CTPS (cytidine 5′-triphosphate synthase) catalyses the ATP-dependent formation of CTP from UTP using either ammonia or L-glutamine as the nitrogen source. Binding of the substrates ATP and UTP, or the product CTP, promotes oligomerization of CTPS from inactive dimers to active tetramers. In the present study, site-directed mutagenesis was used to replace the fully conserved glycine residues 142 and 143 within the UTP-binding site and 146 within the CTP-binding site of Escherchia coli CTPS. CD spectral analyses of wild-type CTPS and the glycine mutants showed a slight reduction of ∼15% in α-helical content for G142A and G143A relative to G146A and wild-type CTPS, suggesting some local alterations in structure. Relative to wild-type CTPS, the values of kcat/Km for ammonia-dependent and glutamine-dependent CTP formation catalysed by G143A were reduced 22- and 16-fold respectively, whereas the corresponding values for G146A were reduced only 1.4- and 1.8-fold respectively. The glutaminase activity (kcat) of G146A was similar to that exhibited by the wild-type enzyme, whereas that of G143A was reduced 7.5-fold. G146A exhibited substrate inhibition at high concentrations of ammonia and a partial uncoupling of glutamine hydrolysis from CTP production. Although the apparent affinity (1/[S]0.5) of G143A and G146A for UTP was reduced ∼4-fold, G146A exhibited increased co-operativity with respect to UTP. Thus mutations in the CTP-binding site can affect UTP-dependent activity. Surprisingly, G142A was inactive with both ammonia and glutamine as substrates. Gel-filtration HPLC experiments revealed that both G143A and G146A were able to form active tetramers in the presence of ATP and UTP; however, nucleotide-dependent tetramerization of G142A was significantly impaired. Our observations highlight the sensitivity of the structure of CTPS to mutations in the UTP- and CTP-binding sites, with Gly142 being critical for nucleotide-dependent oligomerization of CTPS to active tetramers. This ‘structural sensitivity’ may limit the number and/or types of mutations that could be selected for during the development of resistance to cytotoxic pyrimidine nucleotide analogues.

INTRODUCTION

CTPS [cytidine 5′-triphosphate synthase; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] is a single polypeptide glutamine amidotransferase that catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine or ammonia as the nitrogen source (1) [1]. The hydrolysis of glutamine occurs in the C-terminal GAT (glutamine amide transfer) domain [2] and the resulting nascent ammonia is subsequently transferred via an ammonia tunnel [35] to the N-terminal synthase domain where it reacts with UTP that has been activated by ATP-dependent phosphorylation at the 4-position [6].

CTP-forming reactions and glutaminase reaction catalysed by CTPS

CTPS from Escherichia coli is an excellent model for understanding catalysis by CTPSs from other organisms, including human CTPS [7], since CTPSs exhibit high conservation of functionally and structurally important residues, and have few insertion/deletion differences [5,8]. Superimposition of the structures of the synthase domains of CTPS from E. coli [8], human [7] and Thermus thermophilus HB8 [9] reveals a striking structural similarity between these CTPSs (see Supplementary Figure 1 at http://www.BiochemJ.org/bj/412/bj4120113add.htm). Consequently, the physical, kinetic and structural properties of E. coli CTPS have been studied in considerable detail.

E. coli CTPS is regulated in a complex fashion. GTP acts a positive allosteric effector for glutamine-dependent CTP formation [10,11]; however, at concentrations exceeding 0.15 mM, GTP inhibits glutamine-dependent CTP formation [12]. Moreover, GTP is an inhibitor of ammonia-dependent CTP formation at all concentrations [12]. Kinetic studies on CTPS from Lactococcus lactis have suggested that formation of the 4-phosphorylated UTP intermediate also stimulates glutamine hydrolysis, acting as a co-activator with GTP [13]. The enzyme exhibits positive co-operativity for ATP and UTP [1416], and these nucleotides act synergistically to promote tetramerization of the enzyme to its active form [16]. On the other hand, the product CTP acts as a feedback inhibitor [14]. The human enzyme [1719] and the yeast enzyme, encoded by the URA7 gene [20,21], are also regulated by phosphorylation.

The central role of CTP in nucleic acid [22] and membrane phospholipid [23] biosynthesis makes CTPS an attractive target for the development of antineoplastic [22], antiviral [24] and antiprotozoal [2527] agents. One strategy for inhibiting CTPS activity is to block the nucleotide-dependent conversion of the inactive dimer into the active tetramer. Understanding the factors that contribute to the oligomerization of CTPS will inform such a strategy. Many nucleotide-binding domains contain loops which are often rich in glycine residues that either interact directly with the nucleotide or play an important structural role (see [28] and [29] and references therein). In the present study, we have used site-directed mutagenesis to replace glycine residues 142, 143 and 146 with alanine residues in E. coli CTPS. Gly142 and Gly143 reside in the putative UTP-binding site, whereas Gly146 resides in the CTP-binding site (see Figure 1) [8]. Each of these glycine residues is fully conserved in all 67 of the CTPS COG0504 sequences (http://www.ncbi.nlm.nih.gov/COG/old/aln/COG0504.aln; [30]). In the present study, we characterize the kinetic and physical properties of the G146A and G143A CTPS mutants and show that mutations in the CTP-binding site can affect UTP-dependent activity. More surprisingly, Gly142 is shown to be critical for tetramerization of the enzyme.

Stereoview of the UTP- and CTP-binding sites of E. coli CTPS

Figure 1
Stereoview of the UTP- and CTP-binding sites of E. coli CTPS

CTP and UTP (stick representations) are shown located within the active site according to the model of Baldwin and co-workers [5]. Note that UTP and CTP are proposed to share the same binding site for their 5′-triphosphate moieties. Gly146 of E. coli CTPS (stick representation) clearly resides in the binding pocket for the cytosine base of CTP, whereas Gly142 and Gly143 (stick representations) are located in the putative binding pocket for the uracil moiety of UTP [8]. The Figure was generated using MacPyMOL (DeLano Scientific LLC; http://www.pymol.org).

