The Tricarboxylic Acid Cycle

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Chapter 19

• The Tricarboxylic Acid Cycle
• Biochemistry
• by
• Reginald Garrett and Charles Grisham

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Essential Question

• How is pyruvate oxidized under aerobic conditions
• Pyruvate from glycolysis is converted to
  acetyl-CoA and oxidized to CO2 in the
  tricarboxylic acid (TCA) cycle
• What is the chemical logic that dictates how this
  process occurs?

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Hans Krebs showed that the oxidation of acetate
is accomplished by a cycle

• TCA cycle, Citric Acid Cycle or Krebs Cycle
• Pyruvate from glycolysis is oxidatively
  decarboxylated to acetate and then degraded to
  CO2 in TCA cycle
• Some ATP is produced
• More NADH and FADH2 are made (24 electrons)
• NADH and FADH2 go on to make more ATP in electron
  transport and oxidative phosphorylation
  (chapter20)

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Figure 19.1 (a) Pyruvate produced in glycolysis
is oxidized in (b) the tricarboxylic acid (TCA)
cycle. (c) Electrons liberated in this oxidation
flow through the electron-transport chain and
drive the synthesis of ATP in oxidative
phosphorylation. In eukaryotic cells, this
overall process occurs in mitochondria.
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19.1 What Is the Chemical Logic of the TCA
Cycle?

• TCA cycle seems like a complicated way to oxidize
  acetate units to CO2
• Normal ways to cleave C-C bonds in biological
  systems
• cleavage between Carbons ? and ? to a carbonyl
  group (b-cleavage)
• (fructose bisphosphate aldolase)
• ?-cleavage of an ?-hydroxyketone (transketolase
  fig 22.31)

O
CCa Cb
O OH
CCa
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The Chemical Logic of TCA cycle

• Neither of these cleavage strategies is suitable
  for acetate
• Living things have evolved the clever chemistry
  of condensing acetate with oxaloacetate and carry
  out a ?-cleavage.
• TCA combines this ?-cleavage reaction with
  oxidation to form CO2, regenerate oxaloacetate
  and capture all the energy in NADH and ATP

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Figure 19.2The tricarboxylic acid cycle.
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19.2 How Is Pyruvate Oxidatively Decarboxylated
to Acetyl-CoA?

• Pyruvate must enter the mitochondria to enter the
  TCA cycle
• Oxidative decarboxylation of pyruvate is
  catalyzed by the pyruvate dehyrogenase complex
• Pyruvate CoA NAD ? acetyl-CoA CO2 NADH
  H
• Pyruvate dehydrogenase complex is a noncovalent
  assembly of three enzymes
• Five coenzymes are required

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Pyruvate dehydrogenase complex (PDC)

• Three enzymes and five coenzymes
• E1 pyruvate dehydrogenase (24)
• thiamine pyrophosphate
• E2 dihydrolipoyl transacetylase (24)
• lipoic acid
• E3 dihydrolipoyl dehydrogenase (12)
• FAD
• NAD
• CoA
• The product of the first enzyme is pass directly
  to the secondenzyme

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(b) a truncated version of E2
(a) Domain structure of E2 and E3BP subunits
L1 L2 E1BD IC L3 E3BD
30 E1 12 E3
(c) Model of the E2/E3BPE3 core complex
(6 E3BP dimer 48 E2)
(d) Model of human PDC
Figure 19.3 Models of human pyruvate dehydrogenase
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Figure 19.4 The reaction mechanism of the
pyruvate dehydrogenase complex
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(TPP)
Figure 19.5 The mechanism of the first three
steps of the pyruvate dehydrogenase complex
reaction
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The Coenzymes of the Pyruvate Dehydrogenase
Complex
Thiamine pyrophosphate (vitamin B1 analog) TPP
assists in the decarboxylation of a-keto acids
(here) and in the formation and cleavage of
a-hydroxy ketones (as in the transketolase
reaction see Chapter 22).
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The Nicotinamide Coenzymes (vitamin B3, niacin
analog)
NAD/NADH and NADP/NADPH carry out hydride (H-)
transfer reactions. All reactions involving
these coenzymes are two-electron transfers.
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The Flavin Coenzymes (vitamin B2)
FAD/FADH2 Flavin coenzymes can exist in any of
three oxidation states, and this allows flavin
coenzymes to participate in one-electron and
two-electron transfer reactions. Partly because
of this, flavoproteins catalyze many reactions in
biological systems and work with many electron
donors and acceptors.
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• Coenzyme A (vitamin B5, pantothenic acid)
• The two main functions of Co A are
• Activation of acyl groups for transfer by
  nucleophilic attack
• Activation of the a-hydrogen of the acyl group
  for abstraction as a proton

• The reactive sulfhydryl group on CoA mediates
  both of these functions.
• The sulfhydryl group forms thioester linkages
  with acyl groups.
• The two main functions of CoA are illustrated in
  the citrate synthase reaction (see Figure 19.6).

