Steady-state kinetics of the glutaminase reaction of CTP synthase
from
Lactococcus lactis
The role of the allosteric activator GTP in coupling between glutamine hydrolysis
and CTP synthesis
Martin Willemoe¨s
1
and Bent W. Sigurskjold
2
1
Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Copenhagen, Denmark;
2
Department of Biochemistry, August Krogh Institute, University of Copenhagen, Copenhagen, Denmark
CTP synthase catalyzes the reaction glutamine + UTP
+ATP fi glutamate + CTP + ADP + P
i
.Therate
of the reaction is greatly enhanced by the allosteric activator
GTP. We have studied the glutaminase half-reaction of CTP
synthase from Lactococcus lactis and its response to the
allosteric activator GTP and nucleotides that bind to
the active site. In contrast to what has been found for the
Escherichia coli enzyme, GTP activation of the L. lactis
enzyme did not result in similar k
cat
values for the glutami-
nase activity and glutamine hydrolysis coupled to CTP
synthesis. GTP activation of the glutaminase reaction never
reached the levels of GTP-activated CTP synthesis, not even
when the active site was saturated with UTP and the non-
hydrolyzeable ATP-binding analog adenosine 5¢-[c-thio]tri-

shown in Scheme 1A, have been shown now for several
amido transferase enzymes [5–7]. Here the binding of an
already activated substrate, or activation of the substrate on
the enzyme, in this case by phosphorylation, precedes
amination. The overall reaction is as follows:
*
PPPrib
O
HN
N
OPO
3
2-
PPPrib
O
O
HN
N
ATP
NH
3
PPPrib
H
2
N
O
HN
N
OPO
3

N
N
B
*
NH
3
ATP
ADP
P
i
PPPrib
H
2
N
O
HN
N
OH
Scheme 1. Proposed mechanisms of CTP synthesis. The box indicates
an expected transition state like structure. * indicates that the amino
donor can either be free ammonia or ammonia generated from
hydrolysis of glutamine.
Correspondence to M. Willemoe
¨
s, Centre for Crystallographic Studies,
Department of Chemistry, University of Copenhagen,
Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
Fax: + 45 35320299, Tel.: + 45 35320239,
E-mail:
Abbreviations: ADPNP, adenosine 5¢-[b,c-imido]triphosphate;

enhance the rate of chemical steps of the glutaminase
reaction. The finding that the k
cat
value was similar for
glutaminase activity in the presence of GTP, UTP and
ADPNP and CTP synthesis with ATP replacing ADP-
NP seems in agreement with the mechanism in
Scheme 1B, but maybe less so with the mechanism in
Scheme 1A.
We have previously characterized the CTP synthase from
Lactococcus lactis [4]. This enzyme appears to be a more
stable tetramer than the E.coli [10], yeast [11] and
mammalian [12,13] enzymes that all require the presence
of UTP and/or ATP to form tetramers. Therefore, the
L. lactis CTP synthase is an attractive candidate for
mechanistic and structure–function studies since equilibria
between different oligomeric forms of the enzyme will not
interfere with the interpretation of the data.
In this research, we have analyzed the steady state
kinetics of the glutaminase reaction of CTP synthase from
L. lactis in order to distinguish between the effects of
GTP on the glutaminase reaction and the CTP synthesis
reaction. The results from this work suggests that there
are major differences between E.coli and L. lactis CTP
synthase with respect to the regulation of glutamine
hydrolysis.
EXPERIMENTAL PROCEDURES
Materials
Bovine GDH, nucleotides, and all other chemicals were
obtained from Sigma, except ATP-cS which was obtained

)1
Æ
M
)1
was measured as
described previously [4]. For glutaminase activity, a con-
tinuous coupled assay was used where the glutamate
produced by CTP synthase was oxidized by GDH and
monitored by the reduction of NAD
+
to NADH with
De
340
¼ 6300 cm
)1
Æ
M
)1
as described previously [14]. Unless
otherwise stated, the MgCl
2
concentration was 20 m
M
,the
glutamine concentration was 15 m
M
, and the concentration
of each nucleotide when present in the assay was 1 m
M
.

