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Chapter 17
The Citric Acid cycle
Outline
17.1 pyruvate Dehydrogenase Links Glycolysis to the
Citric Acid cycle
17.2 The Citric Acid Cycle Oxides Two-Carbon Units
17.3 Entry to the Citric Acid Cycle and Metabolism
Through It Are Controlled
17.4 The Citric Acid Cycle Is a Source of Biosynthesis
Precusors
17.5 The Glyoxylate cycle Enables Plants and
Bactreria to Grow on Acetate
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醣解作用
葡萄糖
Energy investment
phase能量投資期
果糖1,6雙磷酸
丙酮酸
Energy payoff phase
能量收益期
檸檬酸循環
氧化磷酸化作用
3
Fig 17-1 One molecule of glucose is converted to two molecule of pyruvate
The Central Role of the Citric Acid Cycle in
Metabolism 檸檬酸循環在代謝作用中所扮演的角色
• Organisms can obtain far more energy from nutrients by
aerobic oxidation than by anaerobic oxidation 藉由有氧氧化,生
物體可自營養物中得到比厭氧作用更多的能量
•Glycolysis糖解作用 produces only 2 molecules of ATP for
each molecule of glucose metabolized 每分子葡萄糖經代謝作
用只能產生兩分子的ATP
•In complete aerobic oxidation to CO2 and water在完全的有氧
氧化成二氧化碳與水時
30-32 molecules of ATP can be produced from each molecule
of glucose metabolized 每分子葡萄糖可產生30至32分子的ATP
• three processes of aerobic metabolism 有氧代謝
–The citric acid cycle 檸檬酸循環
–Electron transport 電子傳遞
–Oxidative phosphorylation 氧化磷酸化
•Metabloism 代謝作用
–Catabolism異化作用;分解代謝
•The oxidative breakdown of nutrients
氧化分解營養物質
–Anabolism合成代謝
•Reductive synthesis of biomolecules
還原合成生物分子
•The citric acid cycle is amphibolic雙重代謝
it plays a role in both catabolism and anabolism
It is the central metabolic pathway
•The citric acid cycle also as:
kerbs cycle克氏循環 : Sir Hans Krebs, who first investigated
the pathway (1953 Nobel Prize)
tricarboxylic acid cycle (TCA cycle)三羧酸循環: some of
the molecules involved are acids with three carboxyl groups
• The citric acid cycle, also called tricarboxylic acid
(TCA) cycle
– The final common pathway for the oxidation of
fuel molecules
– Pyruvate  acetyl CoA (key molecule)
– Take place: mitochondria matrix
Fig 17.1 Mitochondria
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The citric acid cycle harvests high-energy
electrons
• Central metabolic hub of the cell
– Gateway to the aerobic metabolism of any molecule
– Important source of precusors for amino acids,
nucleotide bases and porphyrin
– TCA cycle component oxaloacetate is an important
precusor to glucose
3 NADH
1 FADH2
1 ATP
ATP
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Fig 17.2 Overview of the citric acid cycle
ATP
NADH
FADH2
H+
Fig 17.3 Cellular respiration
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產生乙醯輔酶A
乙醯輔酶A氧化作用
電子轉移及
氧化磷酸化
乙醯輔酶A
FIGURE 19.1 The central relationship of the citric acid cycle to
catabolism
In stage 1: Amino acids, fatty acids, and glucose can all produce acetyl-CoA
In stage 2: acetyl-CoA enters the citric acid cycle
Stages 1 and 2 produce reduced electron carriers (e-) 還原電子攜帶者
In stage 3, the electrons enter the electron transport chain, which then
produces ATP
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Fig. 19-1, p.546
Acetly-CoA
ATP
Fig 17.15 The Citric Acid cycle
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17.1 pyruvate Dehydrogenase Links
Glycolysis to the Citric Acid cycle
Aerobic condition 有氧條件下
由糖解作用而來
丙酮酸
丙酮酸去氫酶
•The pyruvate is oxidized to
1.CO2
2.Acetyl group linked to CoA
(Acetyl-CoA 乙醯輔酶)
•NAD+ is reduced to NADH
由脂肪酸的b-氧化作用而來
乙醯輔酶A
Enters the TCA cycle
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Fig 19.3 An overview of the citric acid cycle.
