The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions that produces urea (NH2)2CO from ammonia (NH3). This cycle occurs in ureotelic organisms. The urea cycle converts highly toxic ammonia to urea for excretion. This cycle was the first metabolic cycle to be discovered (Hans Krebs and Kurt Henseleit, 1932), five years before the discovery of the TCA cycle.This cycle was described in more detail later on by Ratner and Cohen. The urea cycle takes place primarily in the liver and, to a lesser extent, in the kidneys.
Amino acid catabolism results in waste ammonia. All animals need a way to excrete this product. Most aquatic organisms, or ammonotelic organisms, excrete ammonia without converting it. Organisms that cannot easily and safely remove nitrogen as ammonia convert it to a less toxic substance such as urea or uric acid via the urea cycle, which occurs mainly in the liver. Urea produced by the liver is then released into the bloodstream where it travels to the kidneys and is ultimately excreted in urine. In species including birds and most insects, the ammonia is converted into uric acid or its urate salt, which is excreted in solid form.
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||NH3 + HCO−
3 + 2ATP
|carbamoyl phosphate + 2ADP + Pi||CPS1||mitochondria|
|2||carbamoyl phosphate + ornithine||citrulline + Pi||OTC, zinc, biotin||mitochondria|
|3||citrulline + aspartate + ATP||argininosuccinate + AMP + PPi||ASS||cytosol|
|4||argininosuccinate||arginine + fumarate||ASL||cytosol|
|5||arginine + H2O||ornithine + urea||ARG1, manganese||cytosol|
- The reactions of the urea cycle
2 carbamoyl phosphate
CPS-1 carbamoyl phosphate synthetase I
OTC Ornithine transcarbamoylase
ASS argininosuccinate synthetase
ASL argininosuccinate lyase
ARG1 arginase 1
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. The carbamoyl phosphate then enters the urea cycle.
Thus, the overall equation of the urea cycle is:
Since fumarate is obtained by removing NH3 from aspartate (by means of reactions 3 and 4), and PPi + H2O → 2 Pi, the equation can be simplified as follows:
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:
We can summarize this by combining the reactions:
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 synthesis of carbamoyl phosphate and the urea cycle are dependent on the presence of N-acetylglutamic acid (NAcGlu), which allosterically activates CPS1. NAcGlu is an obligate activator of carbamoyl phosphate synthetase. Synthesis of NAcGlu by N-acetylglutamate synthase (NAGS) is stimulated by both Arg, allosteric stimulator of NAGS, and Glu, a product in the transamination reactions and one of NAGS's substrates, both of which are elevated when free amino acids are elevated. So Glu not only is a substrate for NAGS but also serves as an activator for the urea cycle.
The remaining enzymes of the cycle are controlled by the concentrations of their substrates. Thus, inherited deficiencies in cycle enzymes other than ARG1 do not result in significant decreases in urea production (if any cycle enzyme is entirely missing, death occurs shortly after birth). Rather, the deficient enzyme's substrate builds up, increasing the rate of the deficient reaction to normal.
The anomalous substrate buildup is not without cost, however. The substrate concentrations become elevated all the way back up the cycle to NH+
4, resulting in hyperammonemia (elevated [NH+
Although the root cause of NH+
4 toxicity is not completely understood, a high [NH+
4] puts an enormous strain on the NH+
4-clearing system, especially in the brain (symptoms of urea cycle enzyme deficiencies include intellectual disability and lethargy). This clearing system involves GLUD1 and GLUL, which decrease the 2-oxoglutarate (2OG) and Glu pools. The brain is most sensitive to the depletion of these pools. Depletion of 2OG decreases the rate of TCAC, whereas Glu is both a neurotransmitter and a precursor to GABA, another neurotransmitter. (p.734)
The urea cycle and the citric acid cycle are independent cycles but are linked. One of the nitrogens in the urea cycle is obtained from the transamination of oxaloacetate to aspartate. The fumarate that is produced in step three is also an intermediate in the citric acid cycle and is returned to that cycle.
