Báo cáo khoa học: Alternative substrates for wild-type and L109A E. coli CTP synthases Kinetic evidence for a constricted ammonia tunnel

doi:10.1111/j.1432-1033.2004.04360.x

Eur. J. Biochem. 271, 4204–4212 (2004) (cid:1) FEBS 2004

Alternative substrates for wild-type and L109A E.coliCTP synthases Kinetic evidence for a constricted ammonia tunnel

Faylene A. Lunn and Stephen L. Bearne

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

CTP synthases to utilize NH3, NH2OH, and NH2NH2 as exogenous substrates, and as nascent substrates generated via the hydrolysis of glutamine, c-glutamyl hydroxamate, and c-glutamyl hydrazide, respectively. We show that the uncoupling of the hydrolysis of c-glutamyl hydroxamate and nascent NH2OH production from N4-hydroxy-CTP for- mation is more pronounced with the L109A enzyme, relative to the wild-type CTP synthase. These results suggest that the NH3 tunnel of L109A, in the presence of bound allosteric effector guanosine 5¢-triphosphate, is not leaky but contains a constriction that discriminates between NH3 and NH2OH on the basis of size.

Keywords: amidotransferase; ammonia tunnel; CTP syn- thase; glutaminase; alternative substrates.

Cytidine 5¢-triphosphate (CTP) synthase catalyses the ATP- dependent formation of CTP from uridine 5¢-triphosphate using either NH3 or L-glutamine as the nitrogen source. The hydrolysis of glutamine is catalysed in the C-terminal glu- tamine amide transfer domain and the nascent NH3 that is generated is transferred via an NH3 tunnel [Endrizzi, J.A., Kim, H., Anderson, P.M. & Baldwin, E.P. (2004) Biochemistry 43, 6447–6463] to the active site of the N-ter- minal synthase domain where the amination reaction occurs. Replacement of Leu109 by alanine in Escherichia coli CTP synthase causes an uncoupling of glutamine hydrolysis and glutamine-dependent CTP formation [Iyengar, A. & Bearne, S.L. (2003) Biochem. J. 369, 497–507]. To test our hypothesis that L109A CTP synthase has a constricted or a leaky NH3 tunnel, we examined the ability of wild-type and L109A

(CTP)

synthase

5¢-triphosphate

Cytidine [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine (Gln) or NH3 as the nitrogen source [1,2]. This Gln amidotransferase is a single polypeptide chain consisting of two domains. The C-terminal Gln amide transfer (GAT) domain utilizes a Cys-His-Glu triad to catalyse the rate-limiting hydrolysis of Gln (glutaminase activity) [3–5], and the nascent NH3 derived from this glutaminase activity is transferred to the N-terminal synthase domain where the amination of a phosphorylated UTP intermediate is catalysed [6,7]. The reactions catalysed by CTPS are summarized in Scheme 1.

CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides. As CTP has a central role in the biosynthesis of nucleic acids [8] and membrane phospho- lipids [9], CTPS is a recognized target for the development of antineoplastic agents [8,10], antiviral agents [10–12], and antiprotozoal agents [13–15]. The Escherichia coli enzyme

synthase

is one of the most thoroughly characterized CTP synthases 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 of the glutaminase activity and Gln-dependent CTP synthesis [3,16] but inhibits CTP synthesis at concentrations > 0.15 mM [17]. In addition, the enzyme is inhibited by the product CTP [18] and displays positive cooperativity for ATP and UTP [18–20]. ATP and UTP act synergistically to promote tetramerization of the enzyme to its active form [20]. Recently, the X-ray crystal structure of E. coli CTPS was solved at a resolution of 2.3 A˚ [21]. The enzyme crystallised as a tetramer, presumably because of the high protein concentrations used as bound nucleotides were not present in the structure (i.e. apo-E. coli CTPS) [21]. The authors identified a solvent-filled (cid:1)vestibule(cid:2) ((cid:1) 230 A˚ 3) that connects the GAT active site and the GAT/synthase interface. This vestibule is connected to a tubular passage that leads into the synthase site. The presence of this vestibule and NH3 tunnel in CTPS is consistent with the identification of NH3 tunnels in the X-ray structures of other amidotransferases inclu- ding carbamoyl phosphate synthase (CPS) [22–24], Gln phosphoribosylpyrophosphate [25,26], GMP synthase [27], glucosamine-6-phosphate [28–30], asparagine synthase B [31], and anthranilate synthase [32,33].

