Satu Pekkala

Ana Isabel Martínez

Belén Barcelona

José Gallego

Elena Bendala

Igor Yefimenko

Vicente Rubio
email: rubio@ibv.csic.es

Javier Cervera
email: cervera@cipf.es

Structural insight on the control of urea synthesis: identification of the binding site for N-acetyl-L-glutamate, the essential allosteric activator of mitochondrial carbamoyl phosphate synthetase

Keywords: acetylglutamate, allosteric site, N-carbamoyl-L-glutamate, urea cycle defects, carbamoyl phosphate synthetase deficiency, carglumic acid

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Carbamoyl phosphate synthetases (CPSs) are complex multidomain enzymes found in virtually all organisms [START_REF] Rubio | The carbamoyl-phosphate synthase family and carbamate kinase: structure-function studies[END_REF]. These allosterically-regulated controlling enzymes synthesize carbamoyl phosphate (CP), a crucial compound for life, in the first committed steps of the routes of synthesis of pyrimidines, arginine, and, in ureotelic vertebrates (mainly amphibians and mammals), urea. In these latter animals (including humans) CPSI, the enzyme that catalyzes the first step of the urea cycle, has an absolute requirement for the allosteric activator N-acetyl-L-glutamate (NAG) [START_REF] Grisolía | Catalytic role of glutamate derivatives in citrulline biosynthesis[END_REF][START_REF] Rubio | Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor[END_REF]. NAG plays a key role in controlling the flow of nitrogen through the urea cycle [START_REF] Meijer | Nitrogen metabolism and ornithine cycle function[END_REF][START_REF] Shigesada | Variations in acetylglutamate level and its possible significance in control of urea synthesis in mammalian liver[END_REF][START_REF] Tatibana | Regulation of urea biosynthesis by the acetylglutamate-arginine system[END_REF], as exemplified by the deficiency of the enzyme making NAG, NAG synthase, a deficiency that causes generally severe neonatal hyperammonaemia [START_REF] Bachmann | N-acetylglutamate synthetase deficiency: a disorder of ammonia detoxication[END_REF] due to secondary CPSI deficiency. Interestingly, there is highly effective substitutive therapy for NAG synthase deficiency, by using the deacylase-resistant NAG analogue N-carbamoyl-L-glutamate [START_REF] Bachmann | N-acetylglutamate synthetase deficiency: a disorder of ammonia detoxication[END_REF][START_REF] Rubio | Treating urea cycle defects[END_REF] (NCG, also called carglumic acid; proprietary drug name, Carbaglu ® ).

Although CPSI activation by NAG is known since very long [START_REF] Grisolía | Catalytic role of glutamate derivatives in citrulline biosynthesis[END_REF], and despite the use of NCG in the treatment of NAGS deficiency for nearly 30 years [START_REF] Bachmann | N-acetylglutamate synthetase deficiency: a disorder of ammonia detoxication[END_REF][START_REF] Rubio | Treating urea cycle defects[END_REF], the CPSI NAG site has remained uncharacterized. Such characterization is a requisite for understanding the mechanism of the extreme allosteric activation of CPSI by NAG, and would have practical interest for identifying CPSI deficiency resulting from NAG site mutations. The patients carrying these mutations might be amenable to treatment with very high doses of NCG or with NCG analogues tailored to bind better to the mutant form of the site.

Until now, the only existing physical information about the NAG site was its gross localization at the CPSI C-terminal domain, deduced from the observation that this domain of ~16 kDa is the only part of CPSI (a polypeptide of ~160 kDa) that is photoaffinity-labelled by the photoactivatable NAG analogue N-chloroacetyl-L-[ 14 C]glutamate ([ 14 C]ClNAG) [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF]. In fact, this C-terminal domain is called the allosteric or regulatory domain in all types of CPS because it is believed to be involved exclusively in effector binding, relaying the regulatory information to the catalytic domains (two homologous 40 kDa domains that catalyze the phosphorylations of bicarbonate and carbamate) [START_REF] Rubio | Structure-activity correlations in carbamoyl phosphate synthetases. In: Carbon dioxide fixation and reduction in biological and model systems[END_REF]. The evidence for this conclusion was based on photoaffinity labelling studies, first performed with CPSI [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF], and then with Escherichia coli CPS (eCPS) [START_REF] Rubio | Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain[END_REF][START_REF] Cervera | Photoaffinity labelling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor[END_REF][START_REF] Cervera | The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry[END_REF][START_REF] Bueso | Location of the binding site for the allosteric activator IMP in the COOH-terminal domain of Escherichia coli carbamyl phosphate synthetase[END_REF][START_REF] Bueso | Photoaffinity labeling with the activator IMP and site-directed mutagenesis of histidine 995 of carbamoyl phosphate synthetase from Escherichia coli demonstrate that the binding site for IMP overlaps with that for the inhibitor UMP[END_REF]; on domain-deletion and domain-exchange experiments carried out with eCPS and with CPSII from hamster CAD [START_REF] Czerwinski | Regulatory changes in the control of carbamoyl phosphate synthetase induced by truncation and mutagenesis of the allosteric binding domain[END_REF][START_REF] Liu | Identification of the regulatory domain of the mammalian multifunctional protein CAD by the construction of an Escherichia coli hamster hybrid carbamyl-phosphate synthetase[END_REF] (CAD catalyzes the three initial steps of pyrimidine biosynthesis); on the observation that phosphorylation of this domain of hamster CAD decreased UTP inhibition of CPSII [START_REF] Carrey | Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD[END_REF]; and, more recently, on the determination of the crystal structures of eCPS in complex with the inhibitor UMP [START_REF] Thoden | Long-range allosteric transitions in carbamoyl phosphate synthetase[END_REF] or with the activators ornithine [START_REF] Thoden | The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution[END_REF] or IMP [START_REF] Thoden | The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase[END_REF]. However, the structural information gathered on eCPS has not allowed to infer where and how does NAG bind to CPSI, given the poor sequence identity ( 20%) between the allosteric domains of CPSI and eCPS [START_REF] Rubio | Structure-activity correlations in carbamoyl phosphate synthetases. In: Carbon dioxide fixation and reduction in biological and model systems[END_REF], and because of the uncertainties on whether the activations of both enzymes occur with similar mechanisms, since eCPS is active in the absence of its effectors [START_REF] Meister | Glutamine-dependent carbamyl phosphate synthetase: catalysis and regulation[END_REF], whereas CPSI is essentially inactive when NAG is not present [START_REF] Rubio | Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor[END_REF]. In addition, the two activators of eCPS, ornithine and IMP, bind to different sites [START_REF] Thoden | The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution[END_REF][START_REF] Thoden | The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase[END_REF], raising the question of which one of these sites, if any, would be analogous to the NAG site if the allosteric domains of eCPS and CPSI were similar.

