The Citric Acid Cycle
chapter
16
1. Balance Sheet for the Citric Acid Cycle The citric acid cycle has eight enzymes: citrate synthase,
aconitase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, succinyl-CoA synthetase, succi-
nate dehydrogenase, fumarase, and malate dehydrogenase.
(a) Write a balanced equation for the reaction catalyzed by each enzyme.
(b) Name the cofactor(s) required by each enzyme reaction.
(c) For each enzyme determine which of the following describes the type of reaction(s) catalyzed:
condensation (carbon–carbon bond formation); dehydration (loss of water); hydration (addition
of water); decarboxylation (loss of CO
2
); oxidation-reduction; substrate-level phosphorylation;
isomerization.
(d) Write a balanced net equation for the catabolism of acetyl-CoA to CO
2
.
Answer
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S-184 Chapter 16 The Citric Acid Cycle
2. Net Equation for Glycolysis and the Citric Acid Cycle Write the net biochemical equation for
the metabolism of a molecule of glucose by glycolysis and the citric acid cycle, including all cofactors.
Answer
3. Recognizing Oxidation and Reduction Reactions One biochemical strategy of many living
organisms is the stepwise oxidation of organic compounds to CO
2
and H
2
O and the conservation of a
major part of the energy thus produced in the form of ATP. It is important to be able to recognize
oxidation-reduction processes in metabolism. Reduction of an organic molecule results from the
hydrogenation of a double bond (Eqn 1, below) or of a single bond with accompanying cleavage (Eqn 2).
Conversely, oxidation results from dehydrogenation. In biochemical redox reactions, the coenzymes NAD
and FAD dehydrogenate/hydrogenate organic molecules in the presence of the proper enzymes.
O
Acetaldehyde
reduction
CCH
3
C
O
O
CH
3
HH
H
HH
O
H
CCH
3
Hoxidation
reduction
oxidation
Acetate
O
Ethanol
reduction
CCH
3
C
O
CH
3
H
H
H
H
H
H
OH
C
(2)
CH
3
Hoxidation
reduction
oxidation
Acetaldehyde
O
H
O
H
H (1)
For each of the metabolic transformations in (a) through (h), determine whether oxidation or reduction
has occurred. Balance each transformation by inserting HOH and, where necessary, H
2
O.
4. Relationship between Energy Release and the Oxidation State of Carbon A eukaryotic cell
can use glucose (C
6
H
12
O
6
) and hexanoic acid (C
6
H
14
O
2
) as fuels for cellular respiration. On the basis
of their structural formulas, which substance releases more energy per gram on complete combustion
to CO
2
and H
2
O?
5. Nicotinamide Coenzymes as Reversible Redox Carriers The nicotinamide coenzymes (see
Fig. 13–24) can undergo reversible oxidation-reduction reactions with specific substrates in the
presence of the appropriate dehydrogenase. In these reactions, NADH H
serves as the hydrogen
Chapter 16 The Citric Acid Cycle S-185
Acetate
O
H
H
CO
2
C
CH
2
O
C
O
O
O
C
CH
3
O
C
O
CH
3
Succinate
Pyruvate
O
OC
O
CH
3
CH
2
CO
O
O
C
O
C
C
H
C
O
O
Toluene
Fumarate
C
H
C
H
H
O
Benzoate
CH
2
C
(h)
OH
H
CH
2
C
H
Methanol
OH OH
O
Glycerol Dihydroxyacetone
CH
2
C
OH
CH
2
OOH
CH
2
C
H
OH
CH
2
OH OH
C
O
O
H
OH
C
O
H
GlyceraldehydeGlycerate
O
C
C
O
H
O
H
Carbon dioxide
Formaldehyde
O
HC
O
Formate
H
Formaldehyde Formate
CH
O
H
OH
(a)
(b)
(c)
(d)
(e)
(f)
(g)
H
H
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S-186 Chapter 16 The Citric Acid Cycle
source, as described in Problem 3. Whenever the coenzyme is oxidized, a substrate must be simulta-
neously reduced:
Substrate NADH H
88z
y88 product NAD
Oxidized Reduced Reduced Oxidized
For each of the reactions in (a) through (f), determine whether the substrate has been oxidized or
reduced or is unchanged in oxidation state (see Problem 3). If a redox change has occurred, balance
the reaction with the necessary amount of NAD
, NADH, H
, and H
2
O. The objective is to recognize
when a redox coenzyme is necessary in a metabolic reaction.
