this reaction the summary reactions for glycolysis through py- both PDH and isocitrate dehydrogenase are
nase are activated by AMP
ruvate (see Reaction 9-16 on page 250) and for the oxidative
decarboxylation of pyruvate to acetyl CoA (Reaction 10-1,
also multiplied by 2), we arrive at the following overall reac-
tion for the entire sequence from glucose through the citric
acid cycle:
glucose + 10NAD+ + 2FAD + 4ADP + 4Pi ¡
6CO2 + 10NADH + 2FADH2 + 4ATP (10-3)
As you consider this summary reaction, two points may
strike you: how modest the ATP yield is thus far and how many
coenzyme molecules are reduced during the oxidation of glu-
cose. However, despite the low ATP yield, we must also recog-
nize the reduced coenzymes NADH and FADH2 as high-energy
compounds. As we will see later in this chapter, the transfer of
electrons from these coenzymes to oxygen is highly exergonic.
Thus, the reoxidation of the 12 reduced coenzyme molecules
shown on the right side of Reaction 10-3 provides the energy
needed to drive the synthesis of most of the 38 ATP molecules
produced during the complete oxidation of glucose.
For the release of that energy, we must look to the remain-
ing stages of respiratory metabolism—electron transport and
oxidative phosphorylation. Before doing so, however, we will
consider several additional features of the citric acid cycle: its
regulation, its central position in energy metabolism, and its
role in other metabolic pathways.
Several Citric Acid Cycle Enzymes Are Subject
to Allosteric Regulation
Like all metabolic pathways, the citric acid cycle must be care-
fully regulated to ensure that its level of activity reflects cellu-
lar needs for its products. The main regulatory sites are shown
in Figure 10-11. Most of the control involves allosteric
regulation of four key enzymes by specific effector molecules
that bind reversibly to them. Effector molecules can be either
inhibitors or activators (see Chapter 6), which are indicated
in Figure 10-11 as red minus signs and green plus signs, re-
spectively. In addition to allosteric regulation, the pyruvate
dehydrogenase complex (PDH) is reversibly inactivated by
phosphorylation and activated by dephosphorylation of one
of its protein components.
To appreciate the logic of citric acid cycle regulation, re-
member that the citric acid cycle uses acetyl CoA, NAD + , FAD,
and ADP as substrates and generates as its products NADH,
FADH2, CO2, and ATP (see Reaction 10-2). Three of these
compounds—NADH, ATP, and acetyl CoA—are important
allosteric effectors of one or more enzymes, as shown in Fig-
ure 10-11. In addition, NAD+, ADP, and AMP each activate
at least one of the regulatory enzymes. In this way, the cycle
is highly sensitive to the redox and energy status of the cell, as
assessed by both the NADH/NAD+ ratio and the relative con-
centrations of ATP, ADP, and AMP.
All four of the NADH-generating dehydrogenases shown
in Figure 10-11 are inhibited by NADH. An increase in the
NADH concentration of the mitochondrion therefore decreas-
es the activities of these dehydrogenases, leading to a reduc-
tion in citric acid cycle activity. In addition, PDH is inhibited
by ATP, which is more abundant when energy is plentiful, and
M10_HARD7694_09_GE_C10.indd 279
and ADP, which are more abundant when energy is needed.
The overall availability of acetyl CoA is determined pri-
marily by the activity of the PDH complex (see Reaction 10-1),
which is allosterically inhibited by NADH, ATP, and acetyl CoA
and is activated by NAD+, AMP, and free CoA (Figure 10-11).
In addition, this enzyme complex is inactivated by phosphory-
lation of one of its protein components when the [ATP]/[ADP]
ratio in the mitochondrion is high and is activated by removal
of the phosphate group when the [ATP]/[ADP] ratio is low.
These phosphorylation and dephosphorylation reactions are
catalyzed by PDH kinase and PDH phosphatase, respectively.
Not surprisingly, ATP is an activator of the kinase and an in-
hibitor of the phosphatase.
Due to these multiple control mechanisms, the genera-
tion of acetyl CoA is sensitive to the [acetyl CoA]/[CoA] and
[NADH]/[NAD+] ratios within the mitochondrion and to the
mitochondrial ATP status as well. In addition to these regula-
tory effects on reactions of the citric acid cycle, feedback con-
trol from the cycle to the glycolytic pathway is provided by the
inhibitory effects of citrate and acetyl CoA on phosphofruc-
tokinase and pyruvate kinase, respectively (see Figure 9-13).
Chapter 10
The Citric Acid Cycle Also Plays a Central Role
in the Catabolism of Fats and Proteins
It is essential to understand the central role of the citric acid
|
cycle in all of aerobic energy metabolism. Thus far, we have
Chemotrophic Energy Metabolism: Aerobic Respiration
regarded glucose as the main substrate for cellular respiration.
In addition to glucose (and other carbohydrates), we must also
note the roles of alternative fuel molecules in cellular energy
metabolism and the citric acid cycle, especially fats and proteins,
which constitute roughly half of a diet based on the most recent
dietary guidelines (carbohydrates = 50%, fats = 30%, proteins
= 20%). Far from being a minor pathway for the catabolism of
a single sugar, the citric acid cycle represents the main conduit
of aerobic energy metabolism in a broad spectrum of organ-
isms ranging from microbes to higher plants and animals.
Fat as a Source of Energy. Fats are highly reduced com-
pounds that liberate more energy per gram upon oxidation
than do carbohydrates (see Chapter 3). For this reason, fats
are an important long-term energy storage form for many or-
ganisms. Fat reserves are especially important in hibernating
animals and migrating birds. In plant seeds, fats are a com-
mon form of energy and carbon storage. Fats are well suited
for this storage function because they allow a maximum num-
ber of calories to be stored compactly.
Most fat is stored as deposits of triacylglycerols, which
are neutral triesters of glycerol and long-chain fatty acids,
which we encountered earlier (see Figure 3-27). Catabolism
of triacylglycerols begins with their hydrolysis to glycerol and
free fatty acids. The glycerol is channeled into the glycolytic
pathway by oxidative conversion to dihydroxyacetone phos-
phate (as shown in Figure 9-10). The fatty acids are linked to
coenzyme A to form fatty acyl CoAs, which are then degraded
by B oxidation, a catabolic process that generates acetyl CoA
and the reduced coenzymes NADH and FADH2.
In bacteria, b oxidation occurs in the cytoplasm, and
in eukaryotes it occurs both in the mitochondria and in
279
13/02/17 6:29 pm
 Acetyl
CoA NADH ATP Enzymes That Catalyze These Reactions
- - -
E1: PDH phosphatase
P Pyruvate
E2: PDH kinase
H2O Pi NAD+ All other enzymes as in Figure 10-9.
Pyruvate E1 Pyruvate
dehydrogenase dehydrogenase NADH
(inactive) E2 (active)
ADP ATP
CO2

