From the
Department of Chemistry, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan and
Harima Institute/SPring-8, The Institute of Physical and Chemical Research (RIKEN), Sayo-gun, Hyogo 679-5148, Japan
Received for publication, December 26, 2002 , and in revised form, March 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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It has been proposed that GMP synthetase, NAD+ 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 (PP-loop) 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+ synthetase (68), 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+ 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 eAsS·ATP and eAsS·ATP·citrulline have been determined by x-ray methods (12). Comparisons of these two complexes with eAsS and eAsS·citrulline·aspartate 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), tAsS·ATP, and tAsS·AMP-PNP·arginine·succinate 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 AMPPNP, 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 Mg2+. 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 eAsS·ATP·citrulline. 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 enzyme-product 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 tAsS·AMP-PNP·arginine·aspartate·Mg2+ at 2.15-Å resolution.
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EXPERIMENTAL PROCEDURES |
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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 x 0.4 x 0.2 mm.
Crystals of the true enzyme-substrate complex (tAsS·ATP· citrulline·aspartate) were obtained at 293 K by soaking the tAsS·ATP crystals in solutions containing 10 mM citrulline, 10 mM aspartate, and 10 mM MgCl2 for 10 min before data collection. Crystals of the true enzyme-product complex (tAsS·AMP·argininosuccinate) were obtained at 293 K by soaking the tAsS·AMP crystals in solutions containing 10 mM argininosuccinate for 10 min before data collection. Crystals of tAsS·AMP-PNP·arginine·aspartate·Mg2+ 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 MgCl2, 100 mM arginine, and 100 mM aspartate.
The x-ray diffraction data sets for the tAsS·ATP·citrulline·aspartate crystal, the tAsS·AMP·argininosuccinate crystal, and the tAsS·AMPPNP·arginine·aspartate·Mg2+ 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 = b = 229.1 and c = 159.8 Å with the space group of R3. There are four subunits in the asymmetric unit, and 72% of the crystal volume is occupied by solvent.
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Structure Determination and RefinementRefinement 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 Rfree values. The Rfactor and Rfree values were decreased after several rounds of refinement and manual rebuilding. When the Rfactor value decreased below 30%, the sigmaA-weighted FoFc 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 sigmaA-weighted FoFc 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 SO4 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 Rfactor and Rfree 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 SO4 range from 0.87 to 1.00 with a mean value of 0.94.
Enzyme AssayEnzymatic 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 MgCl2, 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 ± 0.01 µmol min1 mg1 at 293 K and 0.92 ± 0.02 µmol min1 mg1 at 343 K.
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RESULTS AND DISCUSSION |
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Subunit StructuretAsS, which has been overexpressed in E. coli, has 400 residues per subunit, with a subunit Mr 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 C-terminal) (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 (PP-loop) specific for the pyrophosphate of ATP (4, 5).
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When the C carbon atoms are superimposed between subunits in tAsS·ATP·citrulline·aspartate, 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 tAsS·AMP· argininosuccinate and tAsS·AMP-PNP·arginine·aspartate· Mg2+ 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 tAsS·ATP (13), tAsS·AMP-PNP·arginine·succinate (13), tAsS· ATP·citrulline·aspartate, tAsS·AMP·argininosuccinate, and tAsS· AMP-PNP·arginine·aspartate·Mg2+ 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° (eAsS·ATP) and a 5° (eAsS·ATP·citrulline) rotation of the ATP binding domain toward the synthetase domain compared with that of native eAsS (12). The overall subunit structure of native tAsS or tAsS·ATP·citrulline·aspartate overlaps well with that of eAsS·ATP·citrulline because the subunit -carbon atoms of native tAsS and tAsS complex are fitted to those of the eAsS complex with average r.m.s. deviations of 0.63 and 0.61 Å, except for the loop regions, where the insertion and deletion of amino acid residues occur (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 tAsS·ATP·citrulline·aspartate are superimposed onto those of eAsS·ATP·citrulline, 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.