Ethanol
O
Acetaldehyde
COH(a)
(b)
CH
2
CH
3
CH
3
O
H
CH
2
OH
C
H
2
O
3
PO C
OPO
3
2
1,3-Bisphosphoglycerate
CH
2
H
OH
Glyceraldehyde 3-phosphate
C
H
2
O
3
PO C HPO
4
O
2
(c)
(d)
(e)
(f)
Malate
CH
2
C
Oxaloacetate
COO
H
CO
2
C
Acetaldehyde
O
CH
3
CC
Pyruvate
O
O
CH
3
O
Acetone
OH
CO
2
Acetoacetate
O
OOC
O
CH
2
C COO
O
OOC
H
CO
2
C
Acetate
O
CH
3
CC
Pyruvate
H
O
CH
3
O
O
CH
3
CCH
2
C
O
O
CH
3
CCH
3
O
Answer
6. Pyruvate Dehydrogenase Cofactors and Mechanism Describe the role of each cofactor involved
in the reaction catalyzed by the pyruvate dehydrogenase complex.
7. Thiamine Deficiency Individuals with a thiamine-deficient diet have relatively high levels of pyruvate
in their blood. Explain this in biochemical terms.
8. Isocitrate Dehydrogenase Reaction What type of chemical reaction is involved in the conversion
of isocitrate to ␣-ketoglutarate? Name and describe the role of any cofactors. What other reaction(s) of
the citric acid cycle are of this same type?
9. Stimulation of Oxygen Consumption by Oxaloacetate and Malate In the early 1930s, Albert
Szent-Györgyi reported the interesting observation that the addition of small amounts of oxaloacetate or
malate to suspensions of minced pigeon breast muscle stimulated the oxygen consumption of the prepara-
tion. Surprisingly, the amount of oxygen consumed was about seven times more than the amount
necessary for complete oxidation (to CO
2
and H
2
O) of the added oxaloacetate or malate. Why did the ad-
dition of oxaloacetate or malate stimulate oxygen consumption? Why was the amount of oxygen consumed
so much greater than the amount necessary to completely oxidize the added oxaloacetate or malate?
10. Formation of Oxaloacetate in a Mitochondrion In the last reaction of the citric acid cycle, malate
is dehydrogenated to regenerate the oxaloacetate necessary for the entry of acetyl-CoA into the cycle:
L
-Malate NAD
88n oxaloacetate NADH H
G 30.0 kJ/mol
(a) Calculate the equilibrium constant for this reaction at 25 C.
(b) Because G assumes a standard pH of 7, the equilibrium constant calculated in
(a) corresponds to
K
eq
The measured concentration of
L
-malate in rat liver mitochondria is about 0.20 m
M
when
[NAD
]/[NADH] is 10. Calculate the concentration of oxaloacetate at pH 7 in these mitochondria.
[oxaloacetate][NADH]
[
L
-malate][NAD
]
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S-188 Chapter 16 The Citric Acid Cycle
(c) To appreciate the magnitude of the mitochondrial oxaloacetate concentration, calculate the num-
ber of oxaloacetate molecules in a single rat liver mitochondrion. Assume the mitochondrion is a
sphere of diameter 2.0 mm.
