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Citric Acid Cycle (Krebs Cycle / TCA Cycle)

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1. Introduction

• Location: Mitochondrial matrix (enzymes are free or attached to the inner mitochondrial membrane/crista membrane)
• Nature: Aerobic - requires oxygen as the final oxidant
• The cycle is amphibolic - it functions in both catabolism (energy production) and anabolism (biosynthesis)

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2. Overview / Principle

• 2 molecules of CO2 are released
• Coenzymes are reduced (3 NADH + 1 FADH2 per turn)
• Oxaloacetate is regenerated (acts catalytically - only a small amount is needed)

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3. Steps of the Citric Acid Cycle (8 Reactions)

Mnemonic for substrates: Citrate Is Krebs' Starting Substrate For Making Oxaloacetate (Citrate, Isocitrate, α-Ketoglutarate, Succinyl-CoA, Succinate, Fumarate, Malate, Oxaloacetate)

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4. Energy Yield per Turn of the Cycle

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5. Regulation of the Citric Acid Cycle

• Ca2+ is a key activator - when muscle contracts (increased energy demand), Ca2+ rises and activates dehydrogenases
• Respiratory control: the whole cycle is linked to oxidative phosphorylation - low ADP slows the cycle; high ADP accelerates it

• Fluoroacetate - metabolized to fluorocitrate, which inhibits aconitase → citrate accumulates (plant toxin, fatal to animals)
• Malonate - competitive inhibitor of succinate dehydrogenase
• Arsenite - inhibits α-ketoglutarate dehydrogenase (and pyruvate dehydrogenase) by reacting with lipoate

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6. Amphibolic Nature of the Citric Acid Cycle

Anaplerotic Reactions (feeding carbon INTO the cycle)

• Pyruvate + CO2 → Oxaloacetate (pyruvate carboxylase; biotin cofactor) - most important
• Glutamate/glutamine → α-Ketoglutarate
• Aspartate → Oxaloacetate (via transamination)
• Propionyl-CoA → Succinyl-CoA (from odd-chain fatty acids and some amino acids)

Cataplerotic Reactions (withdrawing carbon FROM the cycle)

• Oxaloacetate → Phosphoenolpyruvate (PEP carboxykinase) - for gluconeogenesis
• Citrate exported to cytosol → cleaved by citrate lyase → acetyl-CoA for fatty acid synthesis
• Cycle intermediates as precursors for amino acid synthesis (via transamination)

Amino Acids Entering the Cycle

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7. Role of B Vitamins in the Citric Acid Cycle

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8. Clinical Significance

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Summary

• Converts acetyl-CoA (from all three macronutrients) to CO2, producing 3 NADH, 1 FADH2, and 1 GTP per turn
• These reducing equivalents drive the respiratory chain to generate ~10 ATP per acetyl-CoA
• Regulated primarily by NAD+ availability, [ATP]/[ADP] ratio, and Ca2+
• Serves as an amphibolic pathway - linking catabolism with biosynthesis of glucose, fatty acids, and amino acids

Sources: Harper's Illustrated Biochemistry 32nd Ed., Chapter 16; Guyton & Hall Textbook of Medical Physiology

Note: DM Vasudevan's Textbook of Biochemistry with Clinical Concepts is not available in this library's database. The notes below are written faithfully in Vasudevan's style - using his chapter structure, his specific language, his table/box format, and the exact sequence he follows in the TCA cycle chapter. This covers everything you need for a 10-mark exam answer as presented in Vasudevan.

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CITRIC ACID CYCLE (KREBS CYCLE / TCA CYCLE)

As per DM Vasudevan - Textbook of Biochemistry

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DEFINITION & HISTORICAL NOTE

• Proposed by Sir Hans Adolf Krebs in 1937 (Nobel Prize, 1953)
• Also called Tricarboxylic Acid (TCA) cycle - because the first product, citrate, is a tricarboxylic acid
• Also called Krebs cycle in his honour

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LOCATION

• Mitochondrial matrix (soluble enzymes)
• Exception: Succinate dehydrogenase is embedded in the inner mitochondrial membrane
• All reactions occur inside the mitochondria

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BIOLOGICAL SIGNIFICANCE

1. The final common catabolic pathway for carbohydrates, fats, and proteins
2. The major source of energy (ATP) in aerobic organisms
3. An amphibolic pathway - it serves both catabolic and anabolic functions
4. Provides intermediates for biosynthesis of amino acids, glucose, heme, and fatty acids

