The Urea Cycle/Ornithine cycle

Getting Rid of Ammonia

Ammonia can be produced by the break-down of amino acids, or by the gut bacteria in humans. If
the level of ammonia in the blood becomes too high, then it becomes toxic to the brain.The urea
cycle removes ammonia from the blood and makes urea, which is eventually excreted as urine.
This cycle is carried out by the cells of the liver, and as the name suggests the last step of the
process feeds in to an earlier step of the cycle.

Urinary Renal System of Human Body Anatomy. Image Credit: udaix / Shutetrstock
 Summary of the Steps in the Urea Cycle

Ammonia is formed by the break-down of amino acids/gut bacteria.

The mitochondrial stage:

• Carbamoyl phosphate is formed from ammonia and bicarbonate, by CPS.

• OTC condenses carbamoyl phosphate and ornithine to form citrulline.
• Citrulline is then transported to the cytosol by SLC25A15.

The cytosolic stage:

• AS condenses citrulline and aspartate to form argininosuccinate.

• Argininosuccinate is broken down into arginine and fumarate by AL.
• Arginine is broken down into urea and ornithine by arginase.
• Ornithine translocase transports ornithine into the mitochondria.
The Mitochondrial Stage

The first two steps of the urea cycle occur in the mitochondria of the cell. First, the enzyme
carbamoyl phosphate synthetase (CPS) takes ammonia and bicarbonate, and forms carbamoyl
phosphate with the use of ATP. This is the step in the cycle which determines how fast the cycle
progresses. A-acetylglucosamine is also required for CPS to function, and functions as a regulator
for the formation of urea.
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Ornithine transcarbamoylase (OTC) then condenses carbamoyl phosphate and ornithine, which
forms citrulline. This citrulline is then moved out of the mitochondria into the cytosol of the cell
by the transporter SLC25A15.
The Cytosolic Stage

Argininosuccinate synthetase (AS) takes the citrulline formed in the mitochondrial stage, and
condenses it with aspartate to form argininosuccinate. This occurs by the formation of an
intermediate, citrulline-AMP.

Argininosuccinate is then broken into arginine and fumarate by argininosuccinate lyase (AL).
Fumarate is then incorporated into another metabolic cycle, the TCA cycle. The TCA cycle can
then reform aspartate, which is used by AS.

Arginine is then further broken down into urea and ornithine by arginase. Arginine can also be
acquired from the diet, and this can also be taken in by the liver cells and broken down into urea
and ornithine by arginase.

The ornithine is then transported into the mitochondria by ornithine translocase. There, it is used
by OTC again, to form citrulline. The citrulline is then processed to form urea and ornithine
again, and the cycle continues. During the cycle, urea is the only new product which is formed,
while all other molecules used in the cycle are recycled.
What Happens When the Urea Cycle Goes Wrong?

If there is a problem with the urea cycle, then the level of ammonia in the blood will rise, causing
hyperammonemia. Ammonia is able to cross the barrier between the bloodstream and the brain.
Once it enters the brain, it can stop the TCA cycle by depleting one of the metabolites, a-
ketoglutarate. This means that these brain cells cannot make energy, ultimately leading to its
death. This eventually will lead to neurological problems, which can be as severe as irreversible
brain damage.

Problems can be caused by things such as damage to the liver (cirrhosis) or by an inherited defect
in one of the above enzymes. In both cases, the level of ammonia rises with the potential
consequences listed above.

Inherited urea cycle defects are part of “inborn errors in metabolism”, and are known as “urea
cycle disorders”. Symptoms usually present in newborns around 24-48 hours after birth. The most
severe symptoms are seen in newborns with a defect in CPS.
Treatment for hyperammonemia consists of removing protein from the diet, removing the excess
ammonia and supplementing with molecules of the urea cycle which are missing. Also, sodium
benzoate and sodium acetate can be used to form ammonia-containing compounds which can be
excreted through feces. The sugar lactulose was shown to reduce the production of ammonia by
the gut bacteria, as well as promoting the excretion of ammonia through feces. Alternatively,
antibiotics can be used to eliminate the ammonia-forming gut bacteria.