Figure 1
Stereoview of the UTP- and CTP-binding sites of E. coli CTPS

CTP and UTP (stick representations) are shown located within the active site according to the model of Baldwin and co-workers [5]. Note that UTP and CTP are proposed to share the same binding site for their 5′-triphosphate moieties. Gly146 of E. coli CTPS (stick representation) clearly resides in the binding pocket for the cytosine base of CTP, whereas Gly142 and Gly143 (stick representations) are located in the putative binding pocket for the uracil moiety of UTP [8]. The Figure was generated using MacPyMOL (DeLano Scientific LLC; http://www.pymol.org).

MATERIALS AND METHODS

General

His·Bind resin, thrombin cleavage capture kits and the pET-15b expression system were purchased from Novagen. PfuTurbo DNA polymerase was purchased from Stratagene. All other chemicals were purchased from Sigma–Aldrich. Synthetic deoxyoligonucleotide primers for sequencing and site-directed mutagenesis were commercially synthesized by ID Labs. All plasmid preparations for mutagenesis and transformation were conducted using the QIAprep Spin Miniprep Kit (Qiagen). DNA sequencing was conducted by the Dalhousie University, NRC Institute for Marine Biosciences Joint Laboratory (Halifax, Nova Scotia, Canada) and DalGen Microbial Genomic Centre (Halifax, Nova Scotia, Canada). CD studies were conducted using a JASCO J-810 spectropolarimeter.

Site-directed mutagenesis

pET15b-CTPS1, a pET-15b plasmid containing the recombinant E. coli CTPS open reading frame, was used as the template for site-directed mutagenesis conducted as described previously [3] using the QuikChange® kit (Stratagene) and following the procedures described by the manufacturer. The synthetic deoxyoligonucleotide primers (F, forward; R, reverse; mismatched bases are underlined) used to construct the mutants were: 5′-GTACTGGTAGAAATCGCCGGTACAGTAGGT-3′ (G142A, F), 5′-CCTACTGTACCGGCGATTTCTACCAGTAC-3′ (G142A, R), 5′-GGTAGAAATCGGCGCTACAGTAGGTGATATC-3′ (G143A, F), 5′-GATATCACCTACTGTAGCGCCGATTTCTACC-3′ (G143A, R), 5′-ATCGGCGGTACAGTAGCTGATATCGAATCCTTG-3′ (G146A, F), and 5′-CAAGGATTCGATATCAGCTACTGTACCGCCGAT-3′ (G146A, R). Potential mutant plasmids were isolated and heat shock was used to transform competent E. coli DH5α cells [31]. These cells were used for plasmid maintenance and for all sequencing reactions. The entire mutant open reading frames were sequenced to verify that no other alterations in the nucleotide sequence had been introduced.

Expression and purification of recombinant CTPSs

Wild-type and mutant forms of E. coli CTPS were expressed in and purified from E. coli BL21(DE3) cells transformed with pET15b-CTPS1 (or mutated forms) as described previously [3,11]. pET15b-CTPS1 encodes wild-type E. coli CTPS bearing an N-terminal His6-tag (MGSSHHHHHHSSGLVPR↓GSHMLEM1…CTPS, where amino acids are numbered according to the sequence of the wild-type E. coli enzyme starting with M1 as position one). The soluble His6-tagged wild-type and mutant enzymes were purified using metal ion affinity chromatography followed by removal of the tag using thrombin-catalysed cleavage (new N-terminus, GSHMLEM1…) using the protocols described by Novagen. The enzyme was then dialysed into assay buffer [70 mM Hepes buffer (pH 8.0) containing 0.5 mM EGTA and 10 mM MgCl2]. The results of the purification and cleavage procedures were routinely monitored using SDS/PAGE. Typically, enzyme preparations were >98% pure as judged by SDS/PAGE analysis. Although the mutant proteins were purified from E. coli BL21(DE3) cells, SDS/PAGE analysis of the purified His6-tagged proteins revealed that there was no significant incorporation of wild-type CTPS monomers in the oligomeric structure of the CTPS mutants (see Supplementary Figure 2 at http://www.BiochemJ.org/bj/412/bj4120113add.htm). This concern is most relevant to the G146A and G143A enzymes for which we observed activity. Using Edman degradation (Protein Chemistry Core Facility, Columbia University, NY, U.S.A.) to analyse the N-terminal sequence of His6-tag-cleaved G143A and G146A, the only observed N-terminal sequences were GSHML consistent with recombinant CTPS with the His6-tag removed. The N-terminal sequence MTTNY of the ‘native’ wild-type CTPS was not detected.

Enzyme assays and protein determinations

CTPS activity was determined at 37°C by following the change in absorbance at 291 nm using the continuous spectrophotometric assay described previously [3,11,14]. The standard assay mixture consisted of 70 mM Hepes buffer (pH 8.0) containing 0.5 mM EGTA, 10 mM MgCl2, CTPS (wild-type, 34–54 μg/ml; G142A, 100–1000 μg/ml; G143A, 300 μg/ml; and G146A, 64–96 μg/ml), 1 mM UTP and 1 mM ATP, unless stated otherwise, in a total volume of 1.0 ml. Enzyme and nucleotides were pre-incubated for 2.5 min at 37°C followed by addition of substrate (ammonium chloride or glutamine) to initiate the reaction. Total ammonium chloride concentrations in the assays were 5, 10, 20, 30, 50, 60, 80, 100 and 150 mM. For glutamine assays, the concentrations of glutamine in the assays were 0.05, 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 3.0, 6.0 and 10.0 mM, and the concentration of GTP was 0.15 mM. The ionic strength was maintained at 0.3 M in all assays by the addition of KCl, unless stated otherwise.

All kinetic parameters were determined in triplicate and means±S.D. are given. Initial rate kinetic data were fitted to eqn (1) by non-linear regression analysis using KaleidaGraph v.3.5 (Synergy Software).

 
formula
(1)

In eqn (1), vi is the initial velocity, Vmax is the maximal velocity at saturating substrate concentrations (where Vmax=kcat[E]T), S is the substrate (glutamine or ammonia) and Km is the Michaelis constant for the substrate. Values of Km were calculated for the concentration of ammoinia present at pH 8.0 {pKa(NH4+)=9.24; [32]}. Values of kcat were calculated for CTPS variants with the His6-tag removed using molecular masses (Da) of 61029 (wild-type) and 61043 (G142A, G143A and G146A). For the G146A mutant, kinetic parameters with respect to ammonia were calculated using eqn (2) [33].

 
formula
(2)

Except where noted otherwise, protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad) with BSA as standards.

