Lecture 6: Urea Cycle

Activation of Carbamoyl phosphate synthetase - 1 (CPS-1)

CPS-1 is activated and converts ammonia and bicarbonate ion into carbamoyl phosphate, requires two ATP, gives us entry into the urea cycle

NH4+ + HCO3- → carbamoyl phosphate

Step one: formation of Citrulline

Ornithine Transcarbamoylase converts carbamoyl phosphate and ornithine into citrulline, Carbamoyl phosphate donates its active carbonyl group to the amine of ornithine e to form citrulline and the phosphate group is released, ornithine leaves the mitochondria

Step two: formation of Argininosuccinate

argininosuccinate synthetase condenses Citrulline and aspartate to form argininosuccinate

citrulline + aspartate → argininosuccinate

Step three: formation of arginine

Argininosuccinate lysis/argininosuccinase cleaves Argininosuccinate to produce arginine and fumarate

argininosuccinate → fumarate + arginine

Step four: formation of urea

Arginase hydrolyzes arginine into urea and Orthinine common Orthinine is transported back into the mitochondria to repeat the urea cycle

arginine → urea + ornithine

Regulation

-A high protein diet includes urea cycle enzyme synthesis, glucagon stimulates the biosynthesis of all urea cycle enzymes after a protein rich meal, amino acids including alanine, glycine, methionine, and cysteine are effective in stimulating glucagon release, amino acid catabolism decreases ammonia load and increases Oxaloacetate and alpha KetoGlutarate for gluconeogenesis

-CPS-1 is the first committed step and is highly regulated, it is allosterically inhibited by NAG and when amino acid catabolism increases, glutamate and NAG also increase

N-Acetyl-Glutamate (NAG)

Activates CPS-1, is not consumed by the urea cycle, is catalyzed by the enzyme NAG synthase, substrates are glutamate and acetyl-CoA, activator is arginine

Glutamate + Acetyl-CoA (+Arginine) → NAG

Ammonia production

Produced in all tissues during trans deamination and amino acid catabolism ammonia assimilation is catalyzed by 4 enzymes:

• glutamate dehydrogenase

• glutamine synthase

• CPS-1

• glycine synthase

glutamate and glutamine provide the critical entry points for incorporation of ammonia into amino acids and other nitrogen containing molecules

Ammonia toxicity

• Ammonia is toxic in our body

• 98% of ammonia is in the form of NH4+

• ammonia assimilation = the metabolic incorporation of ammonia from a reductive amination by GDH and glutamine synthase allow the cells to rid themselves of ammonia

Hyperammonemia

Normal ammonia: 10-30

uMHigh ammonia:

• 80 uM in infants

• 55 uM in children

• 30 uM in adults

causes very bad symptoms

Causes for hyperammonemia

Urea disorders: a deficiency in any of the urea cycle enzymes, results in impaired function and ammonia build up

liver disease and kidney failure can also increase ammonia build up

Treatments

• Aromatic acid benzoate or phenylbutyrate can assimilate excess ammonia

• treating the specific enzyme deficiency

• deficiency in NAGS can affect CPS-1 activation, this can can be treated with Carbamoyl glutamate (an analog to NAG that will activate CPS-1)

Liver’s double filter system

Liver acinus (smallest functional unit of the liver) filter ammonia out of the blood going to the central vein to prevent ammonia from crossing the blood brain barrier

Zone 1 of the liver

• Bulk removal of ammonia

• located near the entrance where the blood comes in

• contains urea cycle enzymes (CPS1, Glutaminase)

• low affinity

• high capacity

Zone 3 of the liver

• Catches the ammonia stragglers

• near exit where blood enters central vein

• glutamine synthase enzyme

• low capacity

• high affinity

Ornithine transcarbamylase deficiency

• Most common and most severe (acute)

• recall that this enzyme condences Carbamoyl phosphate with Orthinine to produce Citrulline

• deficiency causes a build up of carbamoyl phosphate which leads to a high build up of ammonia

Arginase deficiency

• Recall that Arginase cleaves arginine into urea and Orthinine

• causes arginine to build up which causes a moderate build up of ammonia

• chronic but less severe

Ammonia assimilation

GDH (reductive amination)

glutamine synthase: happens in two steps

glu + NH4+ + ATP → gln +ADP + P + H+

glutamine shuttles ammonia safely to the liver

The importance of glutamine

• Glutamine synthase catalyzes the two step reaction of ammonia with glutamate to produce glutamine

• requires ATP

• traps ammonia in the cytosol

• glutamine acts as a nitrogen shuttle and is the source in all biosynthetic reactions

Ammonia scavenging in the brain

• Protects the brain from ammonia toxicity

• compromises energy metabolism by depleting a-kg and pyruvate

• alters glutamate levels (an important neurotransmitter)

Disposition of the carbon skeleton

Glycogenic amino acids can be converted to intermediates of the TCA cycle and gluconeogenesis or ketone bodies

Intermediates of TCA/Gluconeogenesis

Alanine → Pyruvate

Aspartate/Asparagine → Oxaloacetate

Glutamate/Glutamine → Alpha-ketoglutarate

Ketogenic amino acids can be converted to ketone bodies

Leucine and lysine

potentially: alanine, cysteine, glycine, and serine

Amino acids that are both ketogenic and glycogenic

Isoleucine, phenylalanine, tyrosine, tryptophan, threonine