Acetyl CoA

+ + +
CoA NAD+ AMP

Malate
NADH - dehydrogenase Oxaloacetate

CAC-1
CAC-8
Citrate
Malate
NAD+ NADH

CAC-7
CAC-2

Fumarate
FADH2
THE CITRIC ACID CYCLE Isocitrate

CAC-6 NAD+

CAC-3 Isocitrate
FAD - NADH
NADH dehydrogenase
Succinate + ADP
CO2
NADH
CAC-5
NAD+
GTP a-ketoglutarate
Succinyl CoA CO2
CAC-4
ADP GDP Pi

a-ketoglutarate
- NADH
dehydrogenase
ATP - Succinyl CoA

Figure 10-11 Regulation of the Citric Acid Cycle. The pyruvate dehydrogenase reaction and the citric acid
cycle are shown here in outline form, with full names given for regulatory enzymes. Major regulatory effects
are indicated as either activation (+) or inhibition (–).

peroxisomes. In plants and other eukaryotes that do not de-
pend upon fatty acids as an energy source, b oxidation oc-
curs in the peroxisome and can function as a way to recycle
membrane fatty acids. Here we will focus on the process of b
oxidation as it occurs in the mitochondrion of animals using
saturated fatty acids with an even number of carbons as an
energy source.
In animals, most fatty acids derived from dietary fats, like
the pyruvate derived from carbohydrates, are oxidatively con-
verted into acetyl CoA in the mitochondrion, which then can
be further catabolized by the citric acid cycle. The fatty acids
280
are degraded in a series of repetitive cycles, each of which re-
moves two carbons from the fatty acid until it is completely
degraded. This process of fatty acid catabolism to acetyl CoA
is called b oxidation because the oxidative events in each cy-
cle occur on the carbon atom in the b position of the fatty
acid (that is, the second carbon in from the carboxylic acid
group). Each cycle involves the same four steps—oxidation,
hydration, reoxidation, and thiolysis (Figure 10-12)—and
results in the production of one molecule each of FADH2,
NADH, and acetyl CoA as the fatty acid is shortened by two
carbons in each cycle.

