Tricarboxylic Acid Cycle

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Tricarboxylic Acid Cycle

• UNIT II
• Intermediary Metabolism

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Figure 9.1. The tricarboxylic acid cycle shown as
a part of the central pathways of energy
metabolism.
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Overview

• TCA cycle (a.k.a Krebs cycle or citric acid
  cycle) plays several roles in metabolism
• It is the final pathway where oxidative
  metabolism of CHOs, aas fatty acids converge,
  their C skeletons being converted to CO2 H2O.
  This oxidation provides energy for production of
  majority of ATP.
• The cycle occurs in mitoch is in close
  proximity to the reactions of e-transport, which
  oxidize the reduced coenzymes produced by the
  cycle
• The TCA cycle is thus an aerobic pathway, because
  O2 is required as final e-acceptor
• The cycle also participates in a number of
  synthetic reactions. E.g., it functions in
  formation of gluc from C skeletons of some aas,
  it provides building blocks for synthesis of
  some aas heme.
• Intermediates of TCA cycle can also be
  synthesized by catabolism of some aas.
• This cycle should not be viewed as a closed
  circle, but instead as a traffic circle with cpds
  entering leaving as required.

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• II. Reactions of the TCA cycle
• In TCA cycle, OAA is first condensed with an
  acetyl group from acetyl CoA, then is
  regenerated as the cycle is completed. Thus,
  entry of one acetyl CoA into one round of the
  cycle does not lead to the net production or
  consumption of intermediates
• A. Oxidative decarboxylation of pyruvate
• Pyruvate, must be transported to mitoch before it
  can enter TCA cycle. This is accomplished by a
  specific pyruvate transporter
• Once in matrix, pyruvate is converted to acetyl
  CoA by pyruvate dehydrogenase complex
• Note irreversibility of reaction precludes
  formation of pyruvate from acetyl CoA, and
  explains why gluc cant be formed from acetyl CoA
  via gluconeogenesis

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Figure 9.2 Oxidative decarboxylation of pyruvate.
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• Strictly speaking, pyruvate dehydrogenase complex
  is not part of TCA cycle proper, but is a major
  source of acetyl CoA, the 2C substrate of the
  cycle
• 1. Component enzymes
• Pyruvate dehydrogenase complex is a
  multimolecular aggregate of 3 enzs pyruvate
  dehydrogenase (E1, a.k.a a decarboxylase),
  dihydrolipoyl transacetylase (E2),
  dihydrolipoyl dehydrogenase (E3).
• Each is present in multiple copies, and each
  catalyzes a part of the overall reaction.
• Their physical association links the reactions in
  proper sequence without release of intermediates
• In addition to the enzymes participating in
  conversion of pyruvate to acetyl CoA, the complex
  also contains 2 tightly bound regulatory enzymes,
  protein kinase phosphoprotein phosphatase

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Figure 9.3 Mechanism of action of the pyruvate
dehydrogenase complex. TPP thiamine
pyrophosphate L lipoic acid.
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• 2. Coenzymes
• - The pyruvate dehydrogenase complex contains 5
  coenzymes that act as carriers or oxidants for
  the intermediates of the reactions shown in Fig
  9-3.
• E1 requires thiamine pyrophosphate, E2 requires
  lipoic acid coenzyme A, and E3 requires FAD
  NAD
• Note deficiencies of thiamine or niacin can
  cause serious CNS problems. This is because brain
  cells are unable to produce sufficient ATP (via
  TCA cycle) for proper function if pyruvate
  dehydrogenase is inactive
• 3. Regulation of pyruvate dehydrogenase complex
• - The 2 regulatory enzymes that are part of the
  complex alternately activate inactivate E1 the
  cAMP-independent protein kinase phosphorylates
  and, thereby, inhibits E1, whereas phosphoprotein
  phosphatase activates E1