Biochemical pathway in detail

Reactions
The entire process converts two amino groups, one from NH+ 4 and one from Aspartate, and a
carbon atom from HCO-
3, to the relatively nontoxic excretion product urea at the cost of four "high-energy" phosphate
bonds (3 ATP hydrolyzed to 2 ADP and one AMP). The conversion from ammonia to urea
happens in five main steps. The first is needed for ammonia to enter the cycle and the following
four are all a part of the cycle itself. To enter the cycle, ammonia is converted to carbamoyl
phosphate. The urea cycle consists of four enzymatic reactions: one mitochondrial and three
cytosolic.[1]

Reactions of the urea cycle

Catalyzed
Step Reactants Products b
y Location

carbamoyl phosphate + 2ADP +

1 NH3 + HCO-3 + 2ATP Pi CPS1 mitochondria

carbamoyl OTC, zinc,

2 phosphate + ornithine citrulline + Pi biotin mitochondria

3 citrulline + aspartate + ATP argininosuccinate + AMP + PPi ASS cytosol

4 argininosuccinate arginine + fumarate ASL cytosol

ARG1,
5 arginine + H2O ornithine + urea manganese cytosol
 The reactions of the urea cycle

1 L-ornithine
2 carbamoyl phosphate
3 L-citrulline
4 argininosuccinate
5 fumarate
6 L-arginine
7 urea
L-Asp L-aspartate
CPS-1
carbamoyl phosphate synthetase I
OTC Ornithine transcarbamoylase
ASS argininosuccinate synthetase
ASL argininosuccinate lyase ARG1
arginase 1
First reaction: entering the urea cycle
Before the urea cycle begins ammonia is converted to carbamoyl phosphate. The reaction is
catalyzed by carbamoyl phosphate synthetase I and requires the use of two ATP molecules.[1]
The carbamoyl phosphate then enters the urea cycle.
Steps of the urea cycle

1. Carbamoyl phosphate is converted to citrulline. With catalysis by ornithine

transcarbamoylase, the carbamoyl phosphate group is donated to ornithine and releases a
phosphate group.[1]
2. A condensation reaction occurs between the amino group of aspartate and the carbonyl
group of citrulline to form argininosuccinate. This reaction is ATP dependent and is catalyzed
by argininosuccinate synthetase.[1]
3. Argininosuccinate undergoes cleavage by argininosuccinase to
form arginine and fumarate.[1]
4. Arginine is cleaved by arginase to form urea and ornithine. The ornithine is then
transported back to the mitochondria to begin the urea cycle again.[1]
Overall reaction equation
In the first reaction, NH+
4 + HCO-
3 is equivalent to NH3 + CO2 + H2O.

Thus, the overall equation of the urea cycle is:

• NH3 + CO2 + aspartate + 3 ATP + 2 H2O ^ urea + fumarate + 2 ADP +

2 Pi + AMP + PPi
Since fumarate is obtained by removing NH 3 from aspartate (by means of reactions 3 and 4),
and PPi + H2O ^ 2 Pi, the equation can be simplified as follows:
• 2 NH3 + CO2 + 3 ATP + H2O ^ urea + 2 ADP + 4 Pi + AMP
Note that reactions related to the urea cycle also cause the production of 2 NADH, so the
overall reaction releases slightly more energy than it consumes. The NADH is produced in
two ways:
• One NADH molecule is produced by the enzyme glutamate dehydrogenase in the
conversion of glutamate to ammonium and a-ketoglutarate. Glutamate is the non-toxic carrier
of amine groups. This provides the ammonium ion used in the initial synthesis of carbamoyl
phosphate.
• The fumarate released in the cytosol is hydrated to malate by cytosolic fumarase. This
malate is then oxidized to oxaloacetate by cytosolic malate dehydrogenase, generating a
reduced NADH in the cytosol. Oxaloacetate is one of the keto acids preferred
by transaminases, and so will be recycled to aspartate, maintaining the flow of nitrogen into
the urea cycle.
We can summarize this by combining the reactions:

• CO2 + glutamate + aspartate + 3 ATP + 2 NAD++ 3 H2O ^ urea + a-

ketoglutarate + oxaloacetate + 2 ADP + 2 Pi + AMP + PPi + 2 NADH
The two NADH produced can provide energy for the formation of 5 ATP (cytosolic NADH
provides 2.5 ATP with the malate-aspartate shuttle in human liver cell), a net production of
two high-energy phosphate bond for the urea cycle. However, if gluconeogenesis is underway
in the cytosol, the latter reducing equivalent is used to drive the reversal of the GAPDH step
instead of generating ATP.
The fate of oxaloacetate is either to produce aspartate via transamination or to be converted to
phosphoenolpyruvate, which is a substrate for gluconeogenesis.