Eur J Biochem 270, 2195–2206 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03588.x Limited proteolysis of Escherichia coli cytidine 5¢-triphosphate synthase Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis Dave Simard, Kerry A Hewitt, Faylene Lunn, Akshai Iyengar and Stephen L Bearne Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada Cytidine 5¢-triphosphate synthase catalyses the ATPdependent formation of CTP from UTP using either ammonia or L-glutamine as the source of nitrogen When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP) The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP K187A was able to form tetramers in the presence of UTP and ATP Arg429 and Lys432 appear to reside in an CTP synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine or NH3 as the nitrogen source (Scheme 1) [1,2] This glutamine amidotransferase is a single polypeptide chain containing 545 amino acids and consisting of two domains The C-terminal glutamine amide transfer (GAT) domain catalyses the hydrolysis of glutamine, and the nascent NH3 derived from glutamine hydrolysis is transferred to the N-terminal synthase domain where the amination of UTP is catalysed [3,4] CTPS belongs to the Triad family of glutamine amidotransferases [5,6] which utilizes a Cys-His-Glu triad to catalyse glutamine hydrolysis and also includes anthranilate synthase, carbamoyl phosphate synthase, formylglycinamidine synthase, GMP synthase, imidazole glycerol phosphate synthase, and aminodeoxychorismate synthase Correspondence to S L Bearne, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 Tel.: +1 902 494 1974, Fax: + 902 494 1355, E-mail: sbearne@is.dal.ca Abbreviations: CTPS, CTP synthase; GAT, glutamine amide transfer; GF-HPLC, gel-filtration-HPLC; PVDF, poly(vinylidene difluoride) Enzymes: CTP synthase (EC 6.4.3.2) (Received 28 February 2003, revised 17 March 2003, accepted 21 March 2003) exposed loop in the glutamine amide transfer (GAT) domain Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage The R429A and R429A/ K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced % 20-fold and % 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding Keywords: activation; amidotransferase; CTP synthase; glutaminase; proteolysis; site-directed mutagenesis Scheme CTP-forming reactions catalysed by CTPS CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides Because CTP has a central role in the biosynthesis of nucleic acids [7] and membrane phospholipids [8], CTPS is a recognized target for the development of antineoplastic agents [7,9], antiviral agents [9,10], and antiprotozoal agents [11–13] Recently, CTP synthase inhibition has been shown to potentiate the cytotoxic effects of the anticancer drug 1-b-D-arabinofuranosylcytosine [14] and anti-HIV therapies [15] CTPS from E coli is the most thoroughly characterized CTPS with respect to its physical and kinetic properties, and is regulated in a complex fashion [1] GTP is required as a positive allosteric effector to increase the efficiency (kcat/Km) of glutamine-dependent CTP synthesis 45-fold but has a negligible effect on the reaction when NH3 is the substrate [16,17] In addition, the enzyme is inhibited by the product CTP [18], exhibits negative cooperativity for glutamine [19], and displays positive cooperativity for ATP and UTP Ó FEBS 2003 2196 D Simard et al (Eur J Biochem 270) [18–20] ATP and UTP act synergistically to promote tetramerization of the enzyme to its active form [20] The structure of CTPS has not yet been determined and hence little is known about the enzyme’s tertiary structure However, analysis of crystal structures of the Triad amidotransferases GMP synthase and carbamoyl phosphate synthase reveal that the structures of the GAT domains are probably closely related among all Triad enzymes [21,22] Site-directed mutagenesis studies and sequence comparisons have revealed structural and catalytic roles of several amino acid residues within the GAT domain of CTPS, including residues of the catalytic triad (Cys379, His515, and Glu517) [3], residues comprising the oxyanion hole (Gly351, Gly377, Gly381, and possibly adjacent hydrophobic residues) [23], and residues between Ala346 and Tyr355 that appear to play an important structural role [4] Recently, Willemoes reported that ă Thr431 and Arg433 in the GAT domain of Lactococcus lactis CTPS play a role in GTP-dependent activation of glutamine hydrolysis [24] Our knowledge about the synthase domain is much more limited Analyses of mutant CTP synthases from Chlamydia trachomatis [25], hamster [26], and yeast [27] have revealed that mutations which render cells resistant to both the cytotoxic effects of cyclopentenylcytosine and feedback inhibition by CTP occur between residues 116 and 229 (E coli numbering), with many of the mutations clustering between residues 146 through 158 Hence, this region of the synthase domain is believed to form part of the CTPbinding site Competitive inhibition experiments have suggested that for E coli CTPS, this site may also be the UTP-binding site [18] The locations of the ATP- and GTPbinding sites have not yet been identified Recent studies from our laboratory have revealed that residues Asp107 and Leu109 in the synthase domain of E coli CTPS facilitate efficient coupling of glutamine hydrolysis to CTP synthesis [28] To learn more about the structure of CTPS, we investigated controlled proteolysis of the enzyme Using limited trypsin-catalysed proteolysis and site-directed mutagenesis, we have identified peptide bonds C-terminal to three basic residues of E coli CTPS that are highly susceptible to proteolysis One residue, Lys187, is located at the CTP/ UTP-binding site within the