Krebs Cycle (Tricarboxylic Acid Cycle / Citric Acid Cycle)

🟢 Lite — Quick Review (1h–1d)

Rapid summary for last-minute revision before your exam.

Krebs Cycle (TCA cycle) is the hub of cellular metabolism — it oxidizes acetyl-CoA to CO₂, generating NADH, FADH₂, and GTP. It occurs in mitochondria and is central to carbohydrate, fat, and protein metabolism. INI CET frequently asks about enzyme names, number of ATP generated per acetyl-CoA, and regulation.

High-Yield Facts for INI CET:

• Location: Mitochondrial matrix
• 8 steps, 8 enzymes, 2 CO₂ released, 3 NADH + 1 FADH₂ + 1 GTP (GTP = ATP equivalent) per acetyl-CoA
• Product per acetyl-CoA: 3 NADH (×2.5 ATP = 7.5 ATP) + 1 FADH₂ (×1.5 ATP = 1.5 ATP) + 1 GTP (1 ATP) = 10 ATP per acetyl-CoA entering the cycle
• Irreversible regulation: Citrate synthase, isocitrate dehydrogenase (rate-limiting step), α-ketoglutarate dehydrogenase
• Anaplerotic reactions: Pyruvate carboxylase, malate enzyme, branched-chain amino acid transaminases — replenish oxaloacetate

⚡ Exam tip: In the TCA cycle, no oxygen is used directly — the NADH and FADH₂ produced are oxidized by the electron transport chain, which requires oxygen. This is why the Krebs cycle is an aerobic process.

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🟡 Standard — Regular Study (2d–2mo)

Standard content for students with a few days to months.

Krebs Cycle — INI CET (AIIMS PG) Study Guide

Overview of the Cycle

The TCA cycle begins when acetyl-CoA (2 carbons) combines with oxaloacetate (4 carbons) to form citrate (6 carbons). Through 8 enzymatic steps, two CO₂ molecules are released, regenerating oxaloacetate. The cycle turns once per acetyl-CoA entering.

Net Reaction: Acetyl-CoA + 3 NAD⁺ + FAD + GDP + Pi + 2 H₂O → 2 CO₂ + 3 NADH + FADH₂ + GTP + CoA + 2 H⁺

The 8 Steps of the Krebs Cycle

Step 1: Citrate Synthase (CS)

• Acetyl-CoA + Oxaloacetate + H₂O → Citrate + CoA
• Reaction type: Aldol condensation
• Citrate synthase is the first enzyme and is highly exergonic (ΔG°’ = -31.5 kJ/mol), making the reaction essentially irreversible — this ensures the cycle doesn’t run in reverse under normal conditions
• Regulation: Inhibited by ATP, NADH, succinyl-CoA, citrate; stimulated by ADP

Step 2: Aconitase (Aconitate Hydratase)

• Citrate ↔ Isocitrate (via cis-aconitate intermediate)
• Reaction type: Isomerization + dehydration + hydration
• Aconitase contains an [4Fe-4S] cluster essential for its function
• Fluorocitrate (produced from fluoroacetate) inhibits aconitase → accumulates citrate → is toxic

Step 3: Isocitrate Dehydrogenase (IDH) — RATE-LIMITING STEP

• Isocitrate + NAD⁺ → α-ketoglutarate + NADH + CO₂
• Reaction type: Oxidative decarboxylation
• This is the rate-limiting step and major regulatory point
• Three isoforms: IDH3 (mitochondrial, NAD⁺-specific, inhibited by ATP/ADP — a key regulatory mechanism), IDH2 (mitochondrial, NADP⁺), IDH1 (cytosolic, NADP⁺)
• Regulation: Activated by ADP, Ca²⁺ (muscle contraction); inhibited by ATP, NADH

Step 4: α-Ketoglutarate Dehydrogenase (α-KGDH)

