Structures of argininosuccinate synthetase in enzyme-ATP substrates and enzyme-AMP product forms: stereochemistry of the catalytic reaction.

Argininosuccinate synthetase reversibly catalyzes the ATP-dependent condensation of a citrulline with an aspartate to give argininosuccinate. The structures of the enzyme from Thermus thermophilus HB8 complexed with intact ATP and substrates (citrulline and aspartate) and with AMP and product (argininosuccinate) have been determined at 2.1- and 2.0-A resolution, respectively. The enzyme does not show the ATP-induced domain rotation observed in the enzyme from Escherichia coli. In the enzyme-substrate complex, the reaction sites of ATP and the bound substrates are adjacent and are sufficiently close for the reaction to proceed without the large conformational change at the domain level. The mobility of the triphosphate group in ATP and the side chain of citrulline play an important role in the catalytic action. The protonated amino group of the bound aspartate interacts with the alpha-phosphate of ATP and the ureido group of citrulline, thus stimulating the adenylation of citrulline. The enzyme-product complex explains how the citrullyl-AMP intermediate is bound to the active site. The stereochemistry of the catalysis of the enzyme is clarified on the basis of the structures of tAsS (argininosuccinate synthetase from T. thermophilus HB8) complexes.

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, substrate analogues (arginine and aspartate) and Mg 2ϩ . The tAsS is shown to have the same overall structure regardless of whether the enzyme is in the native or the complexed form and to have a structure quite similar to (but with a slightly larger rotation of the ATP binding domain toward the synthetase domain than) that of eAsS⅐ATP⅐citrulline. The reaction sites of the ATP and substrates (citrulline and aspartate) bound to tAsS are adjacent and are sufficiently close for the reaction to proceed without the large conformational change at the domain level. The enzymeproduct complex explains how the citrullyl-AMP intermediate is bound to the active site. The detailed stereochemistry of the catalysis has been explained on the basis of the structures of tAsS complexes. We now report x-ray crystallographic studies of the following three forms of tAsS: the enzyme-substrate complex at 2.1-Å resolution, the enzyme-product complex at 2.0-Å resolution, and tAsS⅐AMP-PNP⅐arginine⅐aspartate⅐Mg 2ϩ at 2.15-Å resolution.

EXPERIMENTAL PROCEDURES
Crystallization and Data Collection-The structural gene of tAsS was inserted between NdeI and the BamHI restriction site of plasmid pET11a. E. coli BL21(DE3)pLysE was transformed with the resultant plasmid. The enzyme was purified by incubation at 343 K for 10 min, ammonium sulfate fractionation, and a three-step procedure of column chromatography, first by a Phenyl-5PW column (TOSOH) with a linear gradient from 1 to 0 M ammonium sulfate followed by a Super Q-5PW column (TOSOH) using a linear gradient of 0 to 0.5 M NaCl and, finally, by a HiLoad 26/60 Superdex 200-pg column (Amersham Biosciences) equilibrated with 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM 2-mercaptoethanol.
Crystallization of the enzyme has been reported elsewhere (13). Briefly, the complexes of tAsS were crystallized at 293 K by the vapor diffusion method (14) using 30 mg/ml protein solution and 2.4 M ammonium sulfate, 30% (v/v) glycerol, 100 mM Tris-HCl, pH 8.5, as the reservoir solution. After 3 days, crystals had grown to dimensions of about 0.4 ϫ 0.4 ϫ 0.2 mm.
Crystals of the true enzyme-substrate complex (tAsS⅐ATP⅐ citrulline⅐aspartate) were obtained at 293 K by soaking the tAsS⅐ATP crystals in solutions containing 10 mM citrulline, 10 mM aspartate, and 10 mM MgCl 2 for 10 min before data collection. Crystals of the true enzyme-product complex (tAsS⅐AMP⅐argininosuccinate) were obtained at 293 K by soaking the tAsS⅐AMP crystals in solutions containing 10 mM argininosuccinate for 10 min before data collection. Crystals of tAsS⅐AMP-PNP⅐arginine⅐aspartate⅐Mg 2ϩ were obtained using a drop composed of a 1:1:1 ratio of the reservoir solution, the protein solution, and the additive solution of 10 mM AMP-PNP, 10 mM MgCl 2 , 100 mM arginine, and 100 mM aspartate.
