UREA CYCLE

By Dr. T.O. Agunbiade

What is Urea Cycle?

 The urea cycle, also known as the ornithine cycle, is a vital metabolic pathway
responsible for the conversion of ammonia into urea. This cycle plays a pivotal role in
the body’s mechanism to eliminate excess nitrogen.

 Nitrogen is indispensable for the synthesis of proteins, nucleic acids, and other
essential molecules. However, the degradation of proteins can lead to the
accumulation of ammonia, a substance that is toxic to cells and tissues. Therefore, it
becomes imperative for the body to have a system in place to manage this excess
ammonia.

 The urea cycle offers a solution to this challenge. Through a series of intricate
biochemical reactions, ammonia is transformed into urea, a compound that is far less
harmful. These reactions occur primarily in the liver and are facilitated by both
mitochondrial and cytosolic enzymes.

 Besides its role in humans, the urea cycle is also significant in amphibians and
mammals. These organisms, termed ureotelic, utilize the cycle to dispose of the highly
toxic ammonia by converting it into urea.

 The discovery of the urea cycle can be credited to Hans Krebs and Kurt Henseleit in
1932. Within the body, ammonia is generated from various processes, including
amino acid catabolism, deaminations, and during prolonged periods of starvation.

 Then, it becomes essential for all animals to excrete this ammonia in some form.
Aquatic organisms, for instance, directly excrete ammonia and are thus called
ammonotelic.

 However, mammals and amphibians cannot directly excrete ammonia due to its toxic
nature. Therefore, they transform it into urea, a simpler and less toxic compound.
  Once formed in the liver, urea is transported to the kidneys via the bloodstream. It is
then excreted as a component of urine. This elimination process is of paramount
importance. If nitrogenous waste is not efficiently removed, its buildup can have
detrimental effects on the body.

 In contrast to ammonia, urea is inert, water-soluble, and can be effortlessly excreted in

urine, emphasizing the importance of the urea cycle in maintaining the body’s internal
equilibrium.

Urea Cycle

Definition of Urea Cycle

The urea cycle is a series of biochemical reactions in the liver that convert ammonia, a toxic
byproduct of protein metabolism, into urea, a less harmful compound that is excreted in the
urine.

Urea Cycle Reactions/Urea Cycle Steps

The urea cycle is a critical metabolic pathway that operates primarily in the liver, facilitating
the detoxification of ammonia into urea. This cycle encompasses both the mitochondrial
matrix and the cytosol of the cell. Here is a detailed and sequential elucidation of the steps
involved in the urea cycle:

1. Formation of Carbamoyl Phosphate: Initiated within the mitochondrial matrix,

ammonia, which primarily arises from various metabolic pathways, combines with
carbon dioxide (represented as HCO₃⁻) to produce carbamoyl phosphate. This
 reaction is ATP-dependent and is catalyzed by the enzyme carbamoyl phosphate
synthetase I. It’s essential to note that this mitochondrial enzyme is distinct from its
cytosolic counterpart, which plays a role in pyrimidine biosynthesis.

2. Formation of Citrulline: In this step, the carbamoyl group from carbamoyl

phosphate is transferred to ornithine, producing citrulline and releasing inorganic
phosphate (Pi). This reaction is facilitated by the enzyme ornithine transcarbamoylase.
Ornithine, although not one of the standard amino acids in proteins, is pivotal in
nitrogen metabolism. Once formed, citrulline is then transported from the
mitochondria to the cytosol.

3. Synthesis of Argininosuccinate: The next phase involves the introduction of the

second amino group. Aspartate, generated in the mitochondria and then transported to
the cytosol, combines with citrulline to form argininosuccinate. This condensation
reaction, occurring in the cytosol and catalyzed by argininosuccinate synthetase,
requires ATP and progresses through a citrullyl-AMP intermediate.

4. Formation of Arginine and Fumarate: Argininosuccinate is subsequently cleaved

by the enzyme argininosuccinase to yield arginine and fumarate. The fumarate
produced can be converted to malate and then transported into the mitochondria,
where it integrates with the citric acid cycle intermediates.

5. Production of Urea and Ornithine: The final step of the urea cycle involves the
cleavage of arginine to produce urea and ornithine. This reaction is catalyzed by the
cytosolic enzyme arginase. The ornithine produced is then transported back into the
mitochondria, marking the beginning of another cycle iteration.

It’s worth highlighting that the enzymes involved in the urea cycle appear to be strategically
clustered. The product of one enzymatic reaction is directly channeled to the subsequent
enzyme in the pathway. For instance, the citrulline exiting the mitochondrion is directly
passed to the active site of argininosuccinate synthetase, ensuring efficiency. This channeling
continues for argininosuccinate, arginine, and ornithine, with only urea being released into
the general cytosolic pool of metabolites.