Answer
11. Cofactors for the Citric Acid Cycle Suppose you have prepared a mitochondrial extract that con-
tains all of the soluble enzymes of the matrix but has lost (by dialysis) all the low molecular weight co-
factors. What must you add to the extract so that the preparation will oxidize acetyl-CoA to CO
2
?
12. Riboflavin Deficiency How would a riboflavin deficiency affect the functioning of the citric acid cy-
cle? Explain your answer.
13. Oxaloacetate Pool What factors might decrease the pool of oxaloacetate available for the activity of
the citric acid cycle? How can the pool of oxaloacetate be replenished?
14. Energy Yield from the Citric Acid Cycle The reaction catalyzed by succinyl-CoA synthetase
produces the high-energy compound GTP. How is the free energy contained in GTP incorporated into
the cellular ATP pool?
15. Respiration Studies in Isolated Mitochondria Cellular respiration can be studied in isolated mito-
chondria by measuring oxygen consumption under different conditions. If 0.01
M
sodium malonate is
added to actively respiring mitochondria that are using pyruvate as fuel source, respiration soon stops
and a metabolic intermediate accumulates.
(a) What is the structure of this intermediate?
(b) Explain why it accumulates.
(c) Explain why oxygen consumption stops.
(d) Aside from removal of the malonate, how can this inhibition of respiration be overcome? Explain.
16. Labeling Studies in Isolated Mitochondria The metabolic pathways of organic compounds have
often been delineated by using a radioactively labeled substrate and following the fate of the label.
(a) How can you determine whether glucose added to a suspension of isolated mitochondria is
metabolized to CO
2
and H
2
O?
(b) Suppose you add a brief pulse of [3-
14
C] pyruvate (labeled in the methyl position) to the mito-
chondria. After one turn of the citric acid cycle, what is the location of the
14
C in the oxaloac-
etate? Explain by tracing the
14
C label through the pathway. How many turns of the cycle are
required to release all the [3-
14
C] pyruvate as CO
2
?
Answer
17. Pathway of CO
2
in Gluconeogenesis In the first bypass step of gluconeogenesis, the conversion of
pyruvate to phosphoenolpyruvate (PEP), pyruvate is carboxylated by pyruvate carboxylase to oxaloac-
etate, which is subsequently decarboxylated to PEP by PEP carboxykinase (Chapter 14). Because the
addition of CO
2
is directly followed by the loss of CO
2
, you might expect that in tracer experiments, the
14
C of
14
CO
2
would not be incorporated into PEP, glucose, or any intermediates in gluconeogenesis.
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S-190 Chapter 16 The Citric Acid Cycle
However, investigators find that when a rat liver preparation synthesizes glucose in the presence of
14
CO
2
,
14
C slowly appears in PEP and eventually at C-3 and C-4 of glucose. How does the
14
C label get
into the PEP and glucose? (Hint: During gluconeogenesis in the presence of
14
CO
2
, several of the
four-carbon citric acid cycle intermediates also become labeled.)
18. [1-
14
C]Glucose Catabolism An actively respiring bacterial culture is briefly incubated with [1-
14
C]
glucose, and the glycolytic and citric acid cycle intermediates are isolated. Where is the
14
C in each of
the intermediates listed below? Consider only the initial incorporation of
14
C, in the first pass of
labeled glucose through the pathways.
(a) Fructose 1,6-bisphosphate
(b) Glyceraldehyde 3-phosphate
(c) Phosphoenolpyruvate
(d) Acetyl-CoA
(e) Citrate
(f) a-Ketoglutarate
(g) Oxaloacetate
19. Role of the Vitamin Thiamine People with beriberi, a disease caused by thiamine deficiency, have
elevated levels of blood pyruvate and a-ketoglutarate, especially after consuming a meal rich in glu-
cose. How are these effects related to a deficiency of thiamine?
