Krebs hensilit cycle

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The Krebs Cycle (Tricarboxylic Acid / Citric Acid Cycle)

The cycle has three names used interchangeably:

Krebs cycle - named for Sir Hans Krebs who first described it
Citric acid cycle - because citrate is the first product formed
TCA cycle (tricarboxylic acid cycle) - because citrate and isocitrate carry three carboxyl groups

Location and Purpose

The TCA cycle takes place entirely in the mitochondrial matrix (except succinate dehydrogenase, which is embedded in the inner mitochondrial membrane). It is the final common pathway where catabolism of carbohydrates, fatty acids, and amino acids all converge. Its two main functions are:

Complete oxidation of the acetyl group to CO2, capturing electrons as NADH and FADH2
Providing carbon skeletons for biosynthetic reactions (gluconeogenesis, amino acid synthesis, heme synthesis)

(Lippincott Illustrated Reviews: Biochemistry, p. 332)

Entry Point: Pyruvate to Acetyl-CoA

Before entering the cycle, pyruvate (from glycolysis) is converted to acetyl-CoA by the pyruvate dehydrogenase complex (PDHC):

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

The PDHC requires five coenzymes: TPP (thiamine/B1), lipoic acid, FAD (riboflavin/B2), NAD+ (niacin/B3), and CoA (pantothenic acid/B5).

The 8 Steps of the TCA Cycle

Here is the complete cycle diagram from Guyton & Hall:

Citric acid cycle showing all intermediates, cofactors, and net equation per glucose molecule

Step	Reaction	Enzyme	Products
1	Oxaloacetate (4C) + Acetyl-CoA (2C) → Citrate (6C)	Citrate synthase	CoA released
2	Citrate → cis-Aconitate → Isocitrate	Aconitase	H2O removed then added
3	Isocitrate → α-Ketoglutarate	Isocitrate dehydrogenase	NADH + CO2 (1st CO2)
4	α-Ketoglutarate → Succinyl-CoA	α-Ketoglutarate dehydrogenase complex	NADH + CO2 (2nd CO2)
5	Succinyl-CoA → Succinate	Succinate thiokinase (succinyl-CoA synthetase)	GTP (substrate-level phosphorylation)
6	Succinate → Fumarate	Succinate dehydrogenase	FADH2
7	Fumarate + H2O → Malate	Fumarase	-
8	Malate → Oxaloacetate	Malate dehydrogenase	NADH (3rd NADH)

(Lippincott Illustrated Reviews: Biochemistry, pp. 339-344)

Energy Yield Per Acetyl-CoA (one turn)

Coenzyme	Quantity	ATP equivalent
NADH	3	~7.5 ATP (2.5 each via ETC)
FADH2	1	~1.5 ATP (1.5 via ETC)
GTP	1	1 ATP (direct)
Total		~10 ATP

Since one glucose yields two acetyl-CoA, the TCA cycle contributes ~20 ATP per glucose (the majority of total ATP from aerobic glucose oxidation).

For the full glucose molecule:

Net equation per glucose: 2 Acetyl-CoA + 6H2O + 2ADP → 4CO2 + 16H + 2CoA + 2ATP

(Guyton and Hall Textbook of Medical Physiology, p. [Fig 68.6])

Regulation of the TCA Cycle

Three key regulatory enzymes (all with large negative ΔG°):

TCA cycle regulation showing inhibitors (red) and activators (green) of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase complex

Enzyme	Activated by	Inhibited by
Citrate synthase	Low acetyl-CoA, OAA	Citrate, NADH, succinyl-CoA, ATP
Isocitrate dehydrogenase	ADP, Ca2+	ATP, NADH
α-Ketoglutarate dehydrogenase	Ca2+	Succinyl-CoA, NADH

The overall principle: when energy (ATP/NADH) is abundant, the cycle slows; when energy is low (ADP high), the cycle accelerates.

