CHAPTER 19 Tricarboxylic acid cycle

 Overview
1. pyruvate from glycolysis is converted to acetyl-CoA and oxidized to CO2 in the tricarboxylic
acid (TCA) cycle
2. Beginning with acetate, a series of five reactions produces two molecules of CO2, with four
electrons extracted in the form of NADH and four electrons passed to oxaloacetate to
produce a molecule of succinate
3. pathway becomes a cycle by three additional reactions that accomplish a four-electron
oxidation of succinate back to oxaloacetate.

 What Is the Chemical Logic of the TCA cycle?

1. The TCA Cycle Provides a Chemically Feasible Way of Cleaving a Two-Carbon Compound
 Path from acetate to 2 CO2 requires the C-C cleavage
 Common reactions in biological system：

A. 𝛽-cleavage can’t be used for acetate, which does not have a 𝛽 carbon
B. 𝛼 -cleavage would require hydroxylation of acetate, which is not a favorable
reaction for acetate
 Both reactions are unsuitable for acetate
 Better to condense acetate with oxaloacetate (OAA) and carry out a 𝛽-cleavage
 TCA combines this with oxidation to form CO2, regenerate OAA and capture all energy
 How Is Pyruvate Oxidatively Decarboxylated to Acetyl-CoA?
1. Introduction：
 in eukaryotic cells, glycolysis occurs in the cytoplasm
 TCA cycle reactions and all steps of aerobic metabolism take place in mitochondria
𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 𝑑𝑒ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛𝑎𝑠𝑒
 [Pyruvate + CoA + NAD+→ acetyl-CoA + CO2 + NADH] is the linking

between glycolysis and the TCA cycle

2. Pyruvate dehydrogenase complex (PDC)
 oxidative decarboxylation of pyruvate is catalyzed by the pyruvate dehydrogenase
complex located in matrix of mito.
 PDC is formed from multiple copies of three enzymes：pyruvate dehydrogenase (E1),
dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3).
 五種輔酵素：thiamine pyrophosphate(TPP)、CoASH、Lipoic acid、FAD、NAD+
3. The reaction mechanism of PDC
 Pyruvate 與 TPP(B1)透過 Pyruvate dehydrogenase(PDH)作用形成 HETPP(Hydroxyethyl
TPP)與 CO2 (decarboxylation)
 lipoic acid 與 HETPP 作用時，透過 dihydrolipoyl transacetylase (DLTA)作用，lipoic acid
會將 S-S 鍵打開與 HETPP 相接，之後 CoASH 參與作用，形成 acetyl-CoA
在 Digydrolipoyl dehydrogenase(DLDH)、FAD 作用下將 lipoic acid re-oxidized 同時形成
NADH
 decarboxylation of pyruvate yields hydroxyethyl-TPP, transfer to lipoic acid is followed by
formation of acetyl-CoA

 How Are Two CO2 Molecules Produced from Acetyl-CoA?

1. Introduction：
在反應過程中有 dehydrogenase 出現必有 NADH 生成
有 CO2 出現相伴著 NADH、FADH2；有 NADH 則不一定會有 CO2
 net Rx：Acetyl-CoA + 2H2O + 3NAD+ + FAD + GDP → FADH2 + 2CO2 + 3NADH + CoA-SH + GTP
2. The citrate synthase reaction initiates the TCA cycle

 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA
 the mechanism involves nucleophilic attack by the carbanion of acetyl-CoA join the
carbonyl carbon of oxaloacetate, followed by thioester hydrolysis
 Perkin condensation：a C-C condensation between a ketone or aldehyde and an ester
 citrate synthase reactions has large, negative free energy → initiate TCA cycle(the site
of regulation)
 NADH is allosteric inhibitors
  citrate synthase binding of OAA induces a conformational change that facilitates the
bonding of Acetyl-CoA
3. Citrate is isomerized by aconitase to form isocitrate

 Citrate is isomerized to isocitrate by aconitase in a two-step process involving aconitate

as an intermediate
 Citrate is a poor substrate for oxidation (tertiary alcohol) but isocitrate is great
(secondary alcohol)
 Aconitase Utilizes an Iron–Sulfur Cluster (Binding of iron (Fe2+) to this position activates
aconitase)
 Fluoroacetate Blocks the TCA Cycle