20. Synthesis of Oxaloacetate by the Citric Acid Cycle Oxaloacetate is formed in the last step of the
citric acid cycle by the NAD
-dependent oxidation of
L
-malate. Can a net synthesis of oxaloacetate
from acetyl-CoA occur using only the enzymes and cofactors of the citric acid cycle, without depleting
the intermediates of the cycle? Explain. How is oxaloacetate that is lost from the cycle (to biosyn-
thetic reactions) replenished?
21. Oxaloacetate Depletion Mammalian liver can carry out gluconeogenesis using oxaloacetate as the
starting material (Chapter 14). Would the operation of the citric acid cycle be affected by extensive
use of oxaloacetate for gluconeogenesis? Explain your answer.
22. Mode of Action of the Rodenticide Fluoroacetate Fluoroacetate, prepared commercially for
rodent control, is also produced by a South African plant. After entering a cell, fluoroacetate is
converted to fluoroacetyl-CoA in a reaction catalyzed by the enzyme acetate thiokinase:
Chapter 16 The Citric Acid Cycle S-191
The toxic effect of fluoroacetate was studied in an experiment using intact isolated rat heart. After the
heart was perfused with 0.22 m
M
fluoroacetate, the measured rate of glucose uptake and glycolysis de-
creased, and glucose 6-phosphate and fructose 6-phosphate accumulated. Examination of the citric acid
cycle intermediates revealed that their concentrations were below normal, except for citrate, with a con-
centration 10 times higher than normal.
(a) Where did the block in the citric acid cycle occur? What caused citrate to accumulate and the
other cycle intermediates to be depleted?
(b) Fluoroacetyl-CoA is enzymatically transformed in the citric acid cycle. What is the structure of
the end product of fluoroacetate metabolism? Why does it block the citric acid cycle? How might
the inhibition be overcome?
(c) In the heart perfusion experiments, why did glucose uptake and glycolysis decrease? Why did
hexose monophosphates accumulate?
(d) Why is fluoroacetate poisoning fatal?
23. Synthesis of
L
-Malate in Wine Making The tartness of some wines is due to high concentrations of
L
-malate. Write a sequence of reactions showing how yeast cells synthesize
L
-malate from glucose under
anaerobic conditions in the presence of dissolved CO
2
(HCO
3
). Note that the overall reaction for this fer-
mentation cannot involve the consumption of nicotinamide coenzymes or citric acid cycle intermediates.
CoA-SH ATP
FCH
2
COO
FCH
2
C
O
S-CoA AMP PP
i
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S-192 Chapter 16 The Citric Acid Cycle
24. Net Synthesis of a-Ketoglutarate a-Ketoglutarate plays a central role in the biosynthesis of several
amino acids. Write a sequence of enzymatic reactions that could result in the net synthesis of
a-ketoglutarate from pyruvate. Your proposed sequence must not involve the net consumption of other
citric acid cycle intermediates. Write an equation for the overall reaction and identify the source of each
reactant.
25. Amphibolic Pathways Explain, giving examples, what is meant by the statement that the citric acid
cycle is amphibolic.
26. Regulation of the Pyruvate Dehydrogenase Complex In animal tissues, the rate of conversion of
pyruvate to acetyl-CoA is regulated by the ratio of active, phosphorylated to inactive, unphosphory-
lated PDH complex. Determine what happens to the rate of this reaction when a preparation of rabbit
muscle mitochondria containing the PDH complex is treated with (a) pyruvate dehydrogenase kinase,
ATP, and NADH; (b) pyruvate dehydrogenase phosphatase and Ca
2
; (c) malonate.
27. Commercial Synthesis of Citric Acid Citric acid is used as a flavoring agent in soft drinks, fruit
juices, and many other foods. Worldwide, the market for citric acid is valued at hundreds of millions of
dollars per year. Commercial production uses the mold Aspergillus niger, which metabolizes sucrose
under carefully controlled conditions.