A unique opportunity for identifying the NAG site in CPSI was provided recently when, as a part of a high-throughput structural genomics/proteomics effort, the crystal structure of the C-terminal domain of human CPSI (hCPSI), generated in isolated form in a cell-free protein expression system, was deposited in the Protein Data Bank [START_REF] Xie | Crystal structure of MGS domain of carbamoyl-phosphate synthetase from homo sapiens[END_REF]. Using cavity searching and flexible docking we now identify the binding site for NAG in this structure. From the shape of the binding pocket and from the chemical characteristics of the residues lining this site, we define unambiguously the way in which NAG binds to CPSI. This binding mode fits closely the expectations derived from previous studies on structure-activity relationships for NAG analogues [START_REF] Grisolía | Catalytic role of glutamate derivatives in citrulline biosynthesis[END_REF][START_REF] Schooler | 2-Acetoxyglutarate as an activator of carbamoyl phosphate synthetase[END_REF][START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF], and is supported by experimental evidence reported in this work: the fine-mapping of the [ 14 C]ClNAG photoaffinity labelling site within CPSI, and the results of site-directed mutagenesis experiments involving residues predicted to interact with the bound activator. The implications of our findings for the diagnosis and treatment of CPSI deficiency are discussed, highlighting with one experimental example the possibility of using modified NAG analogues to improve activation of defective CPSI forms carrying specific mutations in the NAG site. Finally, the proposed NAG binding mode is compared with those of the bacterial activators IMP and ornithine, discussing possible evolutionary origins of the NAG site.

EXPERIMENTAL

Identification of the NAG site and docking of the NAG molecule

The structure deposited in the Protein DataBank (PDB) as file 2YVQ [START_REF] Xie | Crystal structure of MGS domain of carbamoyl-phosphate synthetase from homo sapiens[END_REF], corresponding to the C-terminal domain of hCPS (residues 1343-1478) was analyzed for the presence of cavities with VOIDOO (version 3.3.4) [START_REF] Kleywegt | Detection, delineation, measurement and display of cavities in macromolecular structures[END_REF]. NAG was docked into the pocket identified with VOIDOO using the GOLD (version 4.0.1) package [START_REF] Verdonk | Improved protein-ligand docking using GOLD[END_REF], a genetic algorithm for docking flexible ligands into protein binding sites that has been optimized for the prediction of ligand binding positions. Docking calculations were unrestrained and used the GoldScore fitness function. The preference of NAG for binding in this pocket was confirmed by GOLD docking runs involving the entire hCPS C-terminal domain or by docking NAG into alternative hCPS C-terminal domain cavities. GOLD calculations indicated the need for movement of the W1410 residue forming the pocket lid (see below) for full NAG entry. Subsequent docking runs in which the side chains of W1410 and the overlying F1445 and N1413 residues were allowed to rotate freely, yielded a highly stable binding pose in which the W1410 ring had flipped 180 o while maintaining the stacking interaction with the neighbouring F1445 and keeping a commonly observed side chain conformation [START_REF] Lovell | The Penultimate Rotamer Library[END_REF]. Subsequent GOLD calculations using this alternative protein conformation led to very good NAG docking scores, with almost total convergence (95%) of the NAG binding poses (19 of 20 solutions with an rmsd conformational deviation of 1.35 Å or less; see below). The final hCPS-NAG model was generated by further refining the best GOLD solution by restrained energy minimization using the ff03 force field [START_REF] Duan | A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations[END_REF] of AMBER 8.0 [START_REF] Case | The Amber biomolecular simulation programs[END_REF], and a generalized Born model for solvent simulation. This last minimization used as restraints the protein-NAG hydrogen bonds detected in the converged GOLD binding poses (see below).

Structural analysis and preparation of illustrations

Structural superimpositions were performed with the LSQKAB option of the CCP4 suite [START_REF]CCP4 (Collaborative Computational Project 4) (1994) The CCP4 suite: programs for protein crystallography[END_REF]. Solvent accessibility (probe radius, 1.4 Å) of residues within the eCPS (bound to IMP; PDB file 1CE8 [START_REF] Thoden | The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase[END_REF]) and hCPS (PDB file 2YVQ [START_REF] Xie | Crystal structure of MGS domain of carbamoyl-phosphate synthetase from homo sapiens[END_REF]) structures was calculated with the AREAIMol program of CCP4 [START_REF]CCP4 (Collaborative Computational Project 4) (1994) The CCP4 suite: programs for protein crystallography[END_REF]. Surface potential was calculated with GRASP [START_REF] Honig | Classical electrostatics in biology and chemistry[END_REF]. Multiple sequence alignments were prepared with CLUSTALW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position specific gap penalties and weight matrix choice[END_REF], using default parameters. Figures depicting protein structures were prepared with PyMOL [START_REF] Delano | The PyMOL Molecular Graphics System[END_REF].

