Báo cáo khoa học: Aspartate transcarbamylase from the hyperthermophilic archaeonPyrococcus abyssi Insights into cooperative and allosteric mechanisms docx

Aspartate transcarbamylase from the hyperthermophilic
archaeon Pyrococcus abyssi
Insights into cooperative and allosteric mechanisms
Sigrid Van Boxstael
1
, Dominique Maes
2,3
and Raymond Cunin
1
1 Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel, Belgium
2 Ultrastructuur, Vrije Universiteit Brussel, Belgium
3 Vlaams Interuniversitair Instituut voor Biotechnologie, Belgium
Aspartate transcarbamylase (ATCase; EC 2.1.3.2) cata-
lyses the condensation of carbamyl phosphate (CP)
with the amino group of aspartate to form carbamyl
aspartate and inorganic phosphate. In several organ-
isms, this reaction is the first committed step of the
de novo synthesis of pyrimidine nucleotides and, as such,
it is subject to extensive control: transcriptional repres-
sion of ATCase synthesis by the pyrimidines, and
allosteric regulation of ATCase activity by nucleotide
effectors. ATCase activity is present in almost all
organisms from the three kingdoms of life, Bacteria,
Eukarya and Archaea, under a variety of molecular
forms. The best-studied ATCase is that from the bac-
terium Escherichia coli, which has become a paradigm
of cooperative and allosteric enzymes [1–5].
The discovery of microorganisms living in extreme
conditions of, for example temperature and ⁄ or
pH, prompted investigations into the structure and
Keywords

allostery; aspartate transcarbamylase;
cooperativity; inhibition by analogues;
Pyrococcus abyssi
Correspondence
R. Cunin, Erfelijkheidsleer en Microbiologie,
Vrije Universiteit Brussel, Pleinlaan 2,
1050 Brussels, Belgium
Fax: 32 2 629 1473
Tel: 32 2 629 1341
E-mail:
(Received 15 December 2004, revised
1 March 2005, accepted 22 March 2005)
doi:10.1111/j.1742-4658.2005.04678.x
Aspartate transcarbamylase (ATCase) (EC 2.1.3.2) from the hyperthermo-
philic archaeon Pyrococcus abyssi was purified from recombinant Escheri-
chia coli cells. The enzyme has the molecular organization of class B
microbial aspartate transcarbamylases whose prototype is the E. coli
enzyme. P. abyssi ATCase is cooperative towards aspartate. Despite con-
straints imposed by adaptation to high temperature, the transition between
T- and R-states involves significant changes in the quaternary structure,
which were detected by analytical ultracentrifugation. The enzyme is allos-
terically regulated by ATP (activator) and by CTP and UTP (inhibitors).
Nucleotide competition experiments showed that these effectors compete
for the same sites. At least two regulatory properties distinguish P. abyssi
ATCase from E. coli ATCase: (a) UTP by itself is an inhibitor; (b) whereas
ATP and UTP act at millimolar concentrations, CTP inhibits at micro-
molar concentrations, suggesting that in P. abyssi, inhibition by CTP is the
major control of enzyme activity. While V
max
increased with temperature,

cooperative and allosteric effects were little or not affected, showing that
molecular adaptation to high temperature allows the flexibility required to
form the appropriate networks of interactions. In contrast to the same
enzyme in P. abyssi cellular extracts, the pure enzyme is inhibited by the
carbamyl phosphate analogue phosphonacetate; this difference supports the
idea that in native cells ATCase interacts with carbamyl phosphate synthe-
tase to channel the highly thermolabile carbamyl phosphate.
Abbreviations
AEBSF, aminoethylbenzylfluoride; ATCase, aspartate transcarbamylase; CP, carbamyl phosphate; CPSase, carbamyl phosphate synthetase;
CK, carbamate kinase; IPTG, isopropyl-thio-b-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; PALA, N-phosphonacetyl-
L-
aspartate; SDS, sodium dodecylsulfate.
2670 FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS
mechanisms of their ATCases. ATCase from Pyrococ-
cus abyssi was the first archaeal hyperthermophilic
ATCase to be characterized and expressed in recom-
binant E. coli cells. It was found to be a cooperative
and allosteric enzyme consisting of catalytic and regu-
latory subunits. Recently, the crystal structure of the
catalytic subunit was solved to a resolution of 1.8 A
˚
[6]. First attempts to purify this ATCase from either
native or recombinant cells resulted in the irreversible
loss of the regulatory properties, probably due to dis-
sociation of the holoenzyme [7,8]. This study describes
purification of the P. abyssi ATCase holoenzyme from
recombinant E. coli cells without significant alteration
of its regulatory properties, which allowed major
questions regarding its mechanism and regulation of
activity to be addressed. (a) Is the homotropic cooper-

ativity toward aspartate of this enzyme and the attend-
ant transition from a low affinity T-state to a higher
affinity R-state accompanied by a significant structural
change as in the case of the E. coli ATCase? (b) To
what extent is cooperativity affected by temperature?
(c) What is the mode of action of the allosteric effec-
tors? Do activator and inhibitors bind competitively
to the same sites on the regulatory subunits? Is there
instead an additive effect of inhibitors, or even a syn-
ergy as in E. coli?
Furthermore, comparison of the properties of the
pure enzyme with those observed in native P. abyssi
cell-free extracts may reveal the influence of inter-
actions with other cellular constituents such as other
enzymes. Indeed, a channelling of the highly thermo-
labile substrate CP between carbamyl phosphate syn-
thase and ATCase has been reported for P. abyssi [9]
as for other thermophilic and hyperthermophilic
microorganisms [10,11].
Results
Expression, purification and molecular mass
determination
P. abyssi pyrBI genes were expressed from the isopro-
pyl-thio-b-galactopyranoside (IPTG)-inducible trc pro-
moter on the pTrc99A plasmid transformed into the
E. coli ATCase-deficient strain C600
ATC–
. The enzyme
was purified to homogeneity. The different purification
stages, as analysed by sodium dodecyl sulfate–poly-