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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 tAsS·ATP·citrulline·aspartate, respectively. The corresponding numbers are 27, 6, and 0 for native eAsS and 22, 6, and 2 for eAsS·ATP·citrulline, 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 (2831). On the basis of x-ray structures of mesophilic and thermophilic enzymes (3236), 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.
Active Site of tAsS in the Complex with ATP, Citrulline, and AspartateThe stereo structure and the hydrogen-bonding scheme of the active site are shown in Figs. 3A and 4A, respectively. Three positive electron density peaks were revealed on the difference Fourier map based on the diffraction data collected using the tAsS·ATP crystal soaked in solutions containing 10 mM citrulline, aspartate, and MgCl2 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 Mg2+ 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).
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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 µmol min1 mg1 at 343 K, which is that of yeast AsS (4.54 µmol min1 mg1 at 303 K) (38). The specific activity of tAsS drops to 0.06 µmol min1 mg1 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 moiety-protein 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 main-chain nitrogen and oxygen of Ala-33 interact with the N1 and N6 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 O2' and O3' 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 eAsS·ATP·citrulline (12), tAsS·ATP, and tAsS·AMPPNP·arginine·succinate (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+ 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 eAsS·citrulline·aspartate, eAsS·ATP·citrulline (12), and tAsS· AMP-PNP·arginine·succinate (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 2.72.8 Å and to the ureido oxygen of citrulline with a distance of 3.03.1 Å. The distances between the ureido oxygen and the
-P atoms of U-shaped and S-shaped ATPs are 5.0 and 4.8 Å, respectively, which are 0.81.0 Å shorter than the corresponding values in eAsS·ATP·citrulline (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 eAsS·ATP·citrulline.
Active Site of tAsS in the Complex with AMP and ArgininosuccinateThe 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 tAsS·AMP 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 tAsS·ATP·citrulline·aspartate 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 Mg2+The stereo structure and hydrogen-bonding 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 Mg2+ 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 AMPPNP 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 tAsS·ATP·citrulline·aspartate. 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 Mg2+ 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 (1113), 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 tAsS·ATP·citrulline·aspartate were refined to be 53.3 Å2 and 0.93. Considerably high temperature factors for ATP (44.2, 47.3, and 48.3 Å2 in eAsS·ATP, eAsS·ATP·citrulline, and tAsS·ATP, 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+ 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+ 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 tAsS·ATP·citrulline·aspartate is similar to that in eAsS·ATP·citrulline but is different from that of arginine in tAsS·AMP-PNP·arginine·succinate (Fig. 5). The ureido group of citrulline in tAsS·ATP·citrulline·aspartate is at a distance of about 4.8 or 5.0 Å (5.8 Å in eAsS·ATP·citrulline) 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.
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Mechanistic ImplicationThe 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 tAsS·ATP·citrulline·aspartate as the true enzyme-substrate complex and tAsS·AMP·argininosuccinate 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).
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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
[PDB]
).
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 hydrogen-bonding 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 U-shaped 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 U-shaped 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, CD. 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 (N1O2A) and 3.5 Å (N2O3A) (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 eAsS·ATP· 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 4.85.0 and 0.81.0 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 tAsS·AMP-PNP·arginine·succinate 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 interacting 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). Mg2+ 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 Mg2+ coordinated by ATP or ATP analogues.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Scientific Research on Priority Area from the Ministry of Education, Science, Sports, and Culture of Japan B:13125207 (to K. H.) and by a Research Grant from the Japan Society for the Promotion of Science (Category B 13480196 (to K. H.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 81-6-6605-2507; Fax: 81-6605-3131; E-mail: hirotsu{at}sci.osaka-cu.ac.jp.
1 The abbreviations used are: AsS, argininosuccinate synthetase; eAsS, argininosuccinate synthetase from E. coli; tAsS, argininosuccinate synthetase from T. thermophilus HB8; AMP-PNP, adenylyl imidodiphosphate; r.m.s., root mean square.
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