amino acids + urea cycle

week 2

glucogenic

• used to provide glucose via the process of gluconeogenesis

• Most amino acids are glucogenic

• during their breakdown the remaining carbon skeleton can be converted → oxaloacetate → glucose, via gluconeogenesis, if needed

ketogenic amino acids

• used to provide acetyl CoA or acetoacetate equivalents

• amino acids with carbon skeletons that cannot be converted to glucose (because they are broken down to either acetyl CoA or acetoacetyl CoA)

• are shunted towards fat synthesis or ketosis

list of ketogenic amino acids

• Phenylalanine, tyrosine & tryptophan (aromatic)

• Lysine, threonine

• Leucine and Isoleucine (2 branched chain amino acids)

amino acid biosynthesis

• To synthesise non-essential amino acids we need 2 basic components:

  1. A source of carbon (a ketoacid)

  2. An amine group donor (NH₃⁺)

     • Glutamate/Glutamine

     • Aspartate

     • Carbamoyl-P

• carbon skeleton of amino acids can be derived from a variety of metabolic precursors

  • (from pathways such as glycolysis, TCA Cycle, pentose phosphate pathway or breakdown of important biomolecules)

role of amino acid biosynthesis

formed amino acids can be used to synthesise protein or other important biomolecules (neurotransmitters, hormones, haeme etc.)

Metabolic Fate of Dietary & Intracellular Protein

• All cells can ‘remodel’ amino acids but most amino acid metabolism occurs in liver

• Remodelling: removing the amino group & recycling the carbon skeleton

• To metabolise amino acids, the amino group must first be removed (deamination)

• Toxic ammonia is converted, in the liver, to the less toxic compound urea, which is excreted in the urine

catabolic fate of amino acids

• Nearly all carbon skeletons from amino acid metabolism can be converted into intermediates in glycolysis, TCA or lipid metabolism

• Only lysine doesn’t undergo transamination

• generally, deamination is followed by direct metabolism in a central pathway or interconversion to a metabolite in one of the central pathways

1

Dietary amino acids contribute to tissue protein

2

Excess amine converted to urea via Urea Cycle

3

Carbon skeletons feed into TCA Cycle or converted to acetyl CoA which is a precursor for lipids (& ketone bodies, see 5)

4

Carbon skeletons can be broken down to pyruvate (precursor for a variety of molecules & gluconeogenic substrate)

deamination of amino acids in liver

• most amino acids undergo deamination in the liver

• can occur by the action of a range of enzymatic reactions:

  • aminotransferases

  • glutamate dehydrogenase (oxidative deamination)

  • glutaminase

deamination of amino acids in skeletal muscle

• since muscle cannot make urea, the amino groups must be transported (SAFELY) to the liver:

  • Aminotransferases catalyse the transfer of amine groups from amino acids to amine acceptors (ie alpha keto acids like pyruvate) producing the amino acid, alanine

  • Alanine → bloodstream → liver —transaminated→ pyruvate + glutamine

  • glutamine → NH4+ → urea

  • Pyruvate —gluconeogenesis→ glucose

“Glucose-Alanine cycle”

Glucose-Alanine Cycle

• involves the transport of excess nitrogen from muscle (via alanine) to the liver

• In the liver it is converted into glucose via gluconeogenesis which is then exported from liver back to muscles for energy etc.

Glutamate (Glu)

• 5C, one amino acid group

• acts as –NH₃ acceptor (in AA degradation, accepts –NH₃) forming glutamine

• acts as –NH₃ donor (for biosynthetic pathways/excretion) forming alpha-ketoglutarate

alpha keto acid

carbon skeleton

glutamine (Gln) and alanine (Ala)

• key transporters of amino groups between tissues and liver

• levels of these amino acids in blood is higher than all other amino acids

• alanine (3C) → pyruvate (after deamination)

• glutamine (5C, 2 amino acid groups) → glutamate → alpha-ketoglutarate

Aspartate (Asp)

• 4C

• deaminates into oxaloacetate

2 key mechanisms for deamination

1. transamination

2. oxidative deamination

transamination

• Transfer of amino group to a suitable keto acid acceptor (no free amine released)

• involves aminotransaminase enzymes:

