The Citric Acid Cycle (Krebs cycle)

What is the Citric Acid Cycle considered?

The central hub of metabolism

What is the citric acid cycle?

It is a series of enzymatic reactions that generate energy through the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins.

• from glucose to ATP

How do electrons harvested from food drive ATP synthesis, and what molecules can feed into the TCA cycle?

• Role of Reduced Coenzymes: Electrons removed during oxidation of glucose are carried by NADH and FADH2​ to the mitochondria to drive ATP production.

• Acetyl-CoA Sources: Acetyl-CoA can be produced from glucose, as well as the oxidation of Fats and Amino Acids.

What is the concept behind the TCA cycle

• pyruvate is oxidatively decarboxylated to acetyl-CoA

• Acetyl-CoA enters the cycle

• Acetyl group is completely oxidized to 2 Co₂

• Oxaloacetate îs regenerated to accept another acetyl-CoA

What is the problem of the TCA Cycle?

We want to oxidize acetate to CO₂:

• CH₃COO^- → This requires breaking a C-C bond

What is the challenge of the TCA cycle?

C-C cleavage typically occurs between α and β carbons relative to a carbonyl

• acetate has NO β-carbon

What is the solution to the TCA cycle?

• condense acetate with oxalocetate (4C)

• creates citrate (6C) with proper α-β relationship

• Now C-C cleavage can occur

• Two decarboxylations release 2 CO₂

• Oxaloacetate regenerated for next round

What is Step 0 of the TCA cycle (location, overall reaction, and energetics)?

• Process: Oxidative Decarboxylation of Pyruvate to Acetyl-CoA.

• Location: Mitochondrial matrix (pyruvate must cross both mitochondrial membranes)

• Reaction:

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

• Energetics: ΔG′∘=−33.4 kJ/mol (Irreversible).

What are the 3 enzymes that make up the Pyruvate Dehydrogenase Complex and their associated cofactors?

• E1​ (Pyruvate Dehydrogenase): Uses TPP cofactor.

• E2​ (Dihydrolipoyl Transacetylase): Uses Lipoate and CoA

• E3​ (Dihydrolipoyl Dehydrogenase): Uses FAD and NAD⁺

What are the 5 coenzymes required by the Pyruvate Dehydrogenase Complex?

• TPP (Thiamine Pyrophosphate)

• Lipoate (Lipoic acid)

• CoA (Coenzyme A)

• FAD (Flavin Adenine Dinucleotide)

• NAD (Nicotinamide Adenine Dinucleotide)

Pyruvate Dehydrogenase Complex Mechanism

1. Decarboxylation (E1+TPP)

• Pyruvate binds to TPP

• CO₂ released

• Hydroxyethyl TPP formed

2. Oxidation (E1 → E2)

• Hydroxyethyl group transferred to lipoate

• Oxidized to acetyl group

• lipoate reduced to dihydrolipoate

3. Transfer to CoA (E2)

• Acetyl group transferred to CoA

• Acetyl-CoA released

4. Regeneration (E3)

• Dihydrolipoate oxidized by FAD

• FADH₂ oxidized by NAD⁺

• NAD⁺ → NADH

What are the allosteric activators and inhibitors of the Pyruvate Dehydrogenase Complex (PDC)?

• Inhibitors (High Energy / Product Build-up): acetyl-CoA, NADH, and ATP

• Activators (Low Energy / Substrates Available): CoA, NAD⁺, ADP

How is PDC regulated by enzymes (kinase/phosphatase) and hormones (insulin/glucagon)?

Covalent Modification:

• PDH Kinase (PDK): Phosphorylates E1​→ INACTIVE

• PDH Phosphatase (PDP): Dephosphorylates E1​→ ACTIVE

Hormonal Control:

• Insulin: Activates phosphatase → PDC ON

• Glucagon: Activates kinase → PDC OFF

What is the reaction, enzyme, energetics, and mechanism of TCA Step 1?

• Enzyme: Citrate Synthase

• Reaction: Acetyl-CoA (2C)+Oxaloacetate (4C)+H2​O→Citrate (6C)+CoA-SH

• Energetics: ΔG′∘=−31.4 kJ/mol (Irreversible / Committed step)

• Mechanism: Condensation reaction forming a Citryl-CoA intermediate, followed by thioester hydrolysis to release CoA-SH.