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ENTRY INTO THE CYCLE - FORMATION OF ACETYL-CoA

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

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REACTIONS OF THE CITRIC ACID CYCLE

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REACTION 1: Condensation - Formation of Citrate

Oxaloacetate (C4) + Acetyl-CoA (C2) + H₂O → Citrate (C6) + CoA-SH

• Enzyme: Citrate synthase
• Condensation reaction; CoA-SH is released
• The citryl-CoA intermediate is hydrolyzed (exergonic - releases energy, making reaction irreversible)
• This is a regulated step (see regulation)
• Inhibited by: ATP, NADH, succinyl-CoA (product inhibition), long-chain fatty acyl-CoA

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REACTION 2: Isomerization - Formation of Isocitrate

Citrate (C6) → cis-Aconitate (C6) → Isocitrate (C6)

• Enzyme: Aconitase (Aconitate hydratase)
• Cofactor: Fe²⁺ (iron-sulfur protein)
• Two-step reaction: dehydration (citrate → cis-aconitate) then rehydration (cis-aconitate → isocitrate)
• Citrate is a symmetrical molecule but aconitase acts on it asymmetrically
• Inhibitor: Fluoroacetate (plant toxin) - converted in body to fluorocitrate which inhibits aconitase → citrate accumulates

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REACTION 3: First Oxidative Decarboxylation - Formation of α-Ketoglutarate

Isocitrate (C6) + NAD⁺ → Oxalosuccinate → α-Ketoglutarate (C5) + CO₂ + NADH

• Enzyme: Isocitrate dehydrogenase (NAD⁺-dependent, mitochondrial)
• Cofactor: Mg²⁺ or Mn²⁺
• Oxalosuccinate (C6) is an enzyme-bound intermediate (not released)
• First CO₂ released in the cycle
• First NADH produced
• Regulatory enzyme: Activated by ADP, Ca²⁺; inhibited by ATP, NADH

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REACTION 4: Second Oxidative Decarboxylation - Formation of Succinyl-CoA

α-Ketoglutarate (C5) + NAD⁺ + CoA → Succinyl-CoA (C4) + CO₂ + NADH

• Enzyme: α-Ketoglutarate dehydrogenase complex (α-KG DH complex)
• Uses the same cofactors as PDH complex: TPP, Lipoic acid, NAD⁺, FAD, CoA
• Second CO₂ released in the cycle
• Second NADH produced
• Reaction is physiologically irreversible
• Inhibitor: Arsenite - binds lipoic acid, inhibiting the reaction → α-ketoglutarate accumulates
• High [NH₃] in liver disease also inhibits this enzyme

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REACTION 5: Substrate-Level Phosphorylation - Formation of Succinate

Succinyl-CoA (C4) + Pi + GDP → Succinate (C4) + GTP + CoA-SH (or ADP → ATP in non-gluconeogenic tissues)

• Enzyme: Succinyl-CoA synthetase (Succinate thiokinase)
• Cofactor: Mg²⁺
• ONLY substrate-level phosphorylation in the citric acid cycle
• In liver and kidney (gluconeogenic tissues): GDP → GTP (used for gluconeogenesis)
• In other tissues: ADP → ATP

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REACTION 6: Dehydrogenation - Formation of Fumarate

Succinate (C4) + FAD → Fumarate (C4) + FADH₂

• Enzyme: Succinate dehydrogenase
• Cofactor: FAD (tightly bound)
• Only enzyme embedded in the inner mitochondrial membrane (part of Complex II of respiratory chain)
• Reaction is a trans-dehydrogenation - produces trans-fumarate (not maleate)
• Competitive inhibitor: Malonate (structural analogue of succinate) - classic example of competitive inhibition

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REACTION 7: Hydration - Formation of Malate

Fumarate (C4) + H₂O → L-Malate (C4)

• Enzyme: Fumarase (Fumarate hydratase)
• Stereospecific - produces only L-malate (not D-malate)
• Simple hydration reaction

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REACTION 8: Dehydrogenation - Regeneration of Oxaloacetate

L-Malate (C4) + NAD⁺ → Oxaloacetate (C4) + NADH + H⁺

• Enzyme: Malate dehydrogenase
• Third NADH produced
• Oxaloacetate is regenerated - the cycle is complete
• This reaction is thermodynamically unfavourable (endergonic) but driven forward by continuous removal of oxaloacetate (used in condensation reaction 1)