A rare genetic disorder that affects about one in 35,000 people in the United States. Genetic defects in the enzymes involved in the cycle can occur. Mutations lead to deficiencies of the various enzymes and transporters involved in the urea cycle and cause urea cycle disorders. If individuals with a defect in any of the six enzymes used in the cycle ingest amino acids beyond what is necessary for the minimum daily requirements, then the ammonia that is produced will not be able to be converted to urea. These individuals can experience hyperammonemia or the buildup of a cycle intermediate.
Most urea cycle disorders are associated with hyperammonemia, however argininemia and some forms of argininosuccinic aciduria do not present with elevated ammonia.
Arginase (EC 184.108.40.206, arginine amidinase, canavanase, L-arginase, arginine transamidinase) is a manganese-containing enzyme. The reaction catalyzed by this enzyme is: arginine + H2O → ornithine + urea. It is the final enzyme of the urea cycle. It is ubiquitous to all domains of life.Argininemia
Argininemia, is an autosomal recessive urea cycle disorder where a deficiency of the enzyme arginase causes a buildup of arginine and ammonia in the blood. Ammonia, which is formed when proteins are broken down in the body, is toxic if levels become too high; the nervous system is especially sensitive to the effects of excess ammonia.Argininosuccinate lyase
ASL (argininosuccinate lyase, also known as argininosuccinase) is an enzyme that catalyzes the reversible breakdown of argininosuccinate (ASA) producing the amino acid arginine and dicarboxylic acid fumarate. Located in liver cytosol, ASL is the fourth enzyme of the urea cycle and involved in the biosynthesis of arginine in all species and the production of urea in ureotelic species. Mutations in ASL, resulting low activity of the enzyme, increase levels of urea in the body and result in various side effects.
The ASL gene is located on chromosome 7 between the centromere (junction of the long and short arm) and the long (q) arm at position 11.2, from base pair 64,984,963 to base pair 65,002,090.
ASL is related to intragenic complementation.Argininosuccinate synthase
Argininosuccinate synthase or synthetase (ASS; EC 220.127.116.11) is an enzyme that catalyzes the synthesis of argininosuccinate from citrulline and aspartate. In humans, argininosuccinate synthase is encoded by the ASS gene located on chromosome 9.
ASS is responsible for the third step of the urea cycle and one of the reactions of the citrulline-NO cycle.Aspartic acid
Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Similar to all other amino acids it contains an amino group and a carboxylic acid. Its α-amino group is in the protonated –NH+3 form under physiological conditions, while its α-carboxylic acid group is deprotonated −COO− under physiological conditions. Aspartic acid has an acidic side chain (CH2COOH) which reacts with other amino acids, enzymes and proteins in the body. Under physiological conditions (pH 7.4) in proteins the side chain usually occurs as the negatively charged aspartate form, −COO−. It is a non-essential amino acid in humans, meaning the body can synthesize it as needed. It is encoded by all the codons GAU and GAC.
D-Aspartate is one of two D-amino acids commonly found in mammals.
In proteins aspartate sidechains are often hydrogen bonded to form asx turns or asx motifs, which frequently occur at the N-termini of alpha helices.
The L-isomer of Asp is one of the 22 proteinogenic amino acids, i.e., the building blocks of proteins. Aspartic acid, like glutamic acid, is classified as an acidic amino acid, with a pKa of 3.9, however in a peptide this is highly dependent on the local environment, and could be as high as 14. Asp is pervasive in biosynthesis. Because aspartate can be synthesized by the body it is classified as a non-essential amino acid.Carbamoyl phosphate
Carbamoyl phosphate is an anion of biochemical significance. In land-dwelling animals, it is an intermediary metabolite in nitrogen disposal through the urea cycle and the synthesis of pyrimidines. Its enzymatic counterpart, carbamoyl phosphate synthetase I (CPS I), interacts with a class of molecules called sirtuins, NAD dependent protein deacetylases, and ATP to form carbamoyl phosphate. CP then enters the urea cycle in which it reacts with ornithine (a process catalyzed by the enzyme ornithine transcarbamylase) to form citrulline. A defect in the CPS I enzyme, and a subsequent deficiency in the production of carbamoyl phosphate has been linked to hyper-ammonemia in humans.Carbamoyl phosphate synthetase
Carbamoyl phosphate synthetase catalyzes the ATP-dependent synthesis
of carbamoyl phosphate from glutamine (EC 18.104.22.168) or ammonia (EC 22.214.171.124) and bicarbonate. This enzyme catalyzes the reaction of ATP and bicarbonate to produce carboxy phosphate and ADP. Carboxy phosphate reacts with ammonia to give carbamic acid. In turn, carbamic acid reacts with a second ATP to give carbamoyl phosphate plus ADP.
