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 synthases to utilize NH3, NH2OH, and NH2NH2as e
Trang 1Alternative 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 the
ATP-dependent formation of CTP from uridine 5¢-triphosphate
using either NH3orL-glutamine as the nitrogen source The
hydrolysis of glutamine is catalysed in the C-terminal
glu-tamine amide transfer domain and the nascent NH3that is
generated is transferred via an NH3tunnel [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 synthases to utilize NH3, NH2OH, and NH2NH2as 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
NH3tunnel of L109A, in the presence of bound allosteric effector guanosine 5¢-triphosphate, is not leaky but contains
a constriction that discriminates between NH3and NH2OH
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
eitherL-glutamine (Gln) or NH3as 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
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 vestibule ( 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 NH3tunnel
in CTPS is consistent with the identification of NH3tunnels
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 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 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-NH 2 , 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)
Trang 2affinity 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 reacamina-tions 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 NH3with 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 NH3tunnel of E coli CTPS
Experimental procedures
General materials and methods
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 37C, 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
(39 000 g, 20 min, 4C) 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 4C
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)1of target protein After 8 h at 25C, 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 M1as position 1 Cyclization of Gln-OH
The conversion of Gln-OH to 2-pyrrolidone-5-carboxylic acid [36] at 37C was followed using a Bruker AVANCE
500 MHz NMR spectrometer A solution of Gln-OH (20 mM) in deuterated potassium phosphate buffer (100 mM, pD 8.0) was prepared and the ionic strength was adjusted to 0.30Musing KCl At various times (5, 7, 16,
26, 36, and 46 min) the1H 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
DO lock signal.)
L -glutamine
R = H, OH, NH 2; R' = ribose-5'-triphosphate
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 O O
– + H
O
NH 3
O –
+
H N R 2
glutaminase reaction
synthase reaction
R N
N R' O
OPO 3 ATP ADP
–
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.
Trang 3For those experiments utilizing Gln-OH as the substrate,
we found that it was essential to maintain the Gln-OH stock
solution at 4C and add this solution directly to the assay
cocktail to initiate the reaction At 37C, the observed
first order rate constant for cyclization of Gln-OH to
form 2-pyrrolidone-5-carboxylate and NH2OH was
7.7 (± 0.4)· 10)5s)1 (i.e t1/2 2.5 h) at pD 8.0 (data
not shown) Hence, significant production of NH2OH occurs
in Gln-OH solutions at 37C 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
CTPS activity was determined at 37C using a continuous
spectrophotometric assay by following the rate of increase
in absorbance at 291 nm resulting from either the
conver-sion of UTP to CTP (De¼ 1338M )1Æcm)1) [18], to
N4-hydroxy-CTP (De¼ 4023M )1Æcm)1) [37], or to
N4-amino-CTP (De¼ 1364M )1Æcm)1; estimated from the
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 6MKOH 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 37C 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)1for wild-type and 20–24 lgÆmL)1for 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-NH2were 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)1for 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 0.30Min 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)
vi¼ Vmax½S
In Eqn (1), viis the initial velocity, Vmax(¼ kcat[E]T) is the
maximal velocity at saturating substrate concentrations,
[S] is the substrate concentration, and Kmis the Michaelis constant for the substrate Values of Kmfor NH3, NH2OH, and NH2NH2 were calculated using the concentration of these species present at pH 8.0 (i.e pKa(NH4+)¼ 9.24;
pKa(+NH3OH)¼ 5.97; pKa(NH2NH3+)¼ 8.10 [38]) Val-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
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-NH2 with fluores-cence detection [39] Assays were conducted at 37C in HEPES buffer (70 mM, pH 8.0) 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 37C 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
37C 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, 0.1M, pH 6.2) and an aliquot (20 lL) was analysed using reversed-phase HPLC
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 0.1Msodium 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, OH, and
Gln-NH2 with OPA reagent was monitored using a Waters
474 scanning fluorescence detector (kex¼ 343 nm, kem¼
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 usingPEAKSIMPLEsoftware from Mandel Scientific (Guelph,
ON, Canada) Concentrations of glutamate were deter-mined using a standard curve prepared by derivatization of
Trang 4standard 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 usingSPARTAN¢04WINDOWS(Wavefunction,
Inc., Irvine, CA) This software was also used to calculate
the molecular surface areas and volumes of these molecules
Results and discussion
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 NH3tunnel 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 NH3to 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 NH3tunnel [41]
Many amidotransferases can utilize NH2OH and
NH2NH2 in place of NH3 [42–44] Both NH2NH2 and