synthase domain and is essential for catalysis but not for enzyme tetramerization The other two residues, Arg429 and Lys432 appear to reside in an exposed loop that is important for both GTP binding and GTP-dependent allosteric activation of glutamine hydrolysis Materials and methods Materials HisỈBind resin and thrombin cleavage capture kits were from Novagen; broad range protein markers were from New England Biolabs; Pfu Turbo DNA polymerase was from Stratagene Inc.; nucleotides, a-chymotrypsin from bovine pancreas (54 mg)1), Pronase from Streptomyces griseus (4.7 mg)1), protease V8 from Staphylococcus aureus (1000 mg)1), thermolysin from Bacillus thermoproteolyticus rokko (55 mg)1), and TPCK-treated trypsin from bovine pancreas (10 900 mg)1), and all other chemicals were from Sigma-Aldrich Canada Ltd Oligonucleotide primers for DNA sequencing and site-directed mutagenesis were commercially synthesized by ID Laboratories (London, ON, Canada) QIAprep spin plasmid miniprep kit (Qiagen Inc.) was used for the preparation of plasmids for mutagenesis and transformation DNA sequencing was conducted at the Dalhousie University– NRC Institute for Marine Biosciences Joint Laboratory (Halifax, NS, Canada) and the Robarts Research Institute (London, ON, Canada), while the N-terminal amino acid ´ sequencing was carried out at the Eastern Quebec Proteomics Core Facility (Ste-Foy, QC, Canada) Predictions of secondary structure were conducted using the programs 3-D PSSM [29], GOR4 [30], HNN [31], J-PRED [32], PREDATOR [33], PSIPRED [34], and SSPRO [35] Sequence alignments were conducted using CLUSTALW [36] Enzyme expression and purification Wild-type and mutant forms of recombinant E coli CTPS were expressed in and purified from E coli strain BL21(DE3) cells transformed with either mutant or wildtype plasmid pET15b-CTPS1 as described previously [16] This construct encodes the CTPS gene product with an N-terminal hexahistidine tag Thrombin-catalysed cleavage of the histidine tag from soluble enzymes (new N-terminus, GSHMLEM1…) was carried out as described previously [16] The resulting enzyme was dialysed into Hepes buffer (70 mM, pH 8.0) containing EDTA (0.5 mM) and MgCl2 (10 mM) The results of purification and cleavage procedures were routinely monitored using SDS/PAGE The amino acid residues in the recombinant wild-type and mutant enzymes are numbered according to the sequence of the wild-type E coli enzyme starting with M1 as position one Mutagenesis The plasmid pET15b-CTPS1 [16] was used as the template for site-directed mutagenesis Site-directed mutagenesis was conducted using the Quikchange Site-Directed Mutagenesis Kit (Stratagene Inc.) and following the manufacturer’s protocol The synthetic deoxyoligonucleotide forward (F) and reverse (R) primers used to construct the mutants were: 5¢-GCGTCTGGTGAAGTCGCAACCAAACCGACT CAG-3¢ (F, K187A), 5¢-GCTGAGTCGGTTTGGTT GCGACTTCACCAGACGC-3¢ (R, K187A), 5¢-CGG CAACGTTGAAGTTGCTAGCGAGAAGAGCG-3¢ (F, R429A), 5¢-CGCTCTTCTCGCTAGCAACTTCAACGT TGCCG-3¢ (R, R429A), 5¢-GCAACGTTGAAGTT GCTAGCGAGGCGAGCGATCTCG-3¢ (F, R429A/ K432A), 5¢-CGAGATCGCTCGCCTCGCTAGCAACT TCAACGTTGC-3¢ (R, R429A/K432A), where the positions of the mismatches are underlined Potential mutant plasmids were isolated and used to transform competent DH5a cells These cells were used for plasmid maintenance and for all sequencing reactions The entire mutant genes were sequenced to verify that no other alterations of the nucleotide sequence had been introduced Competent E coli strain BL21(DE3) cells were used as the host for target gene expression Ó FEBS 2003 Limited proteolysis of CTP synthase (Eur J Biochem 270) 2197 Enzyme assay and protein determinations CTPS activity was determined at 37 °C using a continuous spectrophotometric assay by following the rate of increase in absorbance at 291 nm resulting from the conversion of UTP to CTP (De ẳ 1338ặM)1ặcm)1) [18] The standard assay mixture consisted of Hepes buffer (70 mM, pH 8.0) containing EDTA (0.5 mM) and MgCl2 (10 mM), CTPS, and saturating concentrations of UTP (1 mM) and ATP (1 mM) in a total volume of mL Enzyme and nucleotides were preincubated together for at 37 °C followed by addition of substrate (NH4Cl or glutamine) to initiate the reaction Total NH4Cl concentrations used in the assays were 5, 10, 20, 30, 50, 60, 80, and 100 mM, and CTPS concentrations were % 3.0 lgỈmL)1 (wild-type), 3.0 lgỈmL)1 (R429A), and 4.0 lgỈmL)1 (R429A/K432A) For glutamine assays, concentrations of glutamine were 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 3.0, and 6.0 mM and CTPS concentrations were % 4.0 lgỈmL)1 (wild-type), 1.4 mgỈmL)1 (R429A), and 0.9 mgỈmL)1 (R429A/ K432A) The concentration of GTP was maintained at 0.25 mM for all assays when glutamine was used as the substrate In addition, the ionic strength was maintained at 0.25 M in all spectrophotometric assays by the addition of KCl The apparent activation constant (KA) for R429A CTPS (0.4 mgỈmL)1) with respect to GTP was determined for glutamine-dependent CTP formation as described previously [28] All kinetic parameters were determined in triplicate and average values are reported Initial rate kinetic data was fit to Eqn (1) by nonlinear regression analysis using the program ENZYMEKINETICS v1.5 (1996) from Trinity Software (Plymouth, NH) In Eqn (1), vi is the initial velocity, Vmax (¼ kcat[E]T) is the maximal velocity at saturating substrate concentrations, [S] is the substrate concentration (glutamine or NH3), and Km is the Michaelis constant for the substrate Values of Km for NH3 were calculated using the concentration of NH3 present at pH 8.0 {pKa (NH4+) ¼ 9.24 [37]} Values of kcat were calculated for CTPS variants with the hexahistidine