase associated with the pyruvate dehydrogenase complex. What is the biochemical rationale for this treatment?
6. Alternative fuels. As we will see (Chapter 26), fatty acid
breakdown generates a large amount of acetyl CoA. What
will be the effect of fatty acid breakdown on pyruvate dehydrogenase complex activity? On glycolysis?
7. Alternative fates. Compare the regulation of the pyruvate dehydrogenase complex in muscle and liver.
8. Mutations. (a) Predict the effect of a mutation that
enhances the activity of the kinase associated with the PDH
complex. (b) Predict the effect of a mutation that reduces
the activity of the phosphatase associated with the PDH
complex.
9. Flaking wallpaper. Claire Boothe Luce, Ambassador to
Italy in the 1950s (and Connecticut congressperson, playwright, editor of Vanity Fair, and the wife of Henry Luce,
founder of Time magazine and Sports Illustrated) became ill
when she was staying at the ambassadorial residence in Italy.
The wallpaper of her bedroom in the ambassadorial residence was colored a mellow green owing to the presence of
cupric arsenite. Suggest a possible cause of Ambassador
Luce’s illness.
10. Energy rich. What are the thioesters in the reaction
catalyzed by PDH complex?
Selected readings for this chapter can be found online at www.whfreeman.com/Tymoczko