NH2OH (and its derivatives NH2OCH3and CH3NHOH)
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-NH2to 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 NH3tunnel Exogenous NH3and its analogues
Exogenous NH3, NH2OH, and NH2NH2 all served as substrates for wild-type and L109A CTP synthases (Table 1) However, both NH2OH and NH2NH2exhibited
kcat/Kmvalues with both enzymes that were approximately 30-fold less than the kcat/Kmvalue for NH3 This reduction
in kcat/Kmwas caused by an increased Kmvalue for NH2OH and NH2NH2 Relative to NH3, the Kmvalue for NH2OH was increased approximately 40-fold while the kcatvalue 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 Kmfor 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 Table 1 Kinetic Parameters for wild-type and L109A CTP synthases –, Not determined.
Substrate
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 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
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
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
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.
Trang 5Scheme 1 Route 1 represents a bimolecular reaction with
the reactive intermediate This route is unlikely as saturation
kinetics are observed when NH3is a substrate, suggesting
the formation of an initial enzyme-NH3complex Routes 2
and 3 involve the binding of NH3at 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
NH3tunnels to shuttle the nascent NH3from 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
NH3as indicated by the similar kcatvalues 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 NH3could access the
active site via a hole 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 NH3or 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 NH3tunnel that is closer
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 Kmvalues for
NH2OH and NH2NH2relative to NH3for both wild-type and L109A CTP synthases) The entrance for exogenous
NH3is 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; volume¼ 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 Kmvalues 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 NH3and 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
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 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 NH 3 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 NH
Trang 6six-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 kcatwhile there
is no change in the Kmvalue 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 Kmvalue 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-NH2as a substrate Consequently, we were
not able to employ nascent NH2NH2 in our analysis for
tunnel constriction
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 NH3and its reaction to
form CTP at both low (i.e kcat/Kmconditions) and high (i.e
kcatconditions) 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 coupling ratios 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]
Subsaturating coupling ratio¼ ðkcat=KmÞCTP formation
ðkcat=KmÞglutaminase activity
ð2Þ Saturating coupling ratio¼ ðkcatÞCTP formation
ðkcatÞglutaminase activity 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 nascent NH3is 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 kcatvalue 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 bottleneck as it traverses the NH3tunnel,
or the kinetic expression for kcatfor 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
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 bottleneck
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 (1.09fi 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 kcatconditions appears to
be limited by a bottleneck that is not affected by replacement of Leu109 by alanine For this reason, only the kcat/Kmdata (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
substrate
Gln
Glu-OH 0.905 ± 0.281
1.03 ± 0.13
0.206 ± 0.081
0.418 ± 0.093
2.03 ± 0.92 1.14 ± 0.38
2.46 ± 0.63
P = 0.0028
P = 0.0144
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).
substrate
Gln
Glu-OH 0.487 ± 0.021
1.09 ± 0.14
0.203 ± 0.050
0.368 ± 0.069
1.81 ± 0.56 2.23 ± 0.31
2.95 ± 0.68
2.40 ± 0.60
P = 0.0008
P = 0.0013
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).
Trang 7hypothesized that this uncoupling could arise from (a) a
leaky NH3tunnel, (b) a constricted NH3tunnel, 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
NH3and 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 NH3analogue 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
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
NH3to 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
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 NH3during catalysis of
Gln-dependent reactions; and the internal NH3 tunnel/
vestibule may become kinked This kink could be
responsible for the bottleneck 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]
This scenario is consistent with the lack of equilibration
of the nascent NH3derived 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 kink 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 the appropriate conformational changes required for catalysis, substitution by alanine might permit too much
of a conformational change because of differences between the packing of the leucine vs alanine side chains with GTP leading to a more significant kink Although the kink/constriction could occur at any point along the route traversed by the nascent NH3, one possible location is the narrow gate 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
gate 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 NH3and 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 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 NH3tunnel 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.