tag removed using the molecular masses (Da) of 61 029 (wild-type), 60 944 (R429A), and 60 887 (R429A/K432A) The reported errors are standard deviations Except where noted otherwise, protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories Ltd.) with BSA standards mi ¼ Vmax ẵS Km ỵ ẵS 1ị Glutaminase activity Values of kcat for the hydrolysis of glutamine, at fixed saturating concentrations of glutamine (6 mM), UTP (1 mM), and ATP (1 mM) were determined as described previously [38] Data describing the dependence of the apparent kcat values on the concentration of GTP were fitted to Eqn (2) for hyperbolic nonessential activation kinetics where KA is the apparent activation constant, ko is the turnover number in the absence of GTP, and kact is the turnover number at saturating concentrations of GTP [39] kapparent cat   ẵGTP ko ỵ kact KA   ẳ ẵGTP 1ỵ KA 2ị Limited proteolysis Initially, wild-type CTPS was subjected to limited proteolysis by several endopeptidases including trypsin, chymotrypsin, pronase, thermolysin and V8 protease Proteolysis was conducted in Hepes buffer (70 mM, pH 8.0) containing EDTA (0.5 mM) and MgCl2 (10 mM) at 37 °C using a CTPS/protease ratio (lg protein) of 60 : in a total volume of mL Limited proteolysis using pronase and thermolysin was also conducted in potassium phosphate buffer (50 mM, pH 7.2) containing EDTA (1 mM) and MgCl2 (10 mM) EDTA was omitted from the buffers for all thermolysincatalysed reactions Proteolysis experiments were conducted in the absence and presence of ATP (10 mM) and UTP (10 mM) During the proteolysis reactions, aliquots (25 lL) were removed from the reaction mixture over the course of h, and transferred to gel loading buffer [25 lL; Tris/HCl (170 mM, pH 6.8) containing dithiothreitol (120 mM), SDS (5.4%, w/v), Bromophenol blue (0.03%, w/v) and glycerol (27.2%, w/v)] to terminate the reaction The samples were then boiled for and the proteolytic fragments were separated using SDS/PAGE (12% gels) Fragments were visualized by staining with Coomassie blue R-250 and subsequent de-staining in a solution of methanol/H2O/ acetic acid (45 : 45 : 10) Detailed studies were subsequently conducted using trypsin as trypsin-catalysed proteolysis gave different fragments depending on whether the nucleotides were absent or present Limited trypsin-catalysed proteolysis of both wild-type and mutant CTP synthases was analysed by monitoring CTPS activity (vide infra) at specific time points and by SDS/PAGE Trypsin proteolysis reactions (1 mL total volume) contained either wild-type or mutant CTPS (0.20 mgỈmL)1), and were conducted for h in Hepes buffer (70 mM, pH 8.0) containing EDTA (0.5 mM) and MgCl2 (10 mM) at 37 °C Reactions were initiated by addition of trypsin (0.1 lgỈmL)1) and aliquots (100 lL) were removed every 10 over h and assayed for activity using NH3 as the substrate The cleavage fragments produced from trypsin-catalysed proteolysis were analysed using SDS/PAGE (12% and 20% gels) Reactions were conducted as described above, with the exception that at 10, 20, 30, and 60 min, aliquots (20 lL) were removed and transferred to gel loading buffer (15 lL) to terminate the reaction A zero time point was obtained using lL of the enzyme stock solution (% 1.5 mgỈmL)1) used for the reaction The ability of various ligands to protect both wildtype and mutant CTP synthases from proteolysis was examined using ATP (10 mM), UTP (10 mM), ATP and UTP together (10 mM each), CTP (0.1, 0.5, and 1.0 mM), GTP (2.5 mM) and L-glutamine (10 mM) Inactivation assays Aliquots from two separate proteolysis reactions were assayed using both NH4Cl (100 mM) and glutamine Ó FEBS 2003 2198 D Simard et al (Eur J Biochem 270) (10 mM) as substrates, as described above Inactivation of CTPS activity followed first-order kinetics, and the apparent first-order rate constants for inactivation were calculated from plots of the percent activity remaining as a logarithmic function of the time of incubation The ability of various ligands to protect both wild-type and mutant CTPS from proteolysis was examined using ATP (0.5, 1.0, 2.0, and 10 mM), UTP (0.5, 1.0, 2.0, and 10 mM), ATP and UTP together (10 mM each), GTP (0.25 mM), and L-glutamine (10 mM) at the concentrations indicated N-Terminal sequence analysis (excitation and emission wavelengths of 285 nm and 335 nm, respectively) using a Waters 474 scanning fluorescence detector GF-HPLC of both wild-type and mutant enzymes was conducted in the absence and presence of ATP (1 mM) and UTP (1 mM), and the retention times were compared with those observed for the wild-type enzyme The column was standardized using the following proteins (0.5 mgỈmL)1): bovine thyroglobulin (669 kDa), b-amylase (200 kDa), BSA (66 kDa), and carbonic anhydrase (29 kDa) Chromatograms were analysed using PEAKSIMPLE software from Mandel Scientific (Guelph, ON, Canada) The retention time of bovine thyroglobulin was used to estimate the column void volume (Vo) Approximately 190 lg total protein containing CTPS and the various trypsin-catalysed cleavage fragments, produced after a 90-min proteolysis reaction, were separated using SDS/PAGE (12% or 20% gels) and subsequently transferred to a poly(vinylidene difluoride) (PVDF) membrane [Immun-Blot 0.2 lm (Bio-Rad Laboratories Ltd) for fragments with molecular masses of 25, 28 and 53 kDa; Immobilon-Psq0.2 lm PVDF (Millipore Ltd) for the fragment with a molecular mass of 10 kDa] as described by Wilson and Yuan [40] Electroblotting was conducted in CAPS buffer (0.01 M, pH 11.0) containing methanol (10%) Whole enzyme and cleavage fragments were located on the PVDF membrane by staining with Coomassie blue followed by destaining in 50% methanol Sections (50 mm2) of