Previously, we reported that amino acid residues between Arg105 and Gly110 of E. coli CTPS are important for efficient coupling of Gln hydrolysis in the GAT domain to CTP formation in the synthase domain. Replacement of the highly conserved Leu109 residue by alanine produced an enzyme that exhibited wild-type levels of NH3-dependent CTP formation, affinity for Gln, glutaminase activity,

Correspondence to S. L. Bearne, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 1X5, Canada. Fax: +1 902 494 1355, Tel.: +1 902 494 1974, E-mail: sbearne@dal.ca Abbreviations: CPS, carbamoyl phosphate synthase; CTPS, CTP synthase; GAT, Gln amide transfer; Gln, L-glutamine; Gln-OH, L-c-glutamyl hydroxamate; Gln-NH2, L-c-glutamyl hydrazide; OPA, o-phthaldialdehyde. Enzyme: CTP synthase (EC 6.3.4.2) (Received 18 August 2004, revised 3 September 2004, accepted 6 September 2004)

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Scheme 1

O

O

O

O

H2O

–

–

R

O–

O

O

N H

glutaminase reaction

NH3 + L-glutamine

NH3 + L-glutamate

H N R

2

nascent H2N–R from leak equilibrates with solvent

[4]

exogenous H2N–R

[3]

tunnel with constriction or leak

[2]

2

–

HN

R

O

OPO3

ATP

ADP

[1]

N

HN

N

synthase reaction

O

N

O

N

O

N

Pi

R'

R'

R'

UTP

phosphorylated UTP intermediate

R = H CTP R = OH N 4-hydroxy CTP R = NH2 N 4-amino CTP

R = H, OH, NH2; R' = ribose-5'-triphosphate

(39 000 g, 20 min, 4 (cid:2)C) and the soluble histidine-tagged CTPS was purified using metal ion affinity chromatography as described in the Novagen protocols [35]. The resulting enzyme solution was dialysed into HEPES buffer (70 mM, pH 8.0) containing EGTA (0.5 mM). All enzyme purifica- tion procedures were conducted at 4 (cid:2)C.

affinity for GTP, and activation by GTP. Most interest- ingly, however, the L109A mutant exhibited impaired Gln-dependent CTP formation. These observations were consistent with the hypothesis that Leu109 plays a role in either the structure or formation of an NH3 tunnel and ensures efficient coupling of the Gln hydrolysis and amina- tion reactions. In the present report, we show that hydroxyl- amine, L-c-glutamyl hydroxamate (Gln-OH), hydrazine, and L-c-glutamyl hydrazide (Gln-NH2) are alternative substrates for E. coli CTPS. Comparison of the kinetic parameters of Gln and NH3 with those of the corresponding bulkier substrates Gln-OH and NH2OH suggests that the impaired Gln-dependent CTP formation exhibited by the L109A mutant is caused by a constriction of the NH3 tunnel. This is the first functional evidence implicating a constriction in the NH3 tunnel of E. coli CTPS.

Scheme 1. Reactions catalysed by E. coli CTP synthase.

Experimental procedures

General materials and methods

Thrombin-catalysed cleavage of the histidine tag from soluble enzyme (new N-terminus, GSHMLEM1…) was conducted in HEPES buffer (70 mM, pH 8.0) containing EGTA (0.5 mM) using a thrombin ratio of 0.5 unitsÆmg)1 of target protein. After 8 h at 25 (cid:2)C, cleavage was complete and the biotinylated thrombin was removed from the reaction mixture using streptavidin agarose resin (Novagen, EMD Biosciences, Inc., Madison, WI, USA) at a ratio of 32 lL settled resin per unit of thrombin following the Novagen protocol [35]. Cleaved CTPS, free of biotinylated thrombin, was then dialysed against HEPES buffer (70 mM, pH 8.0) containing EGTA (0.5 mM) and MgCl2 (10 mM) (assay buffer). The results of the purification and cleavage procedures were routinely monitored using SDS/PAGE. Typically, enzyme preparations were P 98% pure. 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 1.

Cyclization of Gln-OH

All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada), except where mentioned otherwise. For HPLC experiments, 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.

Enzyme expression and purification

Wild-type and L109A recombinant E. coli CTPS were expressed in and purified from E. coli strain BL21(DE3) cells transformed with the plasmid pET15b-CTPS1 or the mutated plasmid as described previously [3,34]. These constructs encode the E. coli pyrG gene product with an N-terminal His6-tag. The BL21(DE3) cells were grown in Luria–Bertani medium at 37 (cid:2)C, induced using isopropyl thio-b-D-galactoside according to the Novagen expression protocol [35], and lysed using sonication on ice (5 · 10 s bursts with 30 s intervals at output setting 5 using a Branson Sonifier 250). The crude lysate was clarified by centrifugation

The conversion of Gln-OH to 2-pyrrolidone-5-carboxylic acid [36] at 37 (cid:2)C was followed using a Bruker AVANCE 500 MHz NMR spectrometer. A solution of Gln-OH in deuterated potassium phosphate buffer (20 mM) (100 mM, pD 8.0) was prepared and the ionic strength was adjusted to 0.30 M using KCl. At various times (5, 7, 16, 26, 36, and 46 min) the 1H NMR spectrum was recorded. The relative concentrations of Gln-OH and 2-pyrrolidone- 5-carboxylic acid were determined by integration of the signals at 3.80 p.p.m. (triplet) and 4.22 p.p.m. (multiplet) corresponding to the proton on the carbon adjacent to the carboxylate carbon on Gln-OH and 2-pyrrolidone-5-carb- oxylic acid, respectively. (Chemical shifts are relative to the D2O lock signal.)