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Lipoic Acid

1. Lipoic Acid functions to couple acyl-group
   transfer and electron transfer during oxidation
   and decarboxylation of a-keto acids.
2. It is found in pyruvate dehydrogenase and
   a-ketoglutarate dehydrogenase.
3. Lipoic acid is covalently bound to relevant
   enzymes through amide bond formation with the
   e-NH2 group of a lysine side chain.

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

• Tricarboxylic acid cycle, Citric acid cycle, and
  Krebs cycle
• Pyruvate is oxidatively decarboxylated to form
  acetyl-CoA
• Citrate (6C)? Isocitrate (6C)? a-Ketoglutarate
  (5C) ? Succinyl-CoA (4C) ? Succinate (4C) ?
  Fumarate (4C) ? Malate (4C) ? Oxaloacetate (4C)

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1. Citrate synthase reaction

• Acetyl-CoA reacts with oxaloacetate in a Perkin
  condensation (A carbon-carbon condensation
  between a ketone or aldehyde and an ester)

Figure 19.6 Citrate is formed in the citrate
synthase reaction from oxaloacetate and
acetyl-CoA. The mechanism involves nucleophilic
attack by the carbanion of acetyl-CoA on the
carbonyl carbon of oxaloacetate, followed by
thioester hydrolysis.
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• Citrate synthase
• is a dimer
• NADH succinyl-CoA are allosteric inhibitors
• Large, negative ?G -- irreversible

Figure 19.7 Citrate synthase in mammals is a
dimer of 49-kD subunits. In the monomer shown
here, citrate (blue) and CoA (red) bind to the
active site, which lies in a cleft between two
domains and is surrounded mainly by a-helical
segments.
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2. Citrate Is Isomerized by Aconitase to Form
Isocitrate

• Citrate is a poor substrate for oxidation because
  it contains a tertiary alcohol
• So aconitase isomerizes citrate to yield
  isocitrate which has a secondary -OH, which can
  be oxidized
• Note the stereochemistry of the reaction
  aconitase removes the pro-R H of the pro-R arm of
  citrate
• Aconitase uses an iron-sulfur cluster (Fig. 19.9)

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Aconitase Utilizes an Iron-Sulfur Cluster
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• Fluoroacetate is an extremely poisonous agent
  that blocks the TCA cycle
• Rodent poison LD50 is 0.2 mg/kg body weight
• Aconitase inhibitor

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3. Isocitrate Dehydrogenase Catalyzes the First
Oxidative Decarboxylation in the Cycle

• Catalyzes the first oxidative decarboxylation in
  the cycle

1. Oxidation of C-2 alcohol of isocitrate with
   concomitant reduction of NAD to NADH
2. followed by a b-decarboxylation reaction that
   expels the central carboxyl group as CO2

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Isocitrate Dehydrogenase

• Isocitrate dehydrogenase links the TCA cycle and
  electron transport pathway because it makes NADH
• Isocitrate dehydrogenase is a regulation reaction
• NADH and ATP are allosteric inhibitor
• ADP acts as an allosteric activator
• ?-ketoglutarate is also a crucial a-keto acid
  for aminotransferase reactions (Chapter 25),
  connecting the TCA cycle (carbon metabolism) with
  nitrogen metabolism

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4. ?-Ketoglutarate Dehydrogenase

• Catalyzes the second oxidative decarboxylation of
  the TCA cycle
• This enzyme is nearly identical to pyruvate
  dehydrogenase - structurally and mechanistically
• a-ketoglutarate dehydrogenase
• Dihydrolipoyl transsuccinylase
• Dihydrolipoyl dehydrogenase (identical to PDC)
• Five coenzymes used - TPP, CoA-SH, Lipoic acid,
  NAD, FAD

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Like pyruvate dehydrogenase, ?-ketoglutarate
dehydrogenase is a multienzyme complex
consisting of ?-ketoglutarate dehydrogenase,
dihydrolipoyl transsuccinylase, and dihydrolipoyl
dehydrogenase. The complex uses five different
coenzymes.
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19.4 How Is Oxaloacetate Regenerated to
Complete the TCA Cycle?
5. Succinyl-CoA Synthetase A substrate-level
phosphorylation
GTP ADP ATP GDP
(nucleotide diphosphate kinase)
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• A nucleoside triphosphate is made
• Its synthesis is driven by hydrolysis of a CoA
  ester
• The mechanism involves a phosphohistidine