A. Fluoroacetate is an extremely poisonous agent that blocks the TCA cycle

B. The action of fluoroacetate has been traced to aconitase, which is inhibited in vivo
by fluorocitrate, which is formed from fluoroacetate
C. Fluorocitrate 不能被 aconitase 代謝掉而讓 TCA cycle 停擺，導致死亡
4. Isocitrate dehydrogenase catalyzes the first oxidative decarboxylation in the cycle
 isocitrate is oxidatively decarboxylated to yield 𝛼-ketoglutarate, with concomitant
reduction of NAD+ to NADH in the isocitrate dehydrogenase reaction
 oxidation of C2 OH(產生 NADH) → 𝛽-decarboxylation (產生 CO2)
 Isocitrate Dehydrogenase Links the TCA Cycle and Electron Transport
 5. 𝛼-Ketoglutarate dehydrogenase catalyzes the second oxidative decarboxylation of the TCA
cycle

 the subunits of 𝛼-Ketoglutarate dehydrogenase are similar to those of Pyruvate

dehydrogenase
 consisting of 𝛼-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase (DLTA) and
dihydrolipoyl dehydrogenase (DLDH)
 five coenzymes (TPP、CoASH、lipoic acid、NAD、FAD) used same to PDH

 How Is Oxaloacetate Regenerated to Complete the TCA Cycle?

1. Succinyl-CoA synthetase catalyzes substrate-level phosphorylation
 The reaction is catalyzed by succinyl-CoA synthetase, sometimes called succinate
Thiokinase (substrate-level phosphorylation)
 succinyl-CoA is itself a high-energy intermediate and is utilized in the next step of the
TCA cycle to drive the phosphorylation of GDP to GTP or ADP to ATP
 The GTP produced by mammals in this reaction can exchange its terminal phosphoryl
group with ADP via the nucleoside diphosphate kinase reaction

 Thioester → [succinyl-P] → [phosphor-histidine] → GTP → ATP

 A two-carbon acetyl group has been introduced as acetyl-CoA and linked to OAA and
two CO2 molecules have been liberated.
  The cycle has produced two molecules of NADH and one of GTP or ATP and has left a
molecule of succinate
2. Succinate dehydrogenase is FAD-dependent：
 oxidation of a single bond to a double bond
 succinate dehydrogenase is identical with the succinate coenzyme Q reductase of the
electron-transport chain
 succinate dehydrogenase is an integral membrane protein tightly associated with the
inner mitochondrial membrane
 succinate dehydrogenase contains three types of Fe-S centers

3. Fumarase catalyzes the hydration of fumarate to form malate： across the double bond
4. Malate dehydrogenase completes the cycle by oxidizing malate to oxaloacetate：oxidation
of the resulting alcohol to a ketone
 the concentration of oxaloacetate in the mitochondrial matrix is usually quite low.
 The reaction, however, is pulled forward by the favorable citrate synthase reaction

→ the trio of reactions will be seen again in fatty acid and amino acid breakdown and synthesis
 Steric Preferences in NAD+-Dependent Dehydrogenases
 What Are the Energetic Consequences of the TCA Cycle?
1. Glycolysis(→pyruvate)：
Glucose + 2H2O + 2NAD+ + 2ADP → 2pyruvate + 2NADH + 2H+ + 2ATP
2. Pyruvate decarboxylation：
Pyruvate + CoASH + 1NAD+ → Acetyl-CoA + CO2 + NADH + H+
3. TCA：Acetyl-CoA + 3NAD+ + FAD + ADP + 2H2O → FADH2 + ATP + CoASH + 2CO2 + 3NADH
4. Combining glycolysis and the TCA cycle gives the net reaction shown

 Can the TCA Cycle Provide Intermediates for Biosynthesis?

1. TCA cycle provides intermediates for biosynthesis (amino acid are highlighted in orange)
2. Acetyl-CoA 是連結各種物質轉換(fatty acid、amino acid、pyrimidine、purine)的重要橋梁
 What Are the Anaplerotic, or Filling Up, Reactions
1. reactions replenish the TCA cycle intermediates, Hans Kornberg proposed that they be
called anaplerotic reactions
2. anaplerotic reactions：PEP carboxylase、pyruvate carboxylase、malic enzyme
正常的糖解作用的最後一步，是從 PEP 經由 pyruvate kinase 作用形成 pyruvate；而 TCA
cycle 則是由 malate dehydrogenase 將 Malate 轉變成 Oxaloacetate
anaperotic reaction 的目的是要補充 OAA，這樣 TCA cycle 才不會停止

 Pyruvate carboxylation：
A. 消耗ATP 與CO2 (1C)將pyruvate(3C)轉成oxaloacetate(4C) by pyruvate carboxylase
B. This is the most important reaction
C. Allosteric activator by Ac-CoA
D. In mitochondria of animal cell
 PEP carboxylation：
A. Converts PEP (3C) to oxaloacetate (4C) with CO2
B. Inhibit by aspartate
C. In yeast, bacteria, higher plant, not in animal
D. PEP carboxykinase could have been an anaplerotuc reaction (→)
E. CO2 binds weakly to the enzyme, but oxaloacetate binds tightly, so the reaction
goes the wrong way (←)
 Malic enzyme：
A. Malic enzyme 將 pyruvate 與 NADPH 轉成 malate
B. In mitochondria or cytosol in animal and plants

