TCA Cycle

• Prokaryotic cells

  • Approx. 1000-fold smaller than eukaryotic cells
  • Can rely on diffusion
• Eukaryotic cells compared to Prokaryotic cells (why bigger isn't always better)

  • Greater size and complexity, hence they cannot rely on diffusion only, hence the compartmentalisation
  • One of the TCA cycle enzymes is an integral membrane protein, so biology has to select a membrane
  • Close tie between TCA cycle (NADH, FADH2 production) and electron transport chain
• Mitochondria are believed to derive from symbiotic bacteria
• Features similar between bacteria and mitochondria

  • Size and shape - bacillus rod shaped, ranging between 1 and 10 microns in length
  • Both replicate by fission
  • Type of DNA - both contain circular DNA plasmids
  • Ribosome and protein synthesis - mitochondria have their own ribosomes more similar to bacterial ribosomes than to ribosomes of eukaryotic cells
  • Membranes - both inner and outer membranes with the outer membrane containing similar proteins such as porins
• Pyruvate - aerobic conditions

  • Outer membranes of mitochondria contain porins - proteins that allow small molecules like pyruvate to enter the intermembraneous space
  • A pyruvate transporter (MPC) transports pyruvate across the impermeable inner mitochondrial membrane
  • The conversion of pyruvate to acetyl (in complex with CoA) the substrate for the TCA cycle, occurs in the mitochondrial matrix, with the loss of 1 carbon in the form of CO2
• Cristae

  Huge increase in surface area, as this is where most of the energy is produced. TCA cycle occurs in the matrix.
• Pyruvate dehydrogenase

  • E1: Pyruvate dehydrogenase which uses thiamine pyrophosphate (TPP) as its prosthetic group
  • E2: Dihydrolipoyl transacetylase which uses lipoamide and coenzyme A (CoA-SH) as its prosthetic groups
  • E3: Dihydrolipoyl dehydrogenase which uses flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD+) as its cofactors
• Prosthetic groups

  Tightly bound, non-polypeptide unit required for enzymatic activity. For pyruvate dehydrogenase these include: TPP, lipoate, FAD, thiamine (vitamin B1)
• Enzyme complexes


  • Speed up reactions, because the products of one reaction can be passed directly to the next enzyme without the newly formed substrate having to diffuse to the next enzyme
  • The product of a reaction may be highly reactive and take part in other unfavourable 'side' reactions if it is not transferred directly to the next enzyme
• TCA Cycle Steps


  • Citrate synthase
  • Aconitase
  • Isocitrate dehydrogenase
  • α-ketoglutarate dehydrogenase
  • Succinyl CoA synthetase
  • Succinate dehydrogenase
  • Fumarase
  • Malate dehydrogenase
• Citrate synthase

  Reaction type: condensation. The first reaction is a synthase reaction, called such since a new molecule is made but ATP is not used.
• Aconitase


  • Reaction type: isomerisation
  • Cofactor: Fe-S complex
  • Note: two step reaction, intermediate is cis-aconitate
• Isocitrate dehydrogenase

  • Reaction type: oxidative decarboxylation (1 of 4 oxid/red reactions). This is the first carbon loss as CO2 from C6 to C5.
  • Cofactor: NAD+ is reduced to NADH + H+
  • Note: two step reaction, intermediate is oxalosuccinate
• α-ketoglutarate dehydrogenase


  • Reaction type: oxidative decarboxylation (2 of 4 oxid/red reactions). This is the second carbon loss as CO2 from C5 to C4.
  • Cofactor: NAD+ is reduced to NADH + H+, CoA-SH
  • Reminds you of pyruvate dehydrogenase, hence this too is a multi-subunit complex
• Succinyl CoA synthetase


  • Reaction type: the energy released from the hydrolysis of the thioester bond provides the energy for the formation of the phosphoanhydride bond of GTP. A substrate level phosphorylation.
  • Cofactor: GDP (+ Pi -> GTP). The GTP can be used to form ATP (nucleoside diphosphate kinase). ATP and GTP are energetically equivalent.
• Succinate dehydrogenase

  • Reaction type: oxidative dehydrogenation of succinate to fumarate (3 of 4 oxid/red reactions).
  • Cofactor: FAD is reduced to FADH2
  • Note: The only Krebs enzyme contained within the inner mitochondrial membrane. Active site faces the mitochondrial matrix.
• Fumarase

  Reaction type: catalyses a stereospecific trans addition of a hydrogen atom and a hydroxyl group
• Malate dehydrogenase

  • Reaction type: oxidative dehydrogenation of malate to oxaloacetate (4 of 4 oxid/red reactions).
  • Cofactor: NAD+ is reduced to NADH + H+
  • Note: In the cell OAA is rapidly depleted by STEP 1 hence driving the reaction forward
• Balance sheet for the complete oxidation of glucose: Glycolysis generates 8 ATP, TCA cycle generates 30 ATP for 2 pyruvates entering the TCA cycle. Total 38 net ATPs.
• Pyruvate dehydrogenase

  a multi-enzyme complex that uses three enzymes
• Where does the oxidation of pyruvate occur?

  in the mitochondria of the cell
• What does Pyruvate dehydrogenase use as its prosthetic group?


  thiamine pyrophosphate (TPP)
• What does Dihydrolipoyl transacetylase use as its prosthetic group?


  lipoamide and coenzyme A (also known as CoA-SH)
• What does Dihydrolipoyl dehydrogenase use as its cofactors?


  flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD+)
• What are prosthetic groups?

  Tightly bound, non-polypeptide unit required for enzymatic activity
• TTP – thiamine pyrophosphate 

  required for pyruvate decarboxylation
• Lipoate

  required for the transfer of the acetyl group to coenzyme A
• FAD – flavin adenine dinucleotide

  required for the regeneration of the oxidised form of lipoate
• What kind of reaction is the 1st step of the TCA cycle?

  Reaction type: condensation. The first reaction is a synthase reaction, called such since a new molecule is made but ATP is not used
• What kind of reaction is the 2nd step of the TCA cycle?

  Reaction type: isomerisation
• What is the cofactor in the 2nd step of the TCA cycle?

  Fe-S complex
• What kind of reaction is the 3rd step of the TCA cycle?

  Reaction type: oxidative decarboxylation 
• When did the first carbon loss occur in the TCA cycle?

  In step 3, from C6 to C5
• What is the cofactor in the 3rd step of the TCA cycle?

  NAD+ is reduced to NADH + H +
• What kind of reaction is the 4th step of the TCA cycle?

  Reaction type: oxidative decarboxylation 
• What is the cofactor in the 4th step of the TCA cycle?

  NAD+ is reduced to NADH + H +, CoA-SH
• What kind of reaction is the 5th step of the TCA cycle?

  Reaction type: A substrate level phosphorylation. The energy released from the hydrolysis of the thioester bond provides the energy for the formation of the phosphoanhidride bond of GTP.
• What is the cofactor of the 5th step of the TCA cycle?

  GDP (+ Pi -> GTP). The GTP can be used to form ATP (nucleoside diphosphate kinase). ATP and GTP are energetically equivalent.
• What kind of reaction is the 6th step of the TCA cycle?



  Reaction type: oxidative dehydrogenation of succinate to fumarate 
• What is the cofactor of the 6th step of the TCA cycle?


  FAD is reduced to FADH2