Fig. 19-3a, p.548
How Pyruvate Is Converted to AcetylCoA 丙酮酸如何轉換成乙醯輔酶A?
• Pyruvate
– From many source, including glycolysis
– Move from cytosol into the mitochondria via a
specific transporter (特定轉運蛋白)
pyruvate + CoA-SH + NAD+  Acetyl-CoA +CO2 + H+ +NADH
exergonic 釋能, NADH is used to generate ATP via electron transport chain
• Enzyme system: pyruvte dehydrogenase
complex 丙酮酸去氫酶複合物
• CoA-SH 輔酶A
– there is an –SH group at one end of the CoA
molecule (acetyl group is attached)
» Acetyl-CoA is a thioester (high energy compounds)
12
Pyruvate dehydrogenase complex
•A large, highly integrated complex of three distinct enzymes
•a family of homologous complexes include α-ketoglutarate
dehydrogenase complex
•Molecular masses range from 4-10 million dalton
13
Mechanism: The synthesis of acetyl coenzyme A from
pyruvate requires three enzyme and five coenzyme
pyruvate dehydrogenase complex
1. Pyruvate dehydrogenase (PDH)
Involved in the conversion
2. Dihydrolipoyl transacetylase
of pyruvate to acetyl-CoA
3. Dihydrolipoyl dehydrogenase
•Thiamine pyrophsophate as coenzyme (TPP; metabolite of Vitamin B1)
塞胺焦磷酸鹽當作輔酶
Lipoic acid (lipoate) 硫辛酸
•一種維他命，不像其他輔酶，是維他命的代謝物
•Act as an oxidizing agent, involves hydrogen transfer
•Also in formation of a thioester linkage with acetyl group
1. 丙酮酸去氫酶
2. 二氫硫辛酸轉乙醯基酶
3. 二氫硫辛酸去氫酶
14
The conversion of pyruvate into acetyl CoA consists of
three steps:
15
1. Decarboxylation
• Pyruvate combines with TPP and is then carboxylated
to yield hydroxyethyl-TPP
• Catalyzed by the pyruvate dehydrogenase component
負碳離子
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Fig 17.6 Mechanism of the E1 decarboxylation reaction
2. Oxidation
• The hydroxyethyl-TPP is oxidized to form an acetyl
group and transfer to lipoamide (lipoic acid linked to
lysine by amide linkage)
– Formation of an energy-rich thioester bond
• Also catalyzed by the pyruvate dehydrogenase
Two sulfhydryl group
component (E1)
Disulfide group
 oxided form
 reduced form
Thioester linkage
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3. Formation of Acetyl CoA
• The acetyl group is transferred from acetyllipoamide
to CoA and form acetyl CoA
• Catalyzed by dihydrolipoyl transacetylase (E2)
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Dihydrolipoamide is oxidized to lipoamide
• Catalyzed by dihydrolipoyl dehydrogenase (E3)
– 此酵素因具FAD, 故稱之為flavoprotein黃素蛋白
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Flexible linkage allow lipoamide to move
between different active sites
lysine
Fig 17.7 Schematic representation of the
pyruvate dehydrogenase complex
8
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Fig 17.8 Structure of the transacetylase (E2) core
• 反應物與酵素彼此間非常的相近，所以反應的不同階段，可以更有效率的發生
• 硫辛酸與它所連結的離胺酸側鏈，有足夠長度可當成「擺動臂」(swinging arm)，
藉此可以移動到反應的每個步驟應有的位置
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Fig 17.9 Reaction of the pyruvate dehydrogenase complex
17.2 The citric acid cycle oxidizes Two-Carbon
Units
Step 1: Formation of Citrate 檸檬酸的形成
•The reaction of acetyl-CoA and oxaloacetate to form citrate and
CoA-SH
– Called a condensation because a new C-C bond formed
– Catalyzede by the citrate synthase 檸檬酸合成酶(condensing
enzyme縮合酵素)
• A synthase is an enzyme that make a new covalent bond, but it does
not require the direct input of ATP
• An exergonic reaction 釋能反應 (hydrolysis of a thioester releases
energy)
aldol
condensation
hydrolysis
22
Mechanism: the mechanism of citrate synthase
prevents undesirable reactions
• Citrate synthase is a dimer of identical 49kd subunit
– Oxaloacetate binds first, followed by acetyl CoA
• Oxaloacetate induces a major structural rearrangement
leading to the creation of a binding site for acetyl CoA
Fig 17.11 Mechanism of synthesis of citryl CoA by citrate synthase
