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The Krebs Cycle (Citric Acid Cycle / TCA Cycle) - A Complete Guide

The Krebs cycle is named after Sir Hans Krebs, who first described the cyclic nature of these reactions (for which he received the Nobel Prize in 1953). It is also called the citric acid cycle (because citrate was one of the first compounds identified in it) or the tricarboxylic acid (TCA) cycle (because citrate and isocitrate carry three carboxyl groups). All three names refer to the exact same pathway.

1. Location

The TCA cycle operates entirely in the mitochondrial matrix. Its enzymes are either free in the matrix or attached to the inner mitochondrial membrane and crista membrane - the same location as the respiratory chain enzymes that reoxidize the NADH and FADH₂ the cycle produces. This proximity is functionally important.

Basic Medical Biochemistry, p. 827
Harper's Illustrated Biochemistry, 32nd Ed, p. 168

2. Overview and Purpose

The primary function of the TCA cycle is energy conservation through oxidation. Fuels (carbohydrates, fats, proteins) are broken down to acetyl-CoA, which is the cycle's entry substrate. The cycle oxidizes the two-carbon acetyl group to CO₂, capturing the released electrons in NADH and FADH₂. These electron carriers then feed into the electron transport chain (ETC), driving oxidative phosphorylation and massive ATP synthesis.

Key input: Acetyl-CoA (2 carbons)
Key outputs per turn: 2 CO₂, 3 NADH, 1 FADH₂, 1 GTP

The cycle is aerobic - it requires oxygen as the ultimate electron acceptor (via the ETC). Without oxygen, NADH and FADH₂ cannot be reoxidized, and the cycle stops.

The cycle also plays an amphibolic role - it functions in both catabolism and anabolism.

Citric acid cycle diagram with respiratory chain coupling

The citric acid cycle and its link to the respiratory chain - Harper's Illustrated Biochemistry

3. Entry Substrate: Acetyl-CoA

Before entering the cycle, fuels must be converted to acetyl-CoA:

Fuel	Pathway to Acetyl-CoA
Glucose/carbohydrates	Glycolysis → Pyruvate → PDC (pyruvate dehydrogenase complex)
Fatty acids	β-oxidation (direct)
Ketone bodies	Direct conversion
Amino acids (Leu, Lys, Ile, Trp, Phe, Tyr)	Catabolism via acetyl-CoA
Ethanol	Acetaldehyde → Acetate → Acetyl-CoA
Alanine/Serine	Pyruvate → PDC

Acetyl-CoA carries an acetyl group (2 carbons) and donates 8 electrons to the TCA cycle.

Basic Medical Biochemistry, p. 843

4. The Eight Reactions of the Krebs Cycle

Here is the complete, step-by-step walkthrough:

Detailed TCA cycle reactions with enzymes and coenzymes

TCA cycle reactions showing enzymes (pink), intermediates, and coenzyme changes - Basic Medical Biochemistry

Step 1 - Condensation: Oxaloacetate + Acetyl-CoA → Citrate

Enzyme: Citrate synthase
Reaction: The methyl carbon of acetyl-CoA condenses with the carbonyl carbon of oxaloacetate (4C), forming a 6-carbon tricarboxylate, citrate, plus free CoA-SH
Cofactor: Water added
Energy: Exergonic (ΔG° = -31.4 kJ/mol); essentially irreversible under cellular conditions
Regulation: Inhibited by high ATP, NADH, succinyl-CoA (product inhibition); activated when oxaloacetate is available

This is the committed step that pulls the cycle forward.

