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

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

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:
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

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

(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)

[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
–

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

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

; 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

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-

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

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

) 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

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

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

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

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

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

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

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

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

=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

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

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

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

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

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. This uncoupling is not caused by a
leaky NH
3
tunnel but arises because of a constriction within
the t unnel a s demonstrated by the ability of L 109A 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 He alth Research (S .L.B.), a Natural Sciences
and E ngineering Research Council (NSERC) of C anada Collaborative
Health Research Project grant (S.L.B.), and a graduate student
fellowship from the Nova Scotia Health Research Foundation

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