(Lippincott Illustrated Reviews: Biochemistry, p. 345-346)

Anaplerotic Reactions

These reactions "fill up" TCA cycle intermediates when they are drawn off for biosynthesis:

Pyruvate carboxylase: Pyruvate + CO2 → Oxaloacetate (main anaplerotic reaction, requires biotin)
Amino acid catabolism feeds in at multiple points (α-ketoglutarate from glutamate, succinyl-CoA from odd-chain fatty acids, fumarate from tyrosine/phenylalanine)
OAA is also made from transamination of aspartate

Vitamins Required by the TCA Cycle

Vitamin	Coenzyme Form	Where used
Thiamine (B1)	TPP	PDHC, α-KG dehydrogenase
Riboflavin (B2)	FAD	Succinate dehydrogenase, PDHC
Niacin (B3)	NAD+	Isocitrate DH, α-KG DH, malate DH
Pantothenic acid (B5)	CoA	Acetyl-CoA, succinyl-CoA
Biotin	Biocytin	Pyruvate carboxylase (anaplerosis)

Deficiency in thiamine (B1) is clinically important - it impairs both PDHC and α-KG dehydrogenase, causing lactic acidosis and neurological damage (Wernicke's encephalopathy).

(Basic Medical Biochemistry - A Clinical Approach, p. 827)

Key memory aid for intermediates (in order):

Oxaloacetate → Citrate → Isocitrate → α-Ketoglutarate → Succinyl-CoA → Succinate → Fumarate → Malate → Oxaloacetate

Mnemonic: "Oh Citrate Is A Silly Stupid Fool's Mistake"

Urea cycle

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The Urea Cycle (Krebs-Henseleit Cycle)

The urea cycle was first described in 1932 by Hans Krebs and his medical student Kurt Henseleit - making it the original "Krebs cycle" before the TCA cycle. It is the primary pathway for nitrogen excretion, converting toxic ammonia into the non-toxic, water-soluble compound urea for renal excretion.

Urea accounts for ~90% of the nitrogen-containing components of urine. (Lippincott Illustrated Reviews: Biochemistry, p. 712)

Where Does It Happen?

The urea cycle is a bicellular pathway - half in the mitochondrial matrix, half in the cytosol - and occurs only in the liver (hepatocytes).

Steps 1-2: Mitochondrial matrix
Steps 3-5: Cytosol

Nitrogen Sources Going Into Urea

Urea has two nitrogen atoms and one carbon atom:

N-1 - from free ammonia (NH3), derived primarily from oxidative deamination of glutamate by glutamate dehydrogenase (GDH)
N-2 - from aspartate (itself made by transamination of oxaloacetate)
Carbon - from bicarbonate (HCO3-)

(Basic Medical Biochemistry, p. 1321)

The 5 Steps of the Urea Cycle

Here are the two cycle diagrams showing both the biochemistry and compartmentalization:

Urea cycle showing mitochondrial and cytosolic compartments, all 5 enzymes, nitrogen origins (highlighted in pink/purple), and the link to fumarate

Urea cycle with numbered steps, chemical structures of all intermediates, and compartment diagram

Step 1 - Carbamoyl Phosphate Formation (Mitochondria)

Enzyme: Carbamoyl phosphate synthetase I (CPS I)

NH3 + HCO3- + 2 ATP → Carbamoyl phosphate + 2 ADP + Pi

Consumes 2 ATP - the most energy-expensive step
Absolute requirement for N-acetylglutamate (NAG) as an allosteric activator
NAG is synthesized from glutamate + acetyl-CoA by NAG synthase, activated by arginine
CPS II (in cytosol) is a different enzyme used in pyrimidine synthesis - not part of the urea cycle

Step 2 - Citrulline Formation (Mitochondria)

Enzyme: Ornithine transcarbamylase (OTC)

Ornithine + Carbamoyl phosphate → Citrulline + Pi

The carbamoyl group is transferred to ornithine
Citrulline exits the mitochondria via an antiporter (exchanges with incoming ornithine)
Ornithine is the carrier molecule analogous to oxaloacetate in the TCA cycle

Step 3 - Argininosuccinate Formation (Cytosol)

Enzyme: Argininosuccinate synthetase

Citrulline + Aspartate + ATP → Argininosuccinate + AMP + PPi

Aspartate donates its amino group (the second nitrogen of urea)
Consumes 1 ATP (cleaved to AMP + PPi = effectively 2 high-energy bonds)
This is the rate-limiting step

