T2 L1: Cell metabolism - Glycolysis and TCA cycle

• Structure of ATP

  Atp in cytosol present as a complex with Mg2+

  Mg2+ interacts with the oxygens of the triphosphate chain making it susceptible to cleavage in the phosphoryl transfer reactions

  A Mg2+ deficiency virtually impairs all metabolism

  
• Bioenergetics

  • Metabolism: integrated set of enzymatic reactions comprising both anabolic and catabolic reactions
  • Anabolism: synthesis of complex molecules from simpler ones (necessary energy usually derived from ATP)
  • Catabolism: breakdown of energy-rich molecules to simpler ones like CO2, H2O and NH3
• Cofactors, coenzymes & prosthetic groups

  Cofactors: non-protein molecules necessary for enzyme activity, eg metal cations

  • most coenzymes are organic molecules derived from vitamins
  • Participate in enzymatic reactions
  • Most cycle between oxidised and reduced forms
  • Co-enzymes / cosubstrates have a loose association with their enzyme, diffuse between enzymes carrying electrons

  
• The redox coenzymes/prosthetic groups

  • Electrons are transferred from dietary material to these carriers; thus reducing these coenzymes
  • Major redox coenzymes/prosthetic groups involved in transduction of energy from dietary foods to ATP: NAD+, FAD, FMN
  • In each case two electrons are transferred but the number of H+ moved varies, eg NAD+ reduced to NADH whereas FAD reduced to FADH2
• Re-oxidation of redox coenzymes

  • Re-oxidation (recycling) of NADH and FADH2 is via the respiratory chain in mitochondria
  • This is coupled to ATP synthesis - process of oxidative phosphorylation
  • ~2.5 molecules of ATP synthesised for each NADH re-oxidised
  • ~1.5 molecules of ATP synthesised for each FADH2 re-oxidised
• Glycolysis - priming stages

  investment of ATP at hexokinase and PFK-1 reactions

  Fill the flow diagram:

  A - hexokinase

  1 - glucose-6-phosphate (G6P)

  B - Isomerase

  2 - fructose-6-phosphate

  C - phosphofructokinase-1 (PFK-1)

  3 - fructose-1,6-bisphosphate (FBP)

  D - Aldolase

  4 - dihydroxyacetone phosphate (DHAP)

  E - Isomerase

  5 - glyceraldehyde-3-phosphate (G3P)

  
• Glycolysis - payoff stages

  recovery of ATP by SLP using 1,3-bisphosphate kinase and pyruvate kinase reactions

  Fill in the flow diagram:

  1 - Glyceraldehyde-3-phosphate (G3P)

  A - GAPDH

  I - NAD+

  II - NADH

  2 - 1,3-bisphosphoglycerate (1,3 BPG)

  B - PGK

  IV - ADP

  V - ATP

  3 - 3-phosphoglycerate

  C - mutase

  4 - 2-phosphoglycerate

  D - enolase

  5 - phosphoenolpyruvate

  E - PK

  VI - ADP

  VII - ATP

  6 - pyruvate

  
• Products of glycolysis

  For each molecule of glucose:

  • 2 x ATP
  • 2 x pyruvate
  • 2 x NADH

  NADH oxidised by the mitochondrial ETC

  BUT:

  • inner mitochondrial membrane is impermeable to NADH
  • there is no carrier in the membrane to transport it across

  So electrons from NADH enter mitochondria via 2 shuttles:

  1. Glycerol-2-phosphate shuttle, especially prevalent in brain and muscle
  2. Malate-aspartate shuttle, in liver and heart

  both shuttles act to regenerate NAD+ and make 1.5/2.5 mols of ATP
• Glycerol-3-phosphate shuttle 


  in brain and muscle

  
• Malate-aspartate shuttle 


  in liver and heart

  
• Role of pyruvate in metabolism

  at crossroads in metabolism:

  1. Lactate via lactate dehydrogenase
  2. Oxaloacetate via pyruvate carboxylase
  3. Alanine via alanine aminotransferase
  4. Acetly-CoA via pyruvate dehydrogenase complex 2
• Possible fates of pyruvate

  when sufficient oxygen available, pyruvate can be oxidised to CO2 and H2O to generate ATP

  in hypoxic conditions, pyruvate can be reduced to lactate
• Transport of pyruvate into the mitochondrion

  in aerobic conditions, occurs via specific carrier protein embedded in the mitochondrial membrane

  Pyruvate undergoes oxidative decarboxylation by the pyruvate dehydrogenase complex to form Acetyl CoA

  Reaction is irreversible and is the link between glycolysis and the citric acid cycle

  
• Tricarboxylic acid (TCA) cycle

  Final common pathway for the oxidation of fuel molecules

  In 8 steps, acetyl residues (CH3-CO-) are oxidised to CO2

  Involves 4 oxidation-reduction reactions (NADH & FADH2 production) and one molecule of ATP is produced directly for each round of the cycle

  
• 8 intermediates of the TCA cycle

  1. citrate
  2. isocitrate
  3. alpha-ketoglutarate
  4. succinyl CoA
  5. succinate
  6. fumarate
  7. L-malate
  8. oxaloacetate

  
• 9 enzymatic steps in the TCA cycle

  1. Citrate synthase
  2. x
  3. Aconitase
  4. Aconitase
  5. Isocitrate dehydrogenase
  6. alpha-ketoglutarate dehydrogenase
  7. Succinyl-CoA-synthetase
  8. Succinate dehydrogenase
  9. Fumarase
  10. Malate dehydrogenase

  
• Regulatory points of the TCA cycle

  these reactions are irreversible and the main regulatory points

  1. pyruvate dehydrogenase complex
  2. citrate synthase
  3. isocitrate dehydrogenase
  4. alpha-ketoglutarate dehydrogenase complex

  
• Products of the TCA cycle

  energy released from oxidations is conserved in the reduction of:

  • 3 x NADH
  • 1 x FADH2
  • 1 x GTP (ATP)
  • 2 x CO2 also produced
• Components of the TCA cycle are important for biosynthetic intermediates:

  Replenished by anaplerotic reactions

  Concentrations of TCA intermediates in dynamic balance
• MA shuttle

  aspartate → oxaloacetate (NADH) → malate → diffuse thru MM → malate (NAD+) → oxaloacetate → aspartate → diffuse out of MM → return to oxaloacetate

  every time oxaloacetate forms malate, alpha-KG forms glutamate

  every time malate forms oxaloacetate, glutamate forms alpha-KG

  glutamate transports aspartate in

  alpha-kg transports malate in
• Glycolysis occurs independent of oxygen concentration.