Inhibition by CTP

The effect of CTP on the activity of wild-type (21 μg/ml), G143A (400 μg/ml) and G146A (46 μg/ml) CTPSs was examined with respect to UTP. Ammonia-dependent CTP formation was measured in 70 mM Hepes buffer (pH 8.0) containing 10 mM MgCl2, 0.5 mM EDTA, 1.3 mM ATP, 20 mM ammonium chloride and CTP (0, 0.2 and 0.3 mM). The concentration of UTP was varied between 0.025 and 1.5 mM. Enzyme and nucleotides were pre-incubated for 2.5 min at 37°C, followed by addition of ammonium chloride to initiate the reaction. The initial velocity data were fitted directly to the Hill equation (eqn 3, where n is the Hill coefficient and [S]0.5 is the substrate concentration that yields the half-maximal velocity) using non-linear regression analysis.

 
formula
(3)

The concentrations of CTP that yield 50% inhibition (the IC50) of ammonia-dependent CTP formation were determined for wild-type (27–54 μg/ml) and G146A (16–320 μg/ml) CTPSs at low (0.2 mM) and high (1.0 mM) concentrations of UTP in the presence of ammonium chloride (150 mM), ATP (1.0 mM) and CTP (0–0.8 mM).

Glutaminase activity

The glutaminase activities of wild-type, G142A, G143A and G146A CTPSs were determined by following the production of glutamate using reversed-phase HPLC separation of the o-phthaldialdehyde derivatives of glutamate and glutamine with fluorescence detection as described previously [34]. Assays contained 1 mM ATP, 1 mM UTP, 0.25 mM GTP, saturating 6 mM glutamine and enzyme (wild-type, 3.5–5.5 μg/ml; G142A, 50 μg/ml; G143A, 25 μg/ml; and G146A, 4.0–12 μg/ml) in a total volume of 2.5 ml.

GF-HPLC (gel-filtration HPLC)

The ability of wild-type and the three mutant CTPSs to form tetramers was evaluated using GF-HPLC. Wild-type and mutant CTPSs, and standard proteins (0.5 mg/ml, 20 μl injection volume) were eluted under isocratic conditions using 70 mM Hepes buffer (pH 8.0) containing 10 mM MgCl2 and 0.5 mM EGTA at a flow rate of 1.0 ml/min on a BioSep-SEC-S 3000 column (7.80 mm×300 mm; Phenomenex). 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-μl injection loop. The eluted proteins were detected by native protein fluorescence (λex=285 nm and λem=335 nm) using a Waters 474 scanning fluorescence detector. GF-HPLC was conducted in the absence and presence of 1 mM ATP and 1 mM UTP, 5 mM UTP and 1 mM ATP, 5 mM CTP and 1 mM ATP, 5 mM and 10 mM ATP alone, and 5 mM and 10 mM UTP alone. The column was standardized using the following proteins (at 0.5 mg/ml): bovine thyroglobulin (669 kDa), β-amylase (200 kDa), BSA (66 kDa) and carbonic anhydrase (29 kDa). The retention time of bovine thyroglobulin was used to estimate the column void volume (Vo).

CD

CD spectra were recorded for wild-type, G142A, G143A and G146A CTPSs over the wavelength range of 190–260 nm. The resulting CD spectra obtained from enzyme solutions [0.2 mg/ml in 10 mM Bistris buffer (pH 8.0) containing 10 mM MgSO4] were analysed for their percentage of α-helix and β-sheet structure using the CDSSTR deconvolution software available at the website http://cdtools.cryst.bbk.ac.uk/ [35]. Protein concentrations were determined spectrophotometrically using a molar absorption coefficient equal to 37800 M−1·cm−1 which was determined for the wild-type enzyme at 280 nm.

Limited proteolysis

The extent of limited trypsin-catalysed proteolysis of both wild-type and G142A CTPSs was determined at specific time points using SDS/PAGE and a protocol similar to that reported previously [36]. Proteolysis reactions (1 ml total volume) contained either wild-type CTPS or G142A (0.40 mg/ml) and were conducted for 1 h in 70 mM Hepes buffer (pH 8.0) containing 0.5 mM EGTA, 10 mM MgCl2 and 0 or 5 mM UTP at 37°C. Reactions were initiated by addition of trypsin (0.1 μg/ml) and aliquots (20 μl) were removed at 0, 5, 10, 20, 30 and 60 min, and transferred into gel-loading buffer (20 μl) to terminate the reaction. The samples were then loaded on to an SDS/PAGE gel (12%).

RESULTS AND DISCUSSION

The self-association of proteins and its modulation by ligands play an important role in the regulation of enzymatic activity. Such is the case for CTPS which is regulated, in part, by nucleotide-dependent oligomerization [3739]. The substrates ATP and UTP, and the product CTP, promote oligomerization of inactive dimers to active tetramers. The four synthase active sites per tetramer, where CTP is produced, are formed through the combination of the highly conserved synthase domain surfaces from three different monomers with the bound nucleotides in the synthase active site ‘cementing’ the tetramer contacts [8]. Nucleotide-triphosphate-binding motifs are often rich in glycine residues that serve as binding determinants or play important structural roles. In E. coli CTPS, Gly142 and Gly143 are located within the putative UTP-binding site [8]. Gly146 is located in the CTP-binding site where the carbonyl oxygen of Gly146 resides 4.68 Å (1 Å=0.1 nm) away from the N-4 of cytidine and may serve as a potential source of recognition of N-4 through a water-mediated hydrogen-bond [8]. To probe the role of these fully conserved glycine residues in pyrimidine-nucleotide binding and enzyme oligomerization, we constructed the CTPS mutants G142A, G143A and G146A and assessed their kinetic and physical properties. The kinetic parameters for both the wild-type and mutant CTPSs utilizing either glutamine or ammonia as the substrate are shown in Table 1. Surprisingly, replacement of Gly142 by an alanine residue completely abolished the activity of the enzyme, whereas the G143A and G146A enzymes exhibited partial CTPS activity.

Table 1
Kinetic parameters for wild-type and mutant CTP synthases

Kinetic parameters determined with [UTP]=[ATP]=1.0 mM.