The x-ray diffraction data sets for the tAsS⅐ATP⅐citrulline⅐aspartate crystal, the tAsS⅐AMP⅐argininosuccinate crystal, and the tAsS⅐AMP-PNP⅐arginine⅐aspartate⅐Mg 2ϩ crystal were collected using a wavelength of 1.00 Å from the Synchrotron Radiation Source at the SPring-8 BL44B2, BL41XU, and BL40B2 (Hyogo, Japan). The crystals were mounted in a 0.5-mm cryoloop (Hampton Research) and flash-frozen in a cold nitrogen stream at 100 K. All the data were processed and scaled using the program HKL2000 (15) ( Table I). The three crystals are isomorphous with the native crystal (13) and have average cell dimensions of a ϭ b ϭ 229.1 and c ϭ 159.8 Å with the space group of R3. There are four subunits in the asymmetric unit, and ϳ72% of the crystal volume is occupied by solvent.
Structure Determination and Refinement-Refinement of each of the three complex structures was initiated using the coordinate of the unliganded tAsS as an initial model (13). Model building was performed by the program O (16). The refinement was performed by simulated annealing and energy minimization using the maximum likelihood target with the program CNS (17). All the subunits of the complexes were refined independently with 10% of the reflections excluded for the calculation of R free values. The R factor and R free values were decreased after several rounds of refinement and manual rebuilding. When the R factor value decreased below 30%, the sigmaA-weighted F o Ϫ F c map was calculated to assign bound ligands to the residual electron density. Water molecules were picked up on the basis of the peak heights (3.0 ) and distance criteria (4.0 Å from protein or solvent) from the sigmaAweighted F o Ϫ F c map. The water molecules whose thermal factors were above the maximum thermal factor of the main chain after refinement were removed from the list. The occupancy factors for ATP, AMP, and SO 4 were refined because the temperature factors of these ligands showed values considerably higher than those of side-chain atoms of the enzyme. Further refinement cycles and model building resulted in the final values of R factor and R free as shown in Table I. The occupancy factors for ATPs range from 0.88 to 0.95 with a mean value of 0.93, those for AMP range from 0.82 to 0.89 with a mean value of 0.85, and those for SO 4 range from 0.87 to 1.00 with a mean value of 0.94.
Enzyme Assay-Enzymatic activity was measured by analyzing a phosphate derived from a released pyrophosphate using a previously reported procedure (18). Measurements were carried out in the same buffer as that for soaking the substrates (100 mM Tris-HCl, 10 mM ATP, 10 mM citrulline, 10 mM aspartate, 10 mM MgCl 2 , pH 8.5) at 293 and 343 K. The inorganic pyrophosphatase was added to the buffer to hydrolyze a pyrophosphate to produce a phosphate. The reactions were initiated by adding purified enzyme and stopped by the color reagent (0.045% malachite green hydrochloride, 4.2% sodium molybdate in 4 N HCl, Tween 20). The protein concentration was estimated by the method of Bradford (19). The enzyme activity was corrected for ATP pyrophosphatase activity, which was measured independently using the buffer freed of substrates, citrulline, and aspartate. The enzyme has a specific activity of 0.06 Ϯ 0.01 mol min Ϫ1 mg Ϫ1 at 293 K and 0.92 Ϯ 0.02 mol min Ϫ1 mg Ϫ1 at 343 K.

RESULTS AND DISCUSSION
Quality of the Structure-The final model of the tAsS⅐ATP⅐ citrulline⅐aspartate complex contains 1544 amino acid residues for all subunits, 4 ATPs, 4 citrullines, 4 aspartates, and 678 water molecules with an R factor of 22.6% at 2.1-Å resolution. The model lacks Pro-165-Pro-171, Phe-365-Gly-370, and Glu-396 -Ala-400, and the average thermal factor of the main-chain atoms is 34 Å 2 . The final model of the tAsS⅐AMP⅐ argininosuccinate complex contains 1544 amino acid residues for all subunits and 4 AMPs, 4 argininosuccinates, 4 sulfate anions, and 862 water molecules with an R factor of 21.2% at 2.0 Å resolution. The model lacks Pro-165-Pro-171, Phe-365-Gly-370, and Glu-396 -Ala-400. The average thermal factor of the main-chain atoms is 30 Å 2 . The final model of the tAsS⅐AMP-PNP⅐arginine⅐aspartate⅐Mg 2ϩ complex contains 1538 amino acid residues for all subunits and 4 AMP-PNPs, 4 arginines, 4 aspartates, 4 sulfate anions, 4 Mg 2ϩ ions, and 567 water molecules with an R factor of 22.6% at 2.15-Å resolution. The model lacks Pro-165-Pro-171, Phe-365-Gly-370 (Arg-359 -Gly-370 in one subunit), and Glu-396 -Ala-400, and the average thermal factor of the main-chain atoms is 32 Å 2 . All models were of good quality with 99.8% of the residues falling in the most favorable SCHEME 1. The carbonyl oxygen of the ureido group in citrulline undergoes a nucleophilic attack on the ␣-P atom of ␣-phosphate in ATP to produce a citrullyl-AMP intermediate and a pyrophosphate as a leaving group. Aspartate then displaces the AMP moiety of the activated citrullyl-AMP intermediate, producing argininosuccinate (2, 3). and additionally allowed region and only 0.2% in the generously allowed region when the stereochemistry was assessed by PROCHECK (20) (Table I). Structure diagrams were drawn using the programs MOLSCRIPT (21), BOBSCRIPT (22), and Raster3D (23).