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• The kinase is allosterically activated by ATP,
  acetyl CoA, and NADH. Therefore, in presence of
  these high-energy signals, the pyruvate
  dehydrogenase complex is turned off
• Acetyl CoA and NADH also allosterically inhibit
  the dephosphorylated (active) form of E1.
• Protein kinase is allosterically inactivated by
  NAD and coenzyme A, low energy signals that thus
  turn pyruvate dehydrogenase on
• Pyruvate is also a potent inhibitor of protein
  kinase. Therefore, if pyruvate concs are
  elevated, E1 will be maximally active
• Calcium is a strong activator of protein
  phosphatase, stimulating E1 activity.
• Note this is particularly important in skeletal
  muscle, where release of Ca2 during contraction
  stimulates the pyruvate dehydrogenase complex,
  and thereby energy production

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Figure 9.4 Regulation of pyruvate dehydrogenase
complex.
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• 4. Pyruvate dehydrogenase deficiency
• A deficiency in the pyruvate dehydrogenase
  complex is the most common biochemical cause of
  congenital lactic acidosis
• This enz deficiency results in an inability to
  convert pyruvate to acetyl CoA ? pyruvate shunted
  to lactic acid via lactate dehydrogenase
• This causes particular problems for the brain,
  which relies on TCA cycle for most its energy,
  is particularly sensitive to acidosis
• The most severe form of this deficiency causes
  overwhelming lactic acidosis with neonatal death
• A 2nd form produces moderate lactic acidosis, but
  causes profound psychomotor retardation, with
  damage to cerebral cortex, basal ganglia and
  brain stem ? death in infancy

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• A 3rd form causes episodic ataxia (an inability
  to coordinate voluntary muscles) that is induced
  by a CHO-rich meal
• The E1 defect is X-linked, but because the
  importance of the enz in the brain, it affects
  both males females. Therefore, the defect is
  classified as X-linked dominant
• There is no proven treatment for pyruvate
  dehydrogenase complex deficiency, although a
  ketogenic diet (one low in CHO enriched in
  fats) has been shown in some cases to be of
  benefit. Such a diet provides an alternate fuel
  supply in form of ketone bodies that can be used
  by most tissues including the brain, but not the
  liver

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• 5. Mechanism of arsenic poisoning
• As previously described, arsenic can interfere
  with glycolysis at glyceraldehyde-3P step,
  thereby decreasing ATP production
• Arsenic poisoning is, however, due primarily to
  inhibition of enzs that require lipoic acid as a
  cofactor, including pyruvate dehydrogenase,
  a-ketoglutarate dehydrogenase, and branched-chain
  a-keto acid dehydrogenase
• Arsenite (the trivalent form of arsenic) forms a
  stable complex with thiol (-SH) groups of lipoic
  acid, making that cpd unavailable to serve as a
  coenzyme
• When it binds to lipoic acid in pyruvate
  dehydrogenase complex, pyruvate (and consequently
  lactate) accumulate. Like pyruvate dehydrogenase
  complex deficiency, this particularly affects
  brain causing neurologic disturbances and death

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• B. Synthesis of citrate from acetyl CoA and OAA
• Condensation of acetyl CoA OAA to form citrate
  is catalyzed by citrate synthase. This aldol
  condensation has an equil far in direction of
  citrate synthesis
• Citrate synthase is allosterically activated by
  Ca2 ADP, inhibited by ATP, NADH, succinyl
  CoA, fatty acyl CoA derivatives
• However, primary mode of regulation is also
  determined by availability of its substrates,
  acetyl CoA OAA
• Note
• - Citrate, in addition to being an intermediate
  in TCA cycle, provides a source of acetyl CoA for
  cytosolic synthesis of fatty acids
• - Citrate also inhibits PFK, the rate-setting enz
  of glycolysis, activates acetyl CoA carboxylase
  (the rate-limiting enz of fatty acid synthesis)