Overall Reaction of the urea cycle

Aspartate + NH3 + HCO3− + 3ATP + H2O → urea+fumarate + 2ADP + AMP + 2Pi + PPi
This equation highlights the conversion of aspartate, ammonia, bicarbonate, and three
molecules of ATP into urea, fumarate, two molecules of ADP, AMP, inorganic phosphate (Pi),
and pyrophosphate (PPi). Notably, the synthesis of urea is an irreversible process due to the
consumption of four high-energy phosphate bonds, resulting in a large, negative change in
Gibbs free energy (∆G).

Furthermore, the nitrogen atoms in the urea molecule have distinct origins. One nitrogen is
derived from free ammonia (NH3), while the other nitrogen is sourced from aspartate. The
amino acid glutamate plays a pivotal role in this process. Glutamate serves as the immediate
precursor for both the ammonia and aspartate nitrogen. Specifically, ammonia is produced
through the oxidative deamination of glutamate by the enzyme glutamate dehydrogenase. On
the other hand, aspartate nitrogen is formed through the transamination of oxaloacetate by
aspartate transaminase (AST).

In essence, both nitrogen atoms of the urea molecule can be traced back to glutamate.
Glutamate, in turn, assimilates nitrogen from other amino acids, emphasizing its central role
in nitrogen metabolism.

Step by Step Reactions of the urea cycle

1. Formation of Carbamoyl Phosphate

 Reactants: Ammonia (NH₃), bicarbonate (HCO₃⁻), and two molecules of

ATP.

 Products: Carbamoyl phosphate, two molecules of ADP, and inorganic

phosphate (Pi).

 Catalyzed by: The enzyme carbamoyl phosphate synthetase 1 (CPS1).

 Location: This reaction takes place in the mitochondria.

 Function: This is the initial and rate-limiting step of the urea cycle, where
ammonia is captured in the form of carbamoyl phosphate.

2. Formation of Citrulline

 Reactants: Carbamoyl phosphate and ornithine.

 Products: Citrulline and inorganic phosphate (Pi).

  Catalyzed by: The enzyme ornithine transcarbamoylase (OTC), with the
assistance of co-factors zinc and biotin.

 Location: This reaction also occurs in the mitochondria.

 Function: Ornithine and carbamoyl phosphate combine to form citrulline,

which is then transported to the cytosol for the next steps.

3. Formation of Argininosuccinate

 Reactants: Citrulline, aspartate, and ATP.

 Products: Argininosuccinate, AMP, and pyrophosphate (PPi).

 Catalyzed by: The enzyme argininosuccinate synthetase (ASS).

 Location: This reaction takes place in the cytosol.

 Function: Citrulline and aspartate combine, using energy from ATP, to form
argininosuccinate.

4. Cleavage of Argininosuccinate

 Reactants: Argininosuccinate.

 Products: Arginine and fumarate.

 Catalyzed by: The enzyme argininosuccinate lyase (ASL).

 Location: This reaction occurs in the cytosol.

 Function: Argininosuccinate is split into arginine and fumarate. Fumarate can

then enter the citric acid cycle.

5. Formation of Urea

 Reactants: Arginine and water (H₂O).

 Products: Ornithine and urea.

 Catalyzed by: The enzyme arginase (ARG1) with the co-factor manganese.

 Location: This reaction takes place in the cytosol.

 Function: Arginine is hydrolyzed to produce urea, the primary nitrogenous
waste product in humans. Ornithine, the other product, is transported back to
the mitochondria to participate in the cycle again
The intricate relationship between the urea cycle and the citric acid cycle, often referred to as
the “Krebs bicycle,” showcases the sophisticated interplay of metabolic pathways within the
cell. This relationship is particularly evident in the liver, where both cycles operate in tandem,
ensuring the efficient processing of amino groups and carbon skeletons of amino acids.

1. Interconnection of the Cycles: The fumarate produced during the argininosuccinase

reaction in the urea cycle is also an intermediate in the citric acid cycle. This
establishes a direct link between the two cycles. Furthermore, the nitrogen-acquiring
reactions in the urea cycle involve the consumption of ATP, emphasizing the energy-
dependent nature of these processes.

2. The Aspartate-Argininosuccinate Shunt: This shunt provides a metabolic bridge

between the pathways that process the amino groups and carbon skeletons of amino
acids. Aspartate, formed in the mitochondria by the transamination of oxaloacetate
and glutamate, is transported to the cytosol. Here, it serves as a nitrogen donor in the
 urea cycle reaction facilitated by argininosuccinate synthetase. This intricate interplay
effectively links the fates of amino groups and carbon skeletons.