4206 F. A. Lunn and S. L. Bearne (Eur. J. Biochem. 271)

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[S] is the substrate concentration, and Km is the Michaelis constant for the substrate. Values of Km for NH3, NH2OH, and NH2NH2 were calculated using the concentration of +) ¼ 9.24; these species present at pH 8.0 (i.e. pKa(NH4 +) ¼ 8.10 [38]). Val- pKa(+NH3OH) ¼ 5.97; pKa(NH2NH3 ues of kcat (per subunit) were calculated for CTPS variants with the His6-tag removed using the molecular masses (Da) of 61 029 (wild-type) and 60 987 (L109A). Protein concen- trations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin standards.

For those experiments utilizing Gln-OH as the substrate, we found that it was essential to maintain the Gln-OH stock solution at 4 (cid:2)C and add this solution directly to the assay cocktail to initiate the reaction. At 37 (cid:2)C, the observed first order rate constant for cyclization of Gln-OH to and NH2OH was form 2-pyrrolidone-5-carboxylate 7.7 (± 0.4) · 10)5 s)1 (i.e. t1/2 (cid:1) 2.5 h) at pD 8.0 (data not shown). Hence, significant production of NH2OH occurs in Gln-OH solutions at 37 (cid:2)C and the resulting NH2OH can complicate kinetic experiments if the Gln-OH solutions are not kept on ice prior to addition to the assay solution.

Enzyme assays and protein determinations

Glutaminase assay

)1Æcm)1)

[18], [37], or

(70 mM, pH 8.0)

The abilities of wild-type and L109A CTP synthases to catalyse Gln hydrolysis were determined by following the production of glutamate using reversed-phase HPLC separation of the o-phthaldialdehyde (OPA) derivatives of glutamate, Gln, Gln-OH, and Gln-NH2 with fluores- cence detection [39]. Assays were conducted at 37 (cid:2)C in HEPES buffer containing EGTA (0.5 mM), MgCl2 (10 mM), ATP (1 mM), UTP (1 mM), GTP (0.25 mM), and either Gln (0.25, 0.50, 1.0, 5.0, and 10.0 mM), Gln-OH (1.0, 3.0, 5.0, 7.0, and 10 mM), or Gln- NH2 (3.0, 7.0, 10.0, 15.0, and 20.0 mM). CTPS concen- trations ranged between 5 and 56 lgÆmL)1 for wild-type and 5–54 lgÆmL)1 for L109A in a total volume of 2.5 mL.

All components were preincubated for 2.5 min at 37 (cid:2)C prior to initiation of the reaction by addition of substrate (Gln, Gln-OH, or Gln-NH2). To minimize cyclization of Gln-OH, stock solutions (1 mL) were prepared at appro- priate concentrations and flash-frozen in liquid nitrogen. These Gln-OH solutions were thawed for 2.5 min at 37 (cid:2)C and then used to initiate the reaction. At various time points (0, 1, 3, 5, 7, and 10 min), aliquots (20 lL) of the assay solution were transferred to 1.5 mL polypropy- lene tubes and reacted immediately with an equal volume of OPA reagent (40 mM) [39]. Derivatization with OPA was shown to effectively terminate the reaction. (Boiling of the reaction led to rapid cyclization of the Gln-OH [36].) After 1 min at room temperature the reaction was neutralized by addition of sodium acetate buffer (160 lL, 0.1 M, pH 6.2) and an aliquot (20 lL) was analysed using reversed-phase HPLC.

CTPS activity was determined at 37 (cid:2)C using a continuous spectrophotometric assay by following the rate of increase in absorbance at 291 nm resulting from either the conver- to sion of UTP to CTP (De ¼ 1338 M )1Æcm)1) N4-hydroxy-CTP (De ¼ 4023 M to )1Æcm)1; estimated from the N4-amino-CTP (De ¼ 1364 M difference of the spectra of uridine and N4-amino cytidine). Substrates (NH4Cl, NH2OH, NH2NH2, Gln, Gln-OH, and Gln-NH2) were dissolved in assay buffer and the pH was adjusted to 8.0 using 6 M KOH. The standard assay mixture consisted of HEPES buffer (70 mM, pH 8.0) containing EGTA (0.5 mM), MgCl2 (10 mM), CTPS, and saturating concentrations of UTP (1 mM) and ATP (1 mM) in a total volume of 1 mL. Enzyme and nucleotides were preincu- bated together for 2.5 min at 37 (cid:2)C followed by addition of substrate to initiate the reaction. Total NH4Cl concentra- tions in the assays were 5, 10, 20, 30, 50, 60, 80, and 100 mM; total NH2OHÆHCl concentrations in the assays were 5, 10, 15, 20, 30, 40, 50, 75, and 100 mM; total NH2NH2Æ2HCl concentrations in the assays were 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM; and CTPS concentrations were 20 lgÆmL)1 for wild-type and 20–24 lgÆmL)1 for L109A. For assays of Gln- or Gln analogue-dependent CTP formation, concentrations of Gln were 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 3.0, and 6.0 mM; concentrations of Gln-OH were 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 5.0, 10.0, and 15.0 mM; concen- trations of Gln-NH2 were 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 40.0, 60.0, 80.0, and 100.0 mM; and CTPS concentrations ranged between 28 and 120 lgÆmL)1 for wild-type and 40–160 lgÆmL)1 for L109A. The concentration of GTP was maintained at 0.25 mM for all assays when Gln or Gln analogues were used as the substrate. For assays conducted using Gln-OH, a freshly prepared stock solution was stored on ice and added cold to each assay. This protocol was necessary to minimize cyclization of Gln-OH with concom- itant production of NH2OH (see above).

