24 - Urea Cycle

Question 1

What is the fate of urea?

Answer 1

Synthesized in the liver by enzymes of the urea cycle. Secreted into the bloodstream and then sequestered in the kidneys for excretion in the urine.

Question 2

What is the overall urea cycle reaction?

Answer 2

Using 3 ATP to convert aspartate to urea and fumarate

Question 3

How do aquatic animals, terrestrial animals and mammals deal with ammonia in the body?

Answer 3

Aquatic animals: Simply excrete NH3

Terrestrial animals: N is removed as uric acid (insoluble white precipitate)

Mammals: N is removed as soluble urea (except in dalmatians)

Question 4

Describe the glucose-alanine cycle

Answer 4

Alanine serves as a carrier of ammonia and of the carbon skeleton of pyruvate (converted to alanine in blood) from skeletal muscle to the liver where:

Alanine + alpha-ketoglutarate -> pyruvate + glutamate

Glutamate is converted to NH4 and the ammonia is excreted through urea cycle and the pyruvate is used to produce glucose, which returned to the muscle.

Question 5

How is the glucose-alanine cycle broken?

Answer 5

During starvation, glucose formed in the liver is also used by peripheral tissues, breaking the cycle.

Muscle is not a gluconeogenic tissue. But during starvation both the amino group and pyruvate come from muscle protein degradation, providing a pathway yielding glucose for other tissues.

Question 6

How is the glucose-alanine cycle broken?

Answer 6

During starvation, glucose formed in the liver is also used by peripheral tissues, breaking the cycle.

Muscle is not a gluconeogenic tissue. But during starvation both the amino group and pyruvate come from muscle protein degradation, providing a pathway yielding glucose for other tissues.

Question 7

How are amino acids broken down in body tissues?

Answer 7

Tissue

• Glutamate converted to glutamine by glutamine synthetase. Glutamine goes to liver.
• Glutamine hydrolyzed back to glutamate via glutaminase

Muscles

• Amino acids are converted to glutamate via glutamate dehydrogenase and the addition of NH4 and alpha ketoglutarate
• Glutamate is converted to alanine with pyruvate and transaminase enzyme
• AND/OR glucose is converted to pyruvate (which feeds into alanine) through glycolysis
• Alanine goes to liver through blood
• Alanine is converted to glutamate and pyruvate with aKG

Liver
- Glutamate converted to urea

Question 8

How are amino acids broken down in body tissues?

Answer 8

Tissue

• Glutamate converted to glutamine by glutamine synthetase. Glutamine goes to liver.
• Glutamine hydrolyzed back to glutamate via glutaminase

Muscles

• Amino acids are converted to glutamate via glutamate dehydrogenase and the addition of NH4 and alpha ketoglutarate
• Glutamate is converted to alanine with pyruvate and transaminase enzyme
• AND/OR glucose is converted to pyruvate (which feeds into alanine) through glycolysis
• Alanine goes to liver through blood
• Alanine is converted to glutamate and pyruvate with aKG

Liver
- Glutamate converted to urea

Question 9

What are the two nitrogen acquiring reactions in urea synthesis?

Answer 9

1. Carbamoyl phosphate synthetase (CPS)
   - Activation of HCO3 by ATP
   - 1st intermediate carboxyphosphate
   - Nucleophilic attack by NH3
   - 2nd intermediate carbamate
   - Phosphorylation of carbamate by ATP
   - Product: carbamoyl phosphate
   - CPSI uses NH2 in mitochondria and CPSII uses glutamine in cytosol for pyrimidine biosynthesis
2. Argininosuccinate synthetase (ASS)
   - activation of ureido oxygen of citrulline by ATP
   - Intermediate citrullyl-AMP
   - displacement of MAP by alpha amino group of aspartate
   - Product: arginosuccinate

Question 10

Recall the three active sites of E coli carbamoyl phosphate synthetase (CPS)

Answer 10

Active site 1
- Generation of NH3 from glutamine

Active site 2
- NH3 + activated HCO3 to carbamate

Active site 3
- Carbamate and ATP to carbamoyl phosphate and ADP

Question 11

What is the purpose of the tunnel in carbamoyl phosphate synthetase (CPS)?

Answer 11

Channeling

• Prevents loss and concentrates NH3
• NH3 transfer, tunnel lined with polar groups that form H-bonds with NH3

Shielding
- Protects highly reactive carbamate and carboxyphosphate

Carbamate transfer: backbone atoms, no charged groups that may hydrolyze carbamate

Question 12

Review the urea cycle

Answer 12

How well do you know it?

Question 13

What does the 1st amino group enter the urea cycle as? 2nd amino acid?

Answer 13

1st: Carbamoyl phosphate, formed in the mitochondria.
2nd: amino group enters as aspartate, formed in the mitochondria by transamination of oxaloacetate and glutamate, catalyzed by aspartate aminotransferase

Question 14

List the four steps of the urea cycle

Answer 14

1. Formation of citrulline in mitochondria, citrulline passes into cytosol
2. Formation of arginosucinate through citrullyl-AMP intermediate; entry of second amino group.
3. Formation of arginine; releases fumarate, which enters citric acid cycle.
4. Formation of urea, regenerates ornithine.

