Structures of Argininosuccinate Synthetase in Enzyme-ATP Substrates and Enzyme-AMP Product Forms

Abstract

In prokaryotes, an arginine is synthesized by eight-step reactions (arginine biosynthetic pathway) using glutamate as a starting material (1). The glutamate is converted to ornithine by five-step reactions, and the ornithine then enters into the urea cycle to produce arginine in three-step reactions. Argininosuccinate synthetase (AsS) 1 catalyzes the seventh step of the arginine biosynthesis (the second step of the urea cycle). AsS reversibly catalyzes the adenosine triphosphate (ATP)-dependent condensation of citrulline with aspartate (Scheme 1).It has been proposed that GMP synthetase, NAD ϩ synthetase, asparagine synthetase, and AsS have a common domain belonging to a new family of "N-type" ATP pyrophosphatases (4, 5). The domain has the modified version of the P-loop (PPloop) of H-Ser-Gly-Gly-X-Asp-Ser/Thr-Ser/Thr (where H is any hydrophobic amino acid and X is any amino acid) specific for a pyrophosphate. X-ray structures of GMP synthetase (4), NAD ϩ synthetase (6 -8), and asparagine synthetase (9, 10) show that ATP binding domains are folded into the same open ␣/␤ structure, and pyrophosphate and the ␤-and ␥-phosphates of ATP interact with the PP-loop in GMP synthetase and NAD ϩ synthetase, respectively.For the first time Escherichia coli AsS (eAsS) and its complex with citrulline and aspartate have been determined as the x-ray structure of AsS (11). The enzyme consists of the ATP binding domain (small domain) and the synthetase domain (large domain). The ATP binding domain of eAsS has the same fold as that of a new family of N-type ATP pyrophosphatases. A corollary of the ATP binding model to the active site is that a conformational change in the enzyme is necessary for the catalytic reaction. To confirm this proposal, the structures of eAsS⅐ATP and eAsS⅐ATP⅐citrulline have been determined by x-ray methods (12). Comparisons of these two complexes with eAsS and eAsS⅐citrulline⅐aspartate revealed that ATP binding induces a rotation of the ATP binding domain toward the synthetase domain. Based on the structural elucidation of eAsS complexes, the observed kinetic properties were explained, and the catalytic mechanism of AsS was proposed.The structures of Thermus thermophilus HB8 AsS (tAsS), tAsS⅐ATP, and tAsS⅐AMP-PNP⅐arginine⅐succinate have been previously determined by us (13) to show an overall structure similar to that of eAsS. No conformational change in the ATP binding domain was observed on binding of ATP and AMP-PNP, implying that the reaction may proceed without the conformational change at the molecular level. ATP (or AMP-PNP) and substrate analogues are bound to the active site with their reaction sites close to one another and located in a geometric orientation favorable to the catalytic action. The mechanism of the reaction was proposed on the basis of the enzyme-substrate complex model.We have determined the structures of tAsS in the complex with intact ATP and substrates (citrulline and aspartate), in the complex with AMP and product (argininosuccinate), and in the complex with AMP-PNP, substrat...

Structure of human argininosuccinate synthetase

“…An ATP molecule was modelled into the nucleotide-binding domain of human argininosuccinate synthetase using the homologous structure from Th. thermophilus (Goto et al, 2003). The distance between the citrulline molecule and the ATP -phosphate is shorter than the corresponding distance in the E. coli enzyme, in which a conformational change brings the two molecules closer upon ATP binding (Lemke & Howell, 2002).…”

“…The crystal structures of argininosuccinate synthetase from two bacterial species, Escherichia coli (Lemke & Howell, 2001, 2002 and Thermus (Tm.) thermophilus (Goto et al, 2002(Goto et al, , 2003, have previously been reported. In addition, one unpublished apo structure from the bacterium Thermotoga (Tt.)…”

Substrate Activation and Conformational Dynamics of Guanosine 5′-Monophosphate Synthetase

“…For example, in NH 3 -dependent NAD + synthetase, a substrate–binding and specificity loop was ordered, juxtaposed to its adenylylated nucleotide intermediate (48). The structures of other family members, such as argininosuccinate synthetase (49) and β-lactam synthetase (50), also suggest that structurally analogous regions of these proteins are responsible for interaction with their respective substrates.…”

Amino Acid Biosynthesis

“…This transformation occurs in a two-step process that exemplifies the third general mechanism of amine transfer in metabolism (after GAT and PLP action) -ligation of the a-amino group of aspartate and then elimination of fumarate, which is recycled via the TCA cycle [147]. Argininosuccinate synthetase is homologous to asparagine synthetase [148] and uses ATP to activate the urea by adenylation [149]; nucleophilic substitution by aspartate produces argininosuccinate. Argininosuccinate lyase, the terminal enzyme of the pathway, is a member of a family of aspartase enzymes that catalyze the elimination of fumarate (which feeds into the TCA cycle) from aspartate and N-aspartyl derivatives [150,151], demonstrating the generality of this two step synthetase/lyase route for the indirect transfer of ammonia -the third general process for transfer of nitrogen between metabolites.…”