M10_HARD7694_09_GE_C10.indd 280 13/02/17 6:29 pm

 B A O b oxidation of a fatty acid begins with an energy-
requiring activation step in the cytosol. The energy of hydro-
CH3 CH2 CH2 CH2 CH2 CH2 CH2 C O-
lysis of ATP drives the attachment of a CoA molecule to the
Fatty acid
fatty acid (reaction FA-1 in Figure 10-12). This forms a fatty
ATP CoA SH
acyl CoA, which is transported into the mitochondrion by a
FA-1 Activation specific translocase located in the inner membrane. The next
AMP + PPi
four enzymatic steps are repeated in a series of cycles until the
O fatty acid is completely degraded, losing two carbons per cycle
CH3 CH2 CH2 CH2 CH2 CH2 CH2 C S CoA as acetyl CoA. The first three of these steps closely resemble
Fatty acyl CoA the oxidation, hydration, and reoxidation steps that convert
FAD succinate to oxaloacetate in the citric acid cycle (CAC-6, 7,
FA-2 Oxidation and 8).
FADH2
First, an integral membrane protein acting as a dehy-
O drogenase oxidizes the fatty acyl CoA, forming a double bond
CH3 CH2 CH2 CH2 CH2 CH CH C S CoA between the a and b carbons (FA-2). The two electrons and
two protons removed during formation of the unsaturated
H2O FA-3 Hydration derivative are transferred to FAD, forming FADH2. In the next
step, water is added across the double bond by a hydratase so
OH O that the a carbon receives a H atom and the b carbon re-
ceives a hydroxyl group (FA-3). Then another dehydrogenase
Cycle CH3 CH2 CH2 CH2 CH2 CH CH2 C S CoA
oxidizes the b carbon (hence the name b oxidation), convert-
1 NAD +
ing the hydroxyl group to a keto group (FA-4). The two elec-
FA-4 Oxidation
NADH + H+ trons and one proton removed in this oxidation are used to

Chapter 10
reduce NAD+ to NADH. In the fourth step of the cycle, the
O O bond between the a and b carbons is broken by a thiolase,
CH3 CH2 CH2 CH2 CH2 C CH2 C S CoA and a two-carbon fragment is transferred to the S atom of a
second molecule of CoA (FA-5). This results in the produc-

|
CoA SH O

Chemotrophic Energy Metabolism: Aerobic Respiration

tion of acetyl CoA and a fatty acyl CoA that is two carbons
CH3 C S CoA shorter than the fatty acyl CoA that entered the four-step
Acetyl CoA
cycle.
FA-5 Thiolysis These four steps are repeated using the shortened fatty
acyl CoA as a substrate until the original fatty acid is com-
O pletely degraded. Most dietary fatty acids have an even num-
CH3 CH2 CH2 CH2 CH2 C S CoA
ber of carbons and are completely degraded to acetyl CoA,
although unsaturated fatty acids require one or two addition-
FADH2 H2O al enzymes. For unusual fatty acids having an odd number of
CoA SH O carbons, the final cycle produces propionyl CoA, which is one
NADH + H+ carbon longer than acetyl CoA. In a three-step side pathway, a
Cycle CH3 C S CoA
2 carbon from bicarbonate is added to propionyl CoA to gener-
O
ate succinyl CoA, which then enters the citric acid cycle.
CH3 CH2 CH2 C S CoA Although glucose is the preferred energy source of most
H2O
cells, fats provide energy when glucose is limiting (as dur-
FADH2
O ing starvation), under conditions of very low carbohydrate
CoA SH
NADH + H+ intake, or following extremely demanding exercise (such as
Cycle CH3 C S CoA running a marathon). In humans, excessive fat breakdown
3 O can deplete free CoA and lead to a condition known as ke-
CH3 C S CoA
tosis. During ketosis, fats cannot be oxidized completely to
CO2, and partial oxidation products known as ketone bodies
(acetone, acetoacetate, and b-hydroxybutyrate) are formed.
In large quantities, they can lower the pH of the blood, re-
Figure 10-12 The B Oxidation Pathway. Following an ATP-
sulting in ketoacidosis, a condition often seen in uncontrolled
dependent activation step that links a fatty acid to CoA, the fatty acyl
CoA derivative is transported into the mitochondrion (or peroxisome)
diabetes. Although ketone bodies can be a source of energy
and degraded in a series of repetitive cycles of oxidation, hydration, for heart and brain cells, there is currently considerable con-
reoxidation, and thiolysis. Each cycle generates one FADH2 and one troversy over whether ketosis is a health risk or simply a side
NADH in the oxidation steps and releases acetyl CoA in the thiolysis effect of a low-carbohydrate diet.
step, shortening the fatty acid by two carbons. The example above is
for the eight-carbon fatty acid octanoate, which requires three cycles Protein as a Source of Energy and Amino Acids.
of b oxidation. (The a and b carbons are shown on the original fatty Although proteins act as enzymes, transport proteins, hor-
acid and reaction numbers and chemical details are shown for the mones, and receptors in the cell, they can also be catabo-
first cycle only.) lized to generate ATP if necessary, especially during fasting
281