Subunit Structure-tAsS, which has been overexpressed in E. coli, has 400 residues per subunit, with a subunit M r of 44,815. The sequence alignment of tAsS with other AsSs by the program ClustalW (24) showed that tAsS has high sequence homology for eukaryotic AsS with identities of 52.7, 46.5, and 29.3% for human AsS, yeast AsS, and eAsS, respectively. The tAsS is folded into a tetrameric form with a noncrystallographic 222 symmetry. The subunit is divided into an ATP binding domain (N-terminal to Pro-165), a synthetase domain (Val-166 to Arg-359), and a C-terminal arm (Gln-360 to Cterminal) (Fig. 1). The fold of the ATP binding domain is similar to that of N-type ATP pyrophosphatases and has the consensus sequence of Tyr/Phe-Ser-Gly-Gly-Leu-Asp-Thr-Ser (PPloop) specific for the pyrophosphate of ATP (4,5).
When the C␣ carbon atoms are superimposed between subunits in tAsS⅐ATP⅐citrulline⅐aspartate, the average r.m.s. deviation is 0.14 Å with a maximum r.m.s. deviation of 0.16 Å. The corresponding average r.m.s. deviations of tAsS⅐AMP⅐ argininosuccinate and tAsS⅐AMP-PNP⅐arginine⅐aspartate⅐ Mg 2ϩ are 0.15 and 0.13 Å, with a maximum r.m.s. deviation of 0.16 and 0.15 Å, respectively. In these complexes, the four subunits have quite a similar structure. The subunit C␣ carbon atoms are fitted between these three complexes to give an average r.m.s. deviation of 0.15 Å with a maximum r.m.s. deviation of 0.19 Å, indicating that the subunit structures are essentially the same in these complexes. The subunit C␣ carbon atoms of the native tAsS can be superimposed onto those of tAsS⅐ATP (13), tAsS⅐AMP-PNP⅐arginine⅐succinate (13), tAsS⅐ ATP⅐citrulline⅐aspartate, tAsS⅐AMP⅐argininosuccinate, and tAsS⅐ AMP-PNP⅐arginine⅐aspartate⅐Mg 2ϩ within r.m.s. deviations of 0.14, 0.19, 0.19, 0.17, and 0.16 Å with maximum displacements of 0.88, 0.73, 0.95, 0.94, and 0.78 Å. Thus, the overall subunit structure of native tAsS is the same as those of its complexes. It was suggested that the cooperativity interaction caused by conformational changes may occur in the tetrameric AsS from bovine liver or yeast based on kinetics analysis (25,26). However, no significant change in the quaternary structure in tAsS was observed on binding of the ligands.
