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

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

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

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

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

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

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

` 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Fig 17.15 The citric acid cycle

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 acetyl CoA

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

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 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

The citric acid cycle is controlled at several points Citrate synthase (in bacteria) ATP Isocitrate dehydrogenase α-ketoglutarate dehydrogenase 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 Fig 19.8 Control points in the conversion of pyruvate to acetyl-CoA and in the citric acid cycle a-ketoglutarate dehydrogenase complex

17.4 The citric acid cycle is a source of biosynthetic precusor Fig 17.20 Biosynthetic roles of the citric acid cycle

FIGURE 19.15 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 19.10.) Fig. 19-15, p.570

The various catabolic pathways that feed into the TCA cycle Mitochondria FIGURE 19.10 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. 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 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)

Pyruvate + CO2 +ATP + H2O oxaloacetate +ADP+ Pi + 2H+ Fig 17.21 Pathway integration: pathway active during exercise after a night’s rest Pyruvate + CO2 +ATP + H2O oxaloacetate +ADP+ Pi + 2H+

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

Fig 17.23 The glyoxylate pathway

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