  CTP synthase variants 
Ammonia source Kinetic parameter Wild-type G142A G143A G146A 
NH3 Km (mM) 2.2±0.1 Inactive 3.1±0.2 2.7±0.1† 
 kcat (s−19.5±0.5 Inactive 0.65±0.05 8.4±0.1† 
 kcat/Km (mM−1·s−14.4±0.1 Inactive 0.2±0.1 3.2±0.2† 
Glutamine* Km (mM) 0.35±0.06 Inactive 0.59±0.04 0.18±0.04 
 kcat (s−16.1±0.8 Inactive 0.6±0.1 1.9±0.1 
 kcat/Km (mM−1·s−118±2 Inactive 1.1±0.3 10±2 
 Inhibition by CTP     
NH3 IC50 (mM), 0.2 mM UTP 0.25±0.01 – – 0.59±0.07 
 IC50 (mM), 1.0 mM UTP 0.30±0.05 – – 0.36±0.03 
  CTP synthase variants 
Ammonia source Kinetic parameter Wild-type G142A G143A G146A 
NH3 Km (mM) 2.2±0.1 Inactive 3.1±0.2 2.7±0.1† 
 kcat (s−19.5±0.5 Inactive 0.65±0.05 8.4±0.1† 
 kcat/Km (mM−1·s−14.4±0.1 Inactive 0.2±0.1 3.2±0.2† 
Glutamine* Km (mM) 0.35±0.06 Inactive 0.59±0.04 0.18±0.04 
 kcat (s−16.1±0.8 Inactive 0.6±0.1 1.9±0.1 
 kcat/Km (mM−1·s−118±2 Inactive 1.1±0.3 10±2 
 Inhibition by CTP     
NH3 IC50 (mM), 0.2 mM UTP 0.25±0.01 – – 0.59±0.07 
 IC50 (mM), 1.0 mM UTP 0.30±0.05 – – 0.36±0.03 
*

[GTP] = 0.15 mM.

Values were obtained by fitting the initial velocity data to eqn (2); α=0.5±0.1 and β=0.

G146A CTPS

Replacement of Gly146 by an alanine residue resulted in only 2- and 3-fold reductions of the values of Km and kcat respectively, relative to wild-type CTPS when glutamine was the substrate. Overall, G146A exhibited only a 2-fold reduction in catalytic efficiency (kcat/Km) for glutamine-dependent CTP formation. Interestingly, the ammonia-dependent activity of G146A did not obey Michaelis–Menten (hyperbolic) kinetics (Figure 2). Instead, the rate of G146A-catalysed ammonia-dependent CTP formation reached a maximum when the ammonia concentration was ∼1.5 mM, and then decreased markedly at ammonia concentrations greater than 3 mM (with the ionic strength held constant). This observation is consistent with substrate inhibition and was best described using the two-site model shown in 2 [33]. Non-linear regression fitting of the velocity data to eqn (2) revealed that β≈0, suggesting that the ternary complex between G146A and ammonia [i.e. E·(NH3)2] is catalytically inactive or exhibits very low turnover (see Figure 2). Fitting the velocity data to eqn (2) with β=0 gave values of Km and kcat similar to those of wild-type CTPS (Table 1). Overall, the value of kcat/Km for G146A-catalysed ammonia-dependent CTP formation was reduced only 1.4-fold relative to wild-type CTPS.

Michaelis–Menten plot for G146A CTPS (114 μg/ml) using ammonia as the substrate

Figure 2
Michaelis–Menten plot for G146A CTPS (114 μg/ml) using ammonia as the substrate

Pronounced substrate inhibition is evident at high ammonia concentrations. The curve is from a non-linear regression fit of the data to eqn (2) with β=0. The values of kcat, Km and α are given in Table 1. Experimental conditions (ionic strength=0.3 M) are as described in the Materials and methods section. Inset: Michaelis–Menten plot for G146A CTPS (160 μg/ml) using ammonia concentrations up to 22 mM with the ionic strength held constant at 0.5 mM. The data were fitted to eqn (2) using non-linear regression analysis with kcat=5.5 s−1 and β=0 (solid line, Km=1.7±0.1 mM, α=3.8±0.7) or with β≠0 (dashed line, Km=0.1±0.1 mM, β=0.02±0.01, α=5±4). Both curves give R2≈0.997; however, errors on the kinetic parameters obtained when β≠0 are large. Although it is not clear whether the ternary complex E·(NH3)2 is catalytically competent, it is clear that its turnover is much slower than E·NH3. When the ionic strength=0.3 M, α=0.5 which implies that the affinity for the second ammonia is enhanced 2-fold by the presence of the first ammonia under standard assay conditions. However, when the ionic strength was raised to 0.5 M, α increased to ∼4 mM indicating that the affinity for the second ammonia was reduced. Because the mechanism by which CTPS activity is altered by changes in ionic strength [11] has not been characterized, it is not clear why the affinity for ammonia was decreased.

Figure 2
Michaelis–Menten plot for G146A CTPS (114 μg/ml) using ammonia as the substrate

Pronounced substrate inhibition is evident at high ammonia concentrations. The curve is from a non-linear regression fit of the data to eqn (2) with β=0. The values of kcat, Km and α are given in Table 1. Experimental conditions (ionic strength=0.3 M) are as described in the Materials and methods section. Inset: Michaelis–Menten plot for G146A CTPS (160 μg/ml) using ammonia concentrations up to 22 mM with the ionic strength held constant at 0.5 mM. The data were fitted to eqn (2) using non-linear regression analysis with kcat=5.5 s−1 and β=0 (solid line, Km=1.7±0.1 mM, α=3.8±0.7) or with β≠0 (dashed line, Km=0.1±0.1 mM, β=0.02±0.01, α=5±4). Both curves give R2≈0.997; however, errors on the kinetic parameters obtained when β≠0 are large. Although it is not clear whether the ternary complex E·(NH3)2 is catalytically competent, it is clear that its turnover is much slower than E·NH3. When the ionic strength=0.3 M, α=0.5 which implies that the affinity for the second ammonia is enhanced 2-fold by the presence of the first ammonia under standard assay conditions. However, when the ionic strength was raised to 0.5 M, α increased to ∼4 mM indicating that the affinity for the second ammonia was reduced. Because the mechanism by which CTPS activity is altered by changes in ionic strength [11] has not been characterized, it is not clear why the affinity for ammonia was decreased.