The eAsS shows the ATP-induced conformational change in the ATP binding domain, which is approximated to be a 3°( eAsS⅐ATP) and a 5°(eAsS⅐ATP⅐citrulline) rotation of the ATP binding domain toward the synthetase domain compared with that of native eAsS (12). The overall subunit structure of native   (27). However, the synthetase domain fitting by C␣ carbon atoms between native tAsS or tAsS complex and eAsS complex indicated that the ATP binding domain in native tAsS or tAsS complex further (but only slightly) rotates toward the synthetase domain to close the active site (Fig. 2). When the synthetase domain ␣-carbon atoms of native tAsS and tAsS⅐ATP⅐citrulline⅐aspartate are superimposed onto those of eAsS⅐ATP⅐citrulline, the synthetase domain shows average r.m.s. deviations of 0.31 and 0.31 Å, whereas the ATP binding domain shows average r.m.s. deviations of 0.68 and 0.67 Å, respectively. The native eAsS presents a wider surface of the active site than native tAsS or tAsS complex and shows the induced fit for the ATP binding domain to move and bind ATP (12). The tAsS does not show its overall conformational change upon binding of ATP or ATP analogue and has a common structure, i.e. a more closed form of the subunit than that of eAsS in the complex with ATP or ATP and citrulline, irrespective of the native or the complex form. There are four independent subunits in the asymmetric unit of tAsS crystals in contrast to one in eAsS crystals. The average numbers of intermolecular hydrogen bonds, ion pairs, and hydrophobic interactions per subunit are 10.5, 2, and 1.5 for native tAsS and 10, 2, and 0.5 for tAsS⅐ATP⅐citrulline⅐aspartate, respectively. The corresponding numbers are 27, 6, and 0 for native eAsS and 22, 6, and 2 for eAsS⅐ATP⅐citrulline, indicating that the closed form of tAsS observed in the crystals might not be due to the crystallographic artifactual intermolecular interactions. The thermophilic enzymes show increased rigidity at room temperature compared with their mesophilic counterparts (28 -31). On the basis of x-ray structures of mesophilic and thermophilic enzymes (32)(33)(34)(35)(36), it was suggested that the thermostable enzymes show a smaller domain movement compared with mesophilic ones (32). This assumption seems to be applicable to the behavior of the ATP binding domain of tAsS, which is different from that of eAsS. The ␣-phosphate of ATP, the ureido group of citrulline, and the amino group of aspartate are adjacent to provide a favorable arrangement for the reaction to proceed. ATPs are bound to crystallographically independent subunits in two conformers where the triphosphate of ATP is U-shaped (green) or S-shaped (red). AMP moieties of both conformers are in the same structure and interact with the active site residues in a similar manner. The U-shaped ATP causes its ␥-phosphate to interact with the PP-loop (red ribbon), whereas the ␥-phosphate of S-shaped ATP approaches the substrate binding sites of the synthetase domain. B, a close-up view of the active site of tAsS⅐AMP⅐argininosuccinate. AMP, the product (argininosuccinate), SO 4 2Ϫ , Arg-92, and Asp-121 are represented by ball-and-stick models. AMP binds to the ATP binding domain in the same manner as the AMP moiety of ATP in tAsS complexes. The ␣-phosphate of AMP is adjacent to the guanidino part of argininosuccinate and interacts with it. C, a close-up view of the active site of tAsS⅐AMP-PNP⅐arginine⅐aspartate⅐Mg 2ϩ . AMP-PNP, arginine, aspartate, SO 4 2Ϫ , Arg-92, and Asp-121 are represented by ball-and-stick models. The triphosphate of AMP-PNP is extended to the synthetase domain to interact with Ser-173. The ␤-phosphate forms salt bridges with the guanidino group of arginine and the protonated amino group of aspartate with the ␣-phosphate free from interactions with these groups. Three phosphate oxygen atoms of ATP and three water molecules are bound to the Mg 2ϩ ion in an octahedral geometry. ing 10 mM citrulline, aspartate, and MgCl 2 for 10 min at 193 K and cooled to 100 K in a cold nitrogen stream. ATP and the substrates (citrulline and aspartate) could be modeled into these peaks (Fig. 3A). This result indicates that the true enzyme-substrate complex of tAsS is trapped and that its structure provides a structural basis for elucidation of the catalytic action of the enzyme, although the Mg 2ϩ ion was not detected near the triphosphate of ATP. When the crystal soaked in the same solution for 20 min was used for data collection, the resolution of the data was lowered to 2.5 Å, and the residual electron density corresponding to the pyrophosphate moiety of ATP was separated into two diffused peaks, implying that the catalytic reaction proceeded (data not shown).
An extremely thermophilic bacterium, T. thermophilus, HB8 can grow at temperatures between 323 and 355 K with its optimum temperature being 348 K (37). The tAsS in solution has a specific activity of 0.92 mol min Ϫ1 mg Ϫ1 at 343 K, which is 1 ⁄5 that of yeast AsS (4.54 mol min Ϫ1 mg Ϫ1 at 303 K) (38). The specific activity of tAsS drops to 0.06 mol min Ϫ1 mg Ϫ1 at 293 K, indicating that at this temperature the subunit turnover number/min is about 1. The crystal seems to slow down the reaction speed to less than one-tenth that observed in solution. The enzyme-substrate complex was, thus, captured in the crystal.
