1. Chapter 17 The Citric Acid cycle

2. 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

3. Energy investment phase能量投資期
醣解作用
Energy investment phase能量投資期
葡萄糖
果糖1,6雙磷酸
Energy payoff phase能量收益期
丙酮酸
檸檬酸循環
氧化磷酸化作用
Fig 17-1 One molecule of glucose is converted to two molecule of pyruvate

4. 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

5. The citric acid cycle is amphibolic雙重代謝
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

6. 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

7. 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
Fig 17.2 Overview of the citric acid cycle

8. Fig 17.3 Cellular respiration
ATP
NADH
FADH2 H+
Fig 17.3 Cellular respiration

9. `
電子轉移及氧化磷酸化
產生乙醯輔酶A
乙醯輔酶A氧化作用
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
FIGURE 19.1 The central relationship of the citric acid cycle to catabolism. Amino acids, fatty acids, and glucose can all produce acetyl-CoA in stage 1 of catabolism. In stage 2, acetyl-CoA enters the citric acid cycle. Stages 1 and 2 produce reduced electron carriers (shown here as e-). In stage 3, the electrons enter the electron transport chain, which then produces ATP.
Fig. 19-1, p.546

10. Fig 17.15 The Citric Acid cycle
Acetly-CoA
ATP
Fig The Citric Acid cycle

11. 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
The pyruvate is oxidized to
CO2
Acetyl group linked to CoA
(Acetyl-CoA 乙醯輔酶)
NAD+ is reduced to NADH
丙酮酸去氫酶
由脂肪酸的b-氧化作用而來
乙醯輔酶A
Enters the TCA cycle 11
Fig 19.3 An overview of the citric acid cycle.
Fig. 19-3a, p.548

12. How Pyruvate Is Converted to Acetyl-CoA 丙酮酸如何轉換成乙醯輔酶A?
From many source, including glycolysis
Move from cytosol into the mitochondria via a specific transporter (特定轉運蛋白)
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)
pyruvate + CoA-SH + NAD+  Acetyl-CoA +CO2 + H+ +NADH
exergonic 釋能, NADH is used to generate ATP via electron transport chain

13. 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

14. Mechanism: The synthesis of acetyl coenzyme A from pyruvate requires three enzyme and five coenzyme
pyruvate dehydrogenase complex
Pyruvate dehydrogenase (PDH)
Dihydrolipoyl transacetylase
Dihydrolipoyl dehydrogenase
Involved in the conversion of pyruvate to acetyl-CoA
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

15. The conversion of pyruvate into acetyl CoA consists of three steps:

16. 1. Decarboxylation
Pyruvate combines with TPP and is then carboxylated to yield hydroxyethyl-TPP
Catalyzed by the pyruvate dehydrogenase component
負碳離子
Fig 17.6 Mechanism of the E1 decarboxylation reaction

17. 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 component (E1)
Two sulfhydryl group
 reduced form
Disulfide group
 oxided form
Thioester linkage

18. 3. Formation of Acetyl CoA
The acetyl group is transferred from acetyllipoamide to CoA and form acetyl CoA
Catalyzed by dihydrolipoyl transacetylase (E2)

19. 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

20. Flexible linkage allow lipoamide to move between different active sites
lysine
Fig 17.7 Schematic representation of the pyruvate dehydrogenase complex 8
Fig 17.8 Structure of the transacetylase (E2) core

21. 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
Fig 17.9 Reaction of the pyruvate dehydrogenase complex

22. 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

23. 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 Mechanism of synthesis of citryl CoA by citrate synthase

24. 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
hydration
dehydration
4Fe-4S iron-sulfur cluster

25. 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

26. 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

27. 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
ADP
ATP
succynyl-CoA synthetase
GTP + ADP  GDP + ATP
Nucleoside diphosphokinase

28. 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 Reaction mechanism of succinyl CoA synthetase

29. Step 6-8: Oxaloacetate is regenerated by the oxidation of succinate
succinate dehydrogenase
fumarase
Hydration
malate dehydrogenase

30. 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

31. Citric acid cycle
Acetyl-CoA + 3NAD+ + FAD + ADP + Pi + 2H2O 
2CO2 + CoA + 3NADH + 2H+ + FADH2 + ATP

32. Fig 17.15 The citric acid cycle

34. 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 From glucose to acetyl CoA

35. At rest, the muscle cell will not have significant energy demands
Fig 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

36. In rest As exercise begins
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, adipose tissue
insulin stimulates the phosphatase
Pyruvate dehydrogenase phosphatase (PDP)
Fig 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

37. The citric acid cycle is controlled at several points
Citrate synthase (in bacteria)
ATP
Isocitrate dehydrogenase
α-ketoglutarate dehydrogenase
Fig Control of the citric acid cycle

38. 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，稱為調節區 (regulatory site)，這種酵素則稱為異位酶 (allosteric enzyme) 。

39. Control mechanism Pyruvate dehydrogenase complex citrate synthase
Isocitrate dehydrogenase
Fig 19.8 Control points in the conversion of pyruvate to acetyl-CoA and in the citric acid cycle
a-ketoglutarate dehydrogenase complex

40. 17.4 The citric acid cycle is a source of biosynthetic precusor
Fig Biosynthetic roles of the citric acid cycle

41. FIGURE A summary of anabolism, showing the central role of the citric acid cycle. Note that there are pathways for the biosynthesis of carbohydrates, lipids, and amino acids. OAA is oxaloacetate, and ALA is -aminolevulinic acid. Symbols are as in Figure )
Fig , p.570

42. The various catabolic pathways that feed into the TCA cycle
Mitochondria
FIGURE A summary of catabolism, showing the central role of the citric acid cycle. Note that the end products of the catabolism of carbohydrates, lipids, and amino acids all appear. (PEP is phosphoenolpyruvate; -KG is  ketoglutarate; TA is transamination;  is a multistep pathway.)
Cytosol
The various catabolic pathways that feed into the TCA cycle
A summary of catabolism, showing the central role of the citric acid cycle
Fig , p.565

43. 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 acetyl-CoA 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 pyruvate carboxylase (丙酮酸羧化酶)

44. Pyruvate + CO2 +ATP + H2O oxaloacetate +ADP+ Pi + 2H+
Fig Pathway integration: pathway active during exercise after a night’s rest
Pyruvate + CO2 +ATP + H2O oxaloacetate +ADP+ Pi + 2H+

45. 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

46. Fig 17.23 The glyoxylate pathway

47. The glyoxylate cycle takes place:
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