the PVDF membrane with adsorbed protein were submitted for N-terminal amino acid sequence analysis CTPS from E coli was subjected to controlled proteolysis by five endopeptidases (pronase, a-chymotrypsin, V8 protease, thermolysin, and trypsin) in the absence or presence of the nucleotides ATP and/or UTP (data not shown) These preliminary experiments revealed that only treatment of CTPS with trypsin produced a limited number of cleavage fragments over the course of h, of which the formation of some fragments was suppressed in the presence of ATP and/ or UTP Thus, trypsin was used in the present study to investigate the accessibility of regions in CTPS to proteolytic cleavage in the presence and absence of various ligands CD spectra Limited trypsin-catalysed cleavage of wild-type CTPS CD spectra were obtained using a JASCO J-810 spectropolarimeter and were recorded for both the wild-type and mutant enzymes (K187A, R429A, R429A/K432A) over the range 190–260 nm in the absence of nucleotides A marked decrease in buffer transparency was observed below 190 nm and therefore all spectra were truncated at this wavelength The resulting CD spectra obtained from enzyme solutions (0.2 mgỈmL)1) in Bis-Tris propane buffer (10 mM, pH 8.0) containing MgSO4 (10 mM) were analysed for percent a-helix and b-sheet structure using CDNN CD Spectra Deconvolution v 2.1 developed by G Bohm [41] ă Protein concentrations were determined spectrophotometrically at 280 nm using an extinction coefficient equal to 38 030ỈM)1Ỉcm)1 for the wild-type, K187A, R429A, and R429A/K432A CTP synthases Limited trypsin-catalysed proteolysis of wild-type CTPS produced different cleavage fragments depending on whether ATP and/or UTP were absent or present in the reaction mixture (Fig 1) In the absence of nucleotides, trypsincatalysed cleavage of CTPS (63 kDa) produced four fragments with molecular masses corresponding to 10, 25, 28, and 53 kDa However, in the presence of either ATP or UTP (data not shown), ATP and UTP together, or CTP, only fragments with molecular masses of 10 and 53 kDa were produced indicating that these nucleotides protected CTPS from cleavage at the site which produced the 25- and 28-kDa fragments Neither glutamine nor GTP protected CTPS from limited trypsin-catalysed digestion The sites of trypsin-catalysed cleavage yielding each of the fragments were identified using Edman degradation to obtain the N-terminal amino acid sequence of each fragment (Table 1), and the known nucleotide sequence encoding the enzyme [3] The N-terminal sequence of the 25- and 53-kDa fragments were the same as that of the whole recombinant wild-type protein indicating that the cleavage site was located at the C-terminus of each of these two fragments The N-terminal sequence of the 28-kDa fragment indicated that one of the cleavage sites was Lys187 N-terminal analysis of the 10-kDa fragment produced two sequences indicating that cleavage occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, respectively The cleavage pattern observed in the absence of any protecting ligands, and the sites identified using N-terminal analysis are summarized in Fig The molecular mass of each polypeptide fragment, calculated using the known Tetramerization of CTPS The ability of the K187A CTPS to form tetramers was evaluated using gel-filtration-HPLC (GF-HPLC) with native tryptophan fluorescence detection Wild-type and mutant CTP synthases, and standard proteins were eluted under isocratic conditions using Hepes buffer (pH 8.0, 0.07 M) containing MgCl2 (10 mM) and EDTA (0.5 mM) at a flow rate of 1.0 mLỈmin)1 on a BioSep–SEC-S 3000 column (7.80 · 300 mm; Phenomenex, Torrance, CA) A Waters 510 pump and 680 controller were used for solvent delivery Injections were made using a Rheodyne 7725i sample injector fitted with a 20-lL injection loop The eluted proteins were detected by native protein fluorescence Results Limited proteolysis of CTPS Ó FEBS 2003 Limited proteolysis of CTP synthase (Eur J Biochem 270) 2199 Table N-terminal amino acid sequences of trypsin-catalysed cleavage fragments Molecular massa (kDa) 63 53 28 25 10 (61) (48) (27) (21) (13.1) (12.8) N-terminal sequence Cleavage site identifiedb GSHMLEM1 … GSHML T188KPTQHSVKE GSHML S430EKSDLGGTM (major)c S433DLGGTMRL (minor)c None None Lys187 None Arg429 Lys432 a Apparent molecular mass for full-length recombinant wild-type E coli CTPS and fragments determined from SDS/PAGE calibration are given The corresponding molecular masses calculated using the known amino acid sequence are given in parentheses b The amino acid listed is that which provides the carbonyl function to the scissile peptide bond The numbers correspond to the numbering for wild-type E coli CTPS c The ratio of the major peptide to minor peptide was 2.6 : and was determined by integration of the HPLC chromatogram peaks corresponding to the phenylthiohydantoin derivatives of the N-terminal serines amino acid sequence [3], is in excellent agreement with the values deduced from SDS/PAGE calibration (Table 1) Limited trypsin-catalysed cleavage of mutant CTP synthases Fig SDS/PAGE analysis of trypsin-catalysed cleavage of recombinant wild-type CTPS in the absence and presence of ligands For each gel: lane contains molecular mass standards and lanes 2–6 contain wild-type CTPS treated with trypsin for 0, 10, 20, 30, and 60 min, respectively In the absence of any ligands (A), the wild-type protein is rapidly cleaved to yield a 10-kDa (not shown except in E) and a 53-kDa fragment, the latter which is subsequently cleaved to yield two fragments with molecular masses of % 25 and % 28 kDa In the presence of ATP and UTP (10 mM each) (B), and CTP (2.5 mM) (C), only production of