Fine-mapping of the rCPSI photoaffinity labelling site

Purification of rat liver CPSI, synthesis of ClNAG (unlabelled or labelled with [ 14 C] in the glutamate moiety) and photoaffinity labelling of the enzyme were already reported [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF]. Limited proteolysis with V8 staphylococcal protease, HPLC isolation of the resulting 18.9-kDa C-terminal labelled fragment, and electrophoresis, fluorography and Edman sequencing were as previously reported for [ 14 C]UMP-labelled eCPS [START_REF] Rubio | Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain[END_REF]. Digestion of the 18.9-kDa labelled fragment with either cyanogen bromide (CNBr) [START_REF] Gross | The cyanogen bromide reaction[END_REF] or trypsin (alone or followed by V8 protease under conditions at which V8 protease cleaves only at glutamate residues [START_REF] Cervera | Photoaffinity labelling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor[END_REF][START_REF] Drapeau | Cleavage at glutamic acid with staphylococcal protease[END_REF]) were also as reported for [ 14 C]UMP-labelled eCPS [START_REF] Cervera | Photoaffinity labelling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor[END_REF]. o-Iodosobenzoic acid cleavage of the 18.9-kDa labelled fragment was carried out in the dark at 21º C for 24 h according to [START_REF] Mahoney | High-yield cleavage of tryptophanyl peptide bonds by o-Iodosobenzoic acid[END_REF], in a volume of 50 µl. The reaction was terminated with 1 µl of 1 mM dithioerythritol and the mixture was desalted by centrifugal gel filtration [START_REF] Penefsky | Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase[END_REF] through a 1-ml Sephadex G-25 column equilibrated with 50% acetonitrile/ 0.1% trifluoroacetic acid (both vol/vol). After removal of the solvent by lyophilisation, the residue was subjected to SDS-urea-phosphate PAGE using a formulation [START_REF] Cervera | Photoaffinity labelling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor[END_REF] that is suitable for small peptides. For determination of the mass of tryptic or tryptic/V8-protease fragments the Sephacryl S200-SDS gel-filtration technique [START_REF] Cervera | Photoaffinity labelling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor[END_REF] was used. The elution position of each component is given as the distribution coefficient, K d =(V e -V 0 )/(V i -V 0 ), taking V 0 , V i , and V e as the volumes of elution of BSA, mercaptoethanol, and the component of interest, respectively.

For mass spectrometric (MS) analysis, the enzyme was subjected as described above to UV-irradiation in the presence of either ClNAG (experimental irradiation) or NAG (control irradiation) followed by limited proteolysis with V8 protease. After SDS-PAGE, the Coomassie-stained 18.9-kDa band corresponding to the C-terminal domain of the enzyme from the experimental and control samples was excised from each gel and was subjected to peptide fingerprinting using trypsin (Promega) [START_REF] Shevchenko | Linking genome 14 and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels[END_REF] and MALDI-TOF MS analysis in a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, USA) used in linear mode, with concomitant MS/MS analysis of relevant peaks to acquire sequence information. The MS and MS/MS information was analyzed with MASCOT [START_REF] Perkins | Probability-based protein identification by searching sequence databases using mass spectrometry data[END_REF]. The mass spectrometric analysis was carried out in the Proteomics Core Facility of the Centro de Investigación Príncipe Felipe (CIPF Valencia)-Proteored (Genoma España).

Recombinant rCPSI production and mutagenesis

Recombinant rat CPSI (rCPSI) was produced using a commercial kit (Bac-to-Bac ® Baculovirus Expression System, from Invitrogen). The rCPSI cDNA carried by pHN3491 [START_REF] Nyunoya | Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat[END_REF] was subcloned into the pFastBac-HTA vector provided in the kit, using standard techniques for DNA manipulation and for engineering the cDNA to replace the CPSI mitochondrial targeting sequence by the 6xHis, 28-residue, N-terminal tag MSYYHHHHHHDYDIPTTENLYFQGAMDP, provided by the vector. After transformation of E. coli Max Efficiency DH10Bac cells (from Invitrogen), the recombinant bacmid was recovered and transfected into Sf9 insect cells using Cellfectin. The resulting virus was then used to infect Sf9 cells. The best expression (monitored by SDS-PAGE) was obtained at a virus-to-cell ratio of 2, by incubation at 27ºC for 65 hours. Subsequent steps were at 4ºC. After centrifugation of a 50-ml culture, the cells were suspended in 3 ml of 50 mM glycylglycine, pH 7/20 mM KCl/20% glycerol/0.1% Triton X-100/5 mM imidazole/0.1% histidine-tagged specific protease inhibitor mixture/5 M cysteine protease irreversible inhibitor E-64 (the last two components were from Sigma). The cell suspension was frozen (in liquid nitrogen) and thawed 2 times, it was centrifuged, and the supernatant was mixed with 0.5 ml packed TALON resin (from Clontech) equilibrated in the same solution without Triton X-100 or protease inhibitors. After 30-min gentle end-to-end mixing and 2-times washing with 5 ml of equilibration solution containing 10 mM imidazole, the resin was packed in a column and was eluted with equilibration solution containing 0.15 M imidazole. Dithiothreitol (1 mM) was added to the column effluent, and the fractions containing CPSI (monitored by SDS-PAGE) were pooled and concentrated to >1 mg protein ml -1 by centrifugal ultrafiltration (100 kDa cut-off Amicon Ultra, from Millipore).

Site-directed mutagenesis was performed by the overlapping extension method using a commercial kit (Quickchange, from Stratagene), the pFastBac-HTA vector containing the CPSI cDNA as template, a high fidelity thermostable DNA polymerase (Pfu Turbo from Stratagene) and the appropriate mutagenic forward and reverse oligonucleotides (Supplementary material Table S1). Mutations were either to alanine (T1394A) or to residues expected to cause the smallest possible structural change (N1437D, N1440D and T1391V). The W1410K mutation introduces the residue found in bacterial CPSs (Figure S1). Mutant plasmids were sequenced to assure the respective presence and absence of the desired and unwanted mutations. Production of the mutant proteins by the insect cells and mutant protein purification were as for the wild type enzyme.