Step 2 - Isomerization: Citrate → Isocitrate (via cis-Aconitate)

Enzyme: Aconitase (contains an iron-sulfur [Fe-S] cluster)
Reaction: Citrate is first dehydrated to cis-aconitate, then rehydrated to isocitrate (6C)
Purpose: Citrate cannot be oxidized directly; rearrangement positions the hydroxyl group for the next dehydrogenase step
Note: Aconitase is inhibited by fluorocitrate (metabolite of fluoroacetate - a deadly rat poison that blocks the cycle at this step)

Step 3 - First Oxidative Decarboxylation: Isocitrate → α-Ketoglutarate + CO₂

Enzyme: Isocitrate dehydrogenase (IDH)
Reaction: Isocitrate (6C) is oxidized to oxalosuccinate (unstable intermediate), then decarboxylated to α-ketoglutarate (5C) + CO₂
Cofactor: NAD⁺ → NADH (first NADH produced)
Regulation: This is a major regulatory step - activated by ADP, Ca²⁺, NAD⁺; inhibited by ATP, NADH (energy sufficiency signals)

Step 4 - Second Oxidative Decarboxylation: α-Ketoglutarate → Succinyl-CoA + CO₂

Enzyme: α-Ketoglutarate dehydrogenase complex (α-KGDH)
Reaction: α-Ketoglutarate (5C) is oxidatively decarboxylated to succinyl-CoA (4C) + CO₂
Cofactor: NAD⁺ → NADH (second NADH produced)
Co-factors required: Thiamine pyrophosphate (TPP/Vitamin B₁), lipoamide, FAD, CoA, NAD⁺ - structurally similar to pyruvate dehydrogenase complex
Regulation: Inhibited by NADH and succinyl-CoA (product inhibition); activated by Ca²⁺

Both CO₂ molecules are released in steps 3 and 4. Note: the carbons lost as CO₂ in the first turn are NOT the same carbon atoms that entered as acetyl-CoA - they come from the oxaloacetate skeleton.

Step 5 - Substrate-Level Phosphorylation: Succinyl-CoA → Succinate + GTP

Enzyme: Succinyl-CoA synthetase (also called succinate thiokinase)
Reaction: The high-energy thioester bond of succinyl-CoA is cleaved, driving phosphorylation of GDP → GTP (which can be converted to ATP by nucleoside diphosphate kinase)
Co-product: CoA-SH released
Significance: Only step in the TCA cycle that directly generates a high-energy phosphate bond (substrate-level phosphorylation)

Step 6 - Oxidation: Succinate → Fumarate

Enzyme: Succinate dehydrogenase (SDH)
Cofactor: FAD → FADH₂
Reaction: Succinate (4C) is oxidized to fumarate (4C) - a trans double bond is introduced
Unique feature: SDH is the only TCA cycle enzyme embedded in the inner mitochondrial membrane (it is also Complex II of the ETC). It passes electrons directly to ubiquinone (coenzyme Q)
Inhibited by: Malonate (competitive analog of succinate - classic biochemistry teaching example of competitive inhibition)

Step 7 - Hydration: Fumarate → Malate

Enzyme: Fumarase (fumarate hydratase)
Reaction: Water is added across the double bond of fumarate to produce L-malate (4C)
Stereospecificity: Fumarase is absolutely stereospecific - produces only the L-isomer
Cofactor: None required

Step 8 - Final Oxidation: Malate → Oxaloacetate

Enzyme: Malate dehydrogenase
Cofactor: NAD⁺ → NADH (third NADH produced)
Reaction: L-malate is oxidized to oxaloacetate (4C), regenerating the starting compound
Thermodynamics: Slightly endergonic (unfavorable) under standard conditions, but the reaction is driven forward because oxaloacetate is continuously consumed by citrate synthase

The cycle is now complete - oxaloacetate is regenerated and ready for another turn.

5. Summary of Products Per Turn

Krebs cycle step-by-step structural diagram from Guyton

Complete TCA cycle reaction steps - Guyton & Hall Medical Physiology

Product	Quantity per turn	Destination
CO₂	2 molecules	Exhaled
NADH	3 molecules	ETC → ~2.5 ATP each
FADH₂	1 molecule	ETC → ~1.5 ATP each
GTP	1 molecule	≈ 1 ATP
Total ATP equivalent	~10 ATP

Net equation per turn:

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

Per molecule of glucose (2 acetyl-CoA enter):