Question 15

List the four steps of the urea cycle

Answer 15

1. Formation of citrulline in mitochondria, citrulline passes into cytosol
2. Formation of arginosucinate through citrullyl-AMP intermediate; entry of second amino group.
3. Formation of arginine; releases fumarate, which enters citric acid cycle.
4. Formation of urea, regenerates ornithine.

Question 16

What is ‘Kreb’s bicycle?’

Answer 16

A link between the urea cycle and citric acid cycle

• Aspartate-argininosuccinate shunt effectively links the fates of the amino groups and C skeletons of the two cycles
• Patients with kidney disease have impaired urea excretion and will have to restrict their protein intake.

Question 17

How is the urea cycle regulated (at two levels)?

Answer 17

Nitrogen flux through the urea cycle varies with diet

1. Allosteric regulation of CPS1
2. Enzyme levels

Question 18

How does the urea cycle respond to the diet?

Answer 18

Protein rich diet

• Carbon skeletons of amino acids become fuel
• Excess amino groups becomes urea

Prolonged starvation
- Breakdown of muscle protein for metabolic energy, urea production also greatly increases

Question 19

Describe allosteric regulation of the urea cycle

Answer 19

Allosteric regulation of CPS1

• CPS1 is activated by N-acetylglutamate, which increases CPS’ affinity for ATP
• N-acetylglutamate is synthesized from acetyl-CoA and glutamate. Thus, when amino acid breakdown increases, N-acetylglutamate level increases and urea production increases.

This gives protection against ammonia toxicity

Question 20

How is the urea cycle regulated by enzyme levels?

Answer 20

• Major changes in diet can alter levels of urea cycle enzymes
• During starvation, enzyme level increase 10-20x in animal models. This permits the organism to cope with the increased ammonia produced during increases breakdown of proteins. During starvation, insulin is low, glucagon is high.

Enzyme levels are increased by glucagon!

• Steps 2, 3, and 4 are activated by substrate concentration increase

Inherited deficiency of specific urea cycle enzymes can lead to hyperammonemia and toxicity

Question 21

Genetic defects in the urea cycle can be life threatening and humans cannot live on a protein-free diet.

What is the consequence of excess amino acid ingestion?

Answer 21

Amino acids ingested in excess of the minimum daily requirements for protein synthesis are deaminated in the liver, producing free ammonia that cannot be converted to urea. Ammonia is toxic.

An individual with genetic defects in any enzyme of the urea cycle cannot tolerate protein-rich diets.

Question 22

Genetic defects in the urea cycle can be life threatening and humans cannot live on a protein-free diet.

What is the consequence of excess amino acid ingestion?

Answer 22

Amino acids ingested in excess of the minimum daily requirements for protein synthesis are deaminated in the liver, producing free ammonia that cannot be converted to urea. Ammonia is toxic.

An individual with genetic defects in any enzyme of the urea cycle cannot tolerate protein-rich diets.

Question 23

Give four treatments for deficiencies in urea cycle enzymes

Answer 23

1. Aromatic acids
   - Benzoate + glycine to hippurate
   - phenylacetate + glutamine to phenylacetylglutamine
   - Hippurate and phenylacetylglutamine are non-toxic and are excreted in the urine
   - Further synthesis of glycine by glycine synthase removes ammonia

Question 24

Give four treatments for deficiencies in urea cycle enzymes

Answer 24

1. Aromatic acids
   - Benzoate + glycine to hippurate
   - phenylacetate + glutamine to phenylacetylglutamine
   - Hippurate and phenylacetylglutamine are non-toxic and are excreted in the urine
   - Further synthesis of glycine by glycine synthase removes ammonia
2. Carbamoyl glutamate (analog of N-acetylglutamate) to activate CPS1 during N-acetylglutamate deficiency
3. Supplementing diet with arginine
   - Total protein intake restricted to yield arginosuccinate (substitute for urea, excreted)
   - In case of arginase deficiency, arginine must be excluded.
4. Conjugate glycine and glutamine
   - Treatment for deficiencies of CPS1 or OTC
   - Formation of citrulline and arginosuccinate is blocked, excess nitrogen accumulates in glycine and glutamine. Thus, treat with aromatic acids. That is, protein-restricted diet + benzoate + phenylacetate

Question 25

What will blockage of CPS1 or any of the 4 urea cycle enzymes cause?

Answer 25

Hyperammonemia

• Increased glutamine
• Reduced aKG
• TCA cycle inhibited
• ATP production down

Most gangerous urea cycle enzyme deficiency is OTC (ornithine transcarbamoylase) , which is also the most common in males (X-linked). Apparent after birth in severe cases, increased glutamine leads to brain swelling.

Question 26

Give the seven approaches to treating urea cycle enzyme deficiency

Answer 26

1. Remove ammonia (eg hemodialysis)
2. Low protein diet
3. Conjugate glycine and glutamine with benzoate and phenylacetate
4. Activate CPS1 with carbamoyl glutamate (if deficient in N-acetylglutamate synthase)
5. Add arginine to promote arginosuccinate excretion (if deficient in arginosuccinase)
6. Add arginine or citrulline to provide substrate to stimulate residual urea cycle enzyme activity (if not arginase deficient)
7. Treatments are usually combined (eg. conjugation + arginine + citrulline)