Enzyme activity assay

CPSI activity was assayed at 37ºC in a solution containing 50 mM glycylglycine (pH 7.4)/0.1 M KCl/0.1 M NH 4 Cl/5 mM ATP/7 mM MgSO 4 /20 mM KHCO 3 /1 mM dithiothreitol/5 mM L-ornithine/10 U ml -1 ornithine transcarbamylase (from Sigma), and NAG as indicated, or, when indicated, N-acetyl-L( )--phenylglutamate (Phe-NAG) (prepared as in [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF]) , determining citrulline after 10 min, and, when indicated, ADP [START_REF] Rubio | Mitochondrial carbamoyl phosphate synthetase activity in the absence of N-acetyl-L-glutamate. Mechanism of activation by this cofactor[END_REF][START_REF] Yefimenko | Understanding carbamoyl phosphate synthetase deficiency: impact of clinical mutations on enzyme functionality[END_REF]. Results of at least duplicate assays were fitted to hyperbolic kinetics with GraphPad Prism (GraphPad Software, San Diego).

Other assays

Circular dichroism (CD) spectra in the far-UV region (195-250 nm) were obtained at 4ºC with a Jasco 810 spectropolarimeter in 0.2 ml of 20 mM Tris-HCl pH 7,4/20 mM KCl/20% glycerol/ 0.5 mM dithiothreitol/1-2 µM protein, in a 0.1 cmpathlength cell. Each spectrum was the average of ten scans. Protein was determined by the Bradford assay [START_REF] Bradford | A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] using bovine serum albumin as standard.

RESULTS AND DISCUSSION

Proposed site and mode of NAG binding in CPSI

Since the structure of the C-terminal domain of hCPS (PDB file 2YVQ [START_REF] Xie | Crystal structure of MGS domain of carbamoyl-phosphate synthetase from homo sapiens[END_REF]) was obtained using the acellularly-produced isolated domain without the natural context provided by the remaining domains of the protein, there could be doubts on whether the structure is the genuine one or an artefact. The latter possibility appears to be excluded by the observation that, despite the poor sequence identity with the allosteric domain of eCPS (20% identity) (see Supplementary material Figure S1), the structure of the two domains is virtually identical (rmsd of 1.65 Å for superimposition of 108 C atoms; Figure S2): an open sandwich in which the central parallel sheet of five elements (strand order, 32145) is covered on one side by helices 2, 1 and 5 and on the other by helices 3 and 4 (Figure 1A).

Previous studies [START_REF] Alonso | Binding of N-acetyl-L-glutamate to rat liver carbamoylphosphate synthetase (ammonia)[END_REF] demonstrated the binding of one NAG molecule per subunit of rat liver CPSI. Using the hCPS C-terminal domain structure [START_REF] Xie | Crystal structure of MGS domain of carbamoyl-phosphate synthetase from homo sapiens[END_REF], and utilizing automated cavity-searching [START_REF] Kleywegt | Detection, delineation, measurement and display of cavities in macromolecular structures[END_REF] and ligand-docking [START_REF] Verdonk | Improved protein-ligand docking using GOLD[END_REF] algorithms, we only detected one pocket having the approximate dimensions required to accommodate the NAG molecule (Figures 1A,B). The pocket, opening at the sheet C-edge, is located between the sheet and the helical layer containing helices 3 and 4 (Figure 1A). In this pocket, the indolic ring of W1410, along with F1445 and K1444, forms a lid separating the cavity from the surrounding solution (Figure 1B). Initial docking experiments indicated that, in the rotamer observed in the crystal structure of the empty pocket, the W1410 side chain would hamper optimal fitting of NAG inside the cavity. Thus, when flexibility of the side chains of W1410 and the overlying F1445 and Q1413 residues was allowed, a highly stable binding pose was detected (Figure 1D) In this conformation, the W1410 aromatic ring has flipped 180 o around the C -C bond relative to the conformation observed in the crystal structure of the hCPS C-terminal domain, while maintaining the stack with F1445 (Figure 1D). This alternative W1410 side-chain conformation is commonly observed in proteins [START_REF] Lovell | The Penultimate Rotamer Library[END_REF]. Subsequent calculations using this alternative protein conformation led to much better NAG docking scores and to almost total convergence (95%) of the NAG binding poses (Figure 1D).

In the converged solutions (Figure 1E), the NAG molecule adopts a largely extended conformation and places its -COO -and acetamido groups oriented towards one wall and the floor of the pocket, respectively, while the glutamate side-chain is oriented towards the pocket entry. This NAG binding mode results in appropriate interactions with the residues lining the cavity: the -COO -is encircled by the N1440 and N1449 side-chain amide groups on one side and by the NH group of the flipped W1410 ring on the other, forming with them a network of hydrogen bonds, while the -NH group forms a hydrogen bond with the T1391 side chain (Figure 1F). The acetamido group has its methyl end in a hydrophobic nest formed by L1363, themethyl of T1391, W1410, I1423 and I1452, (Figure 1E) whereas its carbonyl group points towards the N1437 side-chain amide, with which it forms a hydrogen bond (Figure 1F). The OH groups of T1391 and T1394 are close to the outwards-looking NAG -COO -, donating hydrogen bonds to it (Figure 1F). The positive surface potential at the pocket entry (estimated with GRASP [START_REF] Honig | Classical electrostatics in biology and chemistry[END_REF] ; not illustrated) fits the binding of the negatively charged -COO -. Finally, the C atom of the glutamate side-chain also looks outwards, making contacts with the indolic ring of W1410 (Figure 1E). These proposed NAG-interacting residues are strictly conserved in CPSs, with some of them (W1410, N1440, N1449, Figure S1) being conserved exclusively in NAG-sensitive CPSs, supporting the functional importance of this pocket and its implication in NAG binding.

