Preparation for TCA

• where is pyruvate transported from and to?

  Transported from the cytoplasm and to the mitochondrial matrix.
• Pyruvate is converted to acetyl-CoA in the matrix. Pyruvate (3C) is oxidized and decarboxylated during the formation of Acetyl CoA (2C)
• The acetyl group is linked to the sulfhydryl group of CoA by a thioester bond. The hydrolysis of the thioester bond is highly exergonic so it also has a high acetyl group transfer potential
• Why is the bridge reaction important for determining the fate of pyruvate?

  It decides whether pyruvate will be oxidized by TCA or be used in the synthesis of fatty acids
• Pyruvate dehydrogenase complex also produces CO2 and captures high-transfer potential electrons in the form of NADH
• There are 3 enzymes that are within the pyruvate dehydrogenase complex, what is E1 and what does it do?

  Pyruvate dehydrogenase complex component. Its prosthetic group is TTP and it catalyzes the oxidative decarboxylation of pyruvate
• There are 3 enzymes that are within the pyruvate dehydrogenase complex, what is E2 and what does it do?

  Dihydrolipoyl transacetylase: its prosthetic group is lipoamide and the enzyme catalyzes the transfer of the acetyl group to CoA and preserved the thioester bond.

  • Lipoamide is now in the reduced form and electrons must be transferred to another molecule to generate oxidized lipoamide so the next reaction can occur
• There are 3 enzymes that are within the pyruvate dehydrogenase complex, what is E3 and what does it do?

  Dihydrolipoyl dehydrogenase: prosthetic group is FAD and it catalyzes the regeneration of the oxidized form of lipoamide by the transfer of electrons from dihydrolipoylamide (reduced form of lipoamide) to FAD to make FADH2 and lipoamide (oxidized). Electrons from FADH2 are transferred to NAD+ which regenerates FAD
• What does the conversion of pyruvate to acetyl CoA using pyruvate dehydrogenase involve?

  • Decarboxylation and oxidation of pyruvate to an acetyl group
  • Transfer of the acetyl group to CoA
• What are the five coenzymes of the pyruvate dehydrogenase complex?

  Catalytic coenzymes (partake in catalyzing reaction): TPP, Lipoamide, and FAD

  Stoichiometric coenzymes (required for the function of the enzyme but do not partake in catalyzing the reaction): CoA and NAD+
• The three steps that convert pyruvate into acetyl CoA are coupled to conserve energy made from decarboxylation to drive formation of acetyl CoA and NADH. What are those three steps?


  1. Decarboxylation: pyruvate reacts carbanion of TPP and is decarboxylated. This is the rate-limiting step of the synthesis of acetyl CoA 2. Oxidation: hydroxyl methyl group of TPP oxidized to form acetyl group and transferred to lipoamide 3. Transfer of CoA: acetyl group transferred from lipoamide to CoA
• Flexible linkage allows lipoamide to move between what between what active site?

  Flexible linker allow lipoamide arm to swing the acetyl group made by the oxidation of hydroxyethyl-TPP from E1 to the E2's active site to be transferred to CoA. The lipoamide arm is now reduced so it swings to E3 where it is oxidized by FAD. The final product NADH, is produced with the reoxidation of FADH2 to FAD
• What are the two mechanisms that regulate pyruvate dehydrogenase complex?

  • Allosteric inhibition: high concentration of acetyl CoA inhibits E2 by binding to it. NADH binds and inhibits E3
  • Covalent modification: phosphorylating E1 by pyruvate dehydrogenase kinase switches off the activity of the complex. Deactivation is reversed by pyruvate dehydrogenase phosphatase.
• How is pyruvate dehydrogenase complex regulated when a muscle is becoming active after a period of rest for example?

  At rest, the muscle will have a high energy charge (ATP/ADP ratio), high NADH/NAD+, and high acetyl CoA/CoA ratio. These high ratios stimulate pyruvate dehydrogenase kinase to phosphorylate the complex, thus deactivating it.
• How is pyruvate dehydrogenase complex regulated when exercise begins for example?

  Energy charge decreases, NADH/NAD+ ratio decreases, acetyl CoA/CoA ratio decreases, and pyruvate concentration increases. Both ADP and pyruvate activate the complex by inhibiting pyruvate dehydrogenase kinase, activating pyruvate dehydrogenase complex.