M10_HARD7694_09_GE_C10.indd 281 13/02/17 6:29 pm

 or starvation conditions when carbohydrates and lipid stores
H3N+ O O O
are depleted. In plants, catabolism of proteins to free amino
acids provides building blocks for protein synthesis during the CH3 CH C O -
CH3 C C O-
germination of protein-storing seeds. In addition, when cells
Alanine Pyruvate
degrade proteins and protein-containing structures, the re-
sulting amino acids either can be used to synthesize new pro- (a) CO2
teins or can be degraded oxidatively to yield energy.
Protein catabolism begins with hydrolysis of the peptide Acetyl CoA
bonds that link amino acids together in the polypeptide chain.
The process is called proteolysis, and the enzymes responsi- O O
H3N+ O CAC-1
ble for it are called proteases. The products of proteolytic diges- -
CH C O C C O-
tion are small peptides and free amino acids. Further digestion
of peptides is catalyzed by peptidases, which either hydrolyze CH2 CH2
internal peptide bonds (endopeptidases) or remove successive Citrate
C O C O
amino acids from the end of the peptide (exopeptidases).
Free amino acids, whether ingested as such or obtained O- O-
CAC-2
by the digestion of proteins, can be catabolized for energy.
Aspartate Oxaloacetate
Generally, these alternative substrates are converted to in-
termediates of mainstream catabolism in as few steps as pos- (b) Isocitrate
sible. Despite their number and chemical diversity, all of the
pathways for amino acid catabolism eventually lead to pyru- CAC-3
vate, acetyl CoA, or a few key intermediates in the citric acid
cycle, notably a-ketoglutarate, oxaloacetate, fumarate, and O O
H3N+ O CO2
succinyl CoA. The mitochondrion also participates in the urea
CH C O- C C O-
cycle, a pathway prominent in the liver in which excess amino
groups from degraded amino acids are converted to urea for CH2 CH2
excretion.
CH2 CH2
Of the 20 amino acids found in proteins, three of them
give rise to pyruvate or citric acid cycle intermediates directly: C O C O
alanine, aspartate, and glutamate can be directly converted O- O-
to pyruvate, oxaloacetate, and a-ketoglutarate, respective-
ly (Figure 10-13). All the other amino acids require more Glutamate a-ketoglutarate
complicated pathways, but ultimately all of them have end-
products that are citric acid cycle intermediates. (c)

Figure 10-13 Interconversion of Several Amino Acids and

The Citric Acid Cycle Serves as a Source of
Precursors for Anabolic Pathways
In addition to the catabolic function of the citric acid cycle,
there is a considerable flow of four-, five-, and six-carbon in-
termediates into and out of the cycle in most cells. These side
reactions can replenish the supply of intermediates in the cy-
cle if needed or use intermediates in the cycle for the synthesis
of other compounds in anabolic pathways. Because the cit-
ric acid cycle is a central link between catabolic and anabolic
pathways, it is often called an amphibolic pathway (from
the Greek prefix amphi–, meaning “both”).
Besides its central role in catabolism, the citric acid cycle
is involved in various anabolic processes. For example, the
three reactions shown in Figure 10-13 convert a-keto inter-
mediates of the citric acid cycle into the amino acids alanine,
aspartate, and glutamate. These amino acids are constituents
of proteins, so the citric acid cycle is indirectly involved in
protein synthesis by providing several of the amino acids re-
quired for the process. Other metabolic precursors provided by
the citric acid cycle include succinyl CoA and citrate. Succinyl
CoA is the starting point for the biosynthesis of heme, whereas
citrate can be transported out of the mitochondrion and used
as a source of acetyl CoA for the stepwise synthesis of fatty
acids in the cytosol.
282
Their Corresponding Keto Acids in the Citric Acid Cycle. The
amino acids (a) alanine, (b) aspartate, and (c) glutamate can be con-
verted into the corresponding a-keto acids: pyruvate, oxaloacetate,
and a-ketoglutarate, respectively. Each of these keto acids is an
intermediate in the citric acid cycle, a portion of which is shown to
provide the metabolic context for these reactions. In each case, the
amino group is shown in blue and the keto group in yellow. These
reactions are readily reversible and can occur in either catabolism
(oxidizing amino acids to CO2 and H2O) or anabolism (producing
amino acids for protein synthesis).
The Glyoxylate Cycle Converts Acetyl CoA to
Carbohydrates
Plant species that store carbon and energy reserves in their
seeds as fats face a special metabolic challenge when their
seeds germinate: they must convert the stored fat to sucrose,
the immediate source of carbon and energy for most cells in
the seedling. Many plant species are in this category, includ-
ing such well-known oil-bearing species as soybeans, peanuts,
sunflowers, castor beans, and maize. The fat consists mainly
of triacylglycerols (triglycerides) and is stored in the cell as
lipid bodies. The electron micrograph in Figure 10-14 shows
the prominence of lipid bodies in the cotyledon (embryonic
leaf) of a cucumber seedling.

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