Argininosuccinate synthase or synthetase (ASS; ) is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
that catalyzes the synthesis of
argininosuccinate
Argininosuccinic acid is a non-proteinogenic amino acid that is an important intermediate in the urea cycle. It is a basic amino acid.
Reactions
Some cells synthesize argininosuccinic acid from citrulline and aspartic acid and use it as a prec ...
from
citrulline
The organic compound citrulline is an α-amino acid. Its name is derived from ''citrullus'', the Latin word for watermelon. Although named and described by gastroenterologists since the late 19th century, it was first isolated from watermelon in 1 ...
and
aspartate
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. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
. In humans, argininosuccinate synthase is encoded by the ''
ASS gene'' located on
chromosome 9
Chromosome 9 is one of the 23 pairs of chromosomes in humans. Humans normally have two copies of this chromosome, as they normally do with all chromosomes. Chromosome 9 spans about 138 million base pairs of nucleic acids (the building blocks of D ...
.
ASS is responsible for the third step of the
urea cycle
The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions that produces urea (NH2)2CO from ammonia (NH3). Animals that use this cycle, mainly amphibians and mammals, are called ureotelic.
The urea cycle converts highl ...
and one of the reactions of the
citrulline-NO cycle.
Expression
The expressed ASS gene is at least 65 kb in length, including at least 12
introns
An intron is any nucleotide sequence within a gene that is not expressed or operative in the final RNA product. The word ''intron'' is derived from the term ''intragenic region'', i.e. a region inside a gene."The notion of the cistron .e., gene. ...
.
In humans, ''ASS'' is expressed mostly in the cells of the
liver
The liver is a major Organ (anatomy), organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the Protein biosynthesis, synthesis of proteins and biochemicals necessary for ...
and
kidney
The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blood ...
.
Mechanism
In the first step of the catalyzed reaction,
citrulline
The organic compound citrulline is an α-amino acid. Its name is derived from ''citrullus'', the Latin word for watermelon. Although named and described by gastroenterologists since the late 19th century, it was first isolated from watermelon in 1 ...
attacks the α-phosphate of
ATP to form citrulline adenylate, a reactive intermediate. The attachment of
AMP #REDIRECT Amp
{{Redirect category shell, {{R from other capitalisation{{R from ambiguous page ...
to the ureido (urea-like) group on citrulline activates the carbonyl center for subsequent
nucleophilic attack
In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...
. This activation facilitates the second step, in which the α-amino group of
aspartate
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. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
attacks the ureido group. Attack by aspartate is the
rate-limiting step
In chemical kinetics, the overall rate of a reaction is often approximately determined by the slowest step, known as the rate-determining step (RDS or RD-step or r/d step) or rate-limiting step. For a given reaction mechanism, the prediction of the ...
of the reaction. This step produces free AMP and L-
argininosuccinate
Argininosuccinic acid is a non-proteinogenic amino acid that is an important intermediate in the urea cycle. It is a basic amino acid.
Reactions
Some cells synthesize argininosuccinic acid from citrulline and aspartic acid and use it as a prec ...
.
Thermodynamically, adenylation of the citrulline ureido group is more favorable than the analogous
phosphorylation
In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...
. Additionally, attack by citrulline at the α-phosphate of ATP produces an equivalent of
pyrophosphate
In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a P–O–P linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate (Na2H2P2O7) and tetrasodium pyrophosphate (Na4P2O7), among other ...
, which can be hydrolyzed in a thermodynamically favorable reaction to provide additional energy to drive the adenylation.
Structure
Quaternary
Argininosuccinate synthetase is a homotetramer, with each subunit consisting of 412 residues.
The interfaces between subunits contain a number of
salt bridges and
hydrogen bonds
In chemistry, a hydrogen bond (or H-bond) is a primarily electrostatic force of attraction between a hydrogen (H) atom which is covalently bound to a more electronegative "donor" atom or group (Dn), and another electronegative atom bearing a ...
, and the C-terminus of each subunit is involved in oligomerization by interacting with the C-termini and nucleotide-binding domains of the other subunits.
Active site
X-ray crystal structures have been generated for argininosuccinate synthetase from ''
Thermus thermophilus
''Thermus thermophilus'' is a Gram-negative bacterium used in a range of biotechnological applications, including as a model organism for genetic manipulation, structural genomics, and systems biology. The bacterium is extremely thermophilic, w ...
'', ''
E. coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'', ''Thermotoga maritime'', and ''
Homo sapiens
Humans (''Homo sapiens'') are the most abundant and widespread species of primate, characterized by bipedalism and exceptional cognitive skills due to a large and complex brain. This has enabled the development of advanced tools, culture, ...