It represents the first committed step in pyrimidine and arginine biosynthesis in prokaryotes and eukaryotes, and in the urea cycle in most terrestrial vertebrates. Most prokaryotes carry one form of CPSase that participates in both arginine and pyrimidine biosynthesis, however certain bacteria can have separate forms.
There are three different forms that serve very different functions:
Carbamoyl phosphate synthetase I (mitochondria, urea cycle)
Carbamoyl phosphate synthetase II (cytosol, pyrimidine metabolism).
Carbamoyl phosphate synthetase III (found in fish).Carbamoyl phosphate synthetase I
Carbamoyl phosphate synthetase I is a ligase enzyme located in the mitochondria involved in the production of urea. Carbamoyl phosphate synthetase I (CPS1 or CPSI) transfers an ammonia molecule from glutamine or glutamate to a molecule of bicarbonate that has been phosphorylated by a molecule of ATP. The resulting carbamate is then phosphorylated with another molecule of ATP. The resulting molecule of carbamoyl phosphate leaves the enzyme.Citrulline
The organic compound citrulline is an α-amino acid. Its name is derived from citrullus, the Latin word for watermelon, from which it was first isolated in 1914 by Koga and Odake. It was finally identified by Wada in 1930.
It has the formula H2NC(O)NH(CH2)3CH(NH2)CO2H. It is a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia by converting it into urea. Citrulline is also produced as a byproduct of the enzymatic production of nitric oxide from the amino acid arginine, catalyzed by nitric oxide synthase.Citrullinemia
Citrullinemia is an autosomal recessive urea cycle disorder that causes ammonia and other toxic substances to accumulate in the blood.Two forms of citrullinemia have been described, both having different signs and symptoms, and are caused by mutations in different genes. Citrullinemia belongs to a class of genetic diseases called urea cycle disorders. The urea cycle is a sequence of chemical reactions taking place in the liver. These reactions process excess nitrogen, generated when protein is used for energy by the body, to make urea, which is excreted by the kidneys.Hyperammonemia
Hyperammonemia is a metabolic disturbance characterised by an excess of ammonia in the blood. It is a dangerous condition that may lead to brain injury and death. It may be primary or secondary.
Ammonia is a substance that contains nitrogen. It is a product of the catabolism of protein. It is converted to the less toxic substance urea prior to excretion in urine by the kidneys. The metabolic pathways that synthesize urea involve reactions that start in the mitochondria and then move into the cytosol. The process is known as the urea cycle, which comprises several enzymes acting in sequence.N-Acetylglutamate synthase
N-Acetylglutamate synthase (NAGS) is an enzyme that catalyses the production of N-acetylglutamate (NAG) from glutamate and acetyl-CoA.