2 Acetyl-CoA + 6H₂O + 2ADP → 4CO₂ + 16H + 2CoA + 2ATP

Harper's Illustrated Biochemistry, p. 170; Guyton & Hall, p. (Fig 68.6)

6. Energetics and ATP Yield

From one acetyl-CoA:

3 NADH × 2.5 ATP = 7.5 ATP
1 FADH₂ × 1.5 ATP = 1.5 ATP
1 GTP = 1 ATP
Total = 10 ATP per acetyl-CoA

For comparison:

Glycolysis (glucose → 2 pyruvate): 2 ATP net + 2 NADH
Pyruvate dehydrogenase: 1 NADH per pyruvate
TCA cycle: 10 ATP per acetyl-CoA × 2 = 20 ATP from TCA alone per glucose
Grand total from complete glucose oxidation: ~30-32 ATP

The majority (~90%) of ATP from glucose comes from oxidative phosphorylation driven by TCA-generated NADH and FADH₂, not from substrate-level phosphorylation.

Harper's Illustrated Biochemistry, p. 170

7. Regulation of the TCA Cycle

The cycle is regulated at three main points to match ATP production with energy demand:

7.1 Pyruvate Dehydrogenase Complex (PDC) - Entry Gate

Controls how much acetyl-CoA enters the cycle.

Activated by: ADP, NAD⁺, CoA, Ca²⁺ (in muscle)
Inhibited by: ATP, NADH, acetyl-CoA, fatty acids

7.2 Citrate Synthase - Step 1

Inhibited by: ATP, NADH, succinyl-CoA, citrate (product inhibition)
When energy is high, citrate accumulates → feeds back to inhibit its own synthesis

7.3 Isocitrate Dehydrogenase - Step 3 (Most Important Regulator)

Activated by: ADP, Ca²⁺, NAD⁺ (low energy state)
Inhibited by: ATP, NADH (high energy state)
This is the primary allosteric control point

7.4 α-Ketoglutarate Dehydrogenase - Step 4

Inhibited by: NADH, succinyl-CoA, GTP
Activated by: Ca²⁺ (important during muscle contraction)

General principle: When ATP/NADH levels are high, the cycle slows. When ADP/NAD⁺ levels are high (energy deficit), the cycle accelerates. Ca²⁺ released during muscle contraction simultaneously activates multiple TCA enzymes to match energy production with contractile demand.

Basic Medical Biochemistry, p. 835-843

8. Coenzymes and Vitamins Required

The TCA cycle has an extensive vitamin requirement:

Vitamin	Coenzyme Form	Enzyme(s)
Niacin (B₃)	NAD⁺	Isocitrate DH, α-KG DH, Malate DH
Riboflavin (B₂)	FAD	Succinate DH
Thiamine (B₁)	Thiamine pyrophosphate (TPP)	α-KG DH complex
Pantothenic acid (B₅)	Coenzyme A	Citrate synthase, succinyl-CoA synthetase
Lipoic acid	Lipoamide	α-KG DH complex

Minerals: Mg²⁺, Ca²⁺, Fe²⁺ (in aconitase), phosphate

Clinical point: Thiamine (B₁) deficiency impairs both PDC and α-KGDH, causing pyruvate/lactate accumulation. This underlies Wernicke's encephalopathy and beriberi.

Harper's Illustrated Biochemistry, p. 171; Basic Medical Biochemistry, p. 828

9. Anaplerosis and Cataplerosis

The TCA cycle intermediates are not just oxidized - they are constantly being drawn off for biosynthesis (cataplerosis) and replenished (anaplerosis). These two processes must be balanced to sustain the cycle.