The ionic strength was maintained at 0.30 M in all assays by the addition of KCl. All kinetic parameters were determined in triplicate and average values are reported. The reported errors are standard deviations. Initial rate kinetic data was fit to Eqn (1) by nonlinear regression analysis using the program PRISM (GraphPad Software, Inc., San Diego, CA).

ð1Þ

vi ¼

Vmax½S(cid:2) Km þ ½S(cid:2)

In Eqn (1), vi is the initial velocity, Vmax (¼ kcat[E]T) is the maximal velocity at saturating substrate concentrations,

Separation of the isoindole derivatives of Gln, glutamate, Gln-OH, and Gln-NH2 were conducted using a Synergi Fusion-RP column (4 lm; 80 A˚ ; 50 · 4.6 mm; Pheno- menex, Torrance, CA) eluted under isocratic conditions using 0.1 M sodium acetate (adjusted to pH 6.2 with glacial acetic acid)/methanol/tetrahydrofuran (800 : 190 : 10; v/v/ v) at a flow rate of 1.5 mLÆmin)1. The solvent was degassed prior to use. The fluorescence of the isoindole derivatives formed from reaction of Gln, glutamate, Gln-OH, and Gln- NH2 with OPA reagent was monitored using a Waters 474 scanning fluorescence detector (kex ¼ 343 nm, kem ¼ 440 nm). These derivatives eluted with retention times equal to 5.6, 2.1, 4.4, and 3.8 min, respectively. Peak areas were determined by integration of the resulting chromatograms using PEAKSIMPLE software from Mandel Scientific (Guelph, ON, Canada). Concentrations of glutamate were deter- mined using a standard curve prepared by derivatization of

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standard glutamate solutions (0.025, 0.050, 0.075, 0.100, 0.150, 0.200, and 0.250 mM).

Calculations

Geometry optimizations and electrostatic potential sur- faces were calculated for NH3, NH2OH, and NH2NH2 by performing self-consistent-field calculations at the 6–31 G** level using SPARTAN¢04 WINDOWS (Wavefunction, Inc., Irvine, CA). This software was also used to calculate the molecular surface areas and volumes of these molecules.

Results and discussion

are substrates for CTPS from Ehrlich ascites tumour cells [45], and NH2OH has been shown to be a substrate for E. coli [46] and Lactococcus lactis [47] CTP synthases. With the exception of Lieberman’s work in 1956 [46], little is known about the ability of E. coli CTPS to utilize alter- native NH3 sources. In addition, some amidotransferases such as CPS [40] and asparagine synthase B [48] have been shown to hydrolyse Gln-OH and Gln-NH2 to give rise to NH2OH and NH2NH2, respectively. Although E. coli CTPS has been shown to utilize Gln-OH as a substrate [16], the present study describes the first detailed kinetic characterization of the ability of E. coli CTPS to utilize alternative substrates. We show that replacement of Leu109 by alanine in E. coli CTPS causes the enzyme to discrim- inate between nascent NH3 and the bulkier analogue NH2OH based on size but does not lead to discrimination between exogenous NH3 and bulkier analogues (i.e. NH2OH and NH2NH2). Our findings are consistent with the L109A mutation causing constriction of an NH3 tunnel.

Exogenous NH3 and its analogues

Exogenous NH3, NH2OH, and NH2NH2 all served as for wild-type and L109A CTP synthases substrates (Table 1). However, both NH2OH and NH2NH2 exhibited kcat/Km values with both enzymes that were approximately 30-fold less than the kcat/Km value for NH3. This reduction in kcat/Km was caused by an increased Km value for NH2OH and NH2NH2. Relative to NH3, the Km value for NH2OH was increased approximately 40-fold while the kcat value was slightly greater than that for NH3. This observation is in accord with the slightly greater nucleophilicity of NH2OH relative to NH3 [49,50]. Hence, it appears that once NH2OH is bound it reacts readily with the phosphorylated UTP intermediate. The individual Km and kcat values for NH2NH2 could not be determined for either wild-type CTPS or L109A CTPS because saturation was not observed, indicating that the Km for this substrate was also markedly increased relative to that observed for NH3.