Fig. 1. Isothermal titration calorimetry of the glutaminase activity of
L. lactis CTP synthase. (A) Enthalpogram showing the recording of
steady-state rates for the hydrolysis of glutamine at increasing substrate
concentrations measured as the displacement of the baseline. The peaks
observed at each injection time are derived from the heat of dilution
of glutamine into the reaction cell. (B) The heat generated by hydrolysis
of 0.15 l
M
of glutamine used to determine DH of the reaction.
Ó FEBS 2002 Glutaminase activity of CTP synthase (Eur. J. Biochem. 269) 4773
displacement with each injection was directly obtained from
the data files as the value recorded just prior to the
subsequent injections. Dividing the power with the molar
reaction enthalpy gives the steady-state rate. The assay
conditions were as described above for the spectrophoto-
metric assays. The molar reaction enthalpy, DH,ofgluta-
mine hydrolysis or CTP synthesis under the experimental
conditions as outlined above, was determined by recording
the complete hydrolysis of between 0.05 and 0.15 lmol of
glutamine injected into the reaction cell containing between
1.7 and 17 l
M
CTP synthase and integrating the entire heat
evolvement over time. For the measurement of DH for
CTP synthesis the enzyme was incubated with ATP, UTP,
GTP and MgCl
2
as described above. Although hydrolysis
of ATP will take place under these conditions before and
after injection of glutamine and prior to kinetic experi-

injection was calculated from
½E
j
¼½E
jÀ1
exp À
V
inj
V
cell

ð2Þ
where V
inj
and V
cell
are the volumes of the injectant solution
and the reaction cell, respectively. The corrections for
the dilution of substrate already present in the reaction
cell upon further substrate injection and for the decrease
in substrate concentration with time were calculated from
½S
j
¼½S
jÀ1
exp À
V
inj
V
cell

syringe.
Analysis of initial velocity data
Analysis of saturation curves was performed by nonlinear
regression using
v ¼
k
cat
½E½S
K
M
þ½S
ð4Þ
where k
cat
is the turnover number for the enzyme, [S] is the
substrate concentration and K
m
is the Henri–Michaelis–
Menten constant. Partial inhibition of the glutaminase
activity induced by GTP was analysed using a modification
of the equation by LiCata and Allewell [16]
v ¼
k
cat
½Eþk
cat;inh
½Eð½A=I
0:5
Þ
n

where k
cat,1
and k
cat,2
are the turnover numbers for the
enzyme in the absence of or fully saturated with the
activator, respectively. GTP inhibition of the ammonia
dependent CTP synthesis reaction for DON labeled CTP
synthase was performed using
% Inhibition ¼ 100%
½I
n
K
n
I
þ½I
n
ð7Þ
where K
I
is the concentration of inhibitor that gives rise to
50% inhibition by the inhibitor I and n is the Hill-
coefficient. The standard errors presented are those given by
the computer program (
ULTRAFIT
for the Macintosh vs. 3.0,
BioSoft).
RESULTS
Steady state kinetics using ITC
The use of a calorimetric assay for the study of the L. lactis

enzyme kinetic data. From Fig. 2 it can be seen that there is
an excellent agreement between measured initial velocities
independently of the assay method used.
Steady state kinetics of uncoupled-
and CTP synthesis-coupled glutamine hydrolysis
The ATP analogs, ATP-cS and ADPNP, did not serve as
substrates (data not shown) but inhibited the CTP synthesis
reaction (Fig. 3). ADPNP, reported to inhibit the E.coli
enzyme with a K
i
similar to the dissociation constant for
ATP [9], was a poor inhibitor of L. lactis CTP synthase
compared to ATP-cS. On the basis of these results, ATP-cS
was chosen as a binding analog of ATP. CTP synthesis
requires the presence of both the nucleotide substrates ATP
and UTP. When phosphorylation of UTP is hindered by the
absence of ATP, only glutamine hydrolysis takes place.
GTP alone or in combination with UTP and ATP-cS gave a
Glutamine, mM
Glutamine, mM
A
B
0 1 2 3 4
0
0.05
0.1
0.15
0 1 2 3 4
0
1

Inhibitor, mM
Fig. 3. Inhibition of L. lactis CTP synthase by ADPNP and ATP-cS.
CTP synthesis was measured spectrophotometrically as described in
Experimental Procedures. Inhibition was by ADPNP (circles) and by
ATP-cS (triangles).
Table 1. Kinetic constants for L. lactis CTP synthase from varying glutamine in the presence of various nucleotides
a
.
Reaction
b
Nucleotides present K
m
(m
M
) k
cat
(s
)1
)
Glutaminase none 1.10 ± 0.06 0.084 ± 0.002
Glutaminase GTP
c
1.15 ± 0.05 0.259 ± 0.004
Glutaminase UTP, ATP-cS 1.26 ± 0.09 0.082 ± 0.002
Glutaminase GTP
c
, UTP, ATP-cS 1.0 ± 0.1 0.56 ± 0.02
CTP synthesis UTP, ATP 0.95 ± 0.03 0.139 ± 0.001
CTP synthesis GTP
d

