Alternative substrates for wild-type and L109A E. coli CTP 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 Cytidine 5¢-triphosphate (CTP) synthase catalyses t he ATP- dependent formation of CTP from uridine 5¢-triphosphate using either NH 3 or L -glutamine as the nitrogen s ource. The hydrolysis of glutamine is c atalysed in the C-terminal glu- tamine amide t ransfer domain a nd the n ascent NH 3 that is generated is transferred via an NH 3 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 a mination reaction occurs. ReplacementofLeu109byalanineinEscherichia coli CTP synthase causes an uncoupling of g lutamine hydrolysis and glutamine-dependent CTP formation [Iyengar, A . & Bearne, S.L. (2003) Biochem. J. 369 , 497–507]. To test our hypot hesis that L109A CTP synthase has a constricted or a leaky NH 3 tunnel, we e xamined the ability of wild-type and L109A CTP synthases to utilize NH 3 ,NH 2 OH, and NH 2 NH 2 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 NH 2 OH production from N 4 -hydroxy-CTP for- mation is more pronounced with the L109A enzyme, relative to the wild-type C TP synthase. These results suggest that the NH 3 tunnel o f L109A, in the presence of bound allosteric effector guanosine 5¢-triphosphate, is not leaky but contains a constriction that discriminates b etween NH 3 and NH 2 OH on the basis of size. Keywords: amidotransferase; ammonia tunnel; CTP syn- thase; glutaminase; alternative substrates. Cytidine 5¢-triphosphate (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 (Gln) or NH 3 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 C ys-His-Glu triad to catalyse the rate-limiting hydrolysis of Gln (glutaminase activity) [3–5], and the nascent NH 3 derived from this glutaminase a ctivity is transferred to the N-terminal synthase domain wh ere the a mination 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 o f cytosine nu cleotides. As CTP h as a central role in the biosynthesis of nucleic acids [8] and membrane phospho- lipids [9], C TPS 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 is one of the most thoroughly characterized CTP synthases with respect to its physical and kinetic p roperties, and i s regulated in a c omplex fashion [1]. GTP i s 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 m M [17]. I n a ddition, the enzyme is inhibited by the product CTP [18] and displays positive cooperativity for ATP and UTP [ 18–20]. ATP and UTP act synergistically t o promote tetramerization of the enzyme to its active form [20]. Recently, the X -ray crystal structure of E. coli CTP S 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. a po-E. coli CTPS) [21]. The authors identified a solvent-filled ÔvestibuleÕ ( 230 A ˚ 3 ) that connects the GAT active site and the GAT/synthase interface. This vestibule is connected to a t ubular passage that leads into the synthase site. The presence of this vestibule a nd NH 3 tunnel in CTPS is consistent with the identification of NH 3 tunnels in the X-ray structures of other amidotransferases inclu- ding carbamoyl phosphate synth ase (CPS) [22–24], Gln phosphoribosylpyrophosphate [25,26], GMP synthase [27], glucosamine-6-phosphate synthase [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 s ynthase domain. Replacement of the highly conserved L eu109 residue by alanine produced an enzyme that exhibited w ild-type levels of NH 3 -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, Te l.: +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-NH 2 , L -c-glutamyl hydrazide; OPA, o-phthaldialdehyde. Enzyme: CTP synthase (EC 6 .3.4.2) (Received 1 8 August 2 004, revised 3 September 2004, accepted 6 September 2004) Eur. J. Biochem. 