Three possible routes that exogenous NH3 or its analogues might traverse to reach the site of reaction with the phosphorylated UTP intermediate are shown in

Previously, we reported that replacement of the highly conserved Leu109 residue in E. coli CTPS by alanine yields an enzyme that has kinetic properties similar to those of wild-type CTPS with respect to NH3-dependent CTP formation, affinity for Gln, glutaminase activity, affinity for GTP, and activation by GTP [34]. However, unlike wild- type CTPS, the L109A mutant exhibited impaired Gln- dependent CTP formation. These observations suggested that Leu109 plays a role in either the structure or formation of an NH3 tunnel and ensures efficient coupling of the Gln hydrolysis and amination reactions. In the present study, we use bulky analogues of exogenous NH3 (i.e. NH2OH and NH2NH2) and nascent NH3 (i.e. NH2OH and NH2NH2 derived from the hydrolysis of Gln-OH and Gln-NH2, respectively) to test our hypothesis that the presence of an alanine at position 109 introduces a constriction in the NH3 tunnel of E. coli CTPS. This approach has been used to demonstrate that the G359S mutant of CPS has a partially blocked NH3 tunnel that prevents diffusion of NH2OH while still allowing some NH3 to diffuse through [40]. The hypothesis that replacement of the bulky Leu109 by the smaller alanine could cause a tunnel blockage has precedent. For example, the F334A mutant of glutamine phosphorib- osylpyrophosphate amidotransferase exhibited kinetic prop- erties expected for a blocked or disrupted NH3 tunnel [41]. Many amidotransferases can utilize NH2OH and NH2NH2 in place of NH3 [42–44]. Both NH2NH2 and NH2OH (and its derivatives NH2OCH3 and CH3NHOH)

Table 1. Kinetic Parameters for wild-type and L109A CTP synthases. –, Not determined.

Wild-type CTPS L109A CTPS

)1Æs)1)

)1Æs)1)

Substrate Km (mM) kcat (s)1) kcat/Km (mM Km (mM) kcat (s)1) kcat/Km (mM

2.15 ± 0.14 82.8 ± 6.8 –a 0.354 ± 0.057 0.165 ± 0.017 39.4 ± 0.5 4.43 ± 0.12 0.169 ± 0.016 0.147 ± 0.019 17.8 ± 2.3 2.77 ± 0.28 0.036 ± 0.001 2.17 ± 0.09 75.3 ± 9.8 –a 0.497 ± 0.132 0.250 ± 0.091 – 10.1 ± 0.3 14.1 ± 1.9 –a 1.86 ± 0.34 0.063 ± 0.014 – 4.63 ± 0.04 0.187 ± 0.003 0.128 ± 0.015 3.85 ± 0.82 0.260 ± 0.061 –

a Saturation could not be achieved and kcat/Km values were determined from measurements conducted with [S] << Km. b Activity too low to measure reliably.

0.327 ± 0.002 0.324 ± 0.101 –a 17.2 ± 0.1 3.06 ± 0.90 0.034 ± 0.004 0.550 ± 0.012 0.260 ± 0.061 –b 5.06 ± 0.24 0.310 ± 0.033 –b 9.22 ± 0.62 1.26 ± 0.40 –b Kinetic parameters for CTP formation NH3 9.50 ± 0.53 NH2OH 14.0 ± 1.8 –a NH2NH2 6.10 ± 0.80 Gln 0.453 ± 0.001 Gln-OH Gln-NH2 1.41 ± 0.04 Kinetic parameters for the glutaminase activity 5.62 ± 0.12 Gln 0.930 ± 0.040 Gln-OH –a Gln-NH2

4208 F. A. Lunn and S. L. Bearne (Eur. J. Biochem. 271)

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to the site of Gln hydrolysis so that exogenous substrates entering through the hole bypass the constriction.

It is important to note that both the wild-type and L109A enzymes do discriminate between NH3 and the bulkier substrates in terms of binding (i.e. elevated Km values for NH2OH and NH2NH2 relative to NH3 for both wild-type and L109A CTP synthases). The entrance for exogenous NH3 is approximately 3 A˚ in diameter thereby permitting access of NH3 (surface area ¼ 43.65 A˚ 2; volume ¼ 26.52 A˚ 3) [21]. On the other hand, entrance of bulkier substrates such as NH2OH (surface area ¼ 54.89 A˚ 2; volume ¼ 35.62 A˚ 3) and NH2NH2 (surface area ¼ 60.23 A˚ 2; volume ¼ 40.62 A˚ 3) may be more difficult and require proper orientation of these molecules along their longitudinal axis in order to pass through the hole and avoid unfavourable steric interactions. This requirement for correct orientation could, in part, be responsible for the elevated Km values observed for the bulkier substrates. The electrostatic potential surfaces of NH3, NH2OH, and NH2NH2 (data not shown), and their ability to act as hydrogen bond donors and acceptors are similar, and hence they are expected to behave similarly within the proteins, provided no adverse steric interactions are encountered.