The proposed mode of NAG binding agrees with prior structure-activity studies with NAG analogues

Structure-activity studies concluded [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF] that NAG binds with its glutamate moiety extended in a low-energy conformation, to a CPSI site having the subsites for the -COO -and for the acetamido group of NAG fixed relative to each other (deduced from the abolition of binding by adding a methylene group between the asymmetric C atom and the -COO -of NAG [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF]) whereas the subsite for the -COO -appeared less A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. constrained. All these traits are reflected in our model for NAG binding: the NAG molecule is extended, the subsites for the -COO -and acetamido groups involve the internal regions of the pocket and the -COO -binds at the pocket opening (Figure 1E), which should offer an sterically less constrained context. Prior observations that thiocarbamoylglutamate does not activate CPSI [START_REF] Grisolía | Catalytic role of glutamate derivatives in citrulline biosynthesis[END_REF] but that N-acetoxyglutarate is an activator [START_REF] Schooler | 2-Acetoxyglutarate as an activator of carbamoyl phosphate synthetase[END_REF] also agree with our present proposal (Figures 1E,F), since in our model the O atom of the NAG acetamido group is appropriately placed for forming one hydrogen bond with the enzyme. The inactive N-acetyl, N-methyl-L-glutamate analogue [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF] would be unable to form the proposed hydrogen bond between the N and T1391 (Figures 1E,F), in addition to causing steric clash with the site. The inability of N-butyryl-L-glutamate [START_REF] Grisolía | Catalytic role of glutamate derivatives in citrulline biosynthesis[END_REF] to activate CPSI parallels the observation of lack of space within the binding pocket (Figure 1E) to accommodate the N-butyryl instead of the Nacetyl. In contrast, little reshuffling would be needed for replacing the N-acetyl group of NAG by the modestly larger N-propionyl or N-chloroacetyl groups and, indeed, the corresponding NAG analogues are quite good activators of CPSI [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF]. The structure also accounts for the binding of NCG, given the similar size and overall shape of the carbamoyl and acetyl groups. However, the higher polarity and different geometry of the NH 2 in the carbamoyl group relative to the CH 3 of the acetyl substituent would be expected to impair somewhat the interactions with the hydrophobic cavity that hosts this methyl group of NAG, explaining the 10-fold lesser affinity of CPSI for NCG relative to NAG [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF]. The importance of the hydrophobic interactions with the methyl group are illustrated by the observation that the affinity of CPSI for N-formyl-L-glutamate, which lacks this methyl group, is two orders of magnitude lower than for NAG [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF]. Finally, in agreement with the prediction from our model that the glutamate side-chain of NAG looks towards the pocket entry, and, in particular, that the NAG C atom is partially exposed and contacts the pocket roof residue W1410, the NAG analogue having a phenyl substituent at the C (N-acetyl-L( )--phenylglutamate; abbreviated here Phe-NAG) was a good CPSI activator [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF]. Thus the extra phenyl group of Phe-NAG can be accommodated in this outwards looking part of the site, possibly by reshuffling somewhat the roof over the site. Overall, the evidence derived from structure-reactivity studies of NAG analogues supports the binding of NAG in the way proposed here.

Mapping of the site of CPSI photoaffinity labelling by N-chloroacetyl-L-glutamate supports the proposed mode of NAG binding

Rat liver CPSI (rCPSI), an enzyme that is functionally virtually identical to hCPSI [START_REF] Rubio | Carbamoyl phosphate synthetase I of human liver. Purification, some properties and immunological crossreactivity with the rat liver enzyme[END_REF] and which has 94.4% sequence identity with it [START_REF] Haraguchi | Cloning and sequence of a cDNA encoding human carbamyl phosphate synthetase I: molecular analysis of hyperammonemia[END_REF], is photoaffinity labelled in its ~16-kDa C-terminal domain with the radioactive and photosensitive NAG analogue [ 14 C]ClNAG [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF]. Given its closeness to the methyl group of NAG (Figure 1E), the -OH group of T1391 appears a good candidate for forming the covalent bond with the NAG radical generated upon irradiation-triggered Cl atom loss [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF]. Because of the low efficiency of the labelling ( 5% of enzyme molecules [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF]), direct identification of the labelled residue has not been possible thus far. However, the results of specific protein cleavage techniques agree with the cross-linking of [ 14 C]NAG to T1391. These studies have been carried out with the 18.9-kDa labelled C-terminal fragment produced by limited proteolysis with V8 staphylococcal protease (Figure 2A, B), a fragment which was proven by N-terminal sequencing to begin at M1329 (Figure 2B). Upon cyanogen bromide (CNBr) cleavage at methionine residues [START_REF] Gross | The cyanogen bromide reaction[END_REF] (Figures 2A,C), the radioactive fragment exhibited an electrophoretic mass of 17 kDa, as expected (Figure 2A, second bar) for the fragment that is C-terminal to the most distal methionine, M1350. Upon o-iodosobenzoic treatment to cleave at tryptophan residues [START_REF] Mahoney | High-yield cleavage of tryptophanyl peptide bonds by o-Iodosobenzoic acid[END_REF] , C), the radioactivity migrated according to a mass, estimated by SDS-urea-PAGE [START_REF] Cervera | Photoaffinity labelling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor[END_REF] (a suitable technique for small fragments), of 7.3 kDa. This value fits only the mass (7.72 kDa) of the most N-terminal (residues 1329-W1397) of the three fragments that should be generated upon cleavage at tryptophan residues (additional two fragments, 1398-W1410, and 1411-1500, with respective masses of 1.4 and 10 kDa). Thus, the enzyme is photolabelled upstream of the o-iodosobenzoic cleavage point at W1397 and downstream of the CNBr cleavage point at M1350, in the region comprised between residues 1351 and 1397.

We showed [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF] that exhaustive tryptic digestion of [ 14 C]ClNAG-labelled rCPSI yields a single labelled fragment, judged from HPLC peptide mapping (Figure 2D). The same minimal labelled tryptic peptide was obtained (given the identical elution position of the radioactivity from reversed-phase HPLC, data not shown) by digestion of the whole enzyme and of the 18.9 kDa C-terminal V8 fragment, indicating that this labelled minimal tryptic peptide is contained entirely within the 18.9 kDa C-terminal V8 fragment (as expected, since the enzyme is labelled at some point between residues 1351 and 1397, far from the N-end of the 18.9-kDa V8 fragment). This labelled tryptic peptide behaves in gel filtration [START_REF] Cervera | Photoaffinity labelling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor[END_REF] as expected for the mass (4.16 kDa) of the minimal tryptic peptide (residues 1388-1424) that contains T1391 (Figures 2A,F). All other minimal tryptic peptides covering the 1351-1397 region (Figure 2A, fourth bar) are much smaller. The minimal tryptic peptide that contains T1391 includes two glutamate residues and thus should be cleaved by V8 staphylococcal protease [START_REF] Drapeau | Cleavage at glutamic acid with staphylococcal protease[END_REF]. If the [ 14 C]NAG is cross-linked to T1391, the radioactive peptide produced by sequential tryptic-V8 digestion should have a mass of 0.77 kDa. Both predictions are satisfied by the present experimental evidence (Figures 2E,F), which reveals: 1) a change in the elution of the radioactive peak from the reversed-phase HPLC column upon V8 digestion of the tryptic fragment; and 2) the elution of this radioactivity from the gel filtration column at the expected position for 0.77 kDa. The only other peptide (residues 1393 to 1414) falling in the 1351-1397 region (the region encompassing the label) that is generated by the V8 protease from the minimal tryptic peptide, has a much larger mass (2.56 kDa) and thus does not fit the gel filtration results. Taken together, all these observations add up to render highly likely that T1391 is the site of photoaffinity labelling by [ 14 C]ClNAG.