when compared to
glutamine hydrolysis in the absence of nucleotides (Table 1).
The rate of CTP synthesis was dramatically influenced by
the presence of GTP, and 20 and 41-fold increases in k
cat
were obtained with GTP concentrations of 0.1 m
M
and
1m
M
, respectively. However, only a modest decrease in K
m
for glutamine was observed when compared to the absence
of GTP (Table 1).
Allosteric GTP activation of uncoupled-
and CTP synthesis-coupled glutamine hydrolysis
As was already indicated by the results in Table 1 and
discussed above, the kinetics of GTP activation of the
glutaminase half-reaction differed markedly on whether
the reaction was coupled to CTP synthesis or not (Fig. 4A).
The glutaminase activity in the absence of GTP, represented
by k
cat,1
(Eqn 6) is not obtainable with the calorimetric assay
where GTP is varied, since the heat evolved representing this
activity is included in the baseline of the experiment.
Therefore, the assay only measures the rate increase due to
the addition of GTP with a resulting k
cat
that represents

cSto1m
M
each (Fig. 4B). In either case, as judged from
the values of k
cat,1
and k
cat,2
, the maximal GTP activation
of uncoupled glutamine hydrolysis was about 14-fold
(Table 2).
At UTP and ATP concentrations of 1 m
M
each, a 49-fold
increase in k
cat
was observed with a concomitant decrease in
K
a
for GTP of about sevenfold compared to uncoupled
glutamine hydrolysis (Fig. 4A and Table 2). GTP-activated
CTP synthesis in the presence of low concentrations
(0.1 m
M
each) of ATP and UTP showed a sevenfold
activation and a K
a
value three orders of magnitude lower
than for uncoupled glutamine hydrolysis where ATP-cS
replaced ATP (Fig. 4C and Table 2).
In another experiment similar to that in Fig. 4C, the GTP

(closedcircles)eachofUTPandATP-cS. (C) GTP activation of CTP
synthesis at 0.1 m
M
each of UTP and ATP.
4776 M. Willemoe
¨
s and B. W. Sigurskjold (Eur. J. Biochem. 269) Ó FEBS 2002
ammonia-dependent activity was fully retained, as was also
found for the E.colienzyme [9]. GTP has previously been
reported to inhibit the NH
4
Cl-dependent CTP synthesis
reaction of the DON-labeled E.colienzyme, but not the
unmodified enzyme [8]. This was also the case for the
L. lactis enzyme (Fig. 6). When this inhibition was analysed
as a function of the GTP concentration using Eqn 7 we
obtained a K
I
¼ 0.40 ± 0.05 and n ¼ 0.39 ± 0.02, results
that are very similar to those found for the E.colienzyme
[8]. Interestingly, the inhibitory response to GTP binding in
this experiment shows negative cooperativity in contrast to
the activation experiments presented above, where cooper-
ativity is not observed.
DISCUSSION
The original model for the mechanism of GTP activation of
the E.coliCTP synthase was rather complex, involving both
negative and positive cooperativity of GTP binding [8].
However, we have not found cooperativity associated with
GTP activation in these or previous studies [4] of the

a
Nucleotides present (m
M
) K
a
(mM) k
cat,1
b
(s
)1
) k
cat,2
c
(s
)1
)
Glutaminase UTP 0.1 2.43 ± 0.08 0.078
e
1.20 ± 0.03
(Fig. 4B)
d
ATP-cS 0.1
Glutaminase UTP 1 1.62 ± 0.03 0.076
f
1.054 ± 0.007
(Fig. 4B) ATP-cS1
CTP synthesis UTP 0.1 0.0027 ± 0.0004 0.028 ± 0.005 0.195 ± 0.007
(Fig. 4C) ATP 0.1
CTP synthesis UTP 1 0.22 ± 0.01 0.130
f