Put simply NAGS catalyzes the following reaction:
acetyl-CoA + L-glutamate → CoA + N-acetyl-L-glutamateNAGS, a member of the N-acetyltransferase family of enzymes, is present in both prokaryotes and eukaryotes, although its role and structure differ widely depending on the species. NAG can be used in the production of ornithine and arginine, two important amino acids, or as an allosteric cofactor for carbamoyl phosphate synthase (CPS1). In mammals, NAGS is expressed primarily in the liver and small intestine, and is localized to the mitochondrial matrix.N-Acetylglutamate synthase deficiency
N-Acetylglutamate synthase deficiency is an autosomal recessive urea cycle disorder.Ornithine
Ornithine is a non-proteinogenic amino acid that plays a role in the urea cycle. Ornithine is abnormally accumulated in the body in ornithine transcarbamylase deficiency. The radical is ornithyl.Ornithine transcarbamylase
Ornithine transcarbamylase (OTC) (also called ornithine carbamoyltransferase) is an enzyme (EC 126.96.36.199) that catalyzes the reaction between carbamoyl phosphate (CP) and ornithine (Orn) to form citrulline (Cit) and phosphate (Pi). There are two classes of OTC anabolic and catabolic. This article focuses on anabolic OTC. Anabolic OTC facilitates the sixth step in the biosynthesis of the amino acid arginine in prokaryotes. In contrast, mammalian OTC plays an essential role in the urea cycle whose purpose is to capture toxic ammonia and transform it into less toxic urea nitrogen source for excretion.Ornithine transcarbamylase deficiency
Ornithine transcarbamylase deficiency is the most common urea cycle disorder in humans. It is an inherited disorder which causes toxic levels of ammonia to build up in the blood.Ornithine transcarbamylase, the defective enzyme in this disorder, is the final enzyme in the proximal portion of the urea cycle. It is responsible for converting carbamoyl phosphate and ornithine into citrulline. OTC deficiency is inherited in an X-linked recessive manner, meaning males are more commonly affected than females.
In severely affected individuals, ammonia concentrations increase rapidly, causing ataxia, lethargy, and death without rapid intervention. OTC deficiency is diagnosed using a combination of clinical findings and biochemical testing, while confirmation is often done using molecular genetics techniques.
Once an individual has been diagnosed, the treatment goal is to avoid precipitating episodes that can cause an increased ammonia concentration. The most common treatment combines a low protein diet with nitrogen scavenging agents. Liver transplant is considered curative for this disease. Experimental trials of gene therapy using adenoviral vectors resulted in the death of one participant, Jesse Gelsinger, and have been discontinued.Ornithine translocase
Ornithine translocase is responsible for transporting ornithine from the cytosol into the mitochondria in the urea cycle.Oxaloacetic acid
Oxaloacetic acid (also known as oxalacetic acid) is a crystalline organic compound with the chemical formula HO2CC(O)CH2CO2H. Oxaloacetic acid, in the form of its conjugate base oxaloacetate, is a metabolic intermediate in many processes that occur in animals. It takes part in gluconeogenesis, the urea cycle, the glyoxylate cycle, amino acid synthesis, fatty acid synthesis and the citric acid cycle.Ureohydrolase
A ureohydrolase is a type of hydrolase enzyme.The ureohydrolase superfamily includes arginase (EC 188.8.131.52), agmatinase (EC 184.108.40.206), formiminoglutamase (EC 220.127.116.11) and proclavaminate amidinohydrolase (EC 18.104.22.168). These enzymes share a 3-layer alpha-beta-alpha structure, and play important roles in arginine/agmatine metabolism, the urea cycle, histidine degradation, and other pathways.
Arginase, which catalyses the conversion of arginine to urea and ornithine, is one of the five members of the urea cycle enzymes that convert ammonia to urea as the principal product of nitrogen excretion. There are several arginase isozymes that differ in catalytic, molecular and immunological properties. Deficiency in the liver isozyme leads to argininemia, which is usually associated with hyperammonemia.
Agmatinase hydrolyses agmatine to putrescine, the precursor for the biosynthesis of higher polyamines, spermidine and spermine. In addition, agmatine may play an important regulatory role in mammals.Formiminoglutamase catalyses the fourth step in histidine degradation, acting to hydrolyse N-formimidoyl-L-glutamate to L-glutamate and formamide.
Proclavaminate amidinohydrolase is involved in clavulanic acid biosynthesis. Clavulanic acid acts as an inhibitor of a wide range of beta-lactamase enzymes that are used by various microorganisms to resist beta-lactam antibiotics. As a result, this enzyme improves the effectiveness of beta-lactamase antibiotics.
Urea cycle Metabolic Pathway