References
1 Koshland, D.E Jr & Levitzki, A (1974) CTP Synthetase and Related Enzymes In The Enzymes (Boyer, P.D., ed.), pp 539–559 Academic Press, New York.
Trang 82 Long, C & Koshland, D.E Jr (1978) Cytidine triphosphate
syn-thetase Methods Enzymol 51, 79–83.
3 Bearne, S.L., Hekmat, O & MacDonnell, J.E (2001) Inhibition of
Escherichia coli CTP synthase by glutamate gamma-semialdehyde
and the role of the allosteric effector GTP in glutamine hydrolysis.
Biochem J 356, 223–232.
4 Levitzki, A & Koshland, D.E Jr (1971) Cytidine triphosphate
synthetase Covalent intermediates and mechanisms of action.
Biochemistry 10, 3365–3371.
5 Willemoe¨s, M (2004) Competition between ammonia derived
from internal glutamine hydrolysis and hydroxylamine present
in the solution for incorporation into UTP as catalysed by
Lactococcus lactis CTP synthase Arch Biochem Biophys 424,
105–111.
6 Weng, M., Makaroff, C.A & Zalkin, H (1986) Nucleotide
se-quence of Escherichia coli pyrG encoding CTP synthetase J Biol.
Chem 261, 5568–5574.
7 Weng, M.L & Zalkin, H (1987) Structural role for a conserved
region in the CTP synthetase glutamine amide transfer domain.
J Bacteriol 169, 3023–3028.
8 Hatse, S., De Clercq, E & Balzarini, J (1999) Role of
anti-metabolites of purine and pyrimidine nucleotide metabolism in
tumor cell differentiation Biochem Pharmacol 58, 539–555.
9 Kennedy, E.P (1986) The biosynthesis of phospholipids In Lipids
and Membranes: Past, Present and Future (Op den Kamp, J.A.F.,
Roelofsen, B & Wirtz, K.W.A., eds), pp 171–206 Elsevier
Scientific Publishers, Amsterdam.
10 Kensler, T.W & Cooney, D.A (1989) Inhibitors of the de novo
pyrimidine pathway In Design of Enzyme Inhibitors as Drugs
(Sandler, M & Smith, H.J., eds), pp 379–401 Oxford University
Press, New York.
11 Gao, W.Y., Johns, D.G & Mitsuya, H (2000) Potentiation of the
anti-HIV activity of zalcitabine and lamivudine by a CTP synthase
inhibitor, 3-deazauridine Nucleosides Nucleotides Nucleic Acids
19, 371–377.
12 De Clercq, E (1993) Antiviral agents: characteristic activity
spectrum depending on the molecular target with which they
interact Adv Virus Res 42, 1–55.
13 Hendriks, E.F., O’Sullivan, W.J & Stewart, T.S (1998) Molecular
cloning and characterization of the Plasmodium falciparum
cyti-dine triphosphate synthetase gene Biochim Biophys Acta 1399,
213–218.
14 Hofer, A., Steverding, D., Chabes, A., Brun, R & Thelander, L.
(2001) Trypanosoma brucei CTP synthetase: a target for the
treatment of African sleeping sickness Proc Natl Acad Sci USA
98, 6412–6416.
15 Lim, R.L., O’Sullivan, W.J & Stewart, T.S (1996) Isolation,
characterization and expression of the gene encoding cytidine
triphosphate synthetase from Giardia intestinalis Mol Biochem.
Parasitol 78, 249–257.
16 Levitzki, A & Koshland, D.E Jr (1972) Role of an allosteric
effector Guanosine triphosphate activation in cytosine
triphos-phate synthetase Biochemistry 11, 241–246.