Nascent NH3 and its analogues

The abilities of wild-type and L109A CTP synthases to catalyse the hydrolysis of Gln, Gln-OH, and Gln-NH2 (i.e. glutaminase activity) and to subsequently catalyse the formation of CTP, N4-hydroxy-CTP, and N4-amino-CTP, respectively, were examined (Table 1). Relative to Gln, the kcat/Km values for Gln-OH and Gln-NH2 hydrolysis catalysed by wild-type CTPS were reduced approximately

Scheme 1. Route 1 represents a bimolecular reaction with the reactive intermediate. This route is unlikely as saturation kinetics are observed when NH3 is a substrate, suggesting the formation of an initial enzyme-NH3 complex. Routes 2 and 3 involve the binding of NH3 at a site on CTPS followed by either direct reaction with the phosphorylated UTP intermediate (route 2) or passage through an internal tunnel to its site of reaction with the phosphorylated UTP intermediate (route 3). Although structural studies of many different amidotransferases have suggested the presence of NH3 tunnels to shuttle the nascent NH3 from the site of Gln hydrolysis to the synthase domain [51], it is not always clear what route is followed by exogenous NH3. For any given exogenous substrate (i.e. NH3, NH2OH, or NH2NH2), the kinetic parameters (Km, kcat, and/or kcat/Km) are similar for both wild-type and L109A E. coli CTP synthases. Thus, replacement of Leu109 by alanine does not cause any discrimination between exogenous substrates of a given size with respect to binding affinity, turnover, and efficiency. In addition, once the bulkier, exogenous NH2OH enters the enzyme, it is transferred to the synthase active site and reacts with the phosphorylated UTP intermediate as efficiently as NH3 as indicated by the similar kcat values for either the wild-type or L109A CTP synthases. Based on their recently solved crystal structure of wild-type E. coli CTPS, Endrizzi et al. [21] suggested that exogenous NH3 could access the active site via a (cid:1)hole(cid:2) on the protein’s surface that resides midway between the Gln and UTP binding sites (Fig. 1). Our observations suggest that, after binding to L109A CTPS, passage of exogenous NH3 or its analogues through the NH3 tunnel (i.e. route 3) are not inhibited by a constriction if it is present. Alternatively, a constriction may be present at a location within the NH3 tunnel that is closer

Fig. 1. Location of Leu109 relative to the opening for exogenous NH3 (PDB code 1S1M [21]). (A) Amino acid residues comprising the walls of the entryway for exogenous NH3 include residues 50–55, Val60, Glu68, Lys297, Tyr298, Ala304, Phe353, Gly354, Arg356, Glu403, and Arg468 (shown in green, space-filling representation). The sulphur of the catalytic nucleophile Cys379 is yellow. The loop comprised of residues 104–110 from the adjacent subunit is shown in red with Leu109 shown in space-filling representation. (B) Viewed from the side, relative to (A), Leu109 is poised above the opening for exogenous NH3. GTP is shown modelled into the cleft [21], however, this model probably does not accurately reflect the change in conformation associated with GTP binding. Movement of the 104–110 loop may occur upon GTP binding so that Leu109 is repositioned to pack against bound GTP and perhaps help further seal the entryway for exogenous NH3.

Alternative substrates for E. coli CTP synthase (Eur. J. Biochem. 271) 4209

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wild-type

L109A

substrate

2.46 ± 0.63 P = 0.0028

0.418 ± 0.093

1.03 ± 0.13

Gln

1.14 ± 0.38 P = 0.5229

2.03 ± 0.92 P = 0.0408

4.39 ± 2.20 P = 0.0144

Glu-OH

0.206 ± 0.081

0.905 ± 0.281

six-fold and 500-fold, respectively. The same trend is also observed for wild-type CTPS-catalysed formation of N4-hydroxy-CTP and N4-amino-CTP. Comparison of the Km and kcat values for Gln-OH hydrolysis with those observed for Gln hydrolysis reveals that this reduction in efficiency arises from a six-fold reduction in kcat while there is no change in the Km value. The marked reduction in the efficiency (kcat/Km) of wild-type CTPS-catalysed formation of N4-hydroxy-CTP resulted mainly from a 111-fold increase in the Km value. A similar trend is also observed with L109A CTPS. Unfortunately, we were unable to detect any significant amount of glutaminase activity using L109A CTPS with Gln-NH2 as a substrate. Consequently, we were not able to employ nascent NH2NH2 in our analysis for tunnel constriction.

Fig. 2. Coupling ratios for wild-type and L109A CTP synthases at subsaturating substrate concentrations. Subsaturating coupling ratios (Eqn 2) are shown in boldface. The factors by which the ratios change upon altering either the substrate (vertical arrows) or enzyme (hori- zontal arrows) are shown in italics. The statistical significance of the changes in the coupling ratios is indicated by the corresponding P value based on an unpaired, 2-tailed t-test (P < 0.05 is statistically significant).

wild-type

L109A

substrate

2.95 ± 0.68 P = 0.0013

0.368 ± 0.069

1.09 ± 0.14

Gln

2.23 ± 0.31 P = 0.0018

1.81 ± 0.56 P = 0.0285

2.40 ± 0.60 P = 0.0008

Glu-OH

0.203 ± 0.050

0.487 ± 0.021

The values of kcat/Km and kcat for wild-type CTPS- catalysed Gln hydrolysis and CTP formation are experi- mentally equal. This indicates that there is total coupling of the reactions forming the nascent NH3 and its reaction to form CTP at both low (i.e. kcat/Km conditions) and high (i.e. kcat conditions) concentrations of Gln. However, when Gln- OH is the substrate, N4-hydroxy-CTP formation is only fully coupled to Gln-OH hydrolysis when the concentration of Gln-OH is subsaturating (Table 1). To illustrate how this coupling is altered when either the nature of the substrate or enzyme is altered, we employ two (cid:1)coupling ratios(cid:2) as defined in Eqns 2 and 3, and reported in Figs 2 and 3. Such ratios have been used to characterize the channelling efficiency of amidotransferases [41].