What regulates Citrate Synthase and why is this step significant?

• Inhibitors: ATP, NADH, Succinyl-CoA, Citrate

• Activators: ADP

• Significance: First committed step of the TCA cycle; condenses 2C + 4C to form the 6C citrate needed to allow subsequent C-C bond cleavage

What is the reaction, enzyme, energetics, and structural requirement for TCA Step 2?

• Enzyme: Aconitase

• Reaction: Citrate⇌Isocitrate (via enzyme-bound intermediate cis-aconitate)

• Energetics: ΔG′∘=+6.3 kJ/mol (Reversible – near equilibrium)

• Cofactor: Requires an Iron-Sulfur cluster [4Fe−4S].

• Why? Relocates the −OH group from a tertiary to a secondary carbon so it can be oxidized in Step 3.

How does Fluoroacetate (rat poison) inhibit the TCA cycle at Step 2?

• "Lethal Synthesis" Pathway:

  1. Fluoroacetate → Fluoroacetyl-CoA

  2. Citrate Synthase converts it into Fluorocitrate

• Target: Fluorocitrate potently inhibits Aconitase.

• Consequence: Blocks the TCA cycle, shutting down cellular respiration and leading to death.

What is the reaction, enzyme, and energetics of TCA Step 3?

• Enzyme: Isocitrate Dehydrogenase

• Reaction: Isocitrate+NAD⁺→α-Ketoglutarate+CO2​+NADH+H⁺

• Energetics: ΔG′∘=−8.4 kJ/mol

• Key Milestones:

  • First oxidative decarboxylation of the cycle.

  • Generates the first NADH and releases the first CO₂​.

What is the intermediate in Step 3 and how is Isocitrate Dehydrogenase regulated?

• Mechanism/Intermediate: Oxidation of the secondary alcohol yields an oxalosuccinate intermediate, which decarboxylates into α-ketoglutarate.

• Inhibitors: ATP, NADH (high energy status)

• Activators: ADP, Ca²⁺ (signals energy need / muscle contraction)

What is the reaction and key characteristics of TCA Step 4?

• Enzyme: α-Ketoglutarate Dehydrogenase Complex

• Reaction: α-Ketoglutarate+NAD⁺+CoA→Succinyl-CoA+CO2​+NADH+H⁺

• Energetics: ΔG′∘=−33.5 kJ/mol (Irreversible)

• Key Milestones: Releases the second CO2​ and forms the second NADH.

How is the α-Ketoglutarate Dehydrogenase complex similar to PDC, and how is it regulated?

• Similarity: Mechanistically identical to Pyruvate Dehydrogenase (PDC)—uses 3 enzymes (E1​,E2​,E3​) and the same 5 coenzymes (TPP, Lipoate, CoA, FAD, NAD⁺).

• Inhibitors: Succinyl-CoA, NADH (product inhibition)

• Activators: Ca²⁺

What is the reaction, enzyme, and significance of TCA Step 5?

• Enzyme: Succinyl-CoA Synthetase

• Reaction: Succinyl-CoA+GDP (or ADP)+Pi​→Succinate+GTP (or ATP)+CoA-SH

• Energetics: ΔG′∘=−2.9 kJ/mol

• Significance: The only substrate-level phosphorylation step in the TCA cycle! Hydrolysis of high-energy thioester bond drives GTP/ATP formation via a phospho-histidine intermediate.

What is the reaction for TCA Step 6 and what makes this enzyme unique?

• Enzyme: Succinate Dehydrogenase

• Reaction: Succinate+FAD→Fumarate+FADH₂​ (ΔG′∘=0 kJ/mol)

• Unique Features:

  1. Only FAD-linked dehydrogenase in TCA (oxidation of −CH₂​−CH₂​− to −CH=CH− is not energetic enough to reduce NAD⁺).

  2. Only membrane-bound TCA enzyme (directly embedded in inner mitochondrial membrane as Complex II of ETC).

What compound inhibits Succinate Dehydrogenase (Step 6) and by what mechanism?

• Inhibitor: Malonate

• Mechanism: Competitive inhibition.

• Why? Malonate is a structural analog of succinate (has 3 carbons instead of 4), binding to the active site and blocking succinate binding.