There are two conformers in ATPs bound to the crystallographically independent four subunits; one is the U-shaped conformation of the triphosphate observed in a b or d subunit, and the other is the S-shaped conformation observed in an a or c subunit (Fig. 3A) (12). The S-shaped ATP has AMP moietyprotein interactions similar to those in U-shaped ATP. The adenine ring is sandwiched by the side chain of Ile-95 and the backbone between Tyr-7 and Ser-8 of the PP-loop. The mainchain nitrogen and oxygen of Ala-33 interact with the N 1 and N 6 amino groups, as observed in other ATP pyrophosphatase domains (4, 6, 7, 10). The main-chain oxygen and nitrogen of Ala-6 and Gly-114 forms hydrogen bonds with the O2Ј and O3Ј of the ribose moiety. The O1A of ␣-phosphate is involved in an electrostatic interaction with the guanidino group of Arg-92 with a distance of 3.2 Å in U-shaped ATP and 3.0 Å in S-shaped ATP.
The ␥-phosphate of U-shaped ATP approaches the consensus PP-loop and is coordinated to the loop with a ␤-phosphate free from direct hydrogen bonds, as was observed in x-ray structures of eAsS⅐ATP⅐citrulline (12), tAsS⅐ATP, and tAsS⅐AMP-PNP⅐arginine⅐succinate (13). The ␥-phosphate of S-shaped ATP leaves the PP-loop, loses interactions, approaches the citrulline and aspartate binding sites of the synthetase domain, and forms a hydrogen bond with the synthetase domain residue, Ser-173. Conversely, S-shaped ATP in NAD ϩ synthetase is bound to the ATP pyrophosphatase domain, and its ␤and ␥-phosphate interact with the PP-loop (6).
The substrates (citrulline and aspartate) bound to each of four independent subunits are in the same conformation and interact with the active site residues in a similar manner (Figs. 3A and 4A). The ␣-amino group and ␣-carboxylate of citrulline and both carboxylates of aspartate are bound to the sites specific for citrulline and aspartate, respectively, as observed in eAsS⅐citrulline⅐aspartate, eAsS⅐ATP⅐citrulline (12), and tAsS⅐ AMP-PNP⅐arginine⅐succinate (13). The ureido group of citrulline, the amino group of aspartate, and the ␣-phosphate group of ATP, which are directly involved in the catalytic reaction of AsS, are close to one another. The ␣-amino group of aspartate is hydrogen-bonded to the ␣-phosphate oxygen of ATP with a distance of 2.7-2.8 Å and to the ureido oxygen of citrulline with a distance of 3.0 -3.1 Å. The distances between the ureido oxygen and the ␣-P atoms of U-shaped and S-shaped ATPs are The AMP-PNP, arginine, and aspartate are drawn by thick bonds. The Mg 2ϩ ion is coordinated by three oxygen atoms of the triphosphate and three water molecules (W1, W2, and W3) in a tetrahedral geometry. The arginine and aspartate interact similarly with Thr-116, Asn-122, and Arg-124 to those seen in A. The interaction mode of sulfate anion is similar to that shown in B.
5.0 and 4.8 Å, respectively, which are 0.8 -1.0 Å shorter than the corresponding values in eAsS⅐ATP⅐citrulline (12). This shortening is mainly due to the shift of ␣-phosphate of ATP toward aspartate induced by the formation of salt bridges between the ␣-phosphate of ATP and the protonated amino group of aspartate and the guanidino group of Arg-92 and in part due to the further rotation of the ATP binding domain toward the synthetase domain compared with that in eAsS⅐ATP⅐citrulline.
Active Site of tAsS in the Complex with AMP and Argininosuccinate-The stereo structure and hydrogen-bonding scheme of the active site are shown in Figs. 3B and 4B, respectively. The difference Fourier map was calculated with the data collected using the tAsS⅐AMP crystal soaked in solutions containing 10 mM argininosuccinate for 10 min and cooled 100 K in a cold nitrogen stream, revealing three large peaks to which AMP, argininosuccinate, and sulfate anion could be assigned. This complex is a true enzyme-substrate complex in the reverse reaction. When the diffraction data were collected using the crystal soaked in the same solution for 24 h, the resolution of the data was lowered to 2.6 Å, and the residual electron density corresponding to argininosuccinate was separated into two peaks (data not shown).