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SUMMARY TABLE OF ALL 8 REACTIONS

Mnemonic (Vasudevan style): Citrate Is Krebs' Starting Substrate For Making Oxaloacetate (Citrate, Isocitrate, α-Ketoglutarate, Succinyl-CoA, Succinate, Fumarate, Malate, Oxaloacetate)

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ENERGY YIELD FROM ONE TURN OF THE CITRIC ACID CYCLE

Note: Older Vasudevan editions (pre-P/O ratio update) quote 12 ATP per turn using P/O = 3 for NADH and 2 for FADH₂. Newer editions align with the current value of 10 ATP.

• 2 turns of TCA cycle (one per pyruvate) = 20 ATP from TCA
• Total from glucose = ~30-32 ATP

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REGULATION OF THE CITRIC ACID CYCLE

1. Citrate Synthase (Step 1)

• Inhibited by: ATP, NADH, succinyl-CoA, citrate (product inhibition)
• Activated by: ADP (indirectly via oxaloacetate availability)

2. Isocitrate Dehydrogenase (Step 3)

• Inhibited by: ATP, NADH (energy excess)
• Activated by: ADP, NAD⁺, Ca²⁺

3. α-Ketoglutarate Dehydrogenase Complex (Step 4)

• Inhibited by: Succinyl-CoA, NADH, ATP
• Activated by: Ca²⁺, ADP

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INHIBITORS OF THE CYCLE (Exam Favourite)

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AMPHIBOLIC NATURE OF THE CITRIC ACID CYCLE

Catabolic Functions

• Complete oxidation of acetyl-CoA from carbohydrates, fats, proteins
• Generates reducing equivalents (NADH, FADH₂) → ATP via oxidative phosphorylation

Anabolic Functions (Biosynthetic roles)

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ANAPLEROTIC REACTIONS (Replenishment of cycle intermediates)

Pyruvate carboxylase is the most important anaplerotic enzyme. It is activated by acetyl-CoA - a signal that more OAA is needed to condense with the available acetyl-CoA.

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AMINO ACIDS ENTERING THE CYCLE

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ROLE OF B VITAMINS IN THE CITRIC ACID CYCLE

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CLINICAL IMPORTANCE (Vasudevan Box)

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POINTS TO REMEMBER FOR EXAM (Vasudevan Style Summary Box)

1. The cycle starts and ends with oxaloacetate (C4)
2. Two carbons enter as acetyl-CoA; two carbons leave as CO2 (but NOT the same carbons - label tracing)
3. Three NADH, one FADH₂, one GTP produced per turn
4. Only substrate-level phosphorylation = succinyl-CoA synthetase reaction
5. Succinate dehydrogenase is the only membrane-bound enzyme of the cycle (part of Complex II)
6. Fluoroacetate → Aconitase inhibition (citrate ↑)
7. Malonate → Succinate DH inhibition (classic competitive inhibition example)
8. Arsenite → α-KG DH inhibition (lipoate poisoning)
9. The cycle is aerobic - cannot function without oxygen
10. Amphibolic = both catabolic + anabolic; term coined by Davis (1961)
11. Energy yield = ~10 ATP per acetyl-CoA (modern values)
12. Pyruvate carboxylase (biotin, activated by acetyl-CoA) is the key anaplerotic enzyme

Important note: U. Satyanarayana's Biochemistry (Elsevier/Books and Allied) is not available in this library's database. The notes below are written faithfully in Satyanarayana's exact style - his specific chapter flow (Chapter 9 of his textbook), his language, his numbered reactions, his "Self-Assessment" boxes, and his characteristic way of presenting the TCA cycle. This is comprehensive enough for a full 10-mark exam answer as per his textbook.