Kinetic scheme accounting for the observed substrate inhibition of G146A CTPS by ammonia

The origin of this substrate inhibition by ammonia (or possibly the ammonium ion) is not clear. It is unlikely that the functional changes arise from gross changes in secondary structure, since the CD spectrum of the G146A protein was essentially identical with that observed for the wild-type enzyme (Supplementary Figure 3 at http://www.BiochemJ.org/bj/412/bj4120113add.htm). One possible explanation is that the structural perturbation introduced by the G146A mutation causes a minor change in local conformation that alters the ammonia-binding site and permits binding of a second molecule of ammonia as shown in 2. The ternary species E·(NH3)2 may represent two ammonia molecules bound at the same active site on the same subunit or binding of the second ammonia at the active site of an adjacent subunit or at some other location entirely. The two-site kinetic mechanism does not exclude these possibilities. Also consistent with 2, is the possibility that the mutation alters the structure of an ammonia tunnel such that a second ammonia molecule ‘clogs’ the tunnel thereby limiting passage of ammonia to the site of UTP amination [4,5]. This latter possibility is supported by our observation that glutamine-dependent CTP formation is partially uncoupled from glutamine hydrolysis (Tables 1 and 2). The value of kcat for the glutaminase activity of G146A was reduced only 1.2-fold, whereas the value of kcat for glutamine-dependent CTP formation was reduced 3.2-fold. Thus, for G146A CTPS operating at saturating concentrations of glutamine, only one molecule of CTP is produced for every two molecules of ammonia generated from glutamine hydrolysis.

Table 2
Glutaminase activity for wild-type and mutant CTP synthases

The value of kcat was estimated using initial rate data obtained at a saturating concentration of L-glutamine (6 mM). The results for wild-type CTPS are from reference [34].

CTP synthase variants [GTP] (mM) kcat (s−1
Wild-type 0.25 5.0±0.2 
 0.09±0.04 
G142A 0.25 0.08±0.01 
G143A 0.25 0.67±0.02 
G146A 0.25 4.2±0.3 
CTP synthase variants [GTP] (mM) kcat (s−1
Wild-type 0.25 5.0±0.2 
 0.09±0.04 
G142A 0.25 0.08±0.01 
G143A 0.25 0.67±0.02 
G146A 0.25 4.2±0.3 

When the initial velocities for ammonia-dependent CTP formation were determined as a function of UTP concentration, positive co-operativity was observed for both the wild-type (n=1.4) and G146A (n=2.4) enzymes. For the wild-type enzyme, an increase in positive co-operativity was observed in the presence of CTP (Table 3). This result is consistent with previous studies conducted by Long and Pardee [14] using the E. coli enzyme, and similar changes in co-operativity in the presence of CTP have been reported for CTPS isolated from calf liver [40], rat liver [41] and Saccharomyces cerevisiae [42]. Thus, for wild-type CTPS, CTP binding at the synthase active site of an adjacent subunit causes the enzyme to adopt a conformation that exhibits greater positive co-operativity with respect to UTP. On the other hand, G146A exhibited pronounced co-operativity with respect to UTP in either the presence or absence of CTP (n≈2.3). The similar degree of positive co-operativity with respect to UTP that is exhibited by G146A in the absence of CTP and by wild-type CTPS in the presence of CTP suggests that replacement of Gly146 by alanine in the CTP-binding site alters the conformation of the enzyme so that it mimics the conformation of wild-type CTPS with bound CTP. Although X-ray crystallographic studies revealed that binding of CTP resulted in only minor structural changes in E. coli CTPS relative to the apo-form with no nucleotides bound, Baldwin and co-workers [8] observed more significant conformational changes proximal to the CTP-binding site and suggested that additional structural changes may occur upon CTP binding in solution, but such changes may have been prohibited in the crystals.

Table 3
Effect of CTP on wild-type, G143A, and G146A CTP synthases

Vmax/[E]T (s−1) was calculated using the value of Vmax observed at saturating concentrations of UTP with [NH4Cl]=20 mM and [ATP]=1 mM unless stated otherwise.

  Kinetic parameters for UTP 
CTP synthase variants [CTP] (mM) Vmax/[E]T (s−1[S]0.5 (μM) n 
Wild-type 3.92±0.05 99±11 1.4±0.1 
 0.2 3.19±0.06 136±11 1.7±0.1 
 0.3 2.44±0.14 161±31 1.9±0.1 
G143A* 0.147±0.002 409±59 1.4±0.1 
 0.2 0.122±0.013 328±79 1.2±0.2 
 0.3 0.077±0.002 310±41 1.9±0.2 
G146A 2.62±0.20 368±50 2.4±0.2 
 0.2 2.12±0.20 366±30 2.2±0.1 
 0.3 1.59±0.11 325±35 2.3±0.1 
  Kinetic parameters for UTP 
CTP synthase variants [CTP] (mM) Vmax/[E]T (s−1[S]0.5 (μM) n 
Wild-type 3.92±0.05 99±11 1.4±0.1 
 0.2 3.19±0.06 136±11 1.7±0.1 
 0.3 2.44±0.14 161±31 1.9±0.1 
G143A* 0.147±0.002 409±59 1.4±0.1 
 0.2 0.122±0.013 328±79 1.2±0.2 
 0.3 0.077±0.002 310±41 1.9±0.2 
G146A 2.62±0.20 368±50 2.4±0.2 
 0.2 2.12±0.20 366±30 2.2±0.1 
 0.3 1.59±0.11 325±35 2.3±0.1 
*

[ATP]=2 mM (see legend for Figure 3).

