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• Citric Acid Cycle


  Also known as: Krebs cycle, Tricarboxylic Acid Cycle, TCA cycle
• "Beloved child has many names"
• Fate of pyruvate


  • Requires oxygen
  • Anaerobic
  • Outcome depends on organism
• Why oxidise pyruvate?


  Glucose + 6O2 → 6CO2 + 6H2O

  ΔG0' = -2.9 x103 kJ/mol

  ADP + Pi + H+ → ATP + H2O

  ΔG0' = 30.5 kJ/mol

  Majority of energy is still in the pyruvate molecule

  Release through further oxidation in citric acid cycle
• Stryer


  Ch 17

  No meaningful ATP production

  Little ATP production

  Majority of ATP production
• The sparker effect


  1. Minced liver or pigeon flight breast

  2. Add pyruvate

  3. Little O2 consumption

  4. Add pyruvate + Trace organic acid

  5. O2 consumption higher than required to oxidise organic acids

  6. Organic acids not used up

  7. Organic acids: citrate, fumarate, malate or succinate
• Krebs cycle discovered in 1920s and 1930s: Szent-Györgyi and Krebs (both got Nobel prizes)
• The citric acid cycle

  Pyruvate → Oxaloacetate → Citrate → CO2 → CO2 → CO2

  The cycle is autocatalytic in its operation. One molecule of oxaloacetate (the acceptor) can spark oxidation of an infinite amount of pyruvate.
• Interpretation of the sparker effect


  Without catalyst limited O2 consumption

  With catalyst (organic acids) O2 consumption increases
• Malonate


  Inhibitor of respiration in all animal tissues

  Comparison of structure to succinate
• Malonate was found to be a potent inhibitor of respiration in all animal tissues
• Conclusion: Pathway must be cyclic
• Citric acid cycle is a shared pathway for breakdown of carbohydrates, fats and amino acids
• Citric acid cycle is important for anabolic pathways
• Plants and bacteria but not animals can turn fat into glucose via a glyoxylate cycle
• Preparing pyruvate


  Pyruvate from glycolysis is in cytosol

  Citric acid cycle takes place in the matrix of mitochondria

  Pyruvate translocase

  Cotransport with H+

  Acetyl-CoA

  CO2 + NADH

  NAD+ + CoA-SH

  Citrate

  Oxaloacetate
• Conversion of pyruvate to acetyl CoA


  COO- C=O CH3 + CoA-SH + NAD+ → C=O CH3 S CoA + CO2 + NADH

  Catalysed by the pyruvate dehydrogenase multienzyme complex

  Irreversible
• Pyruvate dehydrogenase complex


  • 4-10 x106 kDa (larger than ribosomes)

  Components: E1, E2, E3
• Thiamine pyrophosphate (TPP)


  Cofactor of E1, derived from vitamin B1

  Found in the outer seed coats of cereals incl. rice

  Deficiency in man results in "beri-beri"

  Participates in the Decarboxylation of pyruvate
• Coenzyme A


  An adenine nucleotide derivative

  Serves as a carrier of activated acyl groups linked via a thioester bond

  Acyl groups linked to Co A have a high transfer potential
• Flavin adenine dinucleotide (FAD)


  A coenzyme for redox reactions

  Structure of the reactive part (isoalloxazine ring)

  Typical reaction: It accepts two electrons and two protons
• Reactions catalysed by the pyruvate dehydrogenase multi-enzyme complex


  E1 decarboxylates pyruvate

  Lipoamide picks up acetyl group

  E2 transfers to CoA

  E3 regenerates lipoamide
• Structure of E1E2 complex


  • Diameter ~475 Å (Hemoglobin: ~55Å)

  E2 forms the core, E1 is the outer layer

  E2 is trimer (three arms)

  E1 active site directly over E2 catalytic domain
• Multienzyme complexes


  Groups of two or more non-covalently associated enzymes that catalyse two or more sequential steps in a metabolic pathway

  Advantages: Product of first reaction remains attached, can channel intermediates, reactions coordinately regulated
• Pyruvate dehydrogenase deficiency is a rare genetic disorder
• Symptoms of pyruvate dehydrogenase deficiency would NOT include: Increased concentration of lactic acid in the blood stream
• The citric acid cycle


  Acetyl CoA + 2H2O + 3NAD+ + FAD + GDP + Pi → 2CO2 + 3NADH + 3H+ + FADH2 + CoASH + GTP

  ΔG0' = - 58 kJ/mol

  Goes round twice for each glucose molecule

  NADH and FADH2 are re-oxidised by O2 during OXIDATIVE PHOSPHORYLATION in which ATP is produced
• Control of the Citric Acid Cycle


  Pyruvate DH is switched OFF by phosphorylation (kinase stimulated by AcetylCoA, ATP and NADH)

  Switched ON by dephosphorylation (kinase inhibited by ADP and NAD+)

  ATP and NADH are the principal negative regulators

  The need for energy and for carbon skeletons is the main positive regulator

  Citrate synthase regulated in some organisms (but not important in human)
• Anaplerotic reactions


  The withdrawal of citric acid cycle intermediates for biosynthesis is potentially damaging

  If oxaloacetate is depleted, the cycle will stop

  Cycle intermediates must be replenished if withdrawal is occurring

  In humans, the main anaplerotic reaction introduces more oxaloacetate into the cycle, via carboxylation of pyruvate: Pyruvate+ CO2+ATP+H2O → oxaloacetate+ADP+Pi+2H+
• Fates of Carbon Skeletons of Amino Acids

  • Glucogenic amino acids can be used in anaplerotic reactions if needed

  Ketogenic amino acids can not