What is the reaction, enzyme, and mechanism of TCA Step 7?

• Enzyme: Fumarase

• Reaction: Fumarate+H₂​O→L-Malate (ΔG′∘=−3.8 kJ/mol)

• Mechanism: Highly stereospecific trans hydration across the double bond.

• Product: Produces exclusively L-malate (does not form D-malate) to add the −OH group needed for final oxidation.

What is the reaction of TCA Step 8, and how does it proceed despite a highly unfavorable standard free energy?

• Enzyme: Malate Dehydrogenase

• Reaction: L-Malate+NAD⁺→Oxaloacetate+NADH+H⁺

• Energetics: ΔG′∘=+29.7 kJ/mol (Highly unfavorable!)

• How it works: Pulled forward continuously because oxaloacetate (OAA) is kept at extremely low concentration, being rapidly consumed by the highly exergonic Citrate Synthase reaction (ΔG′∘=−31.4 kJ/mol) in Step 1.

• Milestone: Generates the third NADH and regenerates OAA to restart the cycle

What is the net reaction of the TCA cycle, and what is the ATP yield per Acetyl-CoA and per Glucose?

• Net Reaction (per Acetyl-CoA):

  Acetyl-CoA+3NAD⁺+FAD+GDP+Pi​+2H₂​O→2CO2​+3NADH+FADH2​+GTP+CoA+3H+

• Yield per Acetyl-CoA (1 turn):

  • 3 NADH≈7.5 ATP

  • 1 FADH2​≈1.5 ATP

  • 1 GTP=1 ATP

  • Total: ∼10 ATP

• Yield per Glucose (2 Acetyl-CoA / 2 turns):

  • 6 NADH+2 FADH_2​+2 GTP→∼20 ATP

What are the 3 major regulated enzymes in the TCA cycle and their specific activators/inhibitors?

• Citrate Synthase (Entry step):

  • Inhibited by: ATP, NADH, Succinyl-CoA, Citrate

  • Activated by: ADP

• Isocitrate Dehydrogenase (1st decarboxylation):

  • Inhibited by: ATP, NADH

  • Activated by: ADP, Ca²⁺

• α-Ketoglutarate Dehydrogenase (2nd decarboxylation):

  • Inhibited by: Succinyl-CoA, NADH

  • Activated by: Ca²⁺

What is the overall regulatory logic governing the speed of the TCA cycle?

• High ATP/ADP ratio: Slows down (the cell has sufficient energy).

• High NADH/NAD+ ratio: Slows down (indicates the electron transport chain is backed up).

• High Ca²⁺ concentration: Speeds up (signals muscle contraction and immediate demand for ATP).

What is the difference between cataplerotic and anaplerotic reactions in the TCA cycle, and what are key examples of each?

• Cataplerotic Reactions (Intermediates LEAVE the cycle):

  • Citrate → Fatty acid synthesis (in cytoplasm)

  • α-Ketoglutarate → Amino acid synthesis (Glutamate, Glutamine)

  • Succinyl-CoA → Heme synthesis

  • Oxaloacetate → Gluconeogenesis & amino acid synthesis

• Anaplerotic Reactions (Intermediates REPLENISH the cycle):

  • Pyruvate Carboxylase: Converts Pyruvate → Oxaloacetate

  • Amino Acid Degradation: Feeds into various intermediates

  • Odd-Chain Fatty Acids: Converts to Succinyl-CoA

How does anaplerosis contribute to insulin secretion in pancreatic β-cells?

• High Enzyme Levels: Pancreatic β-cells maintain high levels of pyruvate carboxylase.

• Pyruvate/Malate Cycle Pathway:

  1. About half the pyruvate from glycolysis is converted to oxaloacetate via pyruvate carboxylase.

  2. Oxaloacetate is converted to malate, which is exported into the cytosol.

• Signal Generation: Cytosolic malate generates NADPH and other metabolites that act as intracellular messengers to trigger insulin secretion.

What role does anaplerosis play in peripheral tissues during exercise, especially in insulin-resistant individuals?

• Exercise Effects: Increases anaplerotic activity in peripheral tissues.

• Metabolic Outcome:

  • Increases fatty acid oxidation.

  • Helps restore insulin sensitivity in insulin-resistant individuals.