Thioester Succinyl-P Phospho-histidine
GTP
Figure 19.11 The mechanism of the succinyl-CoA
synthetase reaction.
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Completion of the TCA Cycle Oxidation of
Succinate to Oxaloacetate

• This process involves a series of three reactions
  
• These reactions include
• Oxidation of a single bond to a double bond
  (FAD/FADH2)
• Hydration across the double bond
• Oxidation of the resulting alcohol to a ketone
  (NAD/NADH)
• These reactions will be seen again in b-oxidation
  of fatty acids

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6. Succinate Dehydrogenase

• The oxidation of succinate to fumarate
• A membrane-bound enzyme is actually part of the
  electron transport chain in the inner
  mitochondrial membrane (succinate-CoQ reductase)
• The reaction is not sufficiently exergonic to
  reduce NAD

(trans-)
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• The electrons transferred from succinate to FAD
  (to form FADH2) are passed directly to ubiquinone
  (UQ) in the electron transport pathway (chapter
  20)
• FAD is covalently bound to the enzyme
• Contains iron-sulfur cluster

Succinate Dehydrogenase contains three types of
iron-sulfur clusters a 4Fe-4S cluster, a 3Fe-4S
cluster, and a 2Fe-2S cluster.
Figure 19.12 The covalent bond between FAD and
succinate dehydrogenase links the C-8a carbon of
FAD and the N-3 of a His residue of the enzyme.
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7. Fumarase

• Hydration across the double bond
• Catalyzes the trans-hydration of fumarate to form
  L-malate
• trans-addition of the elements of water across
  the double bond

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• Possible mechanisms are shown in Figure 19.13

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8. Malate Dehydrogenase

• Completes the Cycle by Oxidizing L-Malate to
  Oxaloacetate
• This reaction is very endergonic, with a ?Go' of
  30 kJ/mol

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19.5 What Are the Energetic Consequences of the
TCA Cycle?

• One acetate through the cycle produces two CO2,
  one ATP, four reduced coenzymes
• Acetyl-CoA 3 NAD FAD ADP Pi 2 H2O ?
• 2 CO2 3 NADH 3 H FADH2 ATP
  CoASH
• 
  DG0 -40kJ/mol
• Glucose 10 NAD 2 FAD 4 ADP 4 Pi 2 H2O
  ?
• 6 CO2 10 NADH 10 H 2
  FADH2 4 ATP
• NADH H 1/2 O2 3 ADP 3 Pi ? NAD 3ATP
  H2O
• FADH2 1/2 O2 2 ADP 2 Pi ? FAD 2ATP H2O

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The Carbon Atoms of Acetyl-CoA Have Different
Fates in the TCA Cycle

• Neither of the carbon atoms of a labeled acetate
  unit is lost as CO2 in the first turn of the
  cycle
• Carbonyl C of acetyl-CoA turns to CO2 only in the
  second turn of the cycle (following entry of
  acetyl-CoA )
• Methyl C of acetyl-CoA survives two cycles
  completely, but half of what's left exits the
  cycle on each turn after that.

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The Carbon Atoms of Acetyl-CoA Have Different
Fates in the TCA Cycle
Figure 19.15The fate of the carbon atoms of
acetate in successive TCA cycles. (a) The
carbonyl carbon of acetyl-CoA is fully retained
through one turn of the cycle but is lost
completely in a second turn of the cycle.
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19.6 Can the TCA Cycle Provide Intermediates
for Biosynthesis?

• The products in TCA cycle also fuel a variety of
  biosynthetic processes
• a-Ketoglutarate is transaminated to make
  glutamate, which can be used to make purine
  nucleotides, Arg and Pro
• Succinyl-CoA can be used to make porphyrins
• Fumarate and oxaloacetate can be used to make
  several amino acids and also pyrimidine
  nucleotides

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Figure 19.16The TCA cycle provides intermediates
for numerous biosynthetic processes in the cell.
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• Citrate can be exported from the mitochondria and
  then broken down by citric lyase to yield
  acetyl-CoA and oxaloacetate (chapter 24)
• Oxaloacetate is rapidly reduced to malate
• Malate can be transported into mitochondria or
  oxidatively decarboxylated to pyruvate by malic
  enzyme
• Oxaloacetate can also be decarboxylated to yield
  phosphoenolpyruvate

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19.7 What Are the Anaplerotic, or Filling Up,
Reactions?

