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

Abstract

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...

Proteomic and Physiological Analysis of the Response of Oat (Avena sativa) Seeds to Heat Stress under Different Moisture Conditions

“…According to GO, COG functional classification (Supplementary Table 1 ) and KEGG pathway analysis (Supplementary Table 2 ), ASS was classified under amino acid transport and metabolism. Proline synthesis occurs via two routes, involving glutamic acid and ornithine respectively, and ASS could catalyze the synthesis of ornithine (Kumar et al, 1985 ; Husson et al, 2003 ). It has been proven that the ornithine pathway in proline biosynthesis could be activated by salt stress in barley ( Hordeum vulgare ) seedlings (Zhao et al, 2001 ).…”

Argininosuccinate Synthetase is Reversibly Inactivated by S-Nitrosylation in Vitro and in Vivo

“…Together, these findings suggest that AS and/or AL are subject to inhibition in association with nitrosative stress and thiol depletion. Because AS (20), but not AL (30), has been shown to contain a redoxsensitive thiol that inhibits activity when covalently modified, we considered whether this thiol may be a target for modification by NO. If so, we would predict that NO production from Cit would not fail in the presence of an efficient NO scavenger.…”

“…S-nitrosylation has been implicated in the physiological regulation of a broad spectrum of cellular and physiological activities, including regulation of blood flow (15), skeletal muscle contractility (16), neurotransmission (17), DNA repair, and apoptosis (18,19). Interestingly, the thiol-reactive reagent 5,5Ј-dithiobis-2-nitrobenzoic acid was shown to modify a single undefined Cys residue in AS purified from bovine liver, inactivating catalytic function (20). It was also demonstrated that NO donors inhibit the urea cycle in cultured hepatocytes (21), and urea cycle activity can be increased when NOS is inhibited (22).…”

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins

“…Another example with important active site arginine is given by the argininosuccinate synthase, a key enzyme in urea synthesis, deficiency of which is associated with hyperammonemia [156]. The participation of this arginine in the ATP binding during the catalysis was also identified by chemical modification studies [157].…”