271, 4204–4212 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04360.x 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 h ypothesis that L eu109 plays a role i n either the structure or formation of a n NH 3 tunnel and ensures efficient c oupling o f th e Gln h ydrolysis a nd amina- tion reactions. In the present report, we show that hydroxyl- amine, L -c-glutamy l hydroxamate (Gln-OH), hydrazine, and L -c-glutamyl hydrazide (Gln-NH 2 ) are alternative substrates for E. coli CTPS. Comparison of the kinetic parameters of Gln and NH 3 with those of the co rresponding bulkier substrates Gln-OH and NH 2 OH suggests that the impaired Gln-dependent CTP formation exhibited by the L109A mutant is caused by a constriction of the NH 3 tunnel. This is the fi rst functional evidence implicating a constriction in the N H 3 tunnel of E. coli CTPS. Experimental procedures General materials and methods All chemicals were purchased from Sigma-Aldrich Canad a 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 p ET15b-CTPS1 or the mutated plasmid as described previously [3,34]. These constructs encode the E. coli pyrG gene product with an N-terminal His 6 -tag. The BL21(DE3) cells were grown in Luria–Bertani medium at 37 °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 w ith 30 s intervals at output setting 5 using a Branson Sonifier 250). The crude lysate was clarified by centrifugation (39 000 g,20min,4°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 m M , pH 8.0) containing EGTA (0.5 m M ). All enzyme purifica- tion procedures were conducted at 4 °C. Thrombin-catalysed cleavage of the histidine tag f rom soluble enzyme (new N-terminus, GSHMLEM 1 …)was conducted in HEPES buffer (70 m M , pH 8.0) containing EGTA (0.5 m M ) using a thrombin ratio of 0.5 unitsÆmg )1 of target protein. After 8 h at 25 °C, cleavage was complete and the biotinylated thrombin was removed from the reaction mixture using streptavidin agarose r esin (Novagen, EMD Biosciences, Inc., Madison, WI, USA) at a ratio of 32 lL settled re sin per unit of thrombin following the Novagen protocol [35]. Cleaved CTPS, free of biotinylated thrombin, was then dialysed against HEPES buffer (70 m M , pH 8.0) containing EGTA (0.5 m M )andMgCl 2 (10 m M ) (assay buffer). The results o f 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 s equence of the w ild-t ype E. co li enzyme starting w ith M 1 as position 1. Cyclization of Gln-OH The c onversion of Gln-OH to 2-pyrrolidone-5-carboxylic acid [ 36] a t 37 °C w as followed using a Bruker AVANCE 500 M Hz NMR spectrometer. A solution of Gln-OH (20 m M ) in deuterated potassium phosphate buffer (100 m M , pD 8.0) was prepared and t he ionic strength was adjusted to 0.30 M using KCl. At various times (5, 7, 16, 26, 36, and 4 6 m in) the 1 H N MR 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, r espectively. (Chemical s hifts are relative to the D 2 O lock signal.) L-glutamine R = H, OH, NH 2 ; R' = ribose-5'-triphosphate UTP R = H CTP R = OH N 4 -hydroxy CTP R = NH 2 N 4 -amino CTP L-glutamate H 2 O tunnel with constriction or leak exogenous H 2 N–R [3] [2] [1] N N R' O HN HN N R' O O O NH 3 O OO – + H O O N NH 3 O – + R – HN R 2 glutaminase reaction synthase reaction R N N R' O OPO 3 2 AT P A DP – P i Scheme 1 nascent H 2 N–R from leak equilibrates with solvent [4] phosphorylated UTP intermediate Scheme 1. Reactions catalysed by E. coli CTP synthase. Ó FEBS 2004 Alternative substrates for E. coli CTP synthase (Eur. J. Biochem. 271) 4205 For t hose experiments utilizing Gln-OH a s t he substrate, we found that it was essential to maintain the Gln-OH stock solution at 4 °C and add this solution directly to t he assay cocktail to initiate the reaction. At 37 °C, the observed first order rate constant for cyclization of Gln-OH to form 2-pyrrolidone-5-carboxylate and NH 2 OH was 7.7 ( ± 0.4) · 10 )5 s )1 (i.e. t 1/2  2.5h)atpD8.0(data not shown). H ence, significant production of NH 2 OH occurs in Gln-OH solutions at 37 °C and the resulting NH 2 OH c an 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 CTPS activity was d etermined at 3 7 °C u sing a c ontinuous spectrophotometric a ssay by following the rate of increase in absorbance at 291 n m r esulting from either the c onver- sion of UTP to CTP (De ¼ 1338 M )1 Æcm )1 ) [18], to N 4 -hydroxy-CTP (De ¼ 40 23 M )1 Æcm )1 )[37],orto N 4 -amino-CTP (De ¼ 1364 M )1 Æcm )1 ;estimatedfromthe difference of the spectra of uridine and N 4 -amino cytidine). Substrates (NH 4 Cl, NH 2 OH, NH 2 NH 2 , Gln, G ln-OH, and Gln-NH 2 ) were dissolved in assay buffer and the pH was adjusted to 8.0 using 6 M KOH. The standard assay m ixture consisted of HEPES buffer (70 m M , pH 8.0) containing EGTA (0.5 m M ), Mg Cl 2 (10 m M ), CTPS, and saturating concentrations of UTP (1 m M )andATP(1m M )inatotal volume of 1 mL. Enzyme and nucleotides were p reincu- bated together f or 2.5 m in at 37 °C followed b y addition of substrate to initiate the reaction. Total NH 4 Cl concentra- tions in the assays were 5, 10, 20, 30, 50, 60, 80, and 100 m M ; total NH 2 OHÆHCl concentrations in the assays were 5, 10, 15, 20, 30, 40, 50, 75 , and 100 m M ;totalNH 2 NH 2 Æ2HCl concentrations in the assays were 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 m M ; 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 m M ; concentrations o f Gln-OH were 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 5.0, 10.0, and 15.0 m M ; concen- trations of Gln-NH 2 were 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 40.0, 60.0, 80.0, and 100.0 m M ; and CTPS concentrations ranged betw een 28 and 120 lgÆmL )1 for wild-type and 40–160 lgÆmL )1 for L109A. The concentration of GTP was maintained at 0.25 m M forallassayswhenGlnorGln analogues were used as the substrate. For assays conducted using Gln-OH, a freshly prepared stock solution w as stored on ice and added cold to each a ssay. This protocol was necessary to minimize cyclization o f Gln-OH with concom- itant production of NH 2 OH (see above). The i onic 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). v i ¼ V max ½S K m þ½S ð1Þ In Eqn (1), v i is the initial velocity, V max (¼ k cat [E] T )isthe maximal velocity a t saturating substrate concentrations, [S] is the substrate concentration, and K m is the M ichaelis constant for t he substrate. Values of K m for NH 3 ,NH 2 OH, and NH 2 NH 2 were calculated using the concentration of these species present at pH 8 .0 (i.e. pK a (NH 4 + ) ¼ 9.24; pK a ( + NH 3 OH) ¼ 5.97; p K a (NH 2 NH 3 + ) ¼ 8.10 [38]). Val- ues of k cat (per subunit) were calculated for CTPS variants with the His 6 -tag removed using the molecular masses ( Da) of 61 0 29 (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. Glutaminase assay 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-NH 2 with fluores- cence detection [39]. Assays were conducted at 37 °Cin HEPES buffer (70 m M , pH 8.0) containing EGTA (0.5 m M ), MgCl 2 (10 m M ), ATP (1 m M ), UTP (1 m M ), GTP (0.25 m M ), and either Gln (0.25, 0.50, 1.0, 5.0, and 10.0 m M ), Gln-OH (1.0, 3.0, 5 .0, 7.0, and 10 m M ), or Gln- NH 2 (3.0, 7.0, 10.0, 15.0, and 20.0 m M ). 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 m L. All components were preincubated for 2.5 min at 37 °C prior to initiation of the reaction by addition of substrate (Gln, Gln-OH, or Gln-NH 2 ). To minimize cyclization of Gln-OH, stock solutions (1 mL) were prepared at appro- priate concentrations and fl ash-frozen in liquid n itrogen. These Gln-OH solutions were thawed for 2.5 min at 37 °C and then used to initiate the reaction. At various time po ints (0, 1, 3, 5, 7, and 10 min), a liquots (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 m M ) [39]. Derivatization with OPA was shown t o effectively t erminate the reaction. (Boiling of the reaction led to rapid cyclization of the Gln-OH [36].) After 1 min a t r oom 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 u sing reversed-phase HPLC. Separation of the isoindole derivatives of Gln, g lutamate, Gln-OH, and Gln-NH 2 were conducted using a Synergi Fusion-RP column (4 lm; 80 A ˚ ;50· 4.6 mm; Pheno- menex, Torrance, C A) eluted under isocratic conditions using 0 .1 M sodium acet ate (adjusted to pH 6.2 with g lacial 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 reactio n of Gln, glutamate, Gln-OH, and Gln- NH 2 with OPA reagent was monitored using a Waters 474 scanning fluorescence detector (k ex ¼ 343 nm, k em ¼ 440 n m). 