Citric Acid Cycle / Krebs Cycle / Tricarboxylic Acid Cycle

Session 14

What is described in the following image and points:

• Process in which cells consume O₂ and produce CO

• Provides energy (ATP) from glucose (more than than glycolysis)

• Can capture energy stored in lipids and amino acids

• Evolutionary origin: developed about 2.5 billion years ago

• Used by animals, plants, and many microorganisms

• Citric Acid Cycle + Oxidative Phosphorylation

• Occurs in three major stages:

  • Acetyl CoA production

  • Acetyl CoA oxidation

  • Electron transfer and oxidative phosphorylation

Cellular Respiration

The pyruvate dehydrogenase complex includes 3 types of enzymes that collectively remove a carboxylate group from pyruvate and produce what two molecules?

Acetyl-CoA and NADH

Under aerobic conditions, pyruvate is transported into _________ membrane where it is oxidatively decarboxylated by pyruvate dehydrogenase complex to form Acetyl-CoA

mitochondrial

Pyruvate Dehydrogenase Complex Mechanism: the synthesis of Acetyl-CoA from pyruvate requires 3 enzymes and 5 coenzymes. What are the 3 main steps?

• Decarboxylation

• Oxidation

• Transfer of acetyl group to CoA to form Acetyl-CoA

What are the 5 coenzymes of pyruvate dehydrogenase reaction?

• Catalytic cofactors (3) - capable of catalyzing multiple reactions

  • Thiamine pyrophosphate (TPP)

  • Lipoic acid

  • FAD

• Stoichiometric cofactors (2) - 1 to 1 ratio; one cofactor is needed for one reaction

  • CoA

  • NAD⁺

What are the 3 enzymes of pyruvate dehydrogenase complex?

• Pyruvate dehydrogenase (E1)

• Dihydrolipoyl transacetylase (E2)

• Dihydrolipoyl dehydrogenase (E3)

What is described in the following image?

Summary of the Pyruvate Dehydrogenase Reactions

What is being described by the following points:

• Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH + H⁺

• Irreversible reaction

• Link between glycolysis and the citric acid cycle

• Captures high-transfer-potential electrons in the form of NADH

Pyruvate dehydrogenase complex

What is described by the following points:

• The central metabolic hub of the cell

• The final common pathway for the oxidation of fuel molecules

• Important source of precursors for the building blocks of many biomolecules

  • Ex. amino acids, fatty acids, nucleotide bases, and porphyrins

• Series of oxidation-reduction reactions that result in the oxidation of an acetyl group to 2 CO₂ molecules

• Eight reactions

  • An acetyl group is condensed with oxaloacetate

  • 2 CO₂ are lost

  • Oxaloacetate is regenerated

• Each round generates 3 NADH, 1 FADH₂, and 1 GTP or ATP

• Flux through the citric acid cycle regulated primarily by feedback inhibition at 3 steps

Citric Acid Cycle, Tricarboxylic Acid Cycle (TCA) or Krebs cycle

What is described by the following points:

• Harvests high-energy electrons from carbon fuels

• These electrons will be used to power the synthesis of ATP

• Cellular respiration provides the vast preponderance of energy used by aerobic cells

  • >90% in humans

Overview of the Citric Acid Cycle

The Citric Acid Cycle takes place in the ________ of the mitrochondria

matrix

Explain Step 1 of the Citric Acid Cycle

Enzyme: Citrate Synthase

• Aldol condensation + hydrolysis

• Condenses oxaloacetate (a 4C unit) and acetyl (a 2C unit) group of acetyl-CoA

Explain Step 2 of the Citric Acid Cycle

Enzyme: Aconitase

• Dehydration + hydration

• Citrate is isomerized into isocitrate to enable the 6C unit to undergo oxidative decarboxylation

Explain Step 3 of the Citric Acid Cycle

Enzyme: Isocitrate dehydrogenase

• Oxidative decarboxylation of isocitrate → α-ketoglutarate

• Isocitrate + NAD⁺ → α-ketoglutarate + CO₂ + NADH

Explain Step 4 of the Citric Acid Cycle

α-ketoglutarate dehydrogenase complex

• 2nd oxidative decarboxylation

• Homologous to the pyruvate dehydrogenase complex

• Pyruvate + CoA + NAD⁺ → Acetyl-CoA + CO₂ + NADH

• α-ketoglutarate + NAD⁺ → Succinyl CoA + CO₂ + NADH

Explain Step 5 of the Citric Acid Cycle

Enzyme: Succinyl CoA Synthetase (Succinate Thiokinase)

• Cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of an ADP

• Example of substrate-level phosphorylation because succinyl phosphate, a high phosphoryl-transfer potential compound, donates a phosphate to ADP

Explain Step 6-8 of the Citric Acid Cycle

Reactions of four-carbon compounds constitute the final stage of the CAC: the regeneration of oxaloacetate

• Succinate → Fumarate → Malate → Oxaloacetate

What is shown in the following image?

The Citric Acid Cycle

• Step, Reaction, Enzyme, Prosthetic Group, Type (condensation, dehydration, hydration, decarboxylate, oxidation, substrate-level phosphorylation), and Gibbs free energy

What is the net reaction for the Citric Acid Cycle?

Acetyl CoA + 3NAD⁺ + FAD + ADP + P_i + 2H₂O → 2CO₂ + 3NADH + FADH₂ + ATP + 2H⁺ + CoA

The citric acid cycle is an energy-______ cycle

generating

• 1 NADH = 2.5 ATP

• 1 FADH₂ = 1.5 ATP

• ~32 ATP per glucose

How is the pyruvate dehydrogenase complex regulated?

• Allosterically

• By reversible phosphorylation

• Pyruvate → Acetyl-CoA is irreversible in animal cells

• Acetyl-CoA has 2 principle fates:

  • Metabolism by the citric acid cycle

  • Incorporation into fatty acids

Regulation of Pyruvate Dehydrogenase

• Product inhibition by NADH and Acetyl-CoA

• Covalent modification of E1 (phosphorylation) → inactivation

Regulation of Citric Acid Cycle

• Substrate Availability

  • Acetyl-CoA and Oxaloacetate

• Product Inhibition

  • ATP, NADH, citrate…

• Competitive Feedback succinyl-CoA

• Key control points

  • The reactions catalyzed by isocitrate dehydrogenase and α-ketoglutarate dehydrogenase

What is described by the following points:

• Supplies precursors for the synthesis of other compounds (catabolic; cataplerotic reaction)

  • Intermediates are precursors of other molecules

• Intermediates can be replenished (anabolic; anaplerotic reaction)

  • Mammals lack the enzymes for the net conversion of acetyl-CoA → oxaloacetate or any other CAC intermediates

    • Oxaloacetate is formed by the carboxylation of pyruvate

Amphibolic Functions of the Citric Acid Cycle

What is shown in the following image?

• Example of a cataplerotic Citric Acid Cycle reaction

• α-ketoglutarate (TCA intermediate) → Glutamate (amino acid)

• Glutamate is a precursor of the amino acids glutamine, arginine, and proline

What type of reaction is the following:

Pyruvate + CO₂ + ATP + H₂O → Oxaloacetate + ADP + P_i

Anaplerotic reaction to replenish Citric Acid Cycle intermediates