the 10- and 53-kDa cleavage fragments is observed In the presence of GTP (2.5 mM) (D) and L-glutamine (10 mM) (E), fragments with molecular masses of 10, 25, 28, and 53 kDa are produced All gels are 12% except for (E) which is 20% Fig Fragments generated by limited trypsincatalysed proteolysis of wild-type CTPS Peptide bond cleavage occurs C-terminal to Lys187 in the synthase domain, and Arg429 and Lys432 in the GAT domain The CTP/ UTP-binding site and residues comprising the catalytic triad (Cys379, His515, and Glu517) are also shown To confirm that the cleavage sites identified using N-terminal analysis were indeed correct, site-directed mutagenesis was used to construct two single mutants (K187A and R429A) and one double mutant (R429A/K432A) Limited trypsin-catalysed proteolysis of K187A CTPS in the absence of nucleotides produced only the 10- and 53-kDa fragments (Fig 3A), a cleavage pattern which was identical to that observed for wild-type CTPS in the presence of ATP and UTP (Fig 1B) Proteolysis of R429A in the absence of nucleotides gave fragments with molecular masses of approximately 10, 25, 38, and 53 kDa (Fig 3B) The 38-kDa fragment accumulated because rapid cleavage at Arg429 no longer occurred while cleavage at Ó FEBS 2003 2200 D Simard et al (Eur J Biochem 270) and UTP, proteolysis of R429A/K432A CTPS was completely suppressed (Fig 3E) Inactivation and protection studies Treatment of wild-type and mutant CTP synthases with trypsin produced time-dependent loss of both NH3-dependent activity and glutamine-dependent activity (Fig 4), which followed first-order kinetics up to at least 90% of the reaction The observed first-order inactivation rate constants for CTPS activity assayed using either NH3 or glutamine as the substrate are given in Table In the absence of ligands, the observed first-order rate constant for trypsin-catalysed proteolysis of CTPS was slightly greater when glutamine-dependent CTP formation was measured than when NH3-dependent CTP formation was measured A reduction in the observed first-order rate constants for the inactivation of the NH3-dependent activity was observed for increasing concentrations of ATP, UTP, and CTP consistent with each of these nucleotides providing protection from trypsin-catalysed cleavage Interestingly, in the Fig SDS/PAGE analysis of trypsin-catalysed cleavage of mutant CTP synthases in the absence and presence of ligands (A) Lane 1, molecular mass standards; lane 2, trypsin (at 7000 times the concentration used in the proteolysis reactions); lanes 3–7 contain K187A CTPS treated with trypsin for 0, 10, 20, 30, and 60 min, respectively The wild-type protein is rapidly cleaved to yield a 10- (not shown) and a 53-kDa fragment, the latter which is not cleaved to yield the 25- and 28-kDa fragments For each gel shown in B through E, lane contains molecular mass standards and lanes 2–6 contain mutant CTPS treated with trypsin for 0, 10, 20, 30, and 60 min, respectively Limited proteolysis of R429A CTPS (B) in the absence of nucleotides produced fragments with molecular masses of 10, 25, 38, and 53 kDa However, in the presence of ATP and UTP (10 mM each) (C), only the 10- and 53-kDa fragments are produced Limited proteolysis of R429A/ K432A CTPS (D) in the absence of nucleotides produces fragments with molecular masses of 25 and 38 kDa However, in the presence of ATP and UTP (10 mM each) (E), no cleavage fragments were produced over the course of h indicating that proteolysis was greatly suppressed All gels are 12% Lys187 divided the protein into the 25- and 38-kDa fragments The 10- and 53-kDa fragments were formed in much lower amounts than observed with wild-type CTPS because of slow cleavage at Lys432 In the presence of ATP and UTP, cleavage of R429A at Lys187 was suppressed and only the 10- and 53 kDa fragments were formed because of slow cleavage at Lys432 (Fig 3C) Limited proteolysis of the double mutant (R429A/K432A) in the absence of nucleotides produced only two fragments with molecular masses corresponding to 25 and 38 kDa consistent with cleavage occurring only at Lys187 (Fig 3D) In the presence of ATP Fig Time-dependent inactivation of wild-type CTPS by trypsin (A) Inactivation of CTPS-catalysed NH3-dependent CTP formation in the absence of ligands (s) and in the presence of ATP (10 mM, n), UTP (10 mM, h), and ATP and UTP combined (10 mM each, ,) Panel B shows the inactivation of CTPS-catalysed glutamine-dependent CTP formation in the absence of nucleotides (s) and in the presence of UTP (10 mM, n), ATP (10 mM, h), and ATP and UTP combined (10 mM each, ,) In both panels, the activity of the enzyme in the absence of trypsin is also shown (d) Ó FEBS 2003 Limited proteolysis of CTP synthase (Eur J Biochem 270) 2201 Table Observed rate constants for inactivation of recombinant wildtype and mutant CTP synthasesa ND, Not determined kobs (· 10)2 min)1) Protecting ligand None None (R429A) None (R429A/K432A) ATP UTP ATP and UTP CTP GTP L-glutamine Controlb Concentration NH3 as substrate (mM) 0 0.5 1.0 2.0 10.0 0.5 1.0 2.0 10.0 10 + 10 0.1 0.5 1.0 0.25 10.0 ficant gross perturbations in secondary structure are evident in the mutant proteins, the possibility that the mutations cause a localized perturbation of secondary structure or conformational change cannot be ruled out L-glutamine as substrate Mutant enzyme kinetics ( 2) ± 3.9 ( 0.3) ± 1.9 ( 0.4) ± 9.0 ( 0.3) ± ND ND ( 2) ± 1.7 ( 0.5) ± 0.31 ( 0.04) ± 0.4 ( 0.1) ± 5.6 ( 0.9) ± ( 1) ± ( 1) ± 0.7 ( 0.1) ± 0.15 ( 0.07) ± ( 1) ± 0.26 ( 0.09) ± 0.16 ( 0.03) ± 1.7 ( 0.5) ± 1.5 ( 0.3) ± 0.075 ( 0.009) ± ND ND ND 1.9 ( 0.2) ± ND ND ND 3.9 ( 0.9) ± 0.8 ( 0.2) ± ND ND ND ND ND 0.07 ( 0.04) ± The kinetic parameters kcat and Km