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Figure 9.5 Formation of a-ketoglutarate from
acetyl CoA and oxaloacetate.
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• C. Isomerization of citrate
• Citrate is isomerized to isocitrate by aconitase
• Note
• Aconitase is inhibited by fluoroacetate, a cpd
  that is used as a rat poison. Fluoroacetate is
  converted to fluoroacetyl CoA, which condenses
  with OAA to form fluorocitrate, a potent
  inhibitor of aconitase, resulting in citrate
  accumulation
• D. Oxidation and decarboxylation of isocitrate
• - Isocitrate dehydrogenase catalyzes the
  irreversible oxidative decarboxylation of
  isocitrate, yielding the 1st of three NADH
  molecules produced by the cycle, 1st release of
  CO2
• - This is one of rate-limiting steps of TCA
  cycle. The enz is allosterically activated by ADP
  (low energy signal) and Ca2, and is inhibited by
  ATP and NADH, whose levels are elevated when cell
  has abundant energy stores

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• E. Oxidative decarboxylation of a-KG
• Conversion of a-KG to succinyl CoA is catalyzed
  by the a-KG dehydrogenase complex, which consists
  of 3 enzymatic activities
• The mechanism of this oxidative decarboxylation
  is very similar to that used for conversion of
  pyruvate to acetyl CoA
• The reaction releases the 2nd CO2 and produces
  the 2nd NADH of the cycle.
• The coenzymes required are thiamine
  pyrophosphate, lipoic acid, FAD, NAD, and
  coenzyme A. each functions as part of the
  catalytic mechanism in a way analogous to that
  described for pyruvate dehydrogenase complex

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• The equil of reaction is far in direction of
  succinyl CoA, a high-energy thioester similar to
  acetyl CoA.
• a-KG dehydrogenase complex is inhibited by ATP,
  GTP, NADH, and succinyl CoA, and activated by
  Ca2
• However, it is not regulated by phospho/dephospho
  reactions as described for pyruvate dehydrogenase
  complex
• Note a-KG is also produced by oxidative
  deamination or transamination the aa glu

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• F. Cleavage of succinyl CoA
• Succinate thiokinase (a.k.a succinyl CoA
  synthetase) cleaves the high-energy thioester
  bond of succinyl CoA
• This reaction is coupled to phospho of GDP to
  GTP. GTP and ATP are energetically
  interconvertible by the nucleoside diphosphate
  kinase reaction
• GTP ADP ? GDP ATP
• - Generation of GTP by succinate thiokinase is
  another e.g. of substrate-level phospho.
• Note succinyl CoA is also produced from
  propionyl CoA derived from metabolism of fatty
  acids with an odd of C atoms, from metabolism
  of several aas (e.g., ile, val)

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• G. Oxidation of succinate
• Succinate is oxidized to fumarate by succinate
  dehydrogenase, producing reduced coenzyme FADH2
  (FAD rather than NAD, is the e-acceptor because
  the reducing power of succinate is not sufficient
  to reduce NAD)
• Succinate dehydrogenase is inhibited by OAA
• H. Hydration of fumarate
• Fumarate is hydrated to malate in a freely
  reversible reaction catalyzed by fumarase (
  fumarate hydratase)
• Note fumarate is also produced by urea cycle, in
  purine synthesis, and during catabolism of the
  aas, phe tyr.

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• I. Oxidation of malate
• Malate is oxidized to OAA by malate
  dehydrogenase. This reaction produces 3rd and
  final NADH of the cycle.
• Note OAA is also produced by transamination of
  the aa, Asp.

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Figure 9.7. Formation of oxaloacetate from malate.
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• III. Energy produced by the TCA cycle
• Two C atoms enter the cycle as acetyl CoA leave
  as CO2.
• The cycle does not involve net consumption or
  production of OAA or any other intermediate
• Four pairs of es are transferred during one turn
  of the cycle 3 pairs of es reducing NAD to
  NADH one reducing FAD to FADH2.
• Oxidation of one NADH by ETC leads to formation
  of 3 ATP, whereas oxidation of FADH2 yields 2
  ATP
• Total yield of ATP from oxidation of one acetyl
  CoA is

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Figure 9.8. Number of ATP molecules produced from
the oxidation of one molecule of acetyl CoA
(using both substrate-level and oxidative
phosphorylation).
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IV. Regulation of the TCA cycle