• Pyruvate carboxylase - converts pyruvate to
  oxaloacetate (in animals), is activated by
  acetyl-CoA (chapter 22, gluconeogenesis)
• PEP carboxylase - converts PEP to oxaloacetate
  (in bacteria plants), inhibited by aspartate
• Malic enzyme converts pyruvate into malate
• The catabolism of amino acids provides pyruvate,
  acetyl-CoA, oxaloacetate, fumarate,
  a-ketoglutarate, and succinate (chapter 25).

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• PEP carboxykinase
• Could have been an anaplerotic reaction.
• CO2 binds weakly to the enzyme, whereas
  oxaloacetate binds tightly
• The reaction favors formation of PEP from
  oxaloacetate

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19.8 How Is the TCA Cycle Regulated?

• Citrate synthase
• ATP, NADH and succinyl-CoA inhibit
• Isocitrate dehydrogenase
• ATP and NADH inhibits
• ADP and NAD activate
• ? -Ketoglutarate dehydrogenase
• NADH and succinyl-CoA inhibit
• AMP activates
• Pyruvate dehydrogenase
• ATP, NADH, acetyl-CoA inhibit
• NAD, CoA activate

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Regulation of the TCA cycle.
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19.8 How Is the TCA Cycle Regulated?

• When the ADP/ATP or NAD/NADH ratio is high, the
  TCA cycle is turned on
• Succinyl-CoA is an intracycle regulator,
  inhibiting citrate synthase and a-ketoglutarate
  dehydrogenase
• Acetyl-CoA acts as a signal to the TCA cycle that
  glycolysis and fatty acid break-down is producing
  two-carbon unit
• Activate pyruvate carboxylase
• Feedback inhibit pyruvate dehydrogenase

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Pyruvate dehydrogenase is regulated by
phosphorylation/dephosphorylation

• Animals cannot synthesize glucose from
  acetyl-CoA, so pyruvate dehydrogenase complex
  plays a pivotal role in metabolism
• Allosterically regulation
• Inhibit by Acetyl-CoA (dihydrolipoyl
  transacetylase), or NADH (dihydrolipoyl
  dehydrogenase)
• Covalently modification on pyruvate dehydrogenase
  
• Phosphorylation (pyruvate dehydrogenase kinase)
• Dephosphorylation (pyruvate dehydrogenase
  phosphatase)

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• The pyruvate dehydrogenase kinase (PDK Fig 19.3)
  is associated with the enzyme
• Allosterically activated by NADH and acetyl-CoA
• Phosphorylated pyruvate dehydrogenase subunit is
  inactive

• Reactivation of the enzyme by pyruvate
  dehydrogenase phosphatase
• A Ca2-activated enzyme
• Hydrolyzes the phosphoserine moiety on the
  dehydrogenase subunit
• Insulin and Ca2 activate dephosphorylation
• Pyruvate inhibit dephosphorylation

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19.9 Can Any Organisms Use Acetate as Their
Sole Carbon Source?

• Plant and some bacteria can use acetate as the
  only source of carbon for all the carbon
  compounds
• plants and some bacteria employ a modification of
  the TCA cycle called the glyoxylate cycle to
  produce four-carbon compounds from acetyl-CoA
• The CO2-producting steps are bypassed and an
  extra acetate is utilized
• Isocitrate lyase and malate synthase are the
  short-circuiting enzymes (Fig 19.21)

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Figure 19.21 The glyoxylate cycle.
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Glyoxylate Cycle

• In plants, the glyoxylate cycle is carried out in
  glyoxysomes, but yeast and algae carry out in
  cytoplasm
• Isocitrate lyase
• produces glyoxylate and succinate
• Is similar to the aldolase reaction in glycolysis
  
• Malate synthase
• A Claisen condensation of acetyl-CoA and the
  aldehyde group of glyoxylate to form L-malate
• Is similar to the citrate synthase reaction

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Figure 19.22 The isocitrate lyase reaction.
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• The glyoxylate cycle helps plants grow in the
  dark
• Certain seeds grow underground, where
  photosynthesis is impossible
• Many seeds are rich in lipids
• Once the growing plant begins photosynthesis and
  can fix CO2 to produce carbohydrate, the
  glyoxysomes disappear
• Glyoxysomes must borrow three reactions from
  mitochondria succinate to oxaloacetate
• Succinate dehydrogenase
• Fumarate
• Malate dehydrogenase

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Figure 19.23Glyoxysomes lack three of the
enzymes needed to run the glyoxylate cycle.
Succinate dehydrogenase, fumarase, and malate
dehydrogenase are all borrowed from the
mitochondria in a shuttle in which succinate and
glutamate are passed to the mitochondria, and
a-ketoglutarate and aspartate are passed to the
glyoxysome.