''. In ASS from ''T. thermophilus'', ''E. coli'', and ''H. sapiens'', citrulline and aspartate are tightly bound in the
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
by interactions with
serine
Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...
and
arginine
Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the am ...
residues; interactions of the substrates with other residues in the active site vary by species. In ''T. thermophilus'', the ureido group of citrulline appears to be repositioned during nucleophilic attack to attain sufficient proximity to the α-phosphate of ATP.
In ''E. coli'', it is suggested that binding of ATP causes a conformational shift that brings together the nucleotide-binding domain and the synthetase domain.
An argininosuccinate synthetase structure with a bound ATP in the active site has not been attained, although modeling suggests that the distance between ATP and the ureido group of citrulline is smaller in human argininosuccinate synthetase than in the ''E. coli'' variety, so it is likely that a much smaller conformational change is necessary for catalysis.
The ATP binding domain of argininosuccinate synthetase is similar to that of other N-type ATP
pyrophosphatases.
Function
Argininosuccinate synthetase is involved in the synthesis of
creatine
Creatine ( or ) is an organic compound with the nominal formula (H2N)(HN)CN(CH3)CH2CO2H. It exists in various modifications (tautomers) in solution. Creatine is found in vertebrates where it facilitates recycling of adenosine triphosphate ( ...
, polyamines,
arginine
Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the am ...
,
urea
Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid.
Urea serves an important r ...
, and
nitric oxide
Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
.
Arginine synthesis
The transformation of citrulline into argininosuccinate is the rate-limiting step in arginine synthesis. The activity of argininosuccinate synthetase in arginine synthesis occurs largely in at the outer
mitochondrial
A mitochondrion (; ) is an organelle found in the cells of most Eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is use ...
membrane of periportal
liver
The liver is a major Organ (anatomy), organ only found in vertebrates which performs many essential biological functions such as detoxification of the organism, and the Protein biosynthesis, synthesis of proteins and biochemicals necessary for ...
cells as part of the urea cycle, with some activity occurring in cortical
kidney
The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blood ...
cells.
{
Genetic defects that cause incorrect localization of argininosuccinate synthetase to the outer mitochondrial membrane cause type II citrullinemia.
In
fetuses
A fetus or foetus (; plural fetuses, feti, foetuses, or foeti) is the unborn offspring that develops from an animal embryo. Following embryonic development the fetal stage of development takes place. In human prenatal development, fetal develo ...
and infants, arginine is also produced via argininosuccinate synthetase activity in intestinal cells, presumably to supplement the low level of arginine found in mother's milk. Expression of argininosuccinate synthetase in the intestines ceases after two to three years of life.
It is thought that regulation of argininosuccinate synthetase activity in arginine synthesis occurs primarily at the
transcriptional level in response to
glucocorticoids
Glucocorticoids (or, less commonly, glucocorticosteroids) are a class of corticosteroids, which are a class of steroid hormones. Glucocorticoids are corticosteroids that bind to the glucocorticoid receptor that is present in almost every vertebr ...
,
cAMP
Camp may refer to:
Outdoor accommodation and recreation
* Campsite or campground, a recreational outdoor sleeping and eating site
* a temporary settlement for nomads
* Camp, a term used in New England, Northern Ontario and New Brunswick to descri ...
,
glucagon
Glucagon is a peptide hormone, produced by alpha cells of the pancreas. It raises concentration of glucose and fatty acids in the bloodstream, and is considered to be the main catabolic hormone of the body. It is also used as a Glucagon (medicati ...
, and
insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
.
It has also been demonstrated ''in vitro'' that arginine down-regulates argininosuccinate synthetase expression, while citrulline up-regulates it.
Citrulline-NO cycle
The enzyme
endothelial nitric oxide synthase
Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3) or constitutive NOS (cNOS), is an enzyme that in humans is encoded by the ''NOS3'' gene located in the 7q35-7q36 region of chromosome 7. This enzyme is one of three isoforms tha ...
produces
nitric oxide
Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
from arginine in
endothelial cells
The endothelium is a single layer of squamous endothelial cells that line the interior surface of blood vessels and lymphatic vessels. The endothelium forms an interface between circulating blood or lymph in the lumen and the rest of the vessel ...
.
Argininosuccinate synthetase and
argininosuccinate lyase
The enzyme argininosuccinate lyase (EC 4.3.2.1, ASL, argininosuccinase; systematic name 2-(''N'' ω-L-arginino)succinate arginine-lyase (fumarate-forming)) catalyzes the reversible breakdown of argininosuccinate:
:2-(''N'' ω-L-arginino)succina ...
recycle citrulline, a byproduct of nitric oxide production, into arginine. Since nitric oxide is an important signaling molecule, this role of ASS is important to vascular physiology. In this role, argininosuccinate synthetase activity is regulated largely by inflammatory cellular signal molecules such as
cytokines
Cytokines are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines have been shown to be involved in autocrin ...
