TCA Cycle

Different names- same thing

  • TCA Cycle - Tri-carboxylic Acid Cycle
  • The Krebs Cycles
  • The Citric Acid Cycle

A CENTRAL AND FUNDAMENTAL METABOLIC PATHWAY - FUNDAMENTAL TO LIFE

All of the nutrients (carbs, fats, proteins) generate acetyl-CoA which feeds into the kreb cycle

Krebs cycle

  • Final common pathway for fuel molecules
  • Enter as Acetyl-CoA
  • Provide energy transfer molecules
  • Provide intermediate molecules for biosynthesis - anabolism
  • Occurs in mitochondrial matrix

Citrate was discovered in lemon juice

  • Shown to be widespread
  • Demonstrated that minced muscle tissue could catalyse the transfer of hydrogen atoms (a proton and electron) from some organic acids - succinate, malate and citrate
  • Shown by colour change of the redox methlene blue.
  • Enzymes involved were termed dehydrogenases

Measurement of oxygen uptake rate (usage) by the minced tissues showed that the acids were being rapidly oxidised to carbon dioxide

Szent‐Györgyi (Hungary)

  • Succinate – Fumarate – Malate – Oxaloacetate

Carl Martius and Franz Knoop

  • Citrate – Isocitrate – Ketoglutarate – Succinate


  • Hans Kreb realised that these were linked together as a cycle of reactions

  • He showed the formation of citrate from oxaloacetate and pyruvate acetate

Roles

  • Final oxidative step in the catabolism of carbohydrates, fatty acids and amino acids
  • Provides a flow of simple carbon compounds into anabolic processes
  • Functions as a major source of energy, generating some ATP and a lot of NADH
  • The NADH can be oxidised in the respiratory chain to generate more ATP

Link with Glycolysis

  • End product of glycolysis in the 3 carbon compound pyruvate
  • Pyruvate dehydrogenase converts pyruvate, CoA and NAD+ into Acetyl-CoA, CO2 and NADH
  • Acetyl-CoA feeds the 2 carbon acetyl group into the TCA cycle
  • Pyruvate dehydrogenase is a large complicated molecule. It is a complex consisting of 3 distinct enzymes and needs 5 coenzymes

Pyruvate Dehydrogenase (PDH)

  • Catalyses pyruvate (3C) to acetyle-CoA (2C)
  • Generates NADH
    • CH3‐CO‐COO‐ + CoASH + NAD+ >>> CH3‐CO‐SCoA + CO2 + NADH
    • This is irreversible, key checkpoint in metabolism
  • Enzyme complex inhibited by products; acetyl-CoA and NADH
  • Also inhibited by high [GTP] and stimulated by high [AMP]
  • Regulated by reversible phosphorylation (adding/removing a specific phosphate group)
    • Done by other enzymes
    • High ATP/ADP ratio>triggers phosphorylation > inhibits PDH
    • Insulin triggers dephosphorylation > stimulates PDH
  • In mitochondria matrix
  • Glycolysis occurs in cytosol
  • Pyruvate travels through the mitochondrial membranes via outer membrane via large non-specific anion channels
  • Inner membrane via selective transport protein complex mitochondrial pyruvate carrier (MPC)
  • Actively transported across the membrane

Summary of the TCA cycle
1 - Condensation of acetyl CoA (2C) with oxaloacetate (4C) to form citrate (6C). Enzyme is citrate syntheses


2 - Formation of isocitrate (6C) [via cis-aconitate], OH group moved. Enzyme is aconitase


3 - Oxidative decarboxylation of isocitrate (6C) to ketoglutarate (5C) and CO2.Enzyme is iscocitrate dehydrogenase


4 - Oxidative decarboxylation of ketoglutarate (5C) to succinyl-CoA (4C) and Co2. Enzyme is a-ketoglutarate


5 - Conversion of succinyl-CoA (4C) to succinate (4C). Involves hydrolysis and phosphorylation. Enzyme is succinyl CoA synthetase


