Studies of Rat Liver Argininosuccinate Synthetase

Saheki, Takeyori; Kusumi, Takashi; Takada, Shigeo; Katsunuma, Tsunehiko

doi:10.1093/oxfordjournals.jbchem.a131505

Cited by 27 publications

(11 citation statements)

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“…The enzyme thus appears as a tetramer composed of identical subunits of molecular weight 49000. This conclusion is close to that found for the corresponding liver enzyme [7,8], although Rochovansky et al [7] determined somewhat lower molecular weights for the subunit and the active enzyme.…”

Section: Structural Propertiessupporting

confidence: 85%

“…5 A, for the forward reaction the synthetase of S. cerevisiae displays an optimum pH in Tris-HC1 buffer between 7.5 and 8.0; in imidazole-HCl buffer, it is about 7.5. It is of interest to note that the mammalian enzyme assayed under similar conditions to those used here, showed a closer dependence for the alkaline region [8,13]. However, the maximum velocity in the reverse direction, located between pH 6.0 and 6.5 (Fig.…”

Section: Effect O J P H On the Reaction Velocity In The Forward And Bsupporting

confidence: 64%

“…Urea is degraded to ammonia and COZ via allophanate [5,6]. Yeast argininosuccinate synthetase may thus be expected to have regulatory properties different from those of the mammalian liver enzyme, recently purified and investigated [7,8].…”

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confidence: 99%

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Yeast Argininosuccinate Synthetase

Hilger

Simon

Stalon

1979

European Journal of Biochemistry

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Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228000 as determined by gel sieving. It is composed of identical subunits of M , 49000 as shown by gel electrophoresis.The quaternary structure as determined by cross-linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric.The saturation functions by citrulline and aspartate are hyperbolic ; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and aspartate ; it is increased by argininosuccinate and arginine.Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead-end inhibition studies comply with a rapid-equilibrium random model, except for the interconversion of the central quaternary complexes ; the different kinetic constants have been established on this basis.Yeast arginonosuccinate synthetase has a double metabolic function : anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well-adjusted activity for both roles and to an economic and efficient utilization of ATP.Argininosuccinate synthetase catalyses the penultimate reaction of the arginine biosynthetic pathway :Citrulline + aspartate + ATP + Argininosuccinate + AMP + pyrophosphate.

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Section: Structural Propertiessupporting

confidence: 85%

Section: Effect O J P H On the Reaction Velocity In The Forward And Bsupporting

confidence: 64%

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Yeast Argininosuccinate Synthetase

Hilger

Simon

Stalon

1979

European Journal of Biochemistry

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“…Argininosuccinate synthetase has since been studied extensively at both the protein and genetic levels, and reviews are available (5,6). The enzyme has been purified from bovine (7), rat (8), and human liver (9). The cDNAs for human (10) and rat (11) have been sequenced, and the gene for the human locus has been described (12).…”

mentioning

confidence: 99%

Molecular definition of bovine argininosuccinate synthetase deficiency.

Dennis

Healy

Beaudet

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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Citrullinemia is an inborn error of metabolism due to deficiency of the urea cycle enzyme, argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5]. The disease was first described in humans but was recently reported in dairy cattle in Australia. Here we report the nucleotide sequence of the normal bovine cDNA for argininosuccinate synthetase and the mutation present in animals with citrullinemia. Analysis of DNA from affected animals by Southern blotting did not readily identify the mutation in the bovine gene. RNA (Northern) blotting revealed a major reduction in the steady-state amount of mRNA in the liver of affected animals to <5% of controls. The bovine cDNA was cloned and sequenced and revealed 96% identity with the deduced human sequence at the amino acid level. Starting with mutant bovine liver, the mRNA was reverse-transcribed; the cDNA product was amplified with the polymerase chain reaction, cloned, and sequenced. The sequence revealed a C -+ T transition converting arginine-86 (CGA) to a nonsense codon (TGA). A second C --T transition represented a polymorphism in proline-175 (CCC --> CCT). The mutation and the polymorphism were confirmed by amplification of genomic DNA and demonstration with restriction endonuclease enzymes of both the loss of an Ava II site in DNA from mutant animals at codon 86 and the presence or absence of a Dde I site at codon 175. The loss of the Ava II site can be used for rapid, economical, nonradioactive detection of heterozygotes for bovine citrullinemia.

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“…Argininosuccinate synthetase has been isolated from bovine (1), rat (2), and human (3) liver. The physical properties and amino acid composition ofthe enzyme from three species are similar.…”

Section: Modification Of Argininosuccinate Synthetase With [14c]-mentioning

confidence: 99%

Argininosuccinate synthetase: essential role of cysteine and arginine residues in relation to structure and mechanism of ATP activation.

Kumar¹,

Lennane²,

Ratner³

1985

Proc. Natl. Acad. Sci. U.S.A.

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We have undertaken studies to identify amino acid residues that are involved in the catalytic mechanism of argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.51 and have found that a cysteine residue and an arginine residue are required for activity. The reactive cysteine residues are accessible to solvent and available to react with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Four cysteine residues, one per subunit, are shown by enzymatic assay to be required for catalytic activity, suggesting that a reactive cysteine lies within the active site of argininsuccinate synthetase. In the presence of sodium dodecyl sulfate, 12 cysteine residues react with DTNB; consequently, all of the half-cystine residues in the native enzyme are present in the reduced sulfhydryl form. We also present evidence for the participation of arginine groups in the binding of ATP and PP1. Argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5], one of the five enzymes participating in the biosynthesis of arginine and urea, catalyzes reaction 1 by a mechanism involving activation by ATP (discussed in a later section). ATP + citrulline + aspartate " argininosuccinate + AMP + PP,.[1]The native synthetase of Mr 185,000 is composed of four subunits of M, 46,250 that are identical in size and electrophoretic mobility (1). Little is known concerning the activesite region or the amino acid residues involved in the binding of the six substrates and products of the enzyme. In the present work we have carried out studies on the modification of cysteine and arginine residues of the enzyme to elucidate catalytic function, structure, and mechanism.Argininosuccinate synthetase has been isolated from bovine (1), rat (2), and human (3) liver. The physical properties and amino acid composition ofthe enzyme from three species are similar. The human gene encoding argininosuccinate synthetase has been cloned and the nucleotide sequence has been determined (4). The synthetase is subject to genetic mutation in humans; clinical symptoms become evident neonatally at a rate of 53 new cases annually (5). Knowledge of the critical amino acid residues involved in catalysis is important in characterizing the genetic lesions. The identification of active-site residues is also valuable in the design of in vitro site-directed mutagenesis of the human enzyme.We report here the results of modification studies undertaken to ascertain the role of reactive cysteine and arginine residues. Our results show that cysteine and arginine are essential for catalytic activity and thus lie within the active site. The role of specific arginine residues in binding ATP and PP1 is supported by substrate protection experiments. Protection by ATP and PP1 indicates that arginine residues function in the binding of ATP and PPj. The number of arginine residues bound suggests that ATP and PP, probably bind at the same site, thus promoting the successive step catalysis.MATERIALS AND METHODS Materials. Argininosuccinate wa...

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Studies of Rat Liver Argininosuccinate Synthetase

Cited by 27 publications

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Yeast Argininosuccinate Synthetase

Yeast Argininosuccinate Synthetase

Molecular definition of bovine argininosuccinate synthetase deficiency.

Argininosuccinate synthetase: essential role of cysteine and arginine residues in relation to structure and mechanism of ATP activation.

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