.
In endothelial cells, it has been shown that ASS expression is increased by laminar
shear stress
Shear stress, often denoted by (Greek: tau), is the component of stress coplanar with a material cross section. It arises from the shear force, the component of force vector parallel to the material cross section. ''Normal stress'', on the ot ...
due to pulsative blood flow.
Emerging evidence suggests that ASS may also be subject to regulation by phosphorylation at the Ser-328 residue by
protein kinase C
In cell biology, Protein kinase C, commonly abbreviated to PKC (EC 2.7.11.13), is a family of protein kinase enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and t ...
-α
and by
nitrosylation
Nitrosylation is the general term for covalent incorporation of a nitric oxide "nitrosyl" moiety into another (usually organic) molecule. There are multiple chemical mechanisms by which this can be achieved; including biological enzymes and indust ...
at the Cys-132 residue by nitric oxide synthase.
Role in disease
Citrullinemia
Citrullinemia is an inherited autosomal recessive disease.
At least 50
mutation
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
s that cause type I citrullinemia have been identified in the ASS gene. Most of these mutations substitute one amino acid for another in ASS. These mutations likely affect the structure of the enzyme and its ability to bind to citrulline, aspartate, and other molecules. A few mutations lead to the production of an abnormally short enzyme that cannot effectively play its role in the urea cycle.
Defects in ASS disrupt the third step of the urea cycle, preventing the liver from processing excess nitrogen into urea. As a result, nitrogen (in the form of
ammonia
Ammonia is an inorganic compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogenous was ...
) and other byproducts of the urea cycle (such as citrulline) build up in the bloodstream. Ammonia is toxic, particularly to the
nervous system
In biology, the nervous system is the highly complex part of an animal that coordinates its actions and sensory information by transmitting signals to and from different parts of its body. The nervous system detects environmental changes th ...
. An accumulation of ammonia during the first few days of life leads to poor feeding,
vomiting
Vomiting (also known as emesis and throwing up) is the involuntary, forceful expulsion of the contents of one's stomach through the mouth and sometimes the Human nose, nose.
Vomiting can be the result of ailments like Food-poisoning, foo ...
,
seizure
An epileptic seizure, informally known as a seizure, is a period of symptoms due to abnormally excessive or synchronous neuronal activity in the brain. Outward effects vary from uncontrolled shaking movements involving much of the body with los ...
s, and the other signs and symptoms of type I citrullinemia.
Treatment for this defect includes a low-protein diet and dietary supplementation with
arginine
Arginine is the amino acid with the formula (H2N)(HN)CN(H)(CH2)3CH(NH2)CO2H. The molecule features a guanidino group appended to a standard amino acid framework. At physiological pH, the carboxylic acid is deprotonated (−CO2−) and both the am ...
and
phenylacetate. Arginine allows the urea cycle to complete itself, creating the substrates needed to originally fix ammonia. This will lower blood pH. Additionally, phenylacetate reacts with backed-up glutamine, resulting on phenylacetoglutamine, which can be excreted renally.
Cancer
A lack of argininosuccinate synthetase expression has been observed in several types of cancer cells, including
pancreatic
The pancreas is an Organ (anatomy), organ of the digestive system and endocrine system of vertebrates. In humans, it is located in the abdominal cavity, abdomen behind the stomach and functions as a gland. The pancreas is a mixed or heterocrine ...
cancer, liver cancer,
and melanoma.
For example, defects in ASS have been seen in 87% of pancreatic cancers. Cancer cells are therefore unable to synthesize enough arginine for cellular processes and so must rely on dietary arginine. Depletion of plasma arginine using
arginine deiminase has been shown to lead to regression of tumours in mice.
See also
*
Citrullinemia
Citrullinemia is an autosomal recessive urea cycle disorder that causes ammonia and other toxic substances to accumulate in the blood.Freedberg, et al. (2003). ''Fitzpatrick's Dermatology in General Medicine''. (6th ed.). McGraw-Hill. .
Two for ...
*
Urea cycle
The urea cycle (also known as the ornithine cycle) is a cycle of biochemical reactions that produces urea (NH2)2CO from ammonia (NH3). Animals that use this cycle, mainly amphibians and mammals, are called ureotelic.
The urea cycle converts highl ...
*
Synthetase
In biochemistry, a ligase is an enzyme that can catalyze the joining (Ligation (molecular biology), ligation) of two large molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the ...
References
External links
GeneReviews/NCBI/NIH/UW entry on Argininosuccinate Synthetase Deficiency; ASS Deficiency; Argininosuccinic Acid Synthetase Deficiency; CTLN1; Citrullinemia, Classic
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EC 6.3.4
Urea cycle