17 MacDonnell, J.E., Lunn, F.A & Bearne, S.L (2004) Inhibition of
E coli CTP synthase by the positive allosteric effector GTP.
Biochim Biophys Acta 1699, 213–220.
18 Long, C.W & Pardee, A.B (1967) Cytidine triphosphate
syn-thetase of Escherichia coli B I Purification and kinetics J Biol.
Chem 242, 4715–4721.
19 Levitzki, A & Koshland, D.E Jr (1969) Negative cooperativity in
regulatory enzymes Proc Natl Acad Sci USA 62, 1121–1128.
20 Levitzki, A & Koshland, D.E Jr (1972) Ligand-induced
dimer-to-tetramer transformation in cytosine triphosphate synthetase.
Biochemistry 11, 247–253.
21 Endrizzi, J.A., Kim, H., Anderson, P.M & Baldwin, E.P (2004)
Crystal structure of Escherichia coli cytidine triphosphate
synthe-tase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anti-cancer and antiparasitic drug targets Biochemistry 43, 6447–6463.
22 Holden, H.M., Thoden, J.B & Raushel, F.M (1998) Carbamoyl phosphate synthetase: a tunnel runs through it Curr Opin Struct Biol 8, 679–685.
23 Mullins, L.S & Raushel, F.M (1999) Channeling of ammonia through the intermolecular tunnel contained within carbamoyl phosphate synthetase J Am Chem Soc 121, 3803–3804.
24 Thoden, J.B., Holden, H.M., Wesenberg, G., Raushel, F.M & Rayment, I (1997) Structure of carbamoyl phosphate synthetase:
a journey of 96 A˚ from substrate to product Biochemistry 36, 6305–6316.
25 Krahn, J.M., Kim, J.H., Burns, M.R., Parry, R.J., Zalkin, H & Smith, J.L (1997) Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site Biochemistry 36, 11061–11068.
26 Muchmore, C.R., Krahn, J.M., Kim, J.H., Zalkin, H & Smith, J.L (1998) Crystal structure of glutamine phosphoribosylpyr-ophosphate amidotransferase from Escherichia coli Protein Sci 7, 39–51.
27 Tesmer, J.J., Klem, T.J., Deras, M.L., Davisson, V.J & Smith, J.L (1996) The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families Nat Struct Biol 3, 74–86.
28 Teplyakov, A., Obmolova, G., Badet, B & Badet-Denisot, M.A (2001) Channeling of ammonia in glucosamine-6-phosphate syn-thase J Mol Biol 313, 1093–1102.
29 Teplyakov, A., Obmolova, G., Badet-Denisot, M.A & Badet, B (1999) The mechanism of sugar phosphate isomerization by glu-cosamine 6-phosphate synthase Protein Sci 8, 596–602.
30 Teplyakov, A., Obmolova, G., Badet-Denisot, M.A., Badet, B & Polikarpov, I (1998) Involvement of the C terminus in intramolecular nitrogen channeling in glucosamine 6-phosphate synthase: evidence from a 1.6 A˚ crystal structure of the isomerase domain Structure 6, 1047–1055.
31 Larsen, T.M., Boehlein, S.K., Schuster, S.M., Richards, N.G., Thoden, J.B., Holden, H.M & Rayment, I (1999) Three-dimensional structure of Escherichia coli asparagine synthetase B:
a short journey from substrate to product Biochemistry 38, 16146–16157.
32 Morollo, A.A & Eck, M.J (2001) Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium Nat Struct Biol 8, 243–247.
33 Spraggon, G., Kim, C., Nguyen-Huu, X., Yee, M.C., Yanofsky,
C & Mills, S.E (2001) The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, 1-tryptophan Proc Natl Acad Sci USA
98, 6021–6026.
34 Iyengar, A & Bearne, S.L (2003) Aspartate 107 and leucine 109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by E coli CTP synthase Biochem J 369, 497–507.