(a) The yield of citric acid is strongly dependent on the concentration of FeCl
3
in the culture
medium, as indicated in the graph. Why does the yield decrease when the concentration of Fe
3
is above or below the optimal value of 0.5 mg/L?
(b) Write the sequence of reactions by which A. niger synthesizes citric acid from sucrose. Write an
equation for the overall reaction.
(c) Does the commercial process require the culture medium to be aerated—that is, is this a fermen-
tation or an aerobic process? Explain.
Answer
Chapter 16 The Citric Acid Cycle S-193
12345
90
80
70
60
50
Yield of citric acid (%)
[FeCl
3
] (mg/L)
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28. Regulation of Citrate Synthase In the presence of saturating amounts of oxaloacetate, the activity
of citrate synthase from pig heart tissue shows a sigmoid dependence on the concentration of acetyl-
CoA, as shown in the graph. When succinyl-CoA is added, the curve shifts to the right and the sigmoid
dependence is more pronounced.
No
succinyl-CoA
Activity (% of Vmax)
100
80
60
40
20
20 40 60 80 100 120
[Acetyl-CoA] (M)
Succinyl-CoA
added
On the basis of these observations, suggest how succinyl-CoA regulates the activity of citrate synthase.
(Hint: see Fig. 6–34) Why is succinyl-CoA an appropriate signal for regulation of the citric acid cycle?
How does the regulation of citrate synthase control the rate of cellular respiration in pig heart tissue?
29. Regulation of Pyruvate Carboxylase The carboxylation of pyruvate by pyruvate carboxylase
occurs at a very low rate unless acetyl-CoA, a positive allosteric modulator, is present. If you have just
eaten a meal rich in fatty acids (triacylglycerols) but low in carbohydrates (glucose), how does this
regulatory property shut down the oxidation of glucose to CO
2
and H
2
O but increase the oxidation of
acetyl-CoA derived from fatty acids?
30. Relationship between Respiration and the Citric Acid Cycle Although oxygen does not partici-
pate directly in the citric acid cycle, the cycle operates only when O
2
is present. Why?
31. Effect of [NADH]/[NAD
ⴙ
] on the Citric Acid Cycle How would you expect the operation of the
citric acid cycle to respond to a rapid increase in the [NADH]/[NAD
] ratio in the mitochondrial
matrix? Why?
32. Thermodynamics of Citrate Synthase Reaction in Cells Citrate is formed by the condensation of
acetyl-CoA with oxaloacetate, catalyzed by citrate synthase:
Oxaloacetate acetyl-CoA H
2
O88n citrate CoA H
In rat heart mitochondria at pH 7.0 and 25C, the concentrations of reactants and products are: ox-
aloacetate, 1 m
M
; acetyl-CoA, 1 m
M
; citrate, 220 m
M
; and CoA, 65 m
M
. The standard free-energy change for
the citrate synthase reaction is 32.2 kJ/mol. What is the direction of metabolite flow through the cit-
rate synthase reaction in rat heart cells? Explain.
33. Reactions of the Pyruvate Dehydrogenase Complex Two of the steps in the oxidative decarboxy-
lation of pyruvate (steps 4and 5in Fig. 16–6) do not involve any of the three carbons of pyruvate
yet are essential to the operation of the PDH complex. Explain.
34. Citric Acid Cycle Mutants There are many cases of human disease in which one or another enzyme
activity is lacking due to genetic mutation. However, cases in which individuals lack one of the en-
zymes of the citric acid cycle are extremely rare. Why?
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35. Partitioning between the Citric Acid and Glyoxylate Cycles In an organism (such as E. coli)
that has both the citric acid cycle and the glyoxylate cycle, what determines which of these pathways
isocitrate will enter?
Data Analysis Problem
36. How the Citric Acid Cycle Was Determined The detailed biochemistry of the citric acid cycle was
determined by several researchers over a period of decades. In a 1937 article, Krebs and Johnson sum-
marized their work and the work of others in the first published description of this pathway.