  • alanine aminotransferase (ALT)

  • aspartate aminotransferase (AST)

• Reactions are reversible

• enzyme requires vitamin B6/pyridoxine as a cofactor

oxidative deamination

Oxidative removal of a free amino group forming an a-keto acid + free ammonia via glutamate dehydrogenase

deamination: removal of ammonia

• Glutamate is the only amino acid that doesn’t have to transfer its amino group to another molecule

• Glutamate undergoes oxidative deamination - glutamate dehydrogenase removes the amine group and hydrogens

Glutamate + NAD⁺ + H₂O

↔

α-Ketoglutarate + NADH + H⁺ + NH₄⁺

• ammonium produced is used to form urea

mechanism of toxicity of excess ammonia

• Ammonia (NH₃) readily crosses the BBB by diffusion so any process that increases serum ammonia is potentially dangerous i.e. hyperammonemia

• ammonium toxicity:

  • Increased levels of glutamate (excitatory neurotransmitter/excitotoxin → brain cell damage)

  • Increased levels of glutamine (disrupts BBB letting water/plasma in → oedema)

  • Depletion of ATP (interferes with mitochondrial function, possibly through inc. free radicals)

• Terminal stages → coma, brain swelling & death

urea cycle

• liver & kidneys work together to ensure toxic levels of ammonia do not accumulate

• Urea Cycle combines 2 amino groups into the urea molecule (one from glutamate dehydrogenase reaction, one from aspartate)

• Urea is transported via the blood supply to the kidney where it is excreted, and excreted in sweat

• urea synthesis occurs mostly in the liver

• urea diffuses into the blood and goes to the kidney

• High rates of amino acid breakdown result in elevated glutamate (glutamic acid) concentrations which increases the supply of substrate for the cycle

steps in Urea Cycle

1. mitochondrial matrix:

   ammonia → carbamoyl phosphate

2. carbamoyl phosphate + ornithine → citrulline (crosses from mitochondrion into cytosol)

3. citrulline + aspartate (donates 2nd amine) —eventually→ arginine, arginine → urea + ornithine

4. ornithine reused in cycle

formation of urea -where the amines are derived from

• One amine is derived from NH₄⁺ (produced primarily from glutamate via deamination - the glutamate dehydrogenase reaction)

• enters cycle via carbamoyl phosphate

• Second amine NH₃⁺ is derived from aspartate (formed by the transamination of the α-keto acid oxaloacetate)

  • fumarate is formed by this process & recycled via TCA cycle to oxaloacetate

urea cycle and TCA cycle overlap

Fumarate is formed from the cleavage of arginosuccinate (an intermediate of the citric acid cycle)

Fumarate → malate → oxaloacetate (an a-keto acid)

Oxaloacetate can acquire a second amino group to become aspartate – returns to the urea cycle

regulation of the urea cycle

• Urea cycle increases or decreases in response to high/low protein diet (increases and decreases ammonia)

• Regulation is at the level of the enzyme that synthesises carbamoyl phosphate

• acid-base balance:

  • In acidosis urea synthesis is decreased and NH₄⁺ excretion is increased to excrete protons

  • During fasting increased amino acid metabolism to fuel gluconeogenesis increase urea synthesis

Urea Cycle Disorders

• Genetic disorders resulting from a defect in synthesis/function of one of the urea cycle enzymes

• Symptoms arise in infancy (often triggered by switch from human milk to formula or introduction of solid foods – both higher in protein)

• Symptoms:

  • hyperammonaemia

  • lethargy

  • seizures

  • vomiting

  • hypotonia (poor muscle tone)

  • respiratory alkalosis

  • coma (even death if untreated)

• Severity depends on which enzyme is affected

• Most common is ornithine transcarbamoylase deficiency - severe neonatal symptoms – X-linked inheritance

• Blood tests would show increased blood levels of ammonia and/or build up of one or more urea cycle intermediates

increased serum urea

increased urea in bloodstream

caused by:

• increased urea production

  and/or

• decreased urea elimination

seen in:

• heart failure

• dehydration

• a diet high in protein)

decreased serum urea

decreased urea in bloodstream

caused by: decreased urea production

seen in: liver failure