6 - Oxidation of succinate (4C) to fumarate (4C).Removal of 2 H to leave a double bond. Enzyme is succinate dehydrogenase


7 - Hydration of fumarate (4C) Enzyme is fumarase


8 - Oxidation of malateto oxalooacetate. Enzyme is malate dehydrogenase

The checkpoint enzymes

  • Citrate synthase (step 1) is allosterically inhibited by high [ATP] (Km for acetyl-CoA is increased) DG= -31.5 kj/mol
    • Isocitrate dehydrogenase (step 3) is inhibited by ATP and NADH is stimulated by ADP - DG= -21kj/mol
    • a-ketoglutarate dehydrogenase (step 4) s inhibited by NADH and succinyl-CoA and ATP
  • Krebs cycle does no use oxygen directly
  • Generate NADH and FADH2 which feed into ETC
  • Oxygen is used in ETC as ultimate electron acceptor
  • NADH and FADH2 are re-oxidised back to NAD+ and FAD in ETC so can be used again the Krebs cycle

The cofactors

  • These are GTP (or ATP) and NADH, FADH2 (reduced form)
  • NADH generated at steps 3,4, and 8
  • Step 6 generates FADH2
  • Step 5 generates GTP (or ATP) for one turn of the cycle
  • Regeneration of these cofactors (to oxidised form)
  • NAD+ and FAD are regenerated by the ETC
  • GDP can be regenerated by transferring a phosphate group from
  • GTP to ADP by nucleoside diphosphate kinase
    GTP + ADP ‐‐‐‐‐> GDP + ATP

Lipid metabolism

  • Broken down by beta oxidation into 2C fragments
  • 2C is Acetyl-CoA
  • This feeds into the Kreb cycle
  • Biosynthesis of fatty acids is reversal of this breakdown process

Protein metabolism

  • Proteins are broken down into amino acids
  • mino acis undergo transmination and deamination reactions
  • These generate Krebs cycle intermediates
  • Biosynthesis of amino acid - important precursors are oxaloacetate and a-ketoglutarate

Anaplerotic reactions
If krebs cycle intermediates are used up for biosynthesis they need to be replenished to enable the cycle to continue. Anaplerotic (filling up) reactions are used to replace them

  • Example - pyruvate can be carboxylated to oxaloacetate by pyruvate carboxylase with CO2 and ATP - instead of being broken down to acetyl-CoA
  • Oxaloacetate is used to replenish any intermediates drawn off for biosythesis

Yield of ATP from glucose breakdown


Problem:

  • Inner mitochondrial membrane is impermeable to NADH

Answer

  • Electron carried by NADH are transported to the respiratory chain, not the NADH itself
    • There can be an energetic price to pay for the transport

NADH electron transport shuttles
Glycerophosphate shuttle (used by skeletal muscle)

  • Cytosolic NADH (from glycolysis passes its electrons across the membrane via glycerol-3-phosphate
    • Form FADH2 (not NADH) inside mitochondrial matrix
    • This reduces ATP yield
      Malate-Aspartate shuttle (used by liver, kidney, heart)
    • Malate is used to carry electrons across inner membrane into matrix of mitrochondrion
    • Generates NADH and oxaloacetate using malate dehydrogenase

Yield of ATP from oxidation of one molecule of glucose

  • So ATP yield will depend on the shuttle used to transport the electrons across the membrane
  • Let us assume 1 molecule of NADH generates 2.5 ATP and 1 molecule of FADH2 generates 1.5ATP
  • Numbers used here will be explained ETC and oxidative phosphotylation lecture

Summary

  • Krebs is
    = final oxidative step in the catabolism of carbohydrates, fatty acids and amino acids
    = Provide a flow of simple carbon compounds into anabolic processes:
    • It fuctions as a major source of enegy compounds
      = generating 1 ATP by SLP, 3 NADH (7.5 ATP) and 1 FADH2 (1.5 ATP) for one turn of cycle