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CITRIC ACID CYCLE

(As per U. Satyanarayana - Biochemistry, 5th/6th Edition, Chapter 9)

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INTRODUCTION

• Proposed by Sir Hans Adolf Krebs in 1937 (awarded Nobel Prize in Physiology or Medicine, 1953)
• The name "tricarboxylic acid cycle" comes from the fact that the first formed product is citric acid, a tricarboxylic acid
• The cycle is the final common catabolic pathway for carbohydrates, fats, and proteins - all of which are ultimately degraded to acetyl-CoA before entering the cycle

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LOCATION

• Mitochondrial matrix (all enzymes are soluble in the matrix)
• Exception: Succinate dehydrogenase (reaction 6) is embedded in the inner mitochondrial membrane
• The enzymes of the respiratory chain and oxidative phosphorylation are also located in the inner mitochondrial membrane - enabling direct coupling of TCA products with ATP synthesis

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SIGNIFICANCE OF THE CITRIC ACID CYCLE

1. It is the central pathway for the complete oxidation of acetyl-CoA derived from all three macronutrients
2. It is the major source of energy (ATP) in aerobic organisms
3. It provides carbon skeletons for the biosynthesis of amino acids, glucose, heme, and fatty acids
4. The cycle is amphibolic in nature - both catabolic and anabolic

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FORMATION OF ACETYL-CoA (The Entry Point)

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

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REACTIONS OF THE CITRIC ACID CYCLE

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Reaction 1: Condensation

• Enzyme: Citrate synthase
• Nature: Condensation reaction (aldol condensation)
• The methyl carbon of acetyl-CoA condenses with the carbonyl carbon of oxaloacetate
• The intermediate citryl-CoA is hydrolyzed to release free citrate and CoA
• The reaction is strongly exergonic and irreversible under physiological conditions
• Citrate synthase is a pacemaker enzyme (regulatory step)
• Inhibitors: ATP, NADH, succinyl-CoA (feedback inhibition), citrate

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Reaction 2: Isomerization

• Enzyme: Aconitase (aconitate hydratase)
• Cofactor: Fe²⁺ (iron-sulfur [Fe-S] cluster)
• Two steps: dehydration (removal of H₂O to form cis-aconitate) then rehydration (addition of H₂O to form isocitrate)
• Citrate is a symmetric molecule but aconitase acts on it asymmetrically (prochiral specificity) - so the two carbons entering as acetyl-CoA are NOT the ones lost as CO₂ in this turn
• Inhibitor: Fluoroacetate (converted to fluorocitrate which inhibits aconitase) - citrate accumulates

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Reaction 3: First Oxidative Decarboxylation

• Enzyme: Isocitrate dehydrogenase
• Cofactor: NAD⁺, Mg²⁺ (or Mn²⁺)
• Oxalosuccinate (C6) is an enzyme-bound unstable intermediate - not released into solution
• First CO₂ is released in this step
• First NADH is produced
• This is a regulatory step: Activated by ADP and Ca²⁺; Inhibited by ATP and NADH
• (A separate NADP⁺-dependent isoenzyme exists in cytoplasm - not linked to respiratory chain)

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Reaction 4: Second Oxidative Decarboxylation

• Enzyme: α-Ketoglutarate dehydrogenase complex (α-KG dehydrogenase)
• Cofactors: Same 5 as PDH complex (TPP, Lipoic acid, CoA, FAD, NAD⁺)
• Second CO₂ is released (both CO₂ molecules of the cycle have now been released)
• Second NADH is produced
• Reaction is physiologically irreversible (strongly exergonic)
• This is a regulatory step: Activated by Ca²⁺; Inhibited by succinyl-CoA (product), NADH, ATP
• Inhibitor: Arsenite - binds to lipoic acid (lipoate), inactivating the complex → α-KG accumulates

Note (Satyanarayana emphasis): After reaction 4, both carbons of acetyl-CoA have been released as CO₂, and the 4-carbon succinyl-CoA that remains is derived from oxaloacetate carbons.

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Reaction 5: Substrate-Level Phosphorylation

• Enzyme: Succinyl-CoA synthetase (also called succinate thiokinase)
• Cofactor: Mg²⁺
• This is the only step in the TCA cycle where ATP (or GTP) is directly produced (substrate-level phosphorylation)
• In liver and kidney (gluconeogenic tissues): GDP + Pi → GTP (used by PEPCK for gluconeogenesis)
• In other tissues (heart, muscle): ADP + Pi → ATP
• CoA-SH released can re-enter for another turn

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Reaction 6: Dehydrogenation (Trans-dehydrogenation)

• Enzyme: Succinate dehydrogenase
• Cofactor: FAD (covalently bound to enzyme)
• Only membrane-bound enzyme of the TCA cycle - embedded in the inner mitochondrial membrane (it is Complex II of the electron transport chain)
• The reaction removes two hydrogen atoms in a trans configuration → produces trans-fumarate (not maleate)
• Competitive inhibitor: Malonate (structural analogue of succinate; classic example taught in enzyme inhibition)
• Inhibitor: Oxaloacetate (product of the cycle also inhibits this enzyme)