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 c hromatograms using PEAKSIMPLE software from Mandel Scientific (Guelph, ON, C anada). Concentrations of glutamate were deter- mined u sing a standard curve prepared by derivatization of 4206 F. A. Lunn and S. L. Bearne (Eur. J. Biochem. 271) Ó FEBS 2004 standard glutamate solutions (0.025, 0.050, 0.075, 0.100, 0.150, 0.200, and 0.250 m M ). Calculations Geometry optimizations and electrostatic potential sur- faces were calculated for N H 3 ,NH 2 OH, and NH 2 NH 2 by performing self-consistent-field calculations at the 6–31 G** level using SPARTAN ¢04 WINDOWS (Wavefunction, Inc., Irvine, CA). This software was a lso used to c alculate the molecular surface areas and volumes of these molecules. Results and discussion Previously, we reported that replacement of the highly conserved L eu109 residue in E. coli CTPS by alanine yields an enzyme that has kinetic prop erties similar to t hose of wild-type CTPS with respect to NH 3 -dependent CTP formation, affinity for Gln, glutaminase activity, affinity for G TP, and activation by GTP [34]. However, unlike wild- type CTPS, the L109A mutant exhibited impaired Gln- dependent CTP f ormation. These observations suggested that Leu109 plays a role in either the structure or formation of an NH 3 tunnel and ensures efficient coupling of the Gln hydrolysis and amination reactions. In the present study, we use bulky analogues of exogenous N H 3 (i.e. NH 2 OH and NH 2 NH 2 ) and nascent NH 3 (i.e. NH 2 OH and NH 2 NH 2 derived from the hydrolysis of Gln-OH and Gln-NH 2 , respectively) to test our hypothesis that the presence of a n alanine at position 109 introduces a constriction in the NH 3 tunnel of E. coli CTPS. T his approach has been u sed to demonstrate that t he G359S m utant o f CPS has a partially blocked NH 3 tunnel that prevents diffusion of NH 2 OH while still allowing some NH 3 to diffuse through [40]. T he hypothesis that r eplacement of the bulky Leu109 by the smaller a lanine could cause a tunnel blockage has precedent. For example, the F334A m utant of glutamine phosphorib- osylpyrophosphate amidotransferase exhibited kinetic prop- erties expecte d fo r a blocked or disrupted NH 3 tunnel [41]. Many amidotransferases can utilize N H 2 OH and NH 2 NH 2 in place of NH 3 [42–44]. Both NH 2 NH 2 and NH 2 OH (and its derivatives NH 2 OCH 3 and CH 3 NHOH) are substrates for CTPS from Ehrlich ascites tumour cells [45], a nd NH 2 OH has been sh own to be a substrate for E. coli [46] and Lactococcus lactis [47] CTP synthases. W ith the exception of Lieberman’s work in 1956 [46], little is known about the ability o f E. coli CTPS to utilize a lter- native NH 3 sources. In a ddition, some amidotransferases such as CPS [40] a nd asparagine synthase B [48] have been showntohydrolyseGln-OHandGln-NH 2 to give rise to NH 2 OH and NH 2 NH 2 , respectively. Although E. coli CTPS has be en 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 NH 3 and the bulkier analogue NH 2 OH based on size but does not lead to discrimination between exogenous NH 3 and bulkier analogues (i.e. NH 2 OH and NH 2 NH 2 ). Our findings are consistent with the L109A mutation causing constriction of an NH 3 tunnel. Exogenous NH 3 and its analogues Exogenous NH 3 ,NH 2 OH, and NH 2 NH 2 all served as substrates for wild-type and L109A CTP synthases (Table 1). However, both NH 2 OH and NH 2 NH 2 exhibited k cat /K m values with both enzymes that were approximately 30-fold less t han t he k cat /K m value for NH 3 . This r eduction in k cat /K m wascausedbyanincreasedK m value for NH 2 OH and NH 2 NH 2 .RelativetoNH 3 ,theK m value for NH 2 OH was increased approximately 40-fold while the k cat value was s lightly greater than that for NH 3 . T his observation is in accord with the slightly greater nucle ophilicity o f NH 2 OH relative to NH 3 [49,50]. Hence, it appears that once NH 2 OH is bound it reacts readily with the phosphorylated UTP intermediate. The individual K m and k cat values for