Anaplerotic reactions (filling the cycle):

Pyruvate → Oxaloacetate (by pyruvate carboxylase; activated by acetyl-CoA)
Amino acids → α-ketoglutarate, succinyl-CoA, fumarate, oxaloacetate
Propionyl-CoA → Succinyl-CoA (from odd-chain fatty acids)

Cataplerotic reactions (draining the cycle):

Oxaloacetate → Phosphoenolpyruvate (for gluconeogenesis, by PEPCK)
α-Ketoglutarate → Glutamate (by transamination)
Citrate → Cytosol (for fatty acid synthesis)
Succinyl-CoA → Heme synthesis
Harper's Illustrated Biochemistry, p. 169

10. The TCA Cycle is Amphibolic

The cycle serves both catabolic and anabolic functions (hence "amphibolic"):

TCA cycle amphibolic role showing connections to gluconeogenesis and amino acid metabolism

The TCA cycle's role in gluconeogenesis, amino acid synthesis, and transamination - Harper's Illustrated Biochemistry

Catabolic: Oxidizes acetyl-CoA → CO₂ + ATP
Anabolic roles:

Gluconeogenesis: All intermediates can give rise to oxaloacetate → glucose
Amino acid synthesis: α-Ketoglutarate → Glutamate family; oxaloacetate → aspartate family
Fatty acid synthesis: Citrate exits to cytosol where it provides acetyl-CoA for de novo lipogenesis
Heme synthesis: Succinyl-CoA is a precursor for porphyrin synthesis
Nucleotide synthesis: Aspartate (from oxaloacetate) contributes to purine/pyrimidine rings
Harper's Illustrated Biochemistry, p. 171

11. Clinical Relevance

Condition	TCA Cycle Connection
Thiamine deficiency (Wernicke's, Beriberi)	Blocks α-KGDH and PDC → lactic acidosis, neurological damage
Fluoroacetate poisoning	Blocks aconitase via fluorocitrate
Isocitrate DH (IDH) mutations	Found in gliomas and AML - produce 2-hydroxyglutarate (oncometabolite)
SDH mutations	Associated with hereditary paraganglioma/pheochromocytoma
Fumarate hydratase mutations	Associated with renal cell carcinoma, uterine fibroids
Hypoxia	Blocks ETC → NADH/FADH₂ cannot be reoxidized → TCA cycle stops
Diabetes / starvation	Excess acetyl-CoA from β-oxidation overwhelms oxaloacetate → ketogenesis
Pyruvate carboxylase deficiency	Impairs anaplerosis → hypoglycemia, lactic acidosis

12. Key Enzymes Summary Table

Step	Enzyme	Substrate → Product	Cofactor	Key Regulation
1	Citrate synthase	OAA + Acetyl-CoA → Citrate	H₂O	Inhibited by ATP, NADH, succinyl-CoA
2	Aconitase	Citrate → Isocitrate	Fe-S cluster	Inhibited by fluorocitrate
3	Isocitrate dehydrogenase	Isocitrate → α-KG + CO₂	NAD⁺	Activated by ADP, Ca²⁺; inhibited by ATP, NADH
4	α-KG dehydrogenase	α-KG → Succinyl-CoA + CO₂	NAD⁺, TPP, lipoate, FAD	Inhibited by NADH, succinyl-CoA
5	Succinyl-CoA synthetase	Succinyl-CoA → Succinate	GDP → GTP	-
6	Succinate dehydrogenase	Succinate → Fumarate	FAD → FADH₂	Inhibited by malonate
7	Fumarase	Fumarate → L-Malate	H₂O	-
8	Malate dehydrogenase	L-Malate → OAA	NAD⁺ → NADH	Driven by OAA removal

13. Memory Aid

A classic mnemonic for the cycle intermediates in order:

"Citrate Is Krebs' Starting Substrate For Making Oxaloacetate"

Citrate
Isocitrate
Ketoglutarate (α-)
Succinyl-CoA
Succinate
Fumarate
Malate
Oxaloacetate

Sources:

Basic Medical Biochemistry: A Clinical Approach, 6th Ed - Chapter 23 (TCA Cycle)
Harper's Illustrated Biochemistry, 32nd Ed - Chapter 16 (Citric Acid Cycle)
Guyton and Hall Textbook of Medical Physiology - Chapter 68 (Citric Acid Cycle)
Ganong's Review of Medical Physiology, 26th Ed

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