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Step 2: Isomerization of citrate to Isocitrate
檸檬酸異構化成異檸檬酸
• Citrate is isomerized into isocitrate to undergo
oxidative decarboxylation
– Catalyzed by aconitase
• An iron-sulfur protein, or nonheme-iron protein
dehydration
hydration
4Fe-4S iron-sulfur cluster
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Step 3: Isocitrate is Oxidized and
decarboxylated to α-ketoglutarate
•The first of four oxidation-reduction reactions in the
TCA cycle
•Catalyzed by isocitrate dehydrogenase
Isocitrate + NAD+  α-ketoglutarate +CO2 + NADH
Oxidation
Decarboxylation
Unstable β-ketoacid
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Step 4: succinyl coenzyme A is formed by the
oxidative decarboxylated of α-ketoglutarate
• The oxidative decarboxylation of α-ketoglutae
closely resembles the of pyruvate
α-ketoglutarate
dehydrogenase
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Step 5: Formation of Succinate
• In mammals, there are two isozyme
– One specific for ADP
•Tissues perform large amounts of cellular
respiration, such as skeletal and heart muscle
– One specific for GDP
•Tissues that are perform many anabolic reaction,
such as liver
succynyl-CoA
synthetase
ADP
ATP
GTP + ADP  GDP + ATP
Nucleoside diphosphokinase
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Mechanism: succynyl-CoA synthetase transform
types of biochemical energy
• Energy in the thioester molecule is transfer into
phosphory –roup-transfer potiential
Displace Coenzyme A by
orthophosphate
Fig 17.13 Reaction mechanism of succinyl CoA synthetase
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Step 6-8: Oxaloacetate is regenerated
by the oxidation of succinate
succinate
dehydrogenase
fumarase
Hydration
malate dehydrogenase
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succinate dehydrogenase
• An iron-sulfur protein
– Three kinds of iron-sulfur cluster
•2Fe-2S, 3Fe-4S and 4Fe-4S
– Consists of a 70kd and a 27kd subunit
– An integral protein of the inner mitochondrial
membrane (the other enzymes are in the matrix)
•Directly associated with the electron-transport chain
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• Citric acid cycle
Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O 
2CO2 + CoA + 3NADH + 2H+ + FADH2 + ATP
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Fig 17.15 The citric acid cycle
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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
• High concentration of reaction products inhibit
the reaction
– Acetyl CoA inhibits the transacetylase
component (E2)
– NADH inhibits the dihydrolipoyl dehydrogenase
(E3)
Fig 17.16 From glucose to
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acetyl CoA
Fig 17.18 Response of the pyruvate dehydrogenase complex to the energy charge
•At rest, the muscle cell will not have significant energy demands
– NADH/NAD+, acetyl CoA/CoA, ATP/ADP ratio will high
deactivation of pyruvate dehydrogenase
pyruvate dehydrogenase is switched off when the energy
charge is high
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Pyruvate dehydrogenase kinase I (PDKI) :
Associated with the transacetylase component (E2)
In some tissue, the phosphatase is
regulated by hormones (14.1)
In liver, epinephrine binds to the adrenergic receptor
initiate the phosphatidylinositol
pathway
increase Ca2+ concentration
activates the phosphatase
In fatty acid synthesis tissue—liver,
Pyruvate dehydrogenase phosphatase (PDP) adipose tissue
insulin stimulates the phosphatase
Fig 17.17 Regulation of the pyruvate dehydrogenase complex
• In rest
– NADH/NAD+, acetyl CoA/CoA, ATP/ADP ratio will high
promote phosphorylation