for CTP formation were determined with respect to NH3 and glutamine for each of the mutant enzymes except for K187A CTPS which was inactive (Table 3) Direct examination of the conversion of glutamine to glutamate (glutaminase activity) revealed that K187A CTPS was able to catalyse the hydrolysis of glutamine, but had a value of kcat that was half of that observed for wild-type CTPS in the absence of GTP When the concentration of GTP was increased to mM, the value of kact was increased fivefold, compared to 50-fold for wildtype CTPS (Fig 6) R429A and R429A/K432A CTP synthases displayed similar kinetic properties Each mutant had close to wildtype NH3-dependent activity; however, glutamine-dependent CTP formation was impaired Interestingly, Km for glutamine only increased 1.3- to 1.7-fold indicating that the mutations had little effect on glutamine binding However, kcat was reduced % 15-fold for each mutant so that the catalytic efficiency (kcat/Km) of glutamine-dependent CTP formation was decreased 25- and 19-fold for the R429A and R429A/K432A CTP synthases, respectively The kinetics of R429A CTPS were investigated in detail to determine if the impaired glutamine-dependent CTP formation was caused by an inability of GTP to activate glutamine hydrolysis In the presence of ATP and UTP (1 mM each) and saturating glutamine (6 mM), GTP (0.25 mM) caused a 2.5-fold increase in kcat for glutaminedependent CTP formation catalysed by R429A CTPS compared to a 30-fold increase for wild-type CTPS (data not shown) Concentrations of GTP above 0.25 mM (up to mM) did not enhance the observed rate of CTP formation Direct examination of the glutaminase activity revealed that kcat was reduced approximately 10-fold for the R429A and R429A/K432A enzymes relative to wild-type CTPS with the concentration of GTP equal to 0.25 mM (Table 3) More detailed analysis of the glutaminase activity of R429A CTPS (Fig 6) revealed that GTP binding and kact were reduced approximately 10-fold and sixfold, relative to wildtype CTPS Thus mutation of Arg429 to alanine impairs both GTP binding and allosteric activation of glutamine hydrolysis Comparison of the kcat values for the glutaminase activity and glutamine-dependent CTP formation catalysed by R429A CTPS reveals that ammonia is produced from glutamine hydrolysis at a rate that is slightly higher than the rate at which CTP is formed This observation suggests that there may be a partial uncoupling of the glutaminase and synthase reactions, however, the kcat values for the corresponding reactions catalysed by the R429A/K432A mutant are experimentally equal a All kobs values are for inactivation of wild-type CTPS except where indicated otherwise b Control refers to the inactivation of CTPS that is observed during incubation of the enzyme at 37 °C for h in the absence of added trypsin presence of ATP and UTP, the rate constant for inactivation of glutamine-dependent activity was greater than that observed for inactivation of the NH3-dependent activity This observation is consistent with the cleavage sites in the GAT domain not being protected by these nucleotides and the resulting 53-kDa fragment still possessing NH3-dependent activity Although all CTPS ligands tested (ATP, UTP, CTP, GTP, and glutamine) protected CTPS from inactivation to some degree, the most effective protection was afforded by CTP The observed first-order inactivation rate constants for the NH3-dependent activity of the R429A and R429A/ K432A CTP synthases were less than that observed for wild-type CTPS Apparently, reduced cleavage within the GAT domain results in less rapid cleavage within in the synthase domain (i.e at Lys187) and hence a lower value for the rate constant for the loss of NH3-dependent activity Inactivation of the K187A enzyme could not be studied because this enzyme was inactive (vide infra) CD The secondary structural content of wild-type CTPS and the three mutant enzymes was analysed using CD spectroscopy Fig shows that the secondary structure content of all the mutant proteins is similar to that of the wild-type enzyme, except that the a-helix content of the K187A and R429A/K432A mutants is slightly reduced while the content of antiparallel b-sheet structure is slightly increased, relative to wild-type CTPS Although no signi- Oligomerization of CTPS To determine if the K187A mutant was inactive because it was unable to form tetramers, we investigated the Ó FEBS 2003 2202 D Simard et al (Eur J Biochem 270) Fig CD analysis of wild-type and mutant CTP synthases (A) Spectra for wild-type, K187A, R429A, and R429A/K432A CTP synthases are shown Each spectrum is the average of three scans for each CTPS variant (B) The relative amount of each type of secondary structure is indicated for each CTPS variant Error bars represent the standard deviation of the mean for three independent trials Table Kinetic parameters for wild-type and mutant CTP synthases ND, not determined CTPS variants Reaction (substrate) Kinetic parametera CTP formation (NH3) Km (mM) kcat (s)1) kcat/Km (mM)1Ỉs)1) Km (mM) kcat (s)1) kcat/Km (mM)1s)1) kcat (s)1) [GTP] ¼ 0.25 mM CTP formation (L-glutamine) L-glutamate Wild-type 1.70 7.8 4.5 0.24 5.4 22.6 5.01 ± ± ± ± ± ± ± 0.08 0.1 0.2 0.04 0.8 4.5 0.18 K187A R429A 0.13 ± 0.03 1.60 7.3 4.6 0.40 0.36 0.9 0.48 0.08 0.3 0.3 0.03 0.04 0.1 0.03 1.82 6.5 3.6 0.32 0.39 1.2 0.49 0.07 ± 0.02 –c –c 0.14 ± 0.04 1.19 ± 0.04 0.38 ± 0.04 ND ND ND NAb NA ± ± ± ± ± ± ± R429A/K432A ± ± ± ± ± ± ± 0.04 0.8 0.5 0.08 0.04 0.4 0.07 formation (fixed L-glutamine with varying [GTP]) ko (s)1) kact (s)1) KA (mM) 0.14 ± 0.03 7.1 ± 0.3 0.032 ± 0.006 a Assay conditions are as described in Materials and methods [ATP] ¼ [UTP] ¼ mM b No activity was