• α-Ketoglutarate + NAD⁺ + CoA → Succinyl-CoA + NADH + CO₂
• Reaction type: Oxidative decarboxylation
• Multi-enzyme complex (like pyruvate dehydrogenase complex): requires thiamine pyrophosphate (TPP), lipoic acid, CoA, FAD, NAD⁺
• Regulation: Inhibited by NADH, succinyl-CoA, ATP; activated by ADP, Ca²⁺
• α-KGDH is the second rate-limiting step

Step 5: Succinyl-CoA Synthetase (SCS)

• Succinyl-CoA + GDP + Pi ↔ Succinate + GTP + CoA
• Reaction type: Substrate-level phosphorylation
• Only step in TCA cycle that directly generates a high-energy phosphate bond
• GTP can be converted to ATP: GTP + ADP → GDP + ATP (nucleoside diphosphate kinase)
• Some tissues use ADP instead of GDP (brain uses ADP)
• Succinyl-CoA synthetase is the enzyme; the energy is stored in the thioester bond of succinyl-CoA

Step 6: Succinate Dehydrogenase (SDH)

• Succinate ↔ Fumarate (FADH₂ is produced)
• Reaction type: Oxidation (removal of 2 H atoms)
• SDH is the only membrane-bound enzyme of the TCA cycle — it’s embedded in the inner mitochondrial membrane (Complex II of ETC)
• FAD is tightly bound to SDH (covalently bound FAD as prosthetic group)
• FADH₂ generated here yields ~1.5 ATP (not 2.5 like NADH) because it enters the ETC at Complex II

Step 7: Fumarase (Fumarate Hydratase)

• Fumarate + H₂O → L-Malate
• Reaction type: Hydration (addition of H₂O across the double bond)
• Specifically adds H₂O to the trans double bond of fumarate to form L-malate

Step 8: Malate Dehydrogenase (MDH)

• L-Malate + NAD⁺ ↔ Oxaloacetate + NADH
• Reaction type: Oxidation
• Highly unfavorable under standard conditions (ΔG°’ = +29.7 kJ/mol) — this reaction runs forward because OAA is immediately consumed in step 1 (citrate synthase pulls OAA out)
• Regulation: Inhibited by NADH; activated by ADP
• OAA must be regenerated to keep the cycle running — this is the commitment step

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🔴 Extended — Deep Study (3mo+)

Comprehensive coverage for students on a longer study timeline.

Energy Yield from One Acetyl-CoA

Step	Product	ATP Equivalent
Isocitrate → α-KG	1 NADH	2.5 ATP
α-KG → Succinyl-CoA	1 NADH	2.5 ATP
Succinyl-CoA → Succinate	1 GTP	1 ATP
Succinate → Fumarate	1 FADH₂	1.5 ATP
Malate → OAA	1 NADH	2.5 ATP
Total	3 NADH + 1 FADH₂ + 1 GTP	10 ATP

Total ATP from glucose oxidation (including glycolysis and link reaction):

• Glycolysis: 2 ATP + 2 NADH = ~8 ATP (or ~6 if using glycerol-phosphate shuttle)
• Pyruvate dehydrogenase: 2 NADH = 5 ATP
• TCA cycle (×2): 20 ATP
• Total: ~30-32 ATP per glucose

Regulation of the Krebs Cycle

Overall principle: The cycle is regulated at three key steps to match energy supply with demand.

1. Citrate Synthase:

• Allosterically inhibited by ATP, NADH, succinyl-CoA, citrate
• Activated by ADP

2. Isocitrate Dehydrogenase (primary control point):

• Inhibited by ATP, NADH, NADPH (all indicate “energy surplus”)
• Activated by ADP, Ca²⁺ (Ca²⁺ released during muscle contraction stimulates the cycle)

3. α-Ketoglutarate Dehydrogenase:

• Inhibited by NADH, succinyl-CoA, ATP
• Activated by ADP, Ca²⁺
• Similar regulation to isocitrate dehydrogenase

The Ca²⁺ connection: During muscle contraction, Ca²⁺ released from SR activates both IDH and α-KGDH (and PDH phosphatase), increasing energy production to meet demand.