We also obtained independent evidence using mass spectrometry for the labelling by ClNAG of the minimal tryptic peptide encompassing residues 1388-1424. MS fingerprinting with trypsin (see Experimental section) confirmed the assignment of the 18.9-kDa fragment generated by V8 protease to the C-terminal domain of the enzyme, and identified the tryptic fragment encompassed between residues 1388 and 1424 by both its mass (calculated mass, 3,967.93 Da; experimental mass in two determinations, 3,968.24 and 3,968.49 Da) and by sequence information obtained by MS/MS. This tryptic peptide was detected (Figure 2G) in similar amount (judged from the peak size) both in the fingerprint of the 18.9-kDa fragment generated after control irradiation in the presence of NAG (NAG lacks the Cl substituent and thus it is not photosensitive, not photolabelling the enzyme [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF]) or in the experimental irradiation with ClNAG, in line with the fact that a very small fraction of the protein is labelled with ClNAG [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF]. Nevertheless, in the tryptic digest of the sample that was photoaffinity labelled with ClNAG, but not in that in which the ClNAG was replaced by NAG, a small peak was observed having a mass of 4,155.94 Da, (Figure 2G) which corresponds, within experimental error, to the expected mass (4,155.09 Da) of the 1388-1424 tryptic fragment modified by cross-linking to NAG. The small amount of this peptide, which agrees with the well-known low labelling efficiency by ClNAG [START_REF] Rodríguez-Aparicio | Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain[END_REF], prevented further analysis. Nevertheless, the mass of the peak and its exclusive finding in the experimental sample agrees with the conclusion that the 1388-1424 tryptic peptide is the one where ClNAG labelling takes place.

Site-directed mutagenesis studies confirm NAG site identification

Using a baculovirus-based insect cell expression system, we were successful in producing mature rCPSI (that is, the rat liver enzyme lacking the N-terminal mitochondrial targeting sequence) in active form, engineered to incorporate a 6-His tag at its N-terminus for rapid purification. Under optimal expression conditions (see Experimental section), 0.5 mg of largely homogeneous (Figure 3A) active (specific activity, 1.3±0.08 µmol CP min -1 mg -1 ) rCPSI was obtained from 50 ml of culture after a single step of Co 2+ chelate chromatography. The enzyme exhibited normal substrate kinetics (K m values for ATP, HCO 3 -and NH 4 + , 1.06±0.11 mM, 6.43±0.60 mM and 1.07±0.1 mM, respectively) and NAG activation kinetics (K a NAG 0.11 ± 0.01 mM, Figure 3A).

We generated enzyme forms carrying single amino acid mutations affecting NAG site residues predicted to be important for NAG binding. The mutant proteins were produced in the same way and with similar yield and purity as in the case of wildtype CPSI (Figure 3B), and appeared well folded (judged from the similarity of their far-UV CD spectra relative to the spectrum of wild type CPS; data not shown). In agreement with the fact that the catalytic machinery is not provided by the C-terminal domain [START_REF] Rubio | Structure-activity correlations in carbamoyl phosphate synthetases. In: Carbon dioxide fixation and reduction in biological and model systems[END_REF][START_REF] Cervera | The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry[END_REF][START_REF] Thoden | The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution[END_REF], all the mutants were active in the presence of NAG, producing carbamoyl phosphate and ADP with the normal ADP/CP product ratio of 2 (data not shown). However, all mutations significantly increased the concentrations of NAG required for activation (Figure 3C), as expected for residues involved in NAG binding. Of particular note was the increase of 400-fold and 900-fold in the K a NAG triggered respectively by the T1391V and T1394A mutations, supporting the role of the hydroxyl groups of these threonines in NAG binding (Figures 1E,F). The increase of 70-110fold in the K a NAG caused by the N1437D and N1440D mutations also agrees with the role of these asparagines as hydrogen bond donors to the NAG -COO -and acetamido carbonyl (Figure 1E,F). Finally, the W1410K mutation, chosen because it replaces a key pocket roof residue by the highly conserved K found at this position in bacterial CPSs, increased 60-fold the K a NAG (Figure 3C). This result is consistent with the observed partial hampering of NAG entry by the W1410 ring (see above).

The rate of the enzyme reaction for the T1391V and T1394A mutants, at saturation of NAG, was 1/3 relative to wild-type CPSI (Figure 3C), a decrease that appears not to be due to enzyme inactivation, since these mutant rCPSI forms where as stable as wild type CPS at, 4ºC, 25ºC and 37ºC (data not shown). Thus, the enzyme-NAG complex appears genuinely less active for these mutants than for the wild type enzyme, suggesting the involvement of T1391 and T1394 in allosteric signal transmission to the catalytic domains. Since these residues bind to the -COO -of NAG (Figures 1E,F), their involvement in allosteric signal transmission agrees with the previous conclusion [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF] that the subsite for -COO -is involved in the conformational changes associated with NAG activation.