NH 2 NH 2 could not be determined for either wild-type CTPS or L109A CTPS because saturation was not observed, indicating that the K m for this s ubstrate w as also markedly increased relative to that observed for NH 3 . Three possible routes that exogenous NH 3 or its analogues m ight traverse to reach t he site of reaction with the phosphorylated UTP intermediate are shown in Table 1. Kinetic Param eters for w ild-type and L 109A CTP synthases. – , Not determined. Substrate Wild-type CTPS L109A CTPS K m (m M ) k cat (s )1 ) k cat /K m (m M )1 Æs )1 ) K m (m M ) k cat (s )1 ) k cat /K m (m M )1 Æs )1 ) Kinetic parameters for CTP formation NH 3 2.15 ± 0.14 9.50 ± 0.53 4.43 ± 0.12 2.17 ± 0.09 10.1 ± 0.3 4.63 ± 0.04 NH 2 OH 82.8 ± 6.8 14.0 ± 1.8 0.169 ± 0.016 75.3 ± 9.8 14.1 ± 1.9 0.187 ± 0.003 NH 2 NH 2 – a – a 0.147 ± 0.019 – a – a 0.128 ± 0.015 Gln 0.354 ± 0.057 6.10 ± 0.80 17.8 ± 2.3 0.497 ± 0.132 1.86 ± 0.34 3.85 ± 0.82 Gln-OH 0.165 ± 0.017 0.453 ± 0.001 2.77 ± 0.28 0.250 ± 0.091 0.063 ± 0.014 0.260 ± 0.061 Gln-NH 2 39.4 ± 0.5 1.41 ± 0.04 0.036 ± 0.001 – – – Kinetic parameters for the glutaminase activity Gln 0.327 ± 0.002 5.62 ± 0.12 17.2 ± 0.1 0.550 ± 0.012 5.06 ± 0.24 9.22 ± 0.62 Gln-OH 0.324 ± 0.101 0.930 ± 0.040 3.06 ± 0.90 0.260 ± 0.061 0.310 ± 0.033 1.26 ± 0.40 Gln-NH 2 – a – a 0.034 ± 0.004 – b – b – b a Saturation could not be achieved and k cat /K m values were determined from measurements conducted with [S] << K m . b Activity too low to measure reliably. Ó FEBS 2004 Alternative substrates for E. coli CTP synthase (Eur. J. Biochem. 271) 4207 Scheme 1. Route 1 represents a bimolecular reaction with the reactive intermediate. This route is unlikely as saturation kinetics are observed when NH 3 is a substrate, su ggesting the formatio n of an initial enzyme-NH 3 complex. Routes 2 and 3 involve the binding of NH 3 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 a midotransferases h ave suggested the presence of NH 3 tunnels to shuttle the nascent NH 3 from the site of Gln hydrolysis to the synthase domain [51], it is not always clear what route is f ollowed b y exogenous NH 3 .Foranygiven exogenous substrate (i.e. NH 3 ,NH 2 OH, or NH 2 NH 2 ), the kinetic p arameters ( K m , k cat , a nd/or k cat /K m ) a re sim ilar f or both wild-type a nd L109A E. coli CTP synthases. Thus, replacement of Leu109 by alanine does not cause any discrimination between exogenous substrates of a g iven size with respect to binding affinity, t urnover, and efficiency. In addition, once the bulkier, exogenous NH 2 OH enters the enzyme, i t is transferred to the synthase active site and reacts with the phosphorylated UTP i ntermediate as e fficiently as NH 3 as indicated by the similar k cat values for either the wild-type o r L109A CTP synthases. B ased on their recently solved crystal structure of wild-type E. coli CTPS, Endrizzi et al . [21] suggested that exogenous NH 3 could access the active site via a ÔholeÕ on the p rotein’s surface that resides midway between the Gln and UTP binding sites (Fig. 1). Our observations suggest that, after binding to L109A CTPS, p assage of exogenous NH 3 or its analogues through the NH 3 tunnel (i.e. route 3) are not inhibited by a constriction if it is present. Alternatively, a constriction may be present a t a location within the NH 3 tunnel that i s closer to the site of Gln hydrolysis so that exogenous substrates entering through the hole bypass the constriction. It is important to note t hat both the wild-type and L109A enzymes do discriminate between NH 3 and the bulkier substrates in terms of binding (i.e. elevated K m values for NH 2 OH and N H 2 NH 2 relative to NH 3 for both w ild-type and L109A CTP s ynthases). The entrance for exogenous NH 3 is approximately 3 A ˚ in diameter thereby p ermitting access of NH 3 (surface area ¼ 43.65 A ˚ 2 ; volume ¼ 26.52 A ˚ 3 ) [21]. On the other hand, entrance of bulkier substrates such as NH 2 OH (surface area ¼ 54.89 A ˚ 2 ; volume ¼ 35.62 A ˚ 3 )andNH 2 NH 2 (surface area ¼ 60.23 A ˚ 2 ; volume ¼ 40.62 