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Argininosuccinate SynthetaseArgininosuccinate Synthetase
IntroductionIntroduction
(ASS) catalyses the condensation of citrulline and aspartate to form argininosuccinate, the immediate precursor of arginine. First identified in the liver as the limiting enzyme of the urea cycle, ASS is now recognized as a ubiquitous enzyme in mammalian tissues.[1]Since its discovery, the function of argininosuccinate synthase has been linked almost exclusively to hepatic urea production despite the fact that alternative pathways involving argininosuccinate synthase were defined, such as its role in providing arginine for creatine and for polyamine biosynthesis [2]Argininosuccinate synthase plays an important role as the rate-limiting step in providing arginine for an assortment of metabolic processes, both catabolic and anabolic. Thus, the metabolic pathways in which argininosuccinate synthase participates are linked to the varied uses of the amino acid arginine. There are five major pathways in which argininosuccinate synthase plays a key role. These are (a) urea synthesis,(b) nitric oxide synthesis, (c) polyamine synthesis, (d) creatine synthesis, and (e) the de novosynthesis of arginine to maintain serum levels.[3]
StructureStructure
The overall and subunit structure of tAsS is quite similar to that of E. coli AsS, although the precise structure is different between them, probably because the polypeptide chain of tAsS is 47 amino acid residues shorter than that of E. coli AsS. The tAsS is folded into a tetrameric form with a noncrystallographic D2 symmetry, having the shape of a twisted rectangle with an at each of the four corners. In the center of the molecule, there are clusters of a-helices which are surrounded by B-sheets. One subunit in the tetramer interacts with the other three subunits and the surface areas of the subunit interfaces are 4,390 for a and b, 1201 for a and d, and 923 Å 2 for a and c subunits. The largest of these areas is found between two subunits of a and b or c and d, indicating that the tetramer may be considered to be an assembly of two dimer units (a dimer of dimers) around a 2-fold axis. All the subunit interfaces are
distant from the active site and not essential for the catalytic action. The Calpha carbon atoms of the subunit in the native tAsS can be superimposed onto the corresponding ones in tAsS-ATP and tAsS-AMP-PNP-arginine-succinate within r.m.s. deviations of 0.14 and 0.19 Å with the maximum displacement of 0.88 and 0.73 Å, respectively. Thus, tAsS does not significantly change its conformation upon binding of the ATP (AMP-PNP) and substrate analogues. Similarly, E. coli AsS does not change its overall conformation on substrate binding, although the enzyme shows a few localized conformational changes. The C alpha positions of the native E. coli AsS and the complex one with citrulline and aspartate superimpose within r.m.s. deviations of 0.6 Å. The subunit superposition of tAsS-AMP-PNP-arginine-succinate and E. coli AsS-citrulline-aspartate resulted in 284 equivalenced Calpha atoms with r.m.s. deviations of 0.54 Å with the maximum displacement of 2.98 Å, indicating that the overall structures of both subunits are essentially the
same. The subunit is divided into a small domain (ATP binding domain, N-terminal to Pro 165), a large domain (Val 166 to Arg 359), and a C terminal arm (Gln 360 to C-terminal). The small domain has a typical a/B structure of an open parallel B-sheet and is folded into the structure similar to that of the ATP pyrophosphate domains of the GMP, NAD+, and asparagine synthetases (3, 5, 7). The five B-strands designated as b3, b2, b1, b4, and b5 (all parallel) form a twisted B-sheet structure as a central core surrounded by two a-helices (H1 and H2) from the convex surface side of the sheet and two a-helices (H4 and H5) from the concave side. The residues from Phe 69 to Ala 91 (a-helix H3 a loop a-helix H4) go through the large hole formed at the center of the Cterminal domain, and the loop reaches the exit of the hole to interact with the other subunit of the dimeric unit. Thus, this region of the N-terminal domain behaves like a part of the Cterminal one. The a-helix H7 covers the a-helix H1, and its C-terminal loop goes to the C-terminal domain. The large domain consists of four stranded antiparallel-sheets (b6, b7, b12, and b11), five stranded antiparallel-sheets (b10, b9, b8, b13, and b14), and four a-helices. Two B-sheets of the large domain are connected through the a-helix H8 between the B-strands of b10 and b11 and a succession of three a-helices of H9, H10, and H11 between the B-strands of b12