observed (i.e < 0.5% wild-type CTPS activity) c Values could not be determined accurately because of the low activity Ó FEBS 2003 Limited proteolysis of CTP synthase (Eur J Biochem 270) 2203 Fig Glutaminase activity for mutant CTP synthases The values of apparent kcat for the hydrolysis of glutamine by K187A (d) and R429A (s) apparent CTP synthases are shown Inset: values of kcat for the hydrolysis of glutamine by wild-type CTPS The curves shown are from a fit of the data to Eqn (2) and the values of ko, kact, and KA are given in Table ability of this mutant to form tetramers in the presence of nucleotides using GF-HPLC The observed molecular masses for wild-type CTPS in the absence of nucleotides and in the presence of ATP and UTP were 123 and 251 kDa, respectively These values are similar to the predicted values of 122 and 245 kDa, based on the amino acid sequence of the recombinant mutant protein, and are consistent with wild-type CTPS existing primarily as dimers in the absence of ATP and UTP, and with a shifting of the equilibrium to favour the tetrameric species in the presence of ATP and UTP [42] The K187A mutant had an apparent molecular mass of 178 kDa in the absence of nucleotides This value is slightly higher than that observed for the wild-type enzyme and corresponds to the enzyme existing as % 30% tetramer as calculated using Eqn (3) [20], where X is the fraction of the enzyme in the tetramer form and the molecular masses of the dimer (121 944 Da) and the tetramer (243 888 Da) are those predicted based on the monomer molecular mass of 60 972 Da for recombinant K187A lacking the histidine tag In the presence of ATP and UTP, the observed molecular weight for K187A was 259 kDa Thus it appeared that K187A CTPS was capable of forming tetramers in the presence of saturating concentrations of UTP and ATP, similar to the wild-type enzyme molecular mass ẳ 243 888ị2 X þ ð121 944Þ2 ð1 À XÞ ð3Þ ð243 888ÞX þ ð121 944Þð1 À XÞ Discussion Limited proteolysis has been used to delineate the structural organization of several amidotransferases including aspa- ragine synthase [43], carbamoyl phosphate synthase [44–50], anthranilate synthase [51], and glucosamine-6phosphate synthase [52] This methodology has been particularly useful for identifying both ligand-binding sites and, in the case with glucosamine-6-phosphate synthase, an exposed ÔhingeÕ region that, when cleaved by a-chymotrypsin, led to separation of the enzyme into its GAT and synthase domains Our interest in delineating structural aspects of E coli CTPS led us to examine the susceptibility of CTPS to controlled proteolysis In preliminary experiments with endopeptidases of different specificity, we identified trypsin as the enzyme of choice Trypsin-catalysed cleavage of wild-type CTPS generated four fragments in the absence of ATP and UTP, but only two fragments in the presence of these nucleotides Determination of the N-terminal sequence of these fragments, in conjunction with the known nucleotide sequence of the E coli pyrG gene [3], permitted us to identify three principal cleavage sites: Lys187 in the synthase domain, and Arg429 and Lys432 in the GAT domain A summary of the fragmentation pattern arising from trypsin-catalysed cleavage at these sites is presented in Fig Lys187 resides in a region of the synthase domain that is highly conserved among CTP synthases from different organisms This region, between residues 116 and 229, has been suggested to comprise the CTP/UTP-binding site [18,25–27] Our observation that both CTP and UTP afford effective protection to CTPS from trypsin-catalysed cleavage at Lys187 also supports the notion that this residue is located in the CTP/UTP-binding site Interestingly, ATP also provides protection against cleavage and does so better than UTP Such protection could arise because: (a) ATP binds at an adjacent site and sterically blocks access of trypsin to Lys187; (b) ATP-induced tetramerization yields a quaternary structure in which the Lys187 site is not accessible to trypsin; or (c) ATP induces a conformational change in CTPS to yield a conformation in which Lys187 is no longer exposed to bulk solvent Replacement of Lys187 by an alanine residue yielded a protein that was resistant to limited trypsin-catalysed proteolysis in the synthase domain, supporting our conclusion that cleavage occurred C-terminal to this residue The K187A mutant could not catalyse the formation of CTP, however, it retained the ability to form tetramers in the presence of nucleotides, and exhibited a very low level of GTP-dependent glutaminase activity which was enhanced slightly by GTP Interestingly, in the absence of nucleotides, K187A existed as % 30% tetramer suggesting that neutralization of positive charge at residue 187 might play a role in promoting enzyme tetramerization Indeed, hydrophobic interactions between dimers of E coli CTPS have been suggested to play a role in the formation of tetramers [53] Predictions of the secondary structure of the highly conserved region of amino acid sequence between residues 185 and 192 suggest that Lys187 constitutes part of a conserved loop It is not clear whether nucleotides protect this putative loop from proteolytic cleavage because nucleotide binding directly blocks access of trypsin to the cleavage site or, because nucleotide binding causes a change in the enzyme’s conformation or quaternary structure (i.e tetramerization) that subsequently conceals the cleavage site from trypsin 2204 D Simard et al (Eur J Biochem 270) Finally, we note that studies on the chemical modification of E coli CTPS with thiourea dioxide led Roberston et al [54] to conclude that lysine residues were important for catalysis To our