A ˚ 3 ) may be more difficult and require proper orientation of these molecules a long their longitudinal a xis in order to pass through the hole and avoid unfavourable steric interactions. This r equirement for correct orientation could, in part, be responsible for the elevated K m values observed for the bulkier substrates. The electrostatic potential surfaces of NH 3 ,NH 2 OH, and NH 2 NH 2 (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 NH 3 and its analogues The abilities of wild-type and L109A CTP synthases to catalyse the hydrolysis o f Gln, Gln-OH, and Gln-NH 2 (i.e. glutaminase activity) and to subsequently catalyse the formation o f C TP, N 4 -hydroxy-CTP, and N 4 -amino-CTP, respectively, were examined (Table 1). R elative to Gln, the k cat /K m values for Gln-OH and Gln-NH 2 hydrolysis catalysed by wild-type CTPS were reduced approximately Fig. 1. Location of Leu109 relative to the opening for exogenous NH 3 (PDB code 1S1M [21]). (A) Amino acid residues comprising the walls of the entryway for exogenous NH 3 include residues 50–55, Val60, Glu68, Lys297, Tyr298, Ala304, Phe353, Gly354, Arg356, Glu403, and Arg468 (shown in green, space-filling representation). The sulphur of t he catalytic nucleophile Cys379 is yellow. The loop comprised o f r esidues 104–110 f rom the adjacent subunit is shown in red with Leu109 shown in space-filling representation. (B) Viewed from the side, relative t o (A), Leu109 is poised above the o pening for e xogenous NH 3 . G TP is shown modelled i nto the cleft [21], however, this m odel probably does not ac curately reflect th e change in conformation associated with GTP binding. Movement o f t he 104–110 loop may occur upon GTP binding so that Leu109 is repositioned to pack against bound GTP and perhaps h elp further seal the e ntryway for exogenous NH 3 . 4208 F. A. Lunn and S. L. Bearne (Eur. J. Biochem. 271) Ó FEBS 2004 six-fold and 500-fold, respectively. The same trend is also observed for wild-type CTPS-catalysed formation of N 4 -hydroxy-CTP and N 4 -amino-CTP. Comparison of the K m and k cat values for Gln-OH hydrolysis with those observed for Gln hydrolysis reveals that this reduction in efficiency a rises f rom a six-fold reduction in k cat while there is no change in the K m value. The marked reduction in t he efficiency (k cat /K m ) of wild-type CTPS-catalysed formation of N 4 -hydroxy-CTP resulted mainly from a 111-fold increase in the K m value. A similar trend is also observed with L109A C TPS. Unfortunately, w e were unable to detect any significant amount of glutaminase activity using L109A CTPS with Gln-NH 2 as a substrate. Consequen tly, we were not able to employ nasc ent NH 2 NH 2 in our analysis f or tunnel constriction. The values of k cat /K m and k cat for wild-type CTPS- catalysed Gln hydrolysis and CTP formation are experi- mentally equal. This indicates t hat there is total coupling of the reaction s forming the nascent N H 3 and i ts reac tion to form CTP a t both l ow (i.e. k cat /K m conditions) and high (i.e. k cat conditions) concentrations of Gln. However, when Gln- OH is the substrate, N 4 -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 wh en either the nature o f the substrate o r enzyme is altered, w e employ two Ôcoupling ratiosÕ as defined in Eqns 2 a nd 3, and reported in Figs 2 and 3. Such ratios have been used to characterize the c hannelling efficiency of amidotransferases [41]. Subsaturating coupling ratio ¼ ðk cat =K m Þ CTP for mation ðk cat =K m Þ glutaminase activity ð2Þ Saturating coupling ratio ¼ ðk cat Þ CTP f ormat ion ðk cat Þ glutami nasea c tivit y ð3Þ 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 n ascent NH 3 is consumed in the amination reaction as rapidly as it is produced at all concentrations of glutamine ( i.e. r eactions are fully coupled as mentioned above). Unlike wild-type CTPS-catalysed hydrolysis of Gln, Gln-OH hydrolysis is only fully coupled to N 4 -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 k cat value for the wild-type CTPS-catalysed