AMP is located at the same place as the AMP moieties in tAsS complexes containing ATP or AMP-PNP. The sulfate anion occupies the binding site for ␥-phosphates of U-shaped ATP or AMP-PNP and interacts with the PP-loop. When citrulline and aspartate in tAsS⅐ATP⅐citrulline⅐aspartate are superimposed onto the argininosuccinate, only the side chain atoms of citrulline show significant deviations from the corresponding ones in argininosuccinate (Fig. 3, A and B). The ␣-phosphate of AMP and the guanidino part of argininosuccinate are adjacent, and the O2A and O3A of AMP are hydrogen-bonded to the imino groups of argininosuccinate. Therefore, the carbon atom of the guanidino part in argininosuccinate is accessible to the ␣-phosphate oxygen of AMP to reproduce citrulline and aspartate.
Active Site of tAsS in the Complex with AMP-PNP, Arginine, Aspartate, and Mg 2ϩ -The stereo structure and hydrogenbonding scheme of the active site are shown in Figs. 3C and 4C, respectively. After AMP-PNP, arginine, and aspartate were assigned to the residual electron density peaks, additional peaks remained into which sulfate and Mg 2ϩ ion could be modeled. The adenosine moiety of AMP-PNP assumes the same binding mode as those observed in tAsS complexes containing ATP or ATP-PNP. However, the triphosphate group of AMP-PNP has an extended conformation different from that observed in U-shaped or S-shaped ATP. The ␥-phosphate leaves the PP-loop and forms a hydrogen bond with the synthetase domain residue, Ser-173, as observed in the S-shaped ATP in tAsS⅐ATP⅐citrulline⅐aspartate. The ␤-phosphate of AMP-PNP rather than the ␣-phosphate approaches the synthetase domain to interact with the guanidino group and the ␣-amino group of bound arginine and of aspartate, respectively. The PP-loop binds sulfate in the place of ␥-phosphate. Interestingly, the Mg 2ϩ ion is in octahedral coordination with three oxygen atoms of the triphosphate group and three water molecules.
Conformation and Binding Mode of ATP and Substrates-The AMP moiety of ATP or an ATP analogue binds similarly in all the AsS complexes so far determined by x-ray methods (11)(12)(13), whereas the triphosphate group assumes various conformations. In tAsS complexes, the bound ATP or AMP-PNP has a U-shaped, S-shaped, or an extended conformation. In eAsS complexes, the bound ATP has a U-shaped or another extended conformation (12). The mean temperature factor and occupancy factors of ATP in tAsS⅐ATP⅐citrulline⅐aspartate were refined to be 53.3 Å 2 and 0.93. Considerably high temperature factors for ATP (44.2, 47.3, and 48.3 Å 2 in eAsS⅐ATP, eAsS⅐ATP⅐citrulline, and tAsS⅐ATP, respectively) were also observed in other ATP complexes of tAsS and eAsS (12,13), reflecting the flexibility of the bound ATP. Thus, the triphosphate group of bound ATP is mobile and can easily change its conformation. The conformational flexibility of the triphosphate is probably essential for ATP binding (12) and catalytic action in AsS. The triphosphate of S-shaped ATP bound to tAsS is free from hydrogen bonds with the PP-loop (Fig. 3A), whereas both ␤and ␥-phosphates of S-shaped ATP bound to NAD ϩ synthetase interact with the PP-loop (6). This is mainly because the ␣-phosphate of ATP in tAsS is shifted by 2.5 Å from the PP-loop toward the synthetase domain compared with the corresponding ␣-phosphate in NAD ϩ synthetase complex. The shift is probably induced by the salt bridge interaction of ␣-phosphate with Arg-92 (Arg-106 in eAsS) and the protonated amino group of aspartate to cause the ␣-phosphate to approach the ureido group of citrulline. The Arg-92 and the protonated amino group of bound aspartate play a role, at least in part, to locate ␣-phosphate in a favorable position for a nucleophilic attack by the ureido oxygen of citrulline.