deactivation of pyruvate dehydrogenase
• As exercise begins
– ADP, pyruvate activate the dehydroganse by inhibiting kinase
– Ca2+ stimulate phosphatase
36
The citric acid cycle is controlled at several points
Citrate synthase (in bacteria)
ATP
Isocitrate
dehydrogenase
α-ketoglutarate
dehydrogenase
37
Fig 17.19 Control of the citric acid cycle
Control of the Citric Acid Cycle Proper
•Three control points
– The reaction catalyzed by citrate synthase, isocitrate
dehydrogenase, and the α-ketoglutarate dehydrogenase
complex
– The first regulatory site -- Citrate synthase (In many
bacteria)
•An allosteric enzyme inhibited by ATP, NADH, succinyl-CoA
and its own product citrate
– The second regulatory site -- isocitrate dehydrogenase
•Allosteric activator: ADP, NAD+
•Inhibited by ATP, NADH
– The third regulatory site -- α-ketoglutarate
dehydrogenase complex
•Inhibited by ATP, NADH, succinyl-CoA
有些酵素的活性，會被它下游的產物所調節，是為 迴饋控制 (feedback) 現象；這類酵
素的分子上，除了有活性區可與其基質結合外，還有可與其下游產物結合的位置，稱
38
為調節區 (regulatory site)，這種酵素則稱為異位酶 (allosteric enzyme) 。
Control mechanism
Pyruvate
dehydrogenase
complex
citrate synthase
Isocitrate
dehydrogenase
-ketoglutarate
dehydrogenase
complex
Fig 19.8 Control points in the
conversion of pyruvate to
acetyl-CoA and in the citric
acid cycle
39
17.4 The citric acid cycle is a source of
biosynthetic precusor
Fig 17.20 Biosynthetic roles of the citric acid cycle
40
41
Fig. 19-15, p.570
Mitochondria
Cytosol
The various catabolic
pathways that feed
into the TCA cycle
A summary of catabolism, showing the central role of the citric acid cycle42
Fig. 19-10, p.565
The citric acid cycle must be capable of being
rapidly replenished
• The TCA cycle is a source of starting materials for the
biosynthesis of many important biomolecules
– If a component of the citric acid cycle is taken out for
biosynthesis, it must be replaced 中間產物被利用來合成其他
分子,之後這些中間產物就必須被補充
• [Oxaloacetate] maintained at a level sufficient to allow acetylCoA to enter the cycle
• A reaction that replenishes a citric acid cycle intermediate is
called anaplerotic reaction 回補反應
– 當身體能量過低時，TCA cycle中間產物不足時，體內一些
物質也會分解產生這些中間產物，以維持TCA cycle的活性
– In some organisms, acetyl-CoA can be converted to
oxaloacetate by glyoxylate cycle
– In mammals, oxalocacetate is produced from pyruvate by
43
pyruvate carboxylase (丙酮酸羧化酶)
Fig 17.21 Pathway integration: pathway active during
exercise after a night’s rest
Pyruvate + CO2 +ATP + H2O oxaloacetate +ADP+ Pi + 2H+
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17.5 The Glyoxylate Cycle Enables Plants
and Bacteria to Grow on Acetate
• Some plants and bacteria can produce glucose from
fatty acid – glyoxylate Cycle有些植物可以利用脂肪分
解而得的Acetyl-CoA以生成葡萄糖 --乙醛酸循環
• Two enzyme involved
– Isocitrate lyase 異檸檬酸裂解酶
• Cleaves isocitrate, producing glyoxylate and succinate 將
異檸檬酸分解產生乙醛酸和琥珀酸
– Malate synthase 蘋果酸合成酶
• Catalyzes the reaction of glyoxylate with acetyl-CoA to
produce malate
脂肪分解而得
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Fig 17.23 The glyoxylate pathway
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•The net reaction:
2 Acetyl CoA + NAD+ +2H2O  succinate + 2 CoA +NADH + 2 H+
•The glyoxylate cycle takes place:
– In plants: in glyoxysomes乙醛酸體, specialized
organelles devoted to this cycle
– In yeast and algae: in the cytoplasm
•Helps plants grow in the dark:
– Seeds are rich in lipids, which contain fatty acids
– During germination, plants use the acetyl-CoA
produced in fatty acid oxidation to produce
oxaloacetate and other intermediates for carbohydrate
synthesis
(TCA cycle and glyoxylate cycle can operate simultaneously)
– Once plants begin photosynthesis and can fix CO2,
glyoxysomes disappear
47
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