Subsaturatingcouplingratio¼

ðkcat=KmÞCTP formation ðkcat=KmÞglutaminase activity

ð2Þ

ð3Þ

Saturatingcouplingratio¼

ðkcatÞCTPformation ðkcatÞglutaminaseactivity

six-fold relative to the kcat value for Gln hydrolysis, it appears that the additional reduction in kcat (to 13-fold as mentioned above) that is observed for Gln-OH-dependent N4-hydroxy-CTP formation results from some other limit- ing effect such as a (cid:1)bottleneck(cid:2).

that

Examination of the coupling ratios in Figs 2 and 3 reveals that at all substrate concentrations, L109A CTPS exhibits uncoupling (i.e. coupling ratios < 1). At saturating substrate concentrations (Fig. 3), replacement of Leu109 by alanine causes the coupling ratios to be reduced by factors of 2.95 and 2.40 for the Gln- and Gln-OH-dependent reactions, respectively. Interestingly, the coupling ratios for the Gln- and Gln-OH-dependent reactions are also both reduced approximately two-fold for both the wild-type (1.09 fi 0.487) and L109A (0.368 fi 0.203) enzymes. Hence, L109A is no more sensitive to the increased size of NH2OH than wild-type CTPS when substrate concentra- tions are saturating. Therefore, the rate of transfer of the bulkier, nascent NH2OH under kcat conditions appears to is not affected by be limited by a (cid:1)bottleneck(cid:2) replacement of Leu109 by alanine. For this reason, only the kcat/Km data (Fig. 2) are used to determine if the mutant enzyme is sensitive to the larger size of the nascent NH2OH. Previously, we reported that L109A exhibited uncoup- ling of Gln hydrolysis from CTP formation [34]. We

For wild-type CTPS, these ratios are both unity for Gln and Gln-OH at subsaturating concentrations of the substrate (Fig. 2) indicating that the nascent NH3 is consumed in the amination reaction as rapidly as it is produced at all concentrations of glutamine (i.e. reactions are fully coupled as mentioned above). Unlike wild-type CTPS-catalysed hydrolysis of Gln, Gln-OH hydrolysis is only fully coupled to N4-hydroxy-CTP formation at low substrate concentra- tions (Fig. 2) with uncoupling (coupling ratio ¼ 0.487) being observed at saturating concentrations of Gln-OH (Fig. 3). The kcat value for the wild-type CTPS-catalysed formation of N4-hydroxy-CTP from nascent NH2OH (Gln- OH as the substrate) is reduced 13-fold relative to that for nascent NH3 (Gln as the substrate). The bulkier nascent NH2OH must either encounter some unfavourable steric interactions or a (cid:1)bottleneck(cid:2) as it traverses the NH3 tunnel, or the kinetic expression for kcat for the hydrolysis of Gln- OH contains terms that include rate constants for the hydrolysis reaction, production of NH2OH and Glu, and release of Glu. (The exact kinetic mechanism [i.e. order of addition of substrates] is not known because the coopera- tivity displayed by CTPS makes initial velocity studies difficult to interpret [52] and hence the expression for kcat cannot presently be derived.) However, because the kcat value for wild-type hydrolysis of Gln-OH is reduced only

Fig. 3. Coupling ratios for wild-type and L109A CTP synthases at saturating substrate concentrations. Saturating coupling ratios (Eqn 3) are shown in boldface. The factors by which the ratios change upon altering either the substrate (vertical arrows) or enzyme (horizontal arrows) are shown in italics. The statistical significance of the changes in the coupling ratios is indicated by the corresponding P value based on an unpaired, 2-tailed t-test (P < 0.05 is statistically significant).

4210 F. A. Lunn and S. L. Bearne (Eur. J. Biochem. 271)

(cid:1) FEBS 2004

hypothesized that this uncoupling could arise from (a) a leaky NH3 tunnel, (b) a constricted NH3 tunnel, or (c) the failure of a transient tunnel to form. Our comprehensive kinetic characterization of the ability of wild-type and L109A CTP synthases to utilize bulkier analogues of both NH3 and Gln now permits us to refine our hypothesis. As shown in Fig. 2, L109A CTPS exhibits more pronounced uncoupling with Gln-OH than with Gln. Hence, the uncoupling observed with L109A CTPS appears to depend on the size of the nascent NH3 analogue. This observation is most consistent with the presence of a constricted NH3 tunnel. If a leaky tunnel were present, we would expect the bulkier nascent NH2OH to either leak out to bulk solvent, like the nascent NH3 (route 4 in Scheme 1), and therefore exhibit the same degree of uncoupling, or be retained within the tunnel for steric reasons and subsequently form N4- hydroxy-CTP. In this latter case, less uncoupling would be expected for the L109A enzyme, resulting in a higher coupling ratio for nascent NH2OH relative to nascent NH3.