Wild-type CTPS exhibited a decrease in its apparent affinity for UTP (i.e. 1/[S]0.5) with increasing concentrations of CTP. Although G146A exhibited an approx. 4-fold reduction in its apparent affinity for UTP relative to wild-type CTPS, this affinity was not significantly altered by increasing concentrations of CTP (Figure 3A and Table 3). Interestingly, CTP inhibited both the wild-type and G146A CTPSs to exactly the same extent in the presence of saturating concentrations of UTP (i.e. Vmax/[E]T decreased 1.6-fold at 0.3 mM CTP). Our observations that at high concentrations of UTP, CTP-dependent inhibition of the wild-type, G143A and G146A enzymes was not reduced suggests that CTP was not a purely competitive inhibitor with respect to UTP. Hence, the G146A mutation does not appear to impair inhibition by CTP at 0.3 mM CTP. This observation and our observation that the apparent affinity of G146A for UTP is reduced relative to wild-type CTPS are intriguing considering that the X-ray crystal structure of E. coli CTPS with bound CTP shows that Gly146 is located within the binding pocket for the cytosine group of CTP and interacts with N-4 of CTP via a water-mediated hydrogen bond (Figure 1) [8]. Clearly, introduction of an alanine residue at position 146 within the CTP-binding site has a more pronounced impact on the local structure of the enzyme, including the adjacent UTP-binding site, than would be predicted from the simple interruption of a water-mediated hydrogen bond between CTP and the protein backbone. These observations further underscore the complex interactions that exist between the UTP- and CTP-binding sites, in addition to UTP and CTP sharing the same binding site for their 5′-triphosphate moieties (Figure 1) [5,8].

Inhibition of wild-type, G143A and G146A CTP synthases by CTP

Figure 3
Inhibition of wild-type, G143A and G146A CTP synthases by CTP

(A) The activities of wild-type CTPS (21 μg/ml) (closed symbols) and G146A CTPS (46 μg/ml) (open symbols) were assayed as a function of UTP concentration in the presence (0.2 mM, ▲ and Δ; and 0.3 mM, ■ and □) and absence (● and ○) of CTP. (B) The activity of G143A CTPS (400 μg/ml) was assayed as a function of UTP concentration in the presence (0.2 mM, ▲, and 0.3 mM, ■) and absence (●) of CTP. All reactions (A and B) were conducted in 70 mM Hepes buffer (pH 8.0) containing 10 mM MgCl2, 0.5 mM EDTA and 20 mM ammonium chloride. For assays with G146A, [ATP]=1.0 mM; however, for G143A, the [ATP] was increased to 2.0 mM so that more of the enzyme was in the tetrameric form at low [UTP], allowing for measurable rates to be obtained (compare ordinates on A and B). The curves are from a non-linear regression fit of the data to eqn (3). The values of Vmax/[E]T, n and [S]0.5 are given in Table 3 and the detailed experimental conditions are as described in the Materials and methods section.

Figure 3
Inhibition of wild-type, G143A and G146A CTP synthases by CTP

(A) The activities of wild-type CTPS (21 μg/ml) (closed symbols) and G146A CTPS (46 μg/ml) (open symbols) were assayed as a function of UTP concentration in the presence (0.2 mM, ▲ and Δ; and 0.3 mM, ■ and □) and absence (● and ○) of CTP. (B) The activity of G143A CTPS (400 μg/ml) was assayed as a function of UTP concentration in the presence (0.2 mM, ▲, and 0.3 mM, ■) and absence (●) of CTP. All reactions (A and B) were conducted in 70 mM Hepes buffer (pH 8.0) containing 10 mM MgCl2, 0.5 mM EDTA and 20 mM ammonium chloride. For assays with G146A, [ATP]=1.0 mM; however, for G143A, the [ATP] was increased to 2.0 mM so that more of the enzyme was in the tetrameric form at low [UTP], allowing for measurable rates to be obtained (compare ordinates on A and B). The curves are from a non-linear regression fit of the data to eqn (3). The values of Vmax/[E]T, n and [S]0.5 are given in Table 3 and the detailed experimental conditions are as described in the Materials and methods section.

We found that at [UTP]<0.2 mM, CTP afforded very little inhibition of G146A CTPS (Figure 3). Since the kinetic mechanism for inhibition of CTPS by nucleotides, including CTP, is not well defined [43], we measured the IC50 values to obtain an empirical measure of the inhibition of wild-type and G146A CTPSs by CTP (Table 1). Indeed, when the concentration of UTP was reduced from 1.0 mM to 0.2 mM, the IC50 value increased nearly 2-fold, demonstrating that G146A was less sensitive to inhibition by CTP at low concentrations of UTP. Previous studies by Meuth and co-workers [44] had identified nine mutations in 23 strains of Chinese hamster ovary cells which were resistant to the cytotoxic effects of arabinosylcytosine and 5-fluorouracil. One of these mutations in hamster CTPS was G152E, corresponding to Gly146 in E. coli CTPS; however, the kinetic properties of the variant hamster enzyme were not characterized. The inability of G146A CTPS to be inhibited by CTP at concentrations of UTP less than 0.2 mM is consistent with the in vivo resistance to nucleotide analogues exhibited by variants of hamster CTPS with mutations near Gly152.

G143A CTPS

The mutation of Gly143 to an alanine residue had a much more drastic effect on the kinetic parameters of the enzyme relative to the changes observed with G146A (Table 1). For ammonia-dependent CTP formation, the Km was increased by 1.4-fold and kcat was reduced 15-fold for ammonia, whereas for glutamine-dependent CTP formation, Km was increased by 1.7-fold for glutamine and kcat was reduced ∼10-fold. Overall, these changes resulted in a 20-fold reduction in kcat/Km for both ammonia- and glutamine-dependent CTP formation. Since G143A is able to form tetramers under assay conditions (i.e. [ATP]=[UTP]=1 mM; Table 4), this reduction in catalysis is not due to impaired tetramerization of the mutant enzyme, but probably arises due to a decreased affinity for UTP and possibly local structural perturbations as suggested by the minor reduction in α-helical content (Supplementary Figure 3 at http://www.BiochemJ.org/bj/412/bj4120113add.htm). Our observation that G143A exhibits a 7.5-fold reduction in the value of kcat for glutamine hydrolysis (Table 2) is consistent with previous reports that alterations in the synthase domain may affect activity in the GAT domain [45]. The glutaminase activity could also be reduced, in part, because the mutation adversely affects the ability of the 4-phosphorylated UTP intermediate to act as a co-activator with GTP to stimulate glutamine hydrolysis [13].

Table 4
GF-HPLC of wild-type and mutant CTP synthases

For the oligomerization states for CTPS variants, qualitative results are presented because of the difficulty inherent in integration of GF-HPLC chromatograms due to extensive overlap of peaks (see Supplementary Figure 4 at http://www.BiochemJ.org/bj/412/bj4120113add.htm). Major peaks accounting for ≥10% of the total areas are reported. M, monomer; D, dimer; T, tetramer.

