Argininosuccinate synthase
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Argininosuccinate synthase or synthetase (ASS; ) is an enzyme 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 precur ...
from citrulline 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. ASS is responsible for the third step of the urea cycle 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. In humans, ''ASS'' is expressed mostly in the cells of the liver and kidney.


Mechanism

In the first step of the catalyzed reaction, citrulline attacks the α-phosphate of
ATP ATP may refer to: Companies and organizations * Association of Tennis Professionals, men's professional tennis governing body * American Technical Publishers, employee-owned publishing company * ', a Danish pension * Armenia Tree Project, non ...
to form citrulline adenylate, a reactive intermediate. The attachment of AMP to the ureido (urea-like) group on citrulline activates the carbonyl center for subsequent nucleophilic attack. 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 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 precur ...
. 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, 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''. 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 Pyrophosphatases, also known as diphosphatases, are acid anhydride hydrolases that act upon diphosphate bonds. Examples include: * Inorganic pyrophosphatase, which acts upon the free pyrophosphate ion * Tobacco acid pyrophosphatase, which cataly ...
.


Function

Argininosuccinate synthetase is involved in the synthesis of creatine, 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, 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 cells as part of the urea cycle, with some activity occurring in cortical kidney cells.{ Genetic defects that cause incorrect localization of argininosuccinate synthetase to the outer mitochondrial membrane cause type II citrullinemia. In fetuses 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 Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules calle ...
level in response to glucocorticoids,
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. Argininosuccinate synthetase and argininosuccinate lyase 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 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 ...
and by nitrosylation at the Cys-132 residue by nitric oxide synthase.


Role in disease


Citrullinemia

Citrullinemia is an inherited autosomal recessive disease. At least 50 mutations 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) and other byproducts of the urea cycle (such as citrulline) build up in the bloodstream. Ammonia is toxic, particularly to the nervous system. An accumulation of ammonia during the first few days of life leads to poor feeding, vomiting, seizures, 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 Phenylacetate may refer to: * Phenyl acetate, the ester of phenol and acetic acid * The conjugate base of phenylacetic acid {{Short pages monitor