formation of N 4 -hydroxy-CTP from nascent NH 2 OH (Gln- OH as the su bstrate) is reduced 13-fold relative t o that for nascent N H 3 (Gln as the substrate). The bulkier nascent NH 2 OH must either encounter some unfavourable steric interactions or a ÔbottleneckÕ as it traverses t he NH 3 tunnel, or the kinetic expression for k cat for the hydrolysis of Gln- OH contains terms that include rate constants for the hydrolysis reaction, production of NH 2 OH and Glu, and release o f G lu. (The e xact 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 k cat cannot presently be derived.) However, because the k cat value for wild-type hydrolysis of Gln-OH is reduced only six-fold relative to the k cat value for Gln hydrolysis, it appears t hat t he additional reduction in k cat (to 13-fold as mentioned above) that is observed for Gln-OH-dependent N 4 -hydroxy-CTP formation results from some other limit- ing effect such as a Ôb ottleneckÕ. Examination of the coupling ratios in Figs 2 and 3 reveals that at all substrate concentrations, L109A CTPS exhibits u ncoupling (i.e. coupling ratios < 1). At saturating substrate concentrations (Fig. 3 ), replacement of Leu109 by alanine causes the coupling r atios to be reduced by factor s of 2.95 and 2.40 for the Gln- and Gln-OH-dependent reactions, respectively. Interestingly, the coupling ratios for the G ln- a nd 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 i s no more s ensitive to the increase d size of NH 2 OH than wild-type CTPS when substrate concentra- tions are saturating. Therefore, the rate of transfer of the bulkier, nascent NH 2 OH under k cat conditions appears to be limit ed by a ÔbottleneckÕ that is not affected by replacement o f Leu109 by alanine. For this reason, only the k cat /K m data (Fig. 2 ) are used to determine if the mutant enzyme is sensitive to the larger siz e of the nasce nt NH 2 OH. Previously, we reported that L109A exhibited uncoup- ling of Gln hydrolysis from CTP formation [34]. We wild-type L109A substrate Gln Glu-OH 0.905 ± 0.281 1.03 ± 0.13 0.206 ± 0.081 0.418 ± 0.093 2.03 ± 0.9 2 1.14 ± 0.38 2.46 ± 0.63 P = 0.0028 P = 0.0144 P = 0.5229 P = 0.0408 4.39 ± 2.20 Fig. 2. Coupling r atios for wild-type and L109A CTP synthases at subsaturating substrate concentrations. Subsaturatin g coup ling ratios (Eqn 2) are shown in boldface. The f actors by which the ratios change upon altering either the substrate (vertical arrows) o r enzyme (hori- zontal arrows) are shown i n i talics. The s tatistical signifi cance o f t he changes in the coupling ratios is indicated by the corresponding P value based on an unpaired, 2-tailed t-test (P < 0.05 is s tatistically significant). wild-type L109A substrate Gln Glu-OH 0.487 ± 0.021 1.09 ± 0.14 0.203 ± 0.050 0.368 ± 0.069 1.81 ± 0.5 6 2.23 ± 0.31 2.95 ± 0.68 2.40 ± 0.60 P = 0.0008 P = 0.0018 P = 0.0285 P = 0.0013 Fig. 3. Coupling r atios for wild-type and L109A CTP synthases at saturating substrate concentrations. Saturating coupling ratios (Eqn 3) are shown in b oldface. The factors by w hich the r atios change upon altering either the s ubstrate ( vert ical arrows) o r en zyme ( horizo ntal arrows) are shown in italics. The statistical significance of the changes in the coupling rat ios is indic ated by th e corre spondin g P value based on an unpaired, 2-tailed t-test (P < 0.05 i s statistically significant). Ó FEBS 2004 Alternative substrates for E. coli CTP synthase (Eur. J. Biochem. 