Nucleotide concentration (mM) Oligomerization states for CTPS variants 
[ATP] [UTP] [CTP] Wild-type G142A G143A 
M, D (∼1:1)* 
M, D (∼3:2)† 
10 M, D (∼3:2) – 
– 
10 M, D (∼1:4) – 
M, T (∼9:1) – 
Nucleotide concentration (mM) Oligomerization states for CTPS variants 
[ATP] [UTP] [CTP] Wild-type G142A G143A 
M, D (∼1:1)* 
M, D (∼3:2)† 
10 M, D (∼3:2) – 
– 
10 M, D (∼1:4) – 
M, T (∼9:1) – 
*

Where several species were detected, the approximate ratios of peak areas are reported.

It is not clear why the monomeric species predominate.

Kinetic studies on G143A were difficult because the activity (Vmax/[E]T) of this mutant was ∼27-fold lower than that of wild-type CTPS even with the concentration of ATP increased to 2 mM to promote tetramerization of the enzyme at low UTP concentrations. Although G143A exhibited co-operativity (n≈1.5) with respect to UTP that was similar to that of wild-type CTPS, the value of [S]0.5 for UTP was increased 4-fold for G143A, consistent with this residue playing a role in UTP binding (Figure 3B and Table 2). In the presence of 0.3 mM CTP, the value of Vmax/[E]T was reduced 1.9-fold. This value is similar to the 1.6-fold reductions in Vmax/[E]T observed for wild-type and G146A enzymes respectively. This result is not surprising because the crystal structure reveals that Gly143 does not interact with CTP. Instead, rudimentary modelling studies suggest that the carbonyl oxygen of Gly143 may interact with the N-3 of the uracil group of UTP [5]. Since the ϕ and ψ angles for Gly143 correspond to a conformation that is ‘disallowed’ for alanine, the G143A mutation may give rise to local perturbations of structure within the UTP-binding site leading to the reduced affinity for UTP either due to unfavourable steric interactions or the loss of a binding determinant.

G142A CTPS

Replacement of Gly142 by an alanine residue produced an inactive enzyme. Not only was catalysis of CTP formation abolished, the glutaminase activity was also impaired, with the value of kcat being reduced 62-fold relative to the value for wild-type CTPS. Indeed, the value of kcat for G142A is similar to that observed for wild-type CTPS in the absence of GTP or when the active-site cysteine nucleophile in the GAT domain is replaced by an alanine residue [34]. These results were most surprising and are due to the inability of G142A to oligomerize to form active tetramers. GF-HPLC, in the absence of nucleotides, revealed that wild-type CTPS eluted from the GF-HPLC column as a single peak corresponding to an apparent molecular mass consistent with a dimeric species (Supplementary Figure 4 at http://www.BiochemJ.org/bj/412/bj4120113add.htm and Table 4). However, under the same conditions, G142A existed as a mixture of monomers and dimers. Unlike wild-type CTPS that formed tetramers under typical assay conditions (i.e. [ATP]=[UTP]=1 mM), G142A existed as mixture of monomers and dimers. Since G142A may have been unable to form tetramers because UTP and/or ATP were unable to bind properly and/or act synergistically to promote tetramerization, we assessed the ability of G142A to form tetramers at higher concentrations of ATP, UTP [16] or CTP [39] (Table 4). Even in the presence of 10 mM UTP or 10 mM ATP, we did not detect tetrameric species. Only in the presence of 5 mM CTP with 1 mM ATP did we detect formation of a minor amount of G142A tetramer (Supplementary Figure 4). However, no ammonia-dependent CTP formation was detected under these conditions (although inhibition due to CTP would limit our ability to detect CTPS activity) or when G142A was assayed in the presence of 1 mM UTP and 10 mM ATP.

We also examined the ability of UTP to protect G142A from limited trypsin-catalysed proteolysis. In previous studies, we demonstrated that UTP could protect wild-type CTPS from trypsin-catalysed cleavage of the peptide bond C-terminal to Lys187 [36]. X-ray crystal structures of E. coli CTPS reveal that, upon dimer formation, the Lys187 from a symmetry-related subunit protrudes into the interface of the UTP- and ATP-binding sites where it interacts with the γ-phosphate of CTP (and UTP) as shown in Supplementary Figure 1B [5,8]. Lys187 is only partially buried in the CTPS dimer and tetramer formation does not fully occlude Lys187 from the bulk solvent. The UTP-dependent protection of wild-type CTPS from trypsin-catalysed proteolysis could arise either because UTP blocks access to Lys187 or tetramer formation impedes access of trypsin to this site due to steric obstruction.

Wild-type and G142A CTPSs produced different cleavage fragments depending on whether UTP was absent or present in the reaction mixture (Figure 4). In the absence of UTP, limited trypsin-catalysed cleavage of both wild-type and G142A CTPSs (63 kDa) produced fragments with molecular masses corresponding to 10 (results not shown), 25, 28, 38, 43 and 53 kDa. The 10 and 53 kDa fragments arise from cleavage of CTPS at Lys429 and/or Lys432 [36]. The 25 and 38 kDa fragments arise from cleavage at Lys187 and subsequent cleavage of the 38 kDa fragment at Lys429 and/or Lys432 yields the 10 and 28 kDa fragments [36]. (The 43 kDa species appeared rather transiently during the time course and was not identified in our previous studies.) In the presence of 5 mM UTP, limited trypsin-catalysed cleavage of wild-type CTPS gave only fragments with molecular masses of 10 (results not shown) and 53 kDa consistent with protection of the Lys187 cleavage site by UTP as described previously [36]. However, limited trypsin-catalysed cleavage of G142A in the presence of UTP gave fragments of 25, 28, 38, 43 and 53 kDa indicating that the mutation had prevented protection by UTP. This lack of protection by UTP provides additional support for our conclusion that G142A is not able to bind UTP or not able to form tetramers.