and b13. The four-stranded a-sheet adjacent to the small domain and a-helices H9, H10, and H11 make a large hole going through the center of the large domain. The long C-terminal arm region reaches the small domains of the other subunits with its Cterminal a-helix interacting with them. When the arm is neglected, the surface areas of the subunit interfaces for a and b, a and d, and a and c subunits are reduced to 2,750, 11, and 654 Å 2, respectively, indicating that the arm is extensively involved in the subunit interactions as the joint for the formation of a dimer or a tetramer.[4] Sulfate ion and glycerol are the two found to be on argininosuccinate synthetase.[5]
MechanismMechanism
results have proved that the mechanism for the formation of argininosuccinate consists of at least two distinct chemical steps with the formation of citrulline adenylate as a reactive intermediate. Argininosuccinate synthetase catalyzes the reversible conversion of citrulline, aspartate, and ATP to argininosuccinate, AMP, and inorganic pyrophosphate. Step 1, activated citrulline-adenylate is formed, releasing inorganic pyrophosphate. Step 2, nucleophilic attack by aspartate amino group forms argininosuccinate and releases AMP.[6]
ImplicationsImplications
Arginino Succinate Deficiency
The argininosuccinic acid synthetase (AS) gene is located on chromosome 9q34.1 spanning 63 kb and composed of 16 exons encoding a protein of 412 amino acids. The functional enzyme exists as a homotetramer. Surprisingly, there are at least 14 AS pseudogenes found on various chromosomes, including two pseudogenes on chromosome 9 but distant from the location of the active AS gene. There are at least 22 known mutations in the AS gene that result in argininosuccinate synthetase deficiency (ASD). Mutations include missense, nonsense and exon deletions. The frequency of ASD is approximately 1 per 57,000 live births.
ASD is, like the other neonatal onset forms of UCDs, most severe when presenting in newborn infants. As with each of the four neonatal onset UCDs, ASD is characterized by the accumulation of ammonia and glutamine with clinical manifestations appearing in full-term infants with no prior obstetric risk factors. The classic symptoms appear between 24hrs and 48hrs after birth (but not prior to 24hrs) and include convulsions, hyperventilation, ataxia, hypothermia, lethargy, vomiting and poor feeding. If left untreated the hyperammonemia with result in coma and death. The severe effects of hyperammonemia are described in the Nitrogen Metabolism page. Even though sepsis is a rare event in a normal term infant with no prior obstetric complications, this disorder is misdiagnosed in almost half of neonatal UCD cases. Initial laboratory findings will include respiratory alkalosis which is the earliest objective indication of encephalopathy. The encephalopathy will progress to the point where mechanical ventilation is required. Another routine laboratory finding is reduced serum (blood) urea nitrogen (BUN) which may be as low as 1mg/dl (normal for newborns is 3–12mg/dl). If plasma ammonia levels are not measured the infants' death will be attributed to sepsis, intracranial hemorrhage, or some other disorder that would normally be associated with a pre-term delivery.
ASD patients are treated in much the same ways as for other neonatal UCDs in that protein intake must me highly regulated and the hyperammonemia must be controlled. Hemodialysis is the only effective means to rapidly lower serum ammonia levels in these patients. Acute episodes of hyperammonemia can be treated with intravenous administration of Ammunol® and with oral Buphenyl® for chronic adjunctive therapy of hyperammonemia. Additionally, ASD is treated with oral arginine. The utility of arginine therapy stems from the conversion, ultimately, to citrulline by other enzymes of the urea cycle. The arginine is cleaved to urea and ornithine by the action of arginase. Ornithine and carbamoyl phosphate are condensed to citrulline by the action of ornithine transcarbamoylase (OTC). The citulline is then excreted in the urine. Unlike the utility of oral arginine therapy in the treatment of argininosuccinate lyase deficiency (ALD), which leads the excretion of 2 moles of waste nitrogen as argininosuccinate, citrulline only contains 1 mole of waste nitrogen and excretion of citrulline in the urine is not very efficient. Therefore, it is necessary to include sodium phenylbutyrate (or Buphenyl®) in the treatment regimen.[7]
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