The methods used by these researchers were very different from those of modern biochemistry.
Radioactive tracers were not commonly available until the 1940s, so Krebs and other researchers had
to use nontracer techniques to work out the pathway. Using freshly prepared samples of pigeon breast
muscle, they determined oxygen consumption by suspending minced muscle in buffer in a sealed flask
and measuring the volume (in L) of oxygen consumed under different conditions. They measured
levels of substrates (intermediates) by treating samples with acid to remove contaminating proteins,
then assaying the quantities of various small organic molecules. The two key observations that led
Krebs and colleagues to propose a citric acid cycle as opposed to a linear pathway (like that of gly-
colysis) were made in the following experiments.
Experiment I. They incubated 460 mg of minced muscle in 3 mL of buffer at 40 C for 150 min-
utes. Addition of citrate increased O
2
consumption by 893 L compared with samples without added
citrate. They calculated, based on the O
2
consumed during respiration of other carbon-containing
compounds, that the expected O
2
consumption for complete respiration of this quantity of citrate was
only 302 L.
Experiment II. They measured O
2
consumption by 460 mg of minced muscle in 3 mL of buffer
when incubated with citrate and/or with 1-phosphoglycerol (glycerol 1-phosphate; this was known to
be readily oxidized by cellular respiration) at 40 C for 140 minutes. The results are shown in the
table.
Sample Substrate(s) added L O
2
absorbed
1 No extra 342
2 0.3 mL 0.2
M
1-phosphoglycerol 757
3 0.15 mL 0.02
M
citrate 431
4 0.3 mL 0.2
M
1-phosphoglycerol and
0.15 mL 0.02
M
citrate 1,385
(a) Why is O
2
consumption a good measure of cellular respiration?
(b) Why does sample 1 (unsupplemented muscle tissue) consume some oxygen?
(c) Based on the results for samples 2 and 3, can you conclude that 1-phosphoglycerol and citrate
serve as substrates for cellular respiration in this system? Explain your reasoning.
(d) Krebs and colleagues used the results from these experiments to argue that citrate was
“catalytic”—that it helped the muscle tissue samples metabolize 1-phosphoglycerol more
completely. How would you use their data to make this argument?
(e) Krebs and colleagues further argued that citrate was not simply consumed by these reactions,
but had to be regenerated. Therefore, the reactions had to be a cycle rather than a linear path-
way. How would you make this argument?
Other researchers had found that arsenate (AsO
4
3–
) inhibits ␣-ketoglutarate dehydrogenase and
that malonate inhibits succinate dehydrogenase.
(f) Krebs and coworkers found that muscle tissue samples treated with arsenate and citrate would
consume citrate only in the presence of oxygen; and under these conditions, oxygen was con-
sumed. Based on the pathway in Figure 16–7, what was the citrate converted to in this experi-
ment, and why did the samples consume oxygen?
In their article, Krebs and Johnson further reported the following. (1) In the presence of arsenate,
5.48 mmol of citrate was converted to 5.07 mmol of ␣-ketoglutarate. (2) In the presence of malonate,
citrate was quantitatively converted to large amounts of succinate and small amounts of ␣-ketoglutarate.
(3) Addition of oxaloacetate in the absence of oxygen led to production of a large amount of citrate; the
amount was increased if glucose was also added.
Other workers had found the following pathway in similar muscle tissue preparations:
Succinate 88n fumarate 88n malate 88n oxaloacetate 88n pyruvate
(g) Based only on the data presented in this problem, what is the order of the intermediates in the
citric acid cycle? How does this compare with Figure 16–7? Explain your reasoning.
(h) Why was it important to show the quantitative conversion of citrate to ␣-ketoglutarate?
The Krebs and Johnson article also contains other data that filled in most of the missing components
of the cycle. The only component left unresolved was the molecule that reacted with oxaloacetate to
form citrate.
Answer
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