3. Anaplerosis Plays a Critical Role in Insulin Secretion

 A rise in blood glucose induces an increase in 𝛽-cell glucose metabolism leading to
insulin release
  Pancreatic 𝛽-cells maintain high levels of the anaplerotic enzyme pyruvate carboxylase
(PC) then exported to the cytosol (pyruvate → OAA)
 Cytosolic malic enzyme (MEc) converts malate to pyruvate which is returned to the
mitochondrial matrix
 NADPH and other cytosolic signals produced by this and related cycles provoke insulin
secretion.
 in insulin-resistant individuals, exercise increases anaperotic activity, increase fatty acid
oxidation, and restore insulin sensitivity

4. Fool’s gold and the reductive (reverse) citric acid cycle (require energy)
 energy to drive it? Maybe reaction of FeS with H2S to form FeS2 (iron pyrite)
 iron pyrite, which was plentiful in ancient times, and which is an ancient version of
iron-sulfur cluster
 How Is the TCA Cycle Regulated?
1. Three reactions are the key sites：citrate synthase、isocitrate dehydrogenase、ketoglutarate
dehydrogenase
 also pyruvate dehydrogenase：ATP, NADH, acetyl-CoA inhibit, NAD+, CoA active
2. Pyruvate dehydrogenase is regulated by phosphorylation/dephosphorylation
 pyruvate dehydrogenase complex, which converts pyruvate to acetyl-CoA
 activity of pyruvate dehydrogenase is regulated by dephosphorylation/phosphorylation
of the enzyme complex itself.
 High levels of either product, acetyl-CoA or NADH, allosterically inhibit the pyruvate
dehydrogenase complex
 Acetyl-CoA specially blocks DLTA (dihydrolipoyl transacetylase) and NADH acts on
DLDH(dihydrolipoyl dehydrogenase)
 pyruvate dehydrogenase phosphatase maintains the dehydrogenase in an activated state
 pyruvate dehydrogenase kinase (the stimulate phosphorylation) on the pyruvate DH
blocking the pyruvate dehydrogenase reaction
 Can Any Organisms Use Acetate as Their Sole Carbon Source?

1. The Glyoxylate Cycle Operates in Specialized Organelles

 plants and some bacteria and algae can do glyoxylate cycle in glyoxysome
 enzymes common to both the TCA and glyoxylate pathways exist as isozymes
正常的 TCA cycle 的 isocitrate 會經過兩步驟的 decarboxylation 形成 succinate
在 glyoxylate cycle 中 isocitrate 直接跳過該兩步的 decarboxylation 直接形成 succinate
與 glyoxylate，而 succinate 則是繼續進行 TCA 循環(fumarate→malate→OAA)
  glyoxylate 則是與第二的 acetyl-CoA 反應，形成 malate 最後再變成 OAA
在 isocitrate 形成 succinate 與 glyoxylate 需要 isocitrate lyase 的作用；而 glyoxylate 則需
要 malate synthase 和 acetyl-CoA 的作用才能形成 malate
 isocitrate lyase reaction (aldo cleavage)short-circuits TCA cycle by producing glyoxylate
and succinate
Compared with the TCA cycle, the glyoxylate cycle
A. contains only five steps (as opposed to eight)
B. lacks the CO2-liberating reactions
C. consumes two molecules of acetyl-CoA per cycle
D. produces four-carbon units (oxaloacetate) as opposed to one-carbon units
2. The Glyoxylate Cycle Helps Plants Grow in the Dark
 seed rich in lipid → 𝛽-oxidation → acetyl-CoA in glyocysomes in seeds as germination

3. Glyoxysomes Must Borrow Three Reactions from Mitochondria

 glyoxysomes lack three of enzymes needed to run the glyoxylate cycle
 the succinate DH、fumarase、malate DH are all borrow from the mitochondria
 in a shuttle in which succinate and glutamate are passed to mitochondria, aspartate
and 𝛼-ketoglutarate and are passed to the glyoxysome
 spatially and functionally distinct enzymes operating independently in the two cycles
 Transamination to aspartate follows because oxaloacetate cannot be transported out
of the mitochondria
 Aspartate formed in this way then moves from mitochondria back to the glyoxysomes ,
where a reverse transamination with 𝛼-ketoglutarate forms oxaloacetate