Structural aspects of uncoupling

This scenario is consistent with the lack of equilibration of the nascent NH3 derived from Gln hydrolysis with the bulk solvent [4], the failure of L109F to catalyse glutamine hydrolysis at wild-type rates [34], and the observation that GTP binding inhibits NH3-dependent CTP formation [17]. It is probable that the phenyl group in L109F is too large to pack properly against GTP thereby disrupting the appropriate change in conformation required for full coupling and glutaminase activity [34]. Although it is not clear how the L109A mutation leads to uncoupling, one possibility is that a conformational (cid:1)kink(cid:2) arises via the mechanism mentioned above so that a functional tunnel that efficiently couples the glutaminase and amination reactions is not properly formed. Formation of a competent NH3 tunnel upon ligand binding has been suggested by structural studies on GMP synthase [27,55] and Gln phosphoribosylpyrophosphate amidotransferase [25], and the same may be true for CTPS. While the presence of a phenylalanine at position 109 may impede required for the appropriate conformational changes catalysis, substitution by alanine might permit (cid:1)too much(cid:2) of a conformational change because of differences between the packing of the leucine vs. alanine side chains with GTP leading to a more significant (cid:1)kink(cid:2). Although the kink/constriction could occur at any point along the route traversed by the nascent NH3, one possible location is the narrow (cid:1)gate(cid:2) between Pro54 and Val60, identified by Endrizzi et al. [21], that resides at the base of the proposed entryway for exogenous NH3. Further narrowing of this (cid:1)gate(cid:2) upon GTP binding could lead to a constriction that discriminates between nascent NH3 and the bulkier NH2OH within L109A but does not affect the use of exogenous NH3 and its analogues (at least under kcat/Km conditions). Both explanations are fully consistent with the kinetic properties exhibited by L109A CTPS with alter- native, bulkier substrates.

In the crystal structure of apo-E. coli CTPS, Leu109 is located on a loop (residues 105–114) from an adjacent subunit that extends over a deep cleft that separates the GAT and synthase sites (Fig. 1) [21]. Interestingly, Leu109 is poised over this cleft and above the opening that Endrizzi et al. [21] identified as a putative entry point for exogenous NH3 to access a solvent-filled vestibule that connects the GAT active site and the GAT/synthase interface. Modelling studies conducted by Endrizzi et al. [21] suggest that GTP binds in the cleft that overlies the entry point for exogenous NH3. This finding is in accord with our recent report that GTP binding inhibits CTP formation from exogenous NH3 [17]. Studies also suggest that GTP binding induces a conformational change in E. coli [3,16,17,52,53] and L. lac- tis [54] CTP synthases. In the absence of bound ligands, the structure of apo-E. coli CTPS does not provide much insight into what conformational changes might occur upon GTP binding.

In conclusion, we have shown that L109A CTPS exhibits greater uncoupling with the bulkier, nascent NH2OH, derived from Gln-OH hydrolysis, than with NH3 derived from Gln hydrolysis. This uncoupling is not caused by a leaky NH3 tunnel but arises because of a constriction within the tunnel as demonstrated by the ability of L109A CTPS to discriminate between nascent substrates based on size, relative to the wild-type enzyme.

Acknowledgements

This work was supported, in part, by an operating grant from the Canadian Institutes of Health Research (S.L.B.), a Natural Sciences and Engineering Research Council (NSERC) of Canada Collaborative Health Research Project grant (S.L.B.), and a graduate student fellowship from the Nova Scotia Health Research Foundation (F.A.L.). We express our thanks to Professor Enoch Baldwin (University of California, Davis, CA, USA) for kindly providing us with the PDB file for apo-E. coli CTPS and the coordinates for GTP modelled into the GTP-binding site.

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As replacement of Leu109 by Ala does not affect kcat values for the reaction of bound exogenous substrates, the size discrimination that is observed between nascent NH3 and NH2OH must arise from differences between the conformations that result when GTP is bound to wild-type CTPS relative to L109A CTPS. We propose that upon binding GTP (perhaps concomitant with Gln binding) in the cleft between the GAT and synthase domains, the two domains are drawn together. Consequently, the loop comprised of residues 105–114 would move inward so that Leu109 either packs against the bound GTP and/or helps to occlude the entryway for exogenous NH3 during catalysis of Gln-dependent reactions; and the internal NH3 tunnel/ vestibule may become (cid:1)kinked(cid:2). This (cid:1)kink(cid:2) could be responsible for the (cid:1)bottleneck(cid:2) which leads to uncoupling with wild-type CTPS when NH2OH is the substrate at saturating concentrations (Fig. 3 and see above). Such significant conformational changes would be expected because GTP binding causes conformational changes in the GAT domain to promote stabilization of the tetrahedral intermediates and transition states formed during Gln hydrolysis [3].

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