The ␣-amino and ␣-carboxyl groups of citrulline or its analogue (arginine) are bound to the deep pocket of the synthetase domain by the formation of salt bridges and hydrogen bonds with the active site residues. The side-chain conformation of citrulline in tAsS⅐ATP⅐citrulline⅐aspartate is similar to that in eAsS⅐ATP⅐citrulline but is different from that of arginine in tAsS⅐AMP-PNP⅐arginine⅐succinate (Fig. 5). The ureido group of citrulline in tAsS⅐ATP⅐citrulline⅐aspartate is at a distance of about 4.8 or 5.0 Å (5.8 Å in eAsS⅐ATP⅐citrulline) from the ␣-P atom of ATP ␣-phosphate. On the other hand, the guanidino group of arginine is within a distance of 3.2 Å from the ␣-P atom of ␣-phosphate. The aspartate in tAsS or eAsS complexes is located at the same position, with their ␣and ␤-carboxylates bound to the specific sites of the synthetase domain.
Mechanistic Implication-The first step catalyzed by AsS has been proposed to be the nucleophilic attack of the citrulline ureido oxygen on the ␣-P atom of ATP followed by the release of the pyrophosphate and the formation of a citrullyl-AMP intermediate (Scheme 1) (2,3,38). The activated ureido carbon atom of the citrullyl-AMP intermediate is then attacked by the ␣-amino group of L-aspartate, producing AMP and argininosuccinate. Interestingly, the former step of beef liver AsS is stimulated by the bound aspartate by a factor of 600 (39). We have determined the structures of tAsS⅐ATP⅐citrulline⅐aspartate as the true enzyme-substrate complex and tAsS⅐AMP⅐arginino- succinate as the enzyme-product complex, which will be most important in elucidating the mechanism of catalytic action in view of the stereochemistry. The stereochemistry of catalysis along the reaction pathway is proposed based on both structures described in this paper and previously determined structures (13) (Fig. 6).
First, ATP binds to the ATP binding domain with a U-shaped conformation, and one of the substrates, citrulline, then comes into the active site with its ureido group forming hydrogen bonds with Ser-173 and Ser-182 (Fig. 6A) (40). The ␥-phosphate of ATP is coordinated to the PP-loop, but the ␤-phosphate is free from the interaction with the PP-loop. The distance from the ␣-P atom of ATP to the ureido oxygen of citrulline is 5.8 Å. Arg-92 makes electrostatic interaction with the ␣-phosphate oxygen at a distance of 4.0 Å. Fig. 6A is based on the x-ray structure of the tAsS complex with ATP and citrulline (Protein Data Bank code 1J21).
Another substrate, aspartate, enters into the active site and neighbors citrulline (Fig. 6, A and B). The electrostatic interactions of the ATP ␣-phosphate with the protonated amino group of aspartate and the guanidino group of Arg-92 draw the ␣-phosphate toward the bound citrulline and Arg-92, reducing the distance between the ␣-P atom of ATP and the ureido oxygen of citrulline from 5.8 to 5.0 Å. The ␣-phosphate forms salt bridges of 3.2 and 2.7 Å with the protonated amino group and the guanidino group of Arg-92, respectively. The protonated amino group of aspartate bridges the reaction sites of ATP and citrulline by hydrogen bonds. Thus, these hydrogenbonding interactions may play a role in acceleration of the reaction to yield the ATP-citrullyl intermediate (40). Fig. 6B is based on the x-ray structure of the tAsS complex with Ushaped ATP, citrulline, and aspartate. The ␥-phosphate of ATP leaves the PP-loop, approaches the synthetase domain, and interacts with the carboxylate of Asp-12, the hydroxy group of Ser-173, and two water molecules (Fig. 6, B and C). In this process ATP changes its triphosphate structure from a Ushaped to an S-shaped conformation. The distance between the ␣-P atom of ATP and the ureido oxygen is reduced to 4.8 Å, which is still too long for the ureido oxygen to undertake a nucleophilic attack on the ␣-P atom of ATP. Fig. 6C is based on the x-ray structure of the tAsS complex with S-shaped ATP, citrulline, and aspartate.