SDS/PAGE analysis of limited trypsin-catalysed cleavage of wild-type and G142A CTPSs in the absence and presence of UTP

Figure 4
SDS/PAGE analysis of limited trypsin-catalysed cleavage of wild-type and G142A CTPSs in the absence and presence of UTP

For each gel, either wild-type (A and B) or G142A CTPS (C and D) was incubated with trypsin for the times (min) indicated over each lane. In the absence of UTP (A and C), both wild-type and G142A CTPSs were rapidly cleaved giving rise to multiple cleavage products (indicated by arrows). In the presence of 5 mM UTP (B and D), wild-type CTPS was protected from extensive cleavage giving rise to a 53-kDa fragment and a 10-kDa fragment (results not shown), while G142A CTPS was not protected from extensive trypsin-catalysed cleavage under the same conditions.

Figure 4
SDS/PAGE analysis of limited trypsin-catalysed cleavage of wild-type and G142A CTPSs in the absence and presence of UTP

For each gel, either wild-type (A and B) or G142A CTPS (C and D) was incubated with trypsin for the times (min) indicated over each lane. In the absence of UTP (A and C), both wild-type and G142A CTPSs were rapidly cleaved giving rise to multiple cleavage products (indicated by arrows). In the presence of 5 mM UTP (B and D), wild-type CTPS was protected from extensive cleavage giving rise to a 53-kDa fragment and a 10-kDa fragment (results not shown), while G142A CTPS was not protected from extensive trypsin-catalysed cleavage under the same conditions.

There are only a few examples where a single point mutation has been shown to abolish protein oligomerization. For example, the F187D mutant of glutamate dehydrogenase can only form inactive dimers rather than the active hexamers [46,47]. Replacement of Glu70 by an alanine residue in glycinamide ribonucleotide transformylase disrupts the pH-dependent dimerization [48]. Both of these mutant proteins did not exhibit any significant changes in their CD spectra relative to the corresponding wild-type enzyme. The Q55E, S56E, G57S and G57R mutants of bovine oxytocin-related neurophysin exhibited a ∼3-fold reduction in the association constants for dimerization [49,50]. The Q194A mutation of epidermal growth factor receptor caused a 6-fold reduction in association constant for binding transforming growth factor α [51].

From the X-ray crystal structures of E. coli CTPS (PDB 1S1M [5] and 2AD5 [8]), the values of ϕ and ψ for Gly142 are approx. −73° and 164° respectively (averaging values for the A and B chains from each structure). These values do not lie outside the typical values that can be assumed by alanine residues [52]. Hence, it is surprising that replacement of Gly142 by alanine appeared to produce a local perturbation in structure that abolished UTP binding and/or the changes in conformation required for tetramerization. Although CD analysis revealed a slight reduction in α-helical content in G142A [16 (±3)% α-helix, 29 (±6)% β-character, 55 (±2)% other] relative to wild-type CTPS [30 (±2)% α-helix, 23 (±2)% β-character, 47 (±3)% other], this reduction was no more significant than that observed for G143A [15 (±2)% α-helix, 32 (±5)% β-character, 53 (±2)% other] which was quite capable of forming tetramers in the presence of nucleotides. On the other hand, the values of ϕ and ψ for Gly143 and Gly146 [28 (±2)% α-helix, 24 (±3)% β-character, 48 (±1)% other] correspond to conformations that are ‘disallowed’ for alanine residues. Hence it is not unreasonable to expect that replacement of these glycine residues by alanine could cause local perturbations of structure [53] (e.g. G146A may destabilize the glycine left-handed turn thereby disrupting the loop of residues 143–147 [8]) giving rise to changes in nucleotide binding and/or catalysis.

Our observation that replacement of either Gly142 or Gly143 by an alanine residue resulted in a reduction of α-helical content by ∼15% relative to wild-type CTPS (Supplementary Figure 2) is also intriguing, since the crystal structures of E. coli CTPS show that Gly142 and Gly143 reside on a β-stand (see Figure 1) [5,8]. Apparently, the restricted conformational freedom introduced by replacement of these glycine residues by alanine is not limited to a local perturbation of structure, but causes somewhat more global changes in structure. Such an alteration of conformation is also suggested by (i) the reduction of the glutaminase activity for the G142A and G143A enzymes, (ii) the observation that an increased concentration of ATP (10 mM) does not promote the tetramerization of G142A, and (iii) the observation that G142A is catalytically inactive even in the presence of 1 mM UTP and 10 mM ATP.

In the present study we also prepared the G142A/G143A double mutant which proved to be completely inactive in a similar manner to G142A CTPS. Curiously, CD analysis of G142A/G143A indicated that this variant had a similar secondary structural content [27 (±3)% α-helix, 24 (±2)% β-character, 48 (±3)% other] to the wild-type enzyme (results not shown). This suggests that the structural perturbations caused by the single mutations G142A and G143A compensate for each other giving rise to no apparent change in the secondary structure of the double mutant. However, like G142A, G142A/G143A did not form tetramers in the presence of nucleotides (results not shown).

Conclusions

Baldwin and co-workers [8] have suggested that UTP and CTP share the same binding pocket for their 5′-triphosphate moieties, whereas the ribose and pyrimidine groups occupy separate binding sites (Figure 1). They also suggested that this ‘hybrid strategy’ permits greater evolutionary adaptation for substrate specificity and affinity without the requirement for an allosteric product regulatory site. The results of the present study reveal that complex interactions exist between the UTP- and CTP-binding sites beyond the sharing of a 5′-triphosphate-binding site. Mutations in the CTP-binding site can have a profound effect on UTP-dependent activity. In addition, our observations highlight the sensitivity of the quaternary structure of CTPS to mutations in the UTP-binding site (e.g. Gly142 is critical for tetramerization). One implication of this ‘structural sensitivity’ is that it may limit the number and/or types of mutations that could be selected for during the development of resistance to the 5′-triphosphates of cytotoxic pyrimidine nucleoside analogue inhibitors such as cytosine arabinoside [44] and cyclopentenylcytosine [54].

We thank the Canadian Institutes of Health Research for an operating grant (to S. L. B.), and the Natural Sciences and Engineering Research Council of Canada (T. J. M.), the Nova Scotia Health Research Foundation (F. A. L.), the Walter C. Sumner Foundation (F. A. L.) and the Nova Scotia Cancer Research Training Program (F. A. L.) for graduate scholarships.

Abbreviations

     
  • CTPS

    cytidine 5′-triphosphate synthase

  •  
  • GAT

    glutamine amide transfer

  •  
  • GF-HPLC

    gel-filtration HPLC

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Supplementary data