% inhibition
0 0.1 0.2 0.3 0.4
0
10
20
30
40
50
Fig. 6. GTP inhibition of DON-labeled L. lactis CTP synthase. The
data were fitted to Eqn 7 and kinetic constants are given in the text.
GTP, mM
0 0.025 0.05 0.075 0.1
0
0.05
0.1
0.15
0.2
0.25
v, s
-1
Fig. 5. Coupling of glutaminase activity and CTP synthesis at 0.1 m
M
each of UTP and ATP. Spectrophotometrical measurement of CTP
synthesis (squares) and glutaminase activity (circles) was performed as
described in Experimental procedures. The solid line is calculated on
the basis of data (Table 2) from GTP activation of the glutaminase
activity in the presence of 0.1 m
M
each of UTP and ATP-cSandis
shown for comparison.

that of Scheme 1A.
From Fig. 4 and Table 2 it can be seen that the effect of
saturating the active site with ATP-cS and UTP was a relief
of partial inhibition by GTP at higher concentrations than
1m
M
(Fig. 4B). The exact mechanism behind this inhibition
cannot be resolved from our data, but apart from this
inhibition the kinetics were similar when ATP-cSandUTP
were present at 1 m
M
or 0.1 m
M
(Table 2). The results
presented in Fig. 4B seem to exclude that the lower fold of
GTP activation of the uncoupled glutaminase reaction was
due to subsaturation with nucleotides binding to the active
site. That CTP synthesis in the absence of GTP occurs with
a k
cat
that is higher than for the glutaminase activity under
similar conditions, except that ATP-cS replaced ATP or in
the complete absence of nucleotides (Table 1), seems to
indicate an activation of the glutaminase reaction by the
substrate nucleotides alone. A similar observation was made
with the E.colienzyme except that UTP and ADPNP also
activated the glutaminase reaction, though not to the same
extent as ATP and UTP [9]. For the L. lactis enzyme, a
plausible explanation may be that 4-phosphorylated UTP
by itself acts as a weak activator of glutamine hydrolysis.

GTP, the rate of CTP synthesis is stimulated 3–4-fold from
a level below to the level of uncoupled glutamine
hydrolysis (Fig. 5). This directly illustrates that GTP plays
G N
G N
GN
GN
A
C
B
GTP
Fig. 7. A working model for the structural movements in the L. lactis
CTP synthase monomer. (A) uncoupled glutaminase activity, (B) CTP
synthesis in the absence of GTP and (C) CTP synthesis in the presence
of GTP. (A) Glutamine hydrolysis in the absence of nucleotides takes
place on the enzyme without any large structural changes required. (B)
CTP synthesis in the absence of GTP only occurs at a slow rate due to
an equilibrium between the inactive and active form of the monomer
with respect to CTP synthesis that involves rearrangements of the
monomer that brings together the glutaminase site and the active site.
(C) GTP locks the enzyme in the active form for CTP synthesis and
thereby stimulates the k
cat
of the reaction. Since GTP actually will
activate the uncoupled glutaminase reaction the structure of the
monomer represented by (C) must also include a minor but highly
important rearrangement of sidechains in the active site in response to
the formation of 4-phosphoryl UTP. The ammonia dependent CTP
synthesis could in this model proceed via an enzyme form similar to (A).
G, glutaminase site; N, CTP synthesis site.

inhibition of NH
4
Cl utilization, exerted by glutamate
c-semialdehyde as found with the E.coli enzyme [20].
Glutamate c-semialdehyde is an analog of glutamine that
mimics a tetrahedral reaction intermediate [20].
In our current model (Fig. 7), GTP may act to close a
lid over the active site, a lid that in turn holds or rearranges
catalytically important residues, and residues that enable
the enzyme to perform a concerted glutamine hydrolysis
with the formation of 4-phosphoryl UTP. Maybe GTP
could play a role in the formation of a tunnel for passing
ammonia from the glutaminase site to the active site. Such
tunnels have been demonstrated in several glutamine
amidotransferases [5]. Another enzyme, that also catalyzes
amino transfer from glutamine, is carbamoyl phosphate
synthase (CPS) which is the first enzyme of the de novo
pyrimidine biosynthesis. For CPS, glutamine hydrolysis
has been shown to be greatly stimulated by bicarbonate-
dependent ATP hydrolysis, indicating that for this enzyme
the phosphorylated amino acceptor intermediate, carbonyl
phosphate, triggers an allosteric signal to the glutaminase
site [5]. We imagine the same type of allosteric activation of
glutamine hydrolysis takes place by the phosphorylation of
UTP on CTP synthase, only that for CTP synthase this
allosteric effect exerted by the amino acceptor is strongly
controlled by GTP.
ACKNOWLEDGEMENTS
This work was supported by the Danish National Research
Foundation. We gratefully acknowledge the expert technical assist-

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