knowledge, the present study represents the first identification of a catalytically essential lysine residue in E coli CTPS involved in either amido-/NH3 transfer or UTP phosphorylation Arg429 and Lys432 reside in a region of amino acid sequence within the GAT domain that is partially conserved only among CTP synthases from some sources (Fig 7) In accord with our expectations, nucleotides offered no protection against cleavage at these sites but replacement of these residues by alanine (i.e R429A and R429A/K432A) yielded mutant enzymes that were more resistant to proteolytic cleavage in the GAT domain These mutant enzymes displayed wild-type activity with respect to NH3-dependent CTP formation and wild-type affinity for glutamine, but glutamine-dependent CTP formation was markedly impaired Interestingly, although these mutations in the GAT domain did not impair the enzyme’s ability to utilize NH3 as a substrate (i.e the activity associated with the synthase domain [16]), they did cause the rate of loss of NH3-dependent activity during limited trypsin-catalysed proteolysis to be less than would have been predicted based on the inactivation rate constant observed for wild-type Ó FEBS 2003 CTPS (Table 2) This is consistent with previous reports that suggested interactions between the GAT and synthase domains within the tertiary structure of the enzyme [17,28,55] The existence of such interactions is also supported by our observation that mutation of Lys187 to alanine in the synthase domain severely impairs the glutaminase activity in the GAT domain Our observations that R429A CTPS binds GTP with reduced affinity (% 10-fold) and, at saturating concentrations of GTP, the apparent kcat value for glutaminedependent CTP formation is reduced sixfold suggest that Arg429 plays a role in both binding GTP and the mechanism for allosteric activation of glutamine hydrolysis Secondary structure predictions suggest that Arg429 and Lys432 are located within a region where a b-strand undergoes a transition into a loop structure The ability of trypsin to catalyse cleavage adjacent to these residues suggests that this loop is exposed to bulk solvent Despite the fact that this region is not highly conserved between organisms, it does appear to be required for E coli CTPS to catalyse glutamine turnover Arg429 and Lys432 lie close to a conserved sequence motif [GG(TS)(ML)RLG] within the GAT domain (shaded residues 436–442 in Fig 7) that was recently identied by Willemoes [24] Using site-directed ă mutagenesis experiments on CTPS from L lactis, Fig Sequence comparison of a portion of the C-terminus (GAT domain) of CTP synthases For the protein sequences shown, invariant residues (*), conservative substitutions (:), and semiconservative substitutions (.) are indicated The two residues (Arg429 and Lys432) identified as cleavage sites during limited trypsin proteolysis and mutated in the present study are indicated (›) These residues reside in a region of the primary structure that is not conserved among different organisms The conserved sequence motif (GG[TS][ML]RLG) identied by Willemoes [24] is shaded The proteins ă included in the alignment are as follows (accession numbers in parentheses): Girardia intestinalis (AAB41453.1), Synechococcus (Q54775), Spiroplasma citri (P52200), Synechocystis (P74208), Bacillus subtilis (P13242), Mycobacterium leprae (S72961), Mycobacterium bovis (AAB48045.1), Methanococcus jannaschii (Q58574), Chlamydia trachomatis (Q59321), Haemophilus influenzae (P44341), Neisseria meningitidis (CAB84970.1), Nitrosomonas europaea (AAC33441.1), Azospirillum brasilense (P28595), Campylobacter jejuni (CAB72520.1), Heliobacter pylori (O25116), Borrelia burgdorferi (O51522), Cricetulus griseus (P50547), Mus musculus (P70698), Homo sapiens (NP_001896.1), Arabidopsis thaliana (AAC78703.1), Saccharomyces cerevisiae H (URA-8, P38627), Saccharomyces cerevisiae G (URA-7, P28274), Plasmodium falciparum (AAC36385.1), Lactococcus lactis (CAA09021.2), and Escherichia coli (AAA69290.1) Numbering shown is for the E coli sequence Ó FEBS 2003 Limited proteolysis of CTP synthase (Eur J Biochem 270) 2205 Willemoes demonstrated that Thr431 and Arg433 (Thr438 ă and Arg440 in E coli CTPS) within this motif play a role in GTP-dependent activation of glutamine hydrolysis but concluded that these residues were not involved in GTP binding [24] However, our observation that Arg429 is important for GTP binding is consistent with Willemoes ă observation that R433A L lactis CTPS exhibited a 10–17fold increase in GTP-binding affinity [24] and support the notion that the conserved sequence motif and adjacent residues may also be important for GTP binding 15 16 17 Acknowledgements The authors thank the Canadian Institutes of Health Research for an operating grant (S L B.), the Nova Scotia Health Research Foundation and Cancer Care Nova Scotia for graduate fellowships (A I.), and Cancer Care Nova Scotia for research training grants (K H & D S) The authors also express thanks to Prof M Dobson and J Chew for technical advice and assistance with electroblotting 18 19 20 References Koshland, D.E Jr & Levitzki, A (1974) CTP Synthetase and related enzymes In The Enzymes (Boyer, P.D., ed.), pp 539–559 Academic Press, New York Long, C & Koshland, 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of apparent kcat for the hydrolysis of glutamine by K187A (d) and R429A (s) apparent CTP synthases are shown Inset: values of kcat for the hydrolysis of glutamine. .. parameters kcat and Km for CTP formation were determined with respect to NH3 and glutamine for each of the mutant enzymes except for K187A CTPS which was inactive (Table 3) Direct examination of the conversion