Anaplerotic (Refilling) Reactions

The TCA cycle intermediates are drawn off for biosynthetic reactions — anaplerosis replenishes them.

Key Anaplerotic Reactions:

1. Pyruvate carboxylase: Pyruvate + CO₂ + ATP → Oxaloacetate + ADP + Pi

   • Biotin-containing enzyme; activated by acetyl-CoA
   • Most important anaplerotic reaction
   • Occurs in liver and kidney; essential for gluconeogenesis

2. Malate enzyme: Pyruvate + CO₂ + NADPH → Malate + NADP⁺

   • Provides malate for OAA replenishment

3. Branched-chain amino acid transaminases:

   • Transamination of leucine, isoleucine, valine produces α-ketoglutarate → replenishes cycle

4. Glutamate dehydrogenase:

   • Glutamate + NAD(P)⁺ + H₂O → α-ketoglutarate + NAD(P)H + NH₄⁺
   • Important link between amino acid and TCA cycle metabolism

The Citric Acid Cycle in Context

** amphibolic: Both catabolic and anabolic functions

Catabolic uses:

• Glucose oxidation (via glycolysis → acetyl-CoA → TCA)
• Fatty acid β-oxidation (produces acetyl-CoA)
• Amino acid deamination (transamination produces TCA intermediates)
• Glycerol → DHAP → enters at triose phosphate level

Anabolic uses (drawing off intermediates):

• OAA → PEP → gluconeogenesis (liver, kidney)
• α-KG → glutamate synthesis
• Succinyl-CoA → porphyrin synthesis, heme synthesis
• OAA → aspartate → purine/pyrimidine synthesis
• Citrate → acetyl-CoA → fatty acid synthesis (exits mitochondria as citrate)

The Citrate Shuttle:

• When ATP is abundant and fatty acid synthesis is needed, citrate is transported out of mitochondria
• In cytosol: citrate → acetyl-CoA (ATP-citrate lyase) + oxaloacetate
• Acetyl-CoA → fatty acid synthesis
• OAA → malate → pyruvate (malic enzyme) → back to mitochondria

Key Enzyme Deficiencies

Fumarase deficiency:

• Rare autosomal recessive; accumulation of fumaric acid
• Neurological impairment, developmental delay, facial dysmorphism

Succinate dehydrogenase (Complex II) deficiency:

• Mutations cause Leigh syndrome, mitochondrial complex II deficiency
• Results in accumulation of succinate

α-Ketoglutarate dehydrogenase deficiency:

• Associated with neurodegenerative diseases, 2-hydroxyglutaric aciduria

Pyruvate dehydrogenase complex (PDH) deficiency:

• Cannot convert pyruvate to acetyl-CoA → pyruvate shunted to lactate
• Causes lactic acidosis, neurological dysfunction
• Thiamine-responsive (TPP is a cofactor)

Clinical Correlations

Krebs Cycle and Cancer:

• Many cancer cells rely heavily on aerobic glycolysis (Warburg effect) but also require TCA cycle intermediates for biosynthetic reactions
• Glutamine addiction: Glutamine provides α-ketoglutarate to replenish the cycle and support biosynthesis
• Isocitrate dehydrogenase (IDH1/IDH2) mutations in gliomas and acute myeloid leukemia → produce 2-hydroxyglutarate (2-HG), which interferes with epigenetic regulation

Toxins Affecting the Krebs Cycle:

Toxin	Target	Effect
Fluoroacetate	Aconitase	Forms fluorocitrate → blocks cycle
Arsenite	α-KGDH (dithiol cofactors)	Inhibits α-KGDH
Malonate	Succinate dehydrogenase	Competitively inhibits SDH (succinate accumulates)
Atractyloside	Mitochondrial ADP/ATP translocase	Prevents ADP import → cycle stalls

⚡ INI CET High-Yield: Remember the two CO₂-producing steps are isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. These are both oxidative decarboxylations requiring NAD⁺ as cofactor. The carbons lost as CO₂ come from the acetyl-CoA that entered, not directly from oxaloacetate.

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