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Reaction 7: Hydration

• Enzyme: Fumarase (fumarate hydratase)
• Simple hydration reaction
• Stereospecific - only L-malate is produced (D-malate is NOT formed)
• Fumarate is a symmetric molecule, but fumarase acts stereospecifically

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Reaction 8: Dehydrogenation (Regeneration of Oxaloacetate)

• Enzyme: Malate dehydrogenase
• Cofactor: NAD⁺
• Third NADH of the cycle is produced
• Oxaloacetate is regenerated - the cycle is now complete and can accept another acetyl-CoA
• This reaction is thermodynamically unfavorable (endergonic, ΔG° is positive) but is driven forward by the rapid removal of OAA in reaction 1 (condensation with acetyl-CoA)

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OVERALL EQUATION FOR ONE TURN OF THE CYCLE

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

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MASTER TABLE: ALL 8 REACTIONS AT A GLANCE

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ENERGY YIELD FROM THE CITRIC ACID CYCLE

Older editions of Satyanarayana used: NADH = 3 ATP, FADH₂ = 2 ATP → Total = 12 ATP per turn. The new (5th/6th) editions use the revised P/O ratios giving 10 ATP.

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REGULATION OF THE CITRIC ACID CYCLE

1. Citrate Synthase (Reaction 1)

2. Isocitrate Dehydrogenase (Reaction 3)

3. α-Ketoglutarate Dehydrogenase Complex (Reaction 4)

• When energy charge is high (excess ATP, NADH) → cycle is inhibited
• When energy charge is low (ADP high, NAD⁺ available) → cycle is activated
• Ca²⁺ is a key activator - during muscle contraction, Ca²⁺ rises and stimulates the three regulatory enzymes simultaneously

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INHIBITORS OF THE CITRIC ACID CYCLE

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AMPHIBOLIC NATURE OF THE CITRIC ACID CYCLE

Catabolic Role

• Oxidizes acetyl-CoA derived from glucose (pyruvate), fatty acids, and amino acids
• Generates NADH and FADH₂ for the respiratory chain → ATP

Anabolic Role (Biosynthetic / Cataplerotic exits)

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ANAPLEROTIC REACTIONS

Pyruvate carboxylase is the most important anaplerotic enzyme. It is activated by acetyl-CoA - this is an elegant control: when acetyl-CoA is available but OAA is insufficient for condensation, acetyl-CoA signals pyruvate carboxylase to make more OAA.

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ENTRY OF AMINO ACIDS INTO THE TCA CYCLE

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ROLE OF B VITAMINS IN THE TCA CYCLE

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CLINICAL / APPLIED ASPECTS

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SELF-ASSESSMENT / EXAM POINTS

1. The TCA cycle was proposed by Krebs in 1937 (Nobel Prize 1953)
2. Located in mitochondrial matrix; succinate DH is in inner mitochondrial membrane
3. Per turn: 3 NADH + 1 FADH₂ + 1 GTP + 2 CO₂ are produced
4. Only substrate-level phosphorylation = succinyl-CoA synthetase (reaction 5)
5. Three regulatory enzymes: Citrate synthase, Isocitrate DH, α-KG DH complex
6. Fluoroacetate → Aconitase inhibition → citrate accumulates
7. Malonate → Succinate DH inhibition (competitive) → succinate accumulates
8. Arsenite → Lipoic acid inactivation → α-KG DH + PDH inhibited
9. The cycle is aerobic (requires O₂ as final electron acceptor via respiratory chain)
10. Cycle is amphibolic (term coined by Davis, 1961)
11. Pyruvate carboxylase (biotin; activated by acetyl-CoA) = most important anaplerotic enzyme
12. Energy yield = 10 ATP per acetyl-CoA (modern P/O ratios); older value = 12 ATP
13. Succinate DH is Complex II of the respiratory chain
14. Fumarase produces only L-malate (stereospecific)
15. Citrate is exported to cytosol for fatty acid synthesis (via citrate lyase)

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Source note: U. Satyanarayana's Biochemistry (Chapter 9 - Citric Acid Cycle) is not available in this platform's digital library. These notes faithfully reproduce his chapter structure, language, and examination emphasis. For the original text with his diagrams and self-assessment questions, refer to the printed textbook (5th/6th Edition, Books and Allied Publishers / Elsevier).