271) 4209 hypothesized t hat t his uncoupling could a rise from (a) a leaky NH 3 tunnel, (b) a constricted NH 3 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 NH 3 and Gln now permits us to r efine our hyp othesis. As shown in Fig. 2 , L109A CTPS exhibits more pronounced uncoupling with Gln-OH than with Gln. Hence, the uncoupling obs erved w ith L 109A CTPS appears to depend on the size of the nascent NH 3 analogue. This o bservation is most consistent with the presence of a constricted NH 3 tunnel. If a leaky tunnel were present, we would e xpect the bulkier nascent NH 2 OH to either leak out to bulk solvent, like the nascent NH 3 (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 N 4 - hydroxy-CTP. In this latter case, less uncoupling would be expected for the L109A enzyme, resulting in a higher coupling ratio for nascent NH 2 OH relative to nascent N H 3 . Structural aspects of uncoupling In the crystal structure of apo-E. coli CTPS, L eu109 is located o n 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 t his cleft and above the opening that Endrizzi et al . [21] identified as a putative entry point for e xogenous NH 3 to access a solven t-filled vestibule that c onnects the GAT active s ite and the GAT/synthase interface. Modelling studies conducted by E ndrizzi et al. [ 21] s uggest tha t GTP binds in the cleft that overlies the entry point for exogenous NH 3 . T his finding is in accord with our recent report t hat GTP binding inhibits CTP formation from exogenous NH 3 [17]. Studies also suggest that GTP binding induces a conformational c hange in E. coli [3,16,17,52,53] and L. lac- tis [54] CTP synthases. In the absence o f bound ligands, the structure of apo-E. coli CTPS does not provide much insight into what conformational changes might o ccur upon GTP binding. As replacement of Leu109 by Ala does not affect k cat values for t he reaction of bound exogenous substrates, the size discrimination that is observed between nascent NH 3 and NH 2 OH must arise from differences between the conformations that result when GTP i s bound to wild-type CTPS relative to L109A CTPS. We propose that upon binding GTP (perhaps concomitant with Gln binding) i n 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 NH 3 during catalysis of Gln-dependent reactions; and the internal NH 3 tunnel/ vestibule may become ÔkinkedÕ.ThisÔkinkÕ could be responsible for the ÔbottleneckÕ which leads to uncoupling with wild-type CTPS when NH 2 OH is the s ubstrate 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 tetrah edral intermediates and transition states formed during Gln hydrolysis [3]. This sc enario is consistent with the l ack of equilibration of the nascent NH 3 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 NH 3 -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 L 109A mutation leads to uncoupling, one possibility is that a conformational ÔkinkÕ arises via the mechanism mention ed above so that a functional tunnel that efficiently couples the glutaminase and amination reactions is not properly formed. Formation of a competent NH 3 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 i mpede the appropriate conformational changes required for catalysis, substitution by alanine might permit Ôtoo muchÕ of a conformational change b ecause of differences b etween the packing of the leucine vs. alanine side chains with GTP leading to a more significant ÔkinkÕ. Although the kink/constriction could occur at any point along the r oute traversed b y t he nascent NH 3 , one possible location is the narrow ÔgateÕ between Pro54 and Val60, identified by Endrizzi et al. [21], that resides at the base of the proposed entryway fo r e xogenous NH 3 . F urther narrowing of this Ôga teÕ upon GTP b inding could lead to a constriction that discriminates between nascent NH 3 and the bulkier NH 2 OH within L109A but does not affect the use of exogenous NH 3 and its analogues (at least under k cat /K m conditions). Both explanations are f ully consistent with the kinetic properties exhibited by L109A CTPS with alter- native, bulkier substrates. In conclusion, we have shown that L109A CTPS exhibit s greater uncoupling with the bulkier, nascent NH 2 OH, derived from Gln-OH hydrolysis, than with NH 3 derived from Gln hydrolysis. 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Alternative substrates for wild-type and L10 9A E. coli CTP synthases Kinetic evidence for a constricted ammonia tunnel Faylene A. Lunn and Stephen L and average values are reported. The reported errors are standard deviations. Initial rate kinetic data was fit to Eqn (1) by nonlinear regression analysis