An unexpected finding of the present studies is the observation that the W1410K mutation resulted in a modified specificity for the activator. Thus, with this mutant the affinity was increased when the Phe-NAG was used instead of NAG (Figure 3D). Phe-NAG was reported [START_REF] Britton | A structure-reactivity study of the binding of acetylglutamate to carbamoyl phosphate synthetase I[END_REF] and is confirmed here (Figure 3D) to activate rCPSI with a K a that is ~2-fold higher than for NAG, but the W1410K mutant binds Phe-NAG considerably better than NAG (respective K a values, 0.55 and 6.3 mM; Figure 3D A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. insert). As discussed below, this finding, which supports the interaction of the NAG C atom with W1410, may have practical implications in the treatment of CPSI deficiency.

Implications of the present studies for clinical CPSI deficiency

The diagnosis of genetic diseases ideally aims at establishing the disease-causing potential of each amino acid substitution found in a patient presenting the disease, thus excluding that the mutations found could merely be trivial low-frequency polymorphisms having little impact on enzyme functionality or stability. The robust baculovirus expression/purification system developed here provides a well suited system for carrying out these pathogenicity-testing studies in the case of human CPSI deficiency, and, therefore, can be valuable in the diagnosis and evaluation of this genetic condition. In addition, the present characterization of the NAG site also has practical potential for identifying mutations exclusively affecting NAG activation. A number of amino acid substitutions have been reported already in patients of CPSI deficiency that are candidates to affect NAG regulation, since they fall in the C-terminal domain of the enzyme [START_REF] Summar | Molecular genetic research into carbamoyl phosphate synthetase I: molecular defects and linkage markers[END_REF][48][START_REF] Eeds | The frequent observation of evidence for nonsensemediated decay in RNA from patients with carbamyl phosphate synthetase I deficiency[END_REF]. We have begun a study aiming at applying the present knowledge to predict the potential effects of these C-terminal domain CPSI mutations, carrying out in parallel expression studies to test the quality of our structure-based predictions. Hopefully, the knowledge on the C-terminal domain structure and on the NAG site will permit to tell in the near future whether a mutation is likely to impair NAG binding. In such mutations the CPSI catalytic machinery should be intact and it would appear possible, at least in principle, to restore enzyme activity by the administration of large enough doses of NCG [START_REF] Bachmann | N-acetylglutamate synthetase deficiency: a disorder of ammonia detoxication[END_REF][START_REF] Rubio | Treating urea cycle defects[END_REF]. Moreover, our observation that the modified NAG analogue Phe-NAG is a better activator than NAG for W1410K rCPSI suggests that N-carbamoyl-L--phenylglutamate would be a better treatment than NCG in the case that a CPSI deficiency patient were found to carry the W1410K mutation. Clearly, our findings raise the novel possibility of synthesizing modified NCG analogues specially tailored to fit individual NAG site mutations for the treatment of CPSI deficiency.

The origin of the NAG site of CPS

Comparison of the structures of the allosteric domains of hCPSI and eCPS reveals that the NAG site of CPSI is topographically equivalent to the site for the relatively poor activator of eCPS, IMP (Figures S1, S2, and1B,C,G). The IMP site of eCPS is an angle-shaped open furrow formed by the same structural elements that also form the NAG site in CPSI (Figure 1C). In the bacterial enzyme, the distance between the N-ends of -helix 4 and the 3-3 connector is larger than in hCPSI, leaving a space where the purine base of IMP sits. In hCPSI this space is covered by a roof formed by the mutually stacked bulky side-chains of (from outwards to inwards) K1444, F1445 and W1410 (Figure 1B), of which W1410 is invariant in NAG-sensitive CPSs (Figure S1). Because of this roof, the part of the site that in eCPS binds the purine base of IMP is obliterated and the NAG site becomes a pocket.

The similarity between the NAG and IMP sites extends also to the interactions made by IMP and NAG (Figure 1G). The -COO -of NAG can be equated with the IMP phosphate, a phosphate that, in eCPS, makes hydrogen bonds with the side chains of T974 and T977, corresponding to T1391 and T1394 of CPSI. The two IMP ribose hydroxyls appear to be antecedents of both O atoms of the NAG -COO -, since they are encircled by N1015, T1017 and S1026, corresponding to the three hCPS asparagines, N1437, N1440 and N1449 that encircle in CPSI the NAG -COO -(Figure 1G). Even the indolic ring of W1410 is reminiscent (Figures 1B,C andG) of the purine base of bound IMP, being in an approximately equivalent place, although in a different orientation, than the purine base of the nucleotide effector of bacterial CPS. The similarity of the binding of IMP to eCPS and of NAG to CPSI is highlighted also by the overall coincidence, in the alignment of the sequences of both enzymes, of the residues that change their exposition with the binding of the corresponding effector (Figure S1, black dots).

The structure of eCPS revealed the presence of different sites for its two activators ornithine and IMP [START_REF] Thoden | The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution[END_REF][START_REF] Thoden | The binding of inosine monophosphate to Escherichia coli carbamoyl phosphate synthetase[END_REF]. Our results, by proving the equivalence of the NAG site of CPSI and the IMP site of bacterial CPS, exclude a relation between the NAG and the ornithine sites and indicate that the NAG and IMP sites derive from the same ancestral site. Since phosphoribosyl pyrophosphate (PRPP) is an IMP analogue, sharing with IMP the phosphoribosyl moiety, the PRPP site of the pyrimidine-specific CPSII of CAD [START_REF] Tatibana | Control of pyrimidine biosynthesis in mammalian tissues. V. Regulation of glutamine-dependent carbamoyl phosphate synthetase: activation by 5-phosphoribosyl 1-pyrophosphate and inhibition by uridine triphosphate[END_REF] most likely derives from the same ancestral site. In fact, CAD contains a W residue that aligns with W1410 of CPSI (Figure S1), and thus the PRPP site of CAD might resemble the NAG site of CPSI by being a pocket rather than a furrow and by having this tryptophan indolic ring obliterating the site that in eCPS is occupied by the adenine ring of IMP, a ring that is also missing in PRPP.

7

  Biochemical Journal Immediate Publication. Published on 15 Sep 2009 as manuscript BJ20090888 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090888

(Figures 8 Biochemical

 8 Journal Immediate Publication. Published on 15 Sep 2009 as manuscript BJ20090888 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090888 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

2A

, 10 Biochemical

 10 Journal Immediate Publication. Published on 15 Sep 2009 as manuscript BJ20090888 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090888

Figure 1 .