• A. Regulation by activation and inhibition of
  enzyme activity
• In contrast to glycolysis which is regulated
  primarily by PFK, the TCA cycle is controlled by
  regulation of several enz activities. The most
  important of these are citrate synthase,
  isocitrate dehydrogenase, a-KG dehydrogenase
  complex

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• B. Regulation by availability of ADP
• 1. Effect of elevated ADP
• - Energy consumption as a result of muscular
  contraction, biosynthetic reactions or other
  processes result in hydrolysis of ATP to ADP
  Pi.
• Resulting increase in conc of ADP accelerates
  rate of reactions that use ADP to generate ATP,
  most important of which is oxphos
• Production of ATP increases until it matches rate
  of ATP consumption by energy-requiring reactions

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• 2. Effect of low ADP
• If ADP (or Pi) is present in limiting conc,
  formation of ATP by oxphos decreases as a result
  of the lack of phosphate acceptor (ADP) or
  inorganic phosphate (Pi)
• The rate of oxphos is proportional to
  ADPPi/ATP this is known as respiratory
  control of energy production
• Oxidation of NADH FADH2 by ETC also stops if
  ADP is limiting. This is because the processes of
  oxidation phospho are tightly coupled occur
  simultaneously
• As NADH FADH2 accumulate, their oxidized forms
  become depleted causing oxidation of acetyl CoA
  by the TCA cycle to be inhibited as a result of a
  lack of oxidized coenzymes

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Figure 9.9. A. production of reduced coenzymes,
ATP, and CO2 in TCA cycle. B. inhibitors and
activators of the cycle.
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Summary

• Pyruvate is oxidatively decarboxylated by
  pyruvate dehydrogenase complex producing acetyl
  CoA, which is the major fuel for TCA cycle
• This enz complex requires five coenzymes
  thiamine pyrophosphate, lipoic acid, FAD, NAD,
  and coenzyme-A (which contains the vitamin
  pantothenic acid)
• The reaction is activated by NAD, coenzyme-A,
  and pyruvate, and inhibited by ATP, acetyl CoA,
  NADH, and Ca2.
• Pyruvate dehydrogenase deficiency is the most
  common biochemical cause of congenital lactic
  acidosis. Because the deficiency deprives the
  brain of acetyl CoA, the CNS is particularly
  affected, with profound psychomotor retardation
  death occurring in most patients. The deficiency
  is X-linked dominant
• Arsenic poisoning causes inactivation of pyruvate
  dehydrogenase by binding to lipoic acid

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• Citrate is synthesized fro OAA and acetyl CoA by
  citrate synthase. This enz is allosterically
  activated by ADP, and inhibited by ATP, NADH,
  succinyl CoA, and fatty acyl CoA derivatives
• Citrate is isomerized to isocitrate by aconitase.
  Isocitrate is oxidized decarboxylated by
  isocitrate dehydrogenase to a-KG, producing CO2
  and NADH. The enz is inhibited by ATP NADH,
  is activated by ADP Ca2.
• a-KG is oxidatively decarboxylated to succinyl
  CoA by a-KG dehydrogenase complex, producing CO2
  NADH. The enz is very similar to pyruvate
  dehydrogenase and uses the same coenzymes.
• a-KG dehydrogenase complex is activated by Ca2,
  and inhibited by ATP, GTP, NADH, succinyl CoA.

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• Succinyl CoA is cleaved by succinate thiokinase
  ( succinyl CoA synthetase), producing succinate
  and GTP. This is an e.g. of substrate-level
  phospho.
• Succinate is oxidized to fumarate by succinate
  dehydrogenase, producing FADH2. this enz is
  inhibited by OAA.
• Fumarate is hydrated to malate by fumarase (
  fumarate hydratase), and malate is oxidized to
  OAA by malate dehydrogenase, producing NADH.
• Three NADH, one FADH2, and one GTP (whose
  terminal phosphate can be transferred to ADP by
  nucleoside diphosphate kinase, ? ATP) are
  produced by one round of TCA cycle.
• Oxidation of NADHs and FADH2 by ETC yields 11
  ATPs, making 12 the total of ATPs produced

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Figure 9.10 Key concept map for tricarboxylic
acid cycle.