The substrate citrulline changes its side chain conformation with its ␣-amino and ␣-carboxylate groups fixed (Fig. 6, C and  D). The ureido group of citrulline approaches the ␣-phosphate of ATP and is located at a position favorable to the nucleophilic attack of the ureido oxygen on the ␣-P atom of the ␣-phosphate. Fig. 6D was modeled based on the x-ray structure of the tAsS complex with AMP-PNP, arginine, and succinate (Fig. 5) (13). The citrulline side chain is relocated to have a conformation similar to that of the arginine side chain in the complex because the side chain structure of citrulline is analogous to that of arginine. The ureido oxygen of citrulline is at a distance of 3.0 Å from the ␣-P atom of ATP. The ureido group of this model interacts with the protonated amino group of the substrate aspartate, the ␣-phosphate of ATP, and the carboxylate of Asp-121. These interactions may assist the conformational change in the citrulline side chain. Both the interactions of the protonated amino group of aspartate with the ␣-phosphate and also the ureido oxygen are maintained through the process illustrated in Fig. 6, C-D. The ␣-phosphate P-O bond and the ureido carbonyl group are, thus, polarized to stimulate the adenylation of citrulline.
The ureido oxygen undergoes a nucleophilic attack on the ␣-P atom of ATP to yield an activated citrullyl-AMP intermediate and a pyrophosphate (Fig. 6, D and E). The pyrophosphate as a leaving group might be a candidate to accept a proton from the protonated ␣-amino group of the substrate aspartate (13). The intermediate is formed without changing the structure and location around both ends of the reactants. Fig. 6E was modeled based on the x-ray structure of the tAsS complex with AMP and argininosuccinate (Fig. 3B).
The deprotonated amino group of the substrate aspartate is oriented toward the ureido carbon of the citrullyl-AMP intermediate and undergoes a nucleophilic attack on it to yield argininosuccinate (Fig. 6, E and F). Fig. 6F is based on the x-ray structure of the tAsS complex with AMP and argininosuccinate (Fig. 3B). The guanidino part of argininosuccinate is close to the ␣-phosphate of AMP at distances of 3.0 Å (N1-O2A) and 3.5 Å (N2-O3A) (Fig. 4B). In other words AMP and argininosuccinate are located in a stereochemical orientation favorable to the reverse reaction, where argininosuccinate undergoes a nucleophilic attack by the ␣-phosphate oxygen of AMP to reproduce the citrullyl-AMP intermediate.
The assembly of the ␣-phosphate of ATP, the ureido group of citrulline, and the ␣-amino group of aspartate is most important in the catalytic action of tAsS, indicating that the proximity and orientation effect of these groups are dominant factors in the catalysis. In addition to this, the mobile side chain of citrulline and the flexible triphosphate of ATP play important roles in the catalytic action. In eAsS, the rotation of the ATP binding domain toward the synthetase domain was shown to be necessary for the catalysis (12). The tAsS has a structure similar to but with a more closed form than that of eAsS⅐ATP⅐ citrulline. The tAsS may not necessitate a further rotation of the ATP binding domain for the catalytic action. The distance between the ureido oxygen of citrulline and the ␣-P atom of ATP in the tAsS complex is 4.8 -5.0 and 0.8 -1.0 Å shorter than the corresponding distance in the eAsS complex. On the basis of the side-chain location of the citrulline analog (arginine) in the tAsS⅐AMP-PNP⅐arginine⅐succinate complex, it was suggested that citrulline changes its side-chain direction, and its ureido group approaches the ␣-P atom within a distance of 3.0 Å with its ␣-amino and ␣-carboxyl groups fixed in the deep pocket of the active site. Moreover, the citrullyl-AMP intermediate is successfully modeled into the active site as shown in Fig. 6E, where the adenine moiety and the ␣-amino and ␣-carboxyl groups of the intermediate model have the same locations as those of the corresponding regions in ATP and citrulline in Fig.  3A or AMP and argininosuccinate in Fig. 3B. Thus, thermal fluctuations of the bound substrates and the residues interact-ing with them bring the reaction sites sufficiently close together so that the catalysis may proceed, although the minor conformational change at the domain level cannot be completely excluded.
The magnesium ion is required for maximal activity of AsS (38). Mg 2ϩ coordinated to the triphosphate of ATP was first observed in the x-ray structure of tAsS complexed with arginine (citrulline analogue) and aspartate. However, the active site structure showed that the ␤-phosphate of ATP, not the ␣-phosphate, interacts with the bound arginine and aspartate (Fig. 3C). The access of the arginine side chain to the ␣-phosphate seems to be blocked by the ␤-phosphate unless the triphosphate of ATP changes its conformation. The elucidation of how the structure observed in this complex is related to the catalytic process must await further structural studies of the enzyme complex with Mg 2ϩ coordinated by ATP or ATP analogues.