 1 Figure 1. The C-terminal domain of CPSI and the NAG site (A) Cartoon representation of the hCPS allosteric domain (PDB file 2YVQ), coloured to enhance visibility of the structure. Secondary structure elements are labelled. The pocket identified with VOIDOO is shown in semi-transparent surface representation. An NAG molecule bound to this pocket according to the results of docking with GOLD (see text) is shown in sticks representation. (B) and (C) Surface representations of (B) the NAG site as seen in the crystal structure of hCPS C-terminal domain (PDB file 2YVQ), and (C) the IMP site of eCPS (PDB file 1CE8, [21]), in identical orientations. In (B), the side-chains of the NAG pocket roof residues K1444, F1445 and W1410 are shown in sticks representation to allow visualization of the pocket interior. In (C), the IMP activator is represented with sticks. (D) NAG docking to hCPSI. The side chains proposed to surround the NAG molecule are shown in thin stick representation, in the conformations observed in the crystal structure of the empty NAG site (PDB file 2YVVQ, black), superimposed with the conformations resulting from GOLD flexible docking of NAG (in blue). The converged NAG binding poses are shown in thicker sticks.(E) Stereoview of the proposed hCPS site bound to NAG. Secondary structure elements are labelled. (F) Schematic plot (drawn with LIGPLOT [51]) of the interactions between the protein and NAG. Distances are in Å. (G) Stereoview of the superimposed side chains (thinner sticks) of the residues making the IMP site (green carbons) and the proposed NAG site (black carbons) in eCPS and hCPSI, respectively, with the bound IMP and NAG molecules shown with green and black carbons, respectively.

Figure 2 .

 2 Figure 2. Mapping of the site of photoaffinity labelling of CPSI with the NAG analogue N-chloroacetyl-L-glutamate (ClNAG) Except in panel (G), in which the analogue was not radioactive, all other panels reflect the results obtained after photoaffinity labelling with N-chloroacetyl-L-[ 14 C]glutamate. (A) Cleavage techniques used to identify the region where NAG is bound. Bars represent enzyme polypeptides, cleavage points are pointed with small scissors and are localized with black vertical lines, giving cleavage site residue number below the bar. The radioactive fragment obtained in each step is coloured yellow. Figures within the bars are sequence-deduced fragment masses (in kDa), including the expected mass increase (188 Da) if NAG was cross-linked to the fragment. The shadowed bar regions are those limited by the CNBr and o-Iodosobenzoic cleavage points (see text). Letters on the right of the horizontal bars give the panels illustrating the results of each cleavage technique. (B) HPLC-isolation of the C-terminal labelled fragment obtained after limited digestion with V8 staphylococcal protease of the photoaffinity labelled enzyme. The insets show the results, revealed by Coomassie staining or by fluorography, of SDS-PAGE of selected peaks. (C) SDS-PAGE and fluorography of the cleavage products obtained by treating with o-Iodosobenzoic acid (left) or with cyanogen bromide (CNBr, right) the 18.9-kDa labelled fragment isolated in (B). The minus sign indicates lack of treatment. The masses of the radioactive products obtained are given in red. (D) and (E) HPLC separation of the radioactive fragments obtained by (D) exhaustive tryptic digestion of the 18.9-kDa labelled fragment isolated in (B), and (E) by subsequent digestion with staphylococcal V8 protease. (F) Gel filtration determination (Sephacryl S200-SDS technique) of the mass of the labelled minimal tryptic peptide (filled square) or of the radioactive product generated by subsequent digestion with V8 protease (filled circle). The samples applied to the column are the

Figure 3 .

 3 Figure 3. Production of recombinant CPSI, and site-directed mutagenesis studies(A) Recombinant (reCPSI) and liver-purified (liCPSI) rat CPSI exhibit identical NAG activation kinetics. Velocity is expressed as a fraction of the velocity extrapolated at infinite NAG concentration. The insets illustrate SDS-PAGE analysis (Coomassie staining) of CPSI expression. Left panel, crude centrifuged extracts of the insect cells infected with the baculovirus lacking or carrying the CPSI coding sequence (lanes labelled Control and reCPSI, respectively; St denotes mass standards). Right panel, purified recombinant enzyme (reCPSI), comparing it with the enzyme purified from rat liver (liCPSI). (B) SDS-PAGE (Coomassie staining) of the purified mutant CPSI forms prepared here. (C) Effects of the mutations on CPSI activation by NAG. Velocities are given relative to those for the same concentration of the wild type enzyme. The figures to the right correspond to estimates from each curve for the K a NAG (given in mM units) and for the velocity at infinite NAG concentration (given relative to that of wild-type). (D) Influence of the W1410K mutation (insert) on activation by NAG and by the NAG analogue N-acetyl-(±)--phenylglutamate (Phe-NAG).The larger plot illustrates the activation kinetics for NAG and analogue of the wild type enzyme. The inset reveals that the analogue is a better activator than NAG for the W1410K mutant CPS. Velocities are expressed as for panel (C).

  Fig. 1

  Fig. 2

  Publication. Published on 15 Sep 2009 as manuscript BJ20090888 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090888 A c c e p t e d M a n u s c r i p t

1.42 0.46 1.78 E1392 E1414 V8 protease (limited) 1500 19.06 kDa 16.80 kDa 7.72 kDa 1.55 10.19 kDa 1.04 0.67 4.16 kDa 2.56 kDa 1.25 kDa 0.77 kDa 1329 1500 39 1329 1500 V8 digestion (Glu) 1424 1388

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We thank José Luis Llácer (IBV-CSIC) for help with figures and structural calculations, the Proteomics Core Facility of the Centro de Investigación Príncipe Felipe (CIPF Valencia)-Proteored (Genoma España) for diligent mass spectrometric service, and Prof. Santos Fustero and his group (CIPF-Valencia) for making one lot of non-labelled ClNAG.

FUNDING

Supported by grants BFU2007-66781 and BFU2008-05021 of the Spanish Ministry for Science (MEC and MICINN) and AP-035/08 of the Valencian Government.