Step 4 - Argininosuccinate Cleavage (Cytosol)

Enzyme: Argininosuccinate lyase

Argininosuccinate → Arginine + Fumarate

Arginine is the immediate precursor of urea
Fumarate is a key metabolic link - it enters the TCA cycle (or is hydrated to malate → transaminated to aspartate, replenishing the aspartate needed for step 3)

Step 5 - Urea Release (Cytosol)

Enzyme: Arginase-I (liver-specific)

Arginine + H2O → Urea + Ornithine

Ornithine is regenerated and transported back into the mitochondria to restart the cycle
Urea diffuses from the liver into blood and is excreted by the kidneys
Arginase-I is virtually exclusive to the liver - this is why only the liver can complete urea synthesis

(Lippincott Illustrated Reviews: Biochemistry, pp. 712-715)

Overall Stoichiometry

Aspartate + NH3 + HCO3- + 3 ATP + H2O → Urea + Fumarate + 2 ADP + AMP + 2Pi + PPi

4 high-energy phosphate bonds consumed per urea molecule (2 ATP in step 1; 1 ATP→AMP in step 3)
The reaction is energetically irreversible

Regulation

Regulator	Mechanism	Effect
N-acetylglutamate (NAG)	Allosteric activator of CPS I	Activates whole cycle
Arginine	Activates NAG synthase	Increases NAG → activates CPS I
High protein diet / starvation	Increases expression of all urea cycle enzyme genes in liver	Upregulates cycle capacity
Substrate availability	Availability of NH3, HCO3-, aspartate	Controls flux

The cycle is primarily regulated at CPS I through NAG. When protein catabolism is high (e.g. starvation, high-protein meal), more arginine and glutamate are produced, which stimulate NAG synthesis, which activates CPS I.

Link Between Urea Cycle and TCA Cycle

The fumarate produced in step 4 creates a direct link:

Fumarate (urea cycle)
   ↓ Fumarase
Malate
   ↓ Malate dehydrogenase
Oxaloacetate
   ↓ Transamination (AST)
Aspartate → back into urea cycle (step 3)

This is sometimes called the "urea cycle-TCA cycle bicycle" or the Krebs bicycle.

Urea Cycle Disorders (Hyperammonemia)

All urea cycle enzyme defects lead to hyperammonemia - accumulation of toxic ammonia. The most common is OTC deficiency (X-linked):

Enzyme Defect	Accumulates	Key Features
CPS I deficiency	NH3	No specific marker; hyperammonemia + encephalopathy
OTC deficiency (most common, X-linked)	Orotic acid, NH3	Orotic aciduria distinguishes from CPS I deficiency
Argininosuccinate synthetase (Citrullinemia type I)	Citrulline	Elevated plasma citrulline
Argininosuccinate lyase (Argininosuccinic aciduria)	Argininosuccinate	Elevated argininosuccinate in urine/blood
Arginase deficiency	Arginine	Spastic diplegia, intellectual disability; low urea

Why is ammonia toxic? It causes brain swelling by:

Astrocytes convert NH3 + glutamate → glutamine (osmotic imbalance)
Inhibits α-ketoglutarate dehydrogenase (disrupts TCA cycle)
Inhibits glutaminase (further raises glutamine)
Opens mitochondrial permeability transition pore → cell death

(Basic Medical Biochemistry, p. 1328)

Treatment of hyperammonemia:

Low-protein diet
Sodium benzoate / sodium phenylbutyrate (conjugate with amino acids, excreting nitrogen via kidney)
Arginine supplementation (for defects after citrulline synthesis)
Hemodialysis in acute crises
Liver transplantation (definitive)

Memory Aid - The 5 Intermediates

Ornithine → Citrulline → Argininosuccinate → Arginine → Urea (back to Ornithine)

Mnemonic: "Ordinarily Careless Crappers Are Also Frivolous About Urination" (Ornithine - Carbamoyl phosphate - Citrulline - Aspartate - Argininosuccinate - Fumarate - Arginine - Urea)

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