Tricarboxylic Acid Cycle (TCA), Krebs Cycle

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Tricarboxylic Acid Cycle (TCA), Krebs Cycle

• Occurs totally in mitochondria
• Pyruvate (actually acetate) from glycolysis is
  degraded to CO2
• Some ATP is produced
• More NADH is made
• NADH goes on to make more ATP in electron
  transport and oxidative phosphorylation
• Traffic circle, comp. entering leaving

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Tricarboxylic Acid Cycle (TCA),
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Oxidative Decarboxylation of Pyruvate

• Pyr. from aerobic glycolysis
• is transported to cross inner
• mitochondrial membrane
• by specific transporter.
• In the matrix, pyr. is
• irreversibly decarboxylated
• by a multienzyme complex
• Five coenzymere needed
• See figure

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Oxidative Decarboxylation of Pyruvate

• Pyr is converted to acetyl CoA by pyr
  dehydrogenase (pyr DH) complex , which is a
  multienzyme complex.
• pyr dehydrogenase complex is not part of TCA
  cycle proper, but is a mojor source of acetyl
  CoA.
• The irreversibility of the reaction explains why
  glucose can not be formed from acetyl CoA in
  gluconeogenesis.

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Oxidative Decarboxylation of Pyruvate

• pyr dehydrogenase complex is composed of three
  enzymes
  pyr decarboxylase (E1)
  - dihydrolipoyl transacylase (E2)
  - dihydrolipoyl
  dehydrogenase (E3)
• Each catalyzed a part of the overall reaction
• In addition to two regulatory enzymes protein
  kinase and phosphoprotein phosphatase.

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Oxidative Decarboxylation of Pyruvate

• Coenzymes Pyr DH complex contains 5 coenzyme
  which act as a carriers or oxidant for
  intermediates.
• (1) Thiamine pyrophosphate
• (2)Lipoic acid
• (3) CoA
• (4) FAD
• (5) NAD

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Mechanism of Pyr. decarboxylase
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Regulation of Pyr. Dehydrogenase Complex

• Allosteric activation of kinase Phosphatase
• - Cyclic AMP-independent protein kinase (
  activated)?activates phosphorylated E1 ( inactive
  ) inhibits dephosphorylated ( active ) ?
  inhibit Pyr DH.
• protein kinase allosterically activated by ATP,
  acetyl CoA, NADH ( high energy signals)? inhibit
  Pyr DH (turned off).
• protein kinase allosterically inactivated by NAD
  CoA, ( low energy signals)? activate Pyr DH
  (turned ).
• Pyr is a potent inhibitor of kinase, if pyr
  concentration is elevated so E1 is active
• Ca is strong activator of Phosphatase,
  stimulating E1 activity ( skeletat muscle
  contraction)

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Regulation of Pyr. Dehydrogenase Complex
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Reactions of TCA

• Synthesis of citrate from acetyl CoA and
  oxaloacetate (OAA)
• Irreversible, catalyzed by citrate synthase.
• Aldol condensation reaction.
• citrate synthase is inhibited by ATP, NADH,
  succinyl CoA fatty acyle CoA.
• Function of citrate It provides a source of
  acetyl CoA for fatty acid synthesis it inhibits
  PFK1

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Reactions of TCA

• (3) Isomerisation of citrate to isocitrate by
  aconitase ( reversible reaction), It is inhibited
  by fluroacetate, a compound used for rat
  poisoning(fluroacetate is converted to
  flurocitrate which is a potent inhibitor for
  aconitase)
• (4) Oxidative Decarboxylation of isocitrate
  irreversible oxidative phosphorylation, by
  isocitrate DH to give ? -Ketoglutarate, NADH
  CO2
• -It is rate limiting step
• -isocitrate DH is activated by ADP and Ca 2
  inhibited by ATP, NADH

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Reactions of TCA

• (5) Oxidative Decarboxylation of ?
  -Ketoglutarate by ? -Ketoglutarate DH to give
  succinyle CoA (similar to pyr DH),
• Release of 2nd NADH CO2
• ? -Ketoglutarate DH need coenzymes
  TPP,NAD,FAD,CoA lipoic acid.
• ? -Ketoglutarate DH is inhibited by ATP,NADH,
  GTP succinyle CoA. And activated by Ca 2 .
• However it is not regulated by the
  phosphorylation and de phosphorylation reaction
  that describe in Pyr DH

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Reactions of TCA

• (5) Cleavage of succinyle CoA Cleavage of
  (high-energy thioester dound) succinyle CoA to
  succinate by succinate thiokinase.
• It is coupled by release of GTPwhich
  inter-converted by nucleoside diphosphate kinase
  reaction
• Substrate level phosphorylation.
• succinyle CoA can be produced from Proponyle CoA
  ( metabolism of fatty acids)

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Reactions of TCA

• (6) Oxidation of succinate to fumarate by
  succinate DH, producing FADH2
• (7) Hydration of fumarate to malate by fumarase
• (8)Oxidation of malate By malate DH
• To OAA 3nd NADH.

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Regulation of TCA Cycle
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Intermediates for Biosynthesis

• ? -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
• mitochondrial citrate can be exported to be a
  cytoplasmic source of acetyl-CoA (?F.A in fed
  state) and oxaloacetate ?glucose in fast state

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Biosynthetic Anaplerotic reactions
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Anaplerotic Reactions (filling up reactions)

• PEP carboxylase - converts PEP to oxaloacetate
• Pyruvate carboxylase - converts pyruvate to
  oxaloacetate
• Malic enzyme converts pyruvate to malate
• See fig. Reactions from1-5 is anaplerotic i.e.
  filling up reactions

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Membrane Transport System

• The inner mitochondrial membrane is impermeable
  to the most charged and hydrophilic substances.
  However it contains numerous transport proteins
  that permit the passage of specific molecules.
• 1- ATP-ADP transport, see oxid-phospho,
• Transporter for ADP Pi from cytosol into
  mitochondria by specialized carriers ( adenine
  nucleotide carrier) which transport ADP from
  cytosol into mitochondria, while exporting ATP
  from matrix back into the cytosol .

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Membrane Transport System

• Transport of reducing equivalents from cytosol
  into mitochondria using The inner mitochondrial
  membrane lacks an NADH transport proteins, NADH
  produced in cytosol cannot directly penetrate
  into mitochondria. However two electron of NADH
  ( called reducing equivalents) are transported by
  using shuttle.
• 1. glycerophosphate shuttle ( results in
  synthesis of 2 ATP for each cytosolic NADH
  oxidized )
• 2. malate-aspartate shuttle ( results in
  synthesis of 3 ATP in the mitochondrial matrix
  for each cytosolic NADH oxidized )

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Membrane Transport System
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Pyruvate DH deficiency.

• Pyruvate DH deficiency is the most common
  biochemical cause of congenital lactic acidosis.
• Pyruvate ? cannot to acetyl CoA but to lactate
• The most sever form cause neonatal death.
• The moderate form cause psychomotor retardation
  with damage in cerebral cortx, basal ganglia and
  brain stem and death.
• The third form cause episodic ataxia.

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Energy produced from TCA