Citrate Acid Cycle

• Where does the citric acid cycle occur in?

  The matrix
• All enzymes for TCA are in the matrix except 1, what enzyme is that?

  Succinate dehydrogenase. It is embedded in the inner mitochondrial membrane
• The citric acid cycle does not generate much ATP nor uses oxygen as a reactant, what does it do instead?

  Captures high-energy electrons from citrate and its intermediates to form 3 NADH and 1 FADH2 molecules. These electron carriers yield ATP when oxidized by O2 in oxidative phosphorylation
• In the first stage of TCA...

  Oxidation of two-carbon atoms, gathering energy-rich electrons (reactions 1-4)
• In stage 2 of TCA...

  Oxaloacetate is regenerated to allow cycle to occur again, one ATP is made and energy rich electrons are gathered (reactions 5-8)
• What is the first reaction of TCA?

  A condensation reaction whereby oxaloacetate condenses with acetyl CoA to form citryl CoA. The thioester bond from CoA is cleaved and powers the synthesis of citrate.

  • reaction catalyzed by citrate synthase
• A synthase is an enzyme that catalyzes a synthetic reaction whereby two units are joined together without directly using ATP
• How does citrate synthase prevent hydrolysis of citryl CoA?


  Citrate synthase binds substrate in a sequential, ordered manner: oxaloacetate binds first, then acetyl CoA. This is because oxaloacetate induces a conformational change leading to the formation of a binding site for acetyl CoA.

  • after formation of citryl CoA by condensation, additional conformational changes are induced causing the active site to close and preventing acetyl CoA from leaving.
  • After the enzyme is in a closed state, the thioester bond is cleaved by hydrolysis and the enzyme opens back up
• Citrate synthase is well suited to the hydrolysis of citryl CoA but not acetyl CoA. How is this discrimination accomplished?

  First, acetyl CoA does not bind to the enzyme until oxaloacetate is bound and ready for condensation. Second, the catalytic residues crucial for the hydrolysis of the thioester linkage are not appropriately positioned until citryl CoA is formed. Induced fit prevents undesirable side reactions
• What is the second reaction of TCA?

  oxidative decarboxylation cannot occur in citrate due to the position of the hydroxyl so it must be converted to isocitrate first. This is accomplished by a dehydration reaction followed by a rehydration reaction to change the position of the hydroxyl, thus isomerizing the molecule.

  • catalyzed by aconitase
• What is the third reaction of TCA?

  Isocitrate is oxidized and decarboxylated to alpha-ketoglutarate. Isocitrate is oxidized to form the intermediate, oxalosuccinate, which is unstable, and when bound to the enzyme, it loses CO2 to form alpha-ketoglutarate. The oxidation of isocitrate generates the first high-potential electron carrier of this cycle, NADH.

  • Catalyzed by isocitrate dehydrogenase
• What is the fourth reaction of TCA?

  Alpha-ketoglutarate is oxidized and decarboxylated to form succinyl CoA. The oxidization forms the high-energy carrier NADH from NAD+.

  • Catalyzed by alpha-ketoglutarate dehydrogenase complex
• How is the alpha-ketoglutarate dehydrogenase complex similar to the pyruvate dehydrogenase complex?

  • Identical E3 component
  • Oxidative decarboxylation of an alpha-ketoacid that forms the high-energy electron carrier NADH
  • Formation of a thioester linkage to CoA
• What is the 5th reaction in TCA?

  Succinyl CoA is an energy-rich thioester compound, and the cleavage of the thioester is coupled to the phosphorylation of ADP. Succinate is made from this reaction.

  • catalyzed by succinyl-CoA synthase
• What is the sixth reaction of TCA?

  This reaction is the oxidation of succinate to fumarate which gathers electrons on FAD to form FADH2. FAD is the hydrogen acceptor rather than NAD+ because the free-energy change is insufficient to reduce NAD+.

  • catalyzed by succinate dehydrogenase. This is the only enzyme not in the matrix but is embedded in the inner mitochondrial membrane for easy access to the electron transport chain.
  • FADH2 does not dissociate off of the enzyme, but its two electrons are transfers from FADH2 to coenzyme Q.
• What is the seventh reaction of TCA?

  This reaction is the hydration of fumarate to malate.

  • Catalyzed by fumarase
• What is the eighth reaction of TCA?

  Oxidation of malate to form oxaloacetate which gathers electrons in NADH. Although the free energy of this reaction is significantly positive, the oxidation of malate is driven by the use of oxaloacetate by citrate synthase and NADH in ETC which lower the free energy.

  • Catalyzed by malate dehydrogenase
• The key catabolic function of the citric acid cycle is the production of NADH and FADH2 which will be oxidized in the ETC to generate 2.5 ATP per NADH and 1.5 ATP per FADH2.
• How is TCA regulated?

  Regulated at control points: allosteric enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase.

  • Isocitrate dehydrogenase
  • Allosterically stimulated by ADP (when energy is low)
  • Inhibited by ATP and NADH
  • alpha-ketoglutarate dehydrogenase
  • inhibited by succinyl CoA (product) , NADH, and ATP.
• Inhibition of isocitrate dehydrogenase leads to...?

  Accumulation of citrate, which inhibits phosphofructokinase (glycolysis)