35 Novagen (1997) pET System Manual, 7th edn TB055, 18–64 Novagen, Madison, WI, USA.
36 Levintow, L., Meister, A., Hogeboom, G.H & Kuff, E.L (1955) Studies on the relationship between the enzymatic synthesis of glutamine and the glutamyl transfer reaction J Am Chem Soc.
77, 5304–5308.
37 Willemoe¨s, M & Larsen, S (2003) Substrate inhibition of Lac-tococcus lactis cytidine 5¢-triphosphate synthase by ammonium chloride is enhanced by salt-dependent tetramer association Arch Biochem Biophys 413, 17–22.
38 Jencks, W.P & Regenstein, J (1968) Ionization constants of acids and bases In Handbook of Biochemistry (Sober, H.A., ed.), pp J150–J189, The Chemical Rubber Co, Cleveland, Ohio.
Trang 939 Iyengar, A & Bearne, S.L (2002) An assay for CTP synthetase
glutaminase activity using high performance liquid
chromato-graphy Anal Biochem 308, 396–400.
40 Huang, X & Raushel, F.M (2000) Restricted passage of reaction
intermediates through the ammonia tunnel of carbamoyl
phos-phate synthetase J Biol Chem 275, 26233–26240.
41 Bera, A.K., Smith, J.L & Zalkin, H (2000) Dual role for
the glutamine phosphoribosylpyrophosphate amidotransferase
ammonia channel Interdomain signaling and intermediate
chan-neling J Biol Chem 275, 7975–7979.
42 Buchanan, J.M (1973) The amidotransferases Adv Enzymol.
Relat Areas Mol Biol 39, 91–183.
43 Massiere, F & Badet-Denisot, M.A (1998) The mechanism of
glutamine-dependent amidotransferases Cell Mol Life Sci 54,
205–222.
44 Zalkin, H (1993) The amidotransferases Adv Enzymol Relat.
Areas Mol Biol 66, 203–309.
45 Kizaki, H., Ohsaka, F & Sakurada, T (1987) Synthesis of
N4-substituted CTP by mammalian CTP synthetase Biochem.
Biophys Res Commun 145, 569–574.
46 Lieberman, I (1956) Enzymatic amination of uridine triphosphate
to cytidine triphosphate J Biol Chem 222, 765–775.
47 Willemoe¨s, M (2003) Thr-431 and Arg-433 are part of a conserved
sequence motif of the glutamine amidotransferase domain of CTP
synthases and are involved in GTP activation of the Lactococcus
lactis enzyme J Biol Chem 278, 9407–9411.
48 Boehlein, S.K., Schuster, S.M & Richards, N.G (1996) Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B Bioche-mistry 35, 3031–3037.
49 Pearson, R.G., Sobel, H & Songstad, J (1968) Nucleophilic reactivity constants toward methyl iodide and trans-[Pt(py) 2 Cl 2 ].
J Am Chem Soc 90, 319–326.
50 Bunting, J.W., Mason, J.M & Heo, C.K.M (1994) Nucleophili-city towards a saturated carbon atom: rate constants for the aminolysis of methyl 4-nitrobenzenesulfonate in aqu eous solu-tion A comparison of the n and N + parameters for amine nucleophilicity J Chem Soc Perkin Trans 2, 2291–2300.
51 Huang, X., Holden, H.M & Raushel, F.M (2001) Channeling of substrates and intermediates in enzyme-catalyzed reactions Annu Rev Biochem 70, 149–180.
52 Lewis, D.A & Villafranca, J.J (1989) Investigation of the mechanism of CTP synthetase using rapid quench and isotope partitioning methods Biochemistry 28, 8454–8459.
53 Levitzki, A., Stallcup, W.B & Koshland, D.E Jr (1971) Half-of-the-sites reactivity and the conformational states of cytidine triphosphate synthetase Biochemistry 10, 3371–3378.
54 Willemoe¨s, M & Sigurskjold, B.W (2002) Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis Eur J Biochem 269, 4772–4779.
55 Zalkin, H & Smith, J.L (1998) Enzymes utilizing glutamine as an amide donor Adv Enzymol Relat Areas Mol Biol 72, 87–144.