3. Transport and Conversion: Several enzymes of the citric acid cycle, such as
fumarase and malate dehydrogenase, exist in both the cytosol and the mitochondria as
isozymes. Fumarate, produced in the cytosol, can be converted to malate, which can
either be utilized in the cytosol or transported into the mitochondria to participate in
the citric acid cycle. This interplay is further intertwined with the malate-aspartate
shuttle, a mechanism that imports reducing equivalents into the mitochondrion.

4. The Malate-Aspartate Shuttle: Reducing equivalents, in the form of NADH

produced by various metabolic processes, cannot directly cross the mitochondrial
inner membrane. Instead, these equivalents are transported into the mitochondrion via
the malate-aspartate shuttle. In this process, aspartate is converted to oxaloacetate in
the cytosol, which is then reduced to malate using NADH. The malate is subsequently
transported into the mitochondrial matrix, where it can be converted back to
oxaloacetate, generating NADH in the process.

5. Balance of Amino Acids: Maintaining a balance between the concentrations of

glutamate and aspartate in the cytosol is crucial. The enzyme aspartate
aminotransferase (AAT), also known as glutamate-oxaloacetate transaminase (GOT),
plays a pivotal role in transferring amino groups between these amino acids. This
enzyme is highly active in hepatocytes and is a key indicator in diagnosing various
medical conditions when its levels are detected in the blood due to tissue damage.

In conclusion, the urea and citric acid cycles are not isolated metabolic pathways but are
intricately linked, showcasing the cell’s ability to efficiently manage and utilize its resources.
The interconnected nature of these cycles ensures that the cell can effectively process and
detoxify amino groups, while also managing energy and carbon skeletons.

Genetic Defects in the Urea Cycle

Genetic defects in the urea cycle can have profound implications for an individual’s ability to
metabolize proteins. The urea cycle plays a pivotal role in converting toxic ammonia,
produced during the breakdown of amino acids, into urea, which is then excreted from the
body. A malfunction in this cycle can lead to severe health consequences.

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1. Implications of Urea Cycle Defects: Individuals with genetic defects in any enzyme
of the urea formation process face challenges when consuming protein-rich diets.
When they ingest amino acids beyond the minimum daily requirements for protein
synthesis, these amino acids are deaminated in the liver. This process produces free
ammonia. However, due to the defect in the urea cycle, this ammonia cannot be
converted to urea and released into the bloodstream. As a result, ammonia, which is
highly toxic, accumulates in the body. Depending on the specific enzyme that is
absent, this can lead to hyperammonemia or an accumulation of one or more urea
cycle intermediates. Since most steps in the urea cycle are irreversible, the missing
enzyme activity can often be pinpointed by identifying which cycle intermediate is
present in abnormally high concentrations in the blood or urine.

2. Dietary Considerations: While the breakdown of amino acids can be detrimental to

those with urea cycle deficiencies, completely avoiding proteins is not a viable
solution. This is because humans cannot synthesize half of the 20 common amino
acids. These essential amino acids must be obtained through the diet.

3. Available Treatments: Various treatments have been developed to assist individuals

with urea cycle defects. One approach involves the dietary administration of aromatic
acids like benzoate or phenylbutyrate. These acids help reduce blood ammonia levels.
For instance, benzoate transforms into benzoyl-CoA, which then combines with
glycine to produce hippurate. This process consumes glycine, prompting its
regeneration and the uptake of ammonia. Similarly, phenylbutyrate is converted to
phenylacetate, which then combines with glutamine to form phenylacetylglutamine.
This process also results in the uptake of ammonia. Both hippurate and
phenylacetylglutamine are non-toxic and are excreted through urine.Specific
treatments are tailored to particular enzyme deficiencies. For example, a deficiency in
N-acetylglutamate synthase can be addressed by administering carbamoyl glutamate,
an analog of N-acetylglutamate. This analog effectively activates carbamoyl
phosphate synthetase I. In cases of deficiencies in enzymes like ornithine
transcarbamoylase, argininosuccinate synthetase, and argininosuccinase,
supplementing the diet with arginine proves beneficial. However, these treatments
often necessitate strict dietary control and essential amino acid supplements. In rare
instances of arginase deficiency, arginine must be excluded from the diet, as it is the
substrate for the malfunctioning enzyme.
In conclusion, genetic defects in the urea cycle present significant challenges in protein
metabolism. However, with careful dietary management and specific treatments, these
challenges can be addressed, ensuring the well-being of affected individuals.