acrylamide gel electrophoresis (SDS–PAGE), are
shown in Fig. 1. Twenty grams of wet cells yielded
3 mg of pure ATCase holoenzyme.
The molecular mass of the P. abyssi ATCase mole-
cule was estimated to be 301 ± 15 kDa by calibrated
gel filtration on Superdex 200 pg, using two XK 16 ⁄ 60
columns in series for greater accuracy. An independ-
ent estimate of 304 ± 10 kDa for the molecular
mass of the holoenzyme was obtained by analytical
ultracentifugation. Taken together with the known
structure of the trimeric catalytic subunit (c
3
) [6],
and the molecular masses for the catalytic and regu-
latory polypeptides calculated from the sequence
(34 879 and 16 947 Da, respectively), this value cor-
responds to a [2(c
3
):3(r
2
)] molecular architecture, typ-
ical for class B ATCases.
Thermal stability of activity
The kinetic stability of the P. abyssi holoenzyme was
measured by determining the residual activity at
37 °C after incubation for increasing periods at tem-
peratures between 90 and 98 °C. The measured half-
lives (s
0.5
) are given in Table 1. For a comparison,

the half-life of the isolated catalytic subunit was
80 min at 90 ° C and 5 min at 98 °C under the same
conditions [6].
Fig. 1. Denaturing polyacrylamide gel, presenting successive purifi-
cation stages of Pyrococcus abyssi ATCase. Lane 1: cell-free
extract; lane 2: after the heat-purification step (20 min, 85 °C); lane
3: after anion-exchange chromatography (ResourceQ and MonoQ);
lane 4: after size-exclusion chromatography (Superdex 200 pg);
lane 5: molecular mass markers.
Table 1. Thermostability of Pyrococcus abyssi ATCase. Half-life of
activity (s
0.5
) at different temperatures.
T (°C) s
0.5
(min)
90 260
92 240
94 190
96 190
98 100
ATCase was at 100 lgÆmL
)1
in 50 mM phosphate pH 7.5.
S. Van Boxstael et al. Aspartate transcarbamylase from Pyrococcus abyssi
FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS 2671
Saturation by the substrates
Saturation by aspartate was measured at 30, 37, 45
and 55 °C, in the presence of saturating amounts of
CP. The values obtained for the kinetic parameters are

given in Table 2. Although the optimal growth tem-
perature of P. abyssi is 96 °C under atmospheric pres-
sure, 55 °C was the highest temperature at which
complete saturation experiments were performed
because of the high thermolability of the other sub-
strate CP (a half-life of < 2 s at 100 °C, 4 min at
55 °C) [10]. The enzyme is stable for hours at that
temperature. The saturation curves were sigmoidal at
all temperatures, indicative of a cooperativity toward
aspartate (Fig. 2). The curvature shown by the Eadie–
Hofstee plot (Fig. 2) is characteristic of cooperative
enzymes [12]. The Hill coefficient, taken as an index of
cooperativity, did not vary significantly in the range of
temperatures tested.
V
max
increased sixfold with increasing temperature
in the range tested. In contrast, temperature did not
influence the affinity of the enzyme for aspartate as
indicated by S
0.5
. As a result, the catalytic efficiency,
expressed as V
max
⁄ S
0.5
, also increased by about sixfold
over the 30–55 °C range. For a comparison, the kinetic
parameters of the isolated catalytic subunit measured
at 55 °C are also given (Table 2, bottom line).

Because of the thermolability of CP, saturation by
this substrate was measured at 37 °C only. In the pres-
ence of saturating aspartate (15 mm), the apparent K
m
for CP is 5 ± 2 lm and V
max
is 3.2 ± 0.3 mmolÆ
h
)1
Æmg
)1
ATCase. The saturation curve was hyperbolic
at low (1 mm) and saturating (15 mm) aspartate con-
centrations (Fig. 3). Under the same conditions, E. coli
ATCase shows cooperativity toward CP at high, but
not at low, aspartate concentrations, a consequence of
the cooperativity toward aspartate and of the ordered
binding of the substrates, first CP then aspartate [13].
These results raised the possibility that binding of the
substrates does not follow the same ordered mechan-
ism as in the case of E. coli ATCase, especially as a
loop (80s loop) which contains two residues inter-
acting with the substrates is shorter in P. abyssi
ATCase [6].
Order of substrate binding
In an attempt to see whether the lack of apparent coop-
erativity towards CP reflects a mode of substrate bind-
ing different from that of E. coli ATCase, the inhibition
patterns of N-phosphonacetyl-l-aspartate (PALA)
toward CP and aspartate were analysed. PALA is a

transition state analogue with chemical groups similar
Table 2. Kinetic parameters of the Pyrococcus abyssi enzyme as a
function of temperature. V
max
is the maximal velocity, S
0.5
the con-
centration of aspartate at half the V
max
, n
H
the Hill coefficient and
V
max
⁄ S
0.5
the catalytic efficiency. Units (U) are mmoles carbamyl
aspartate formed per hour and per mg ATCase.
T (°C) V
max
(U) S
0.5
(mM) V
max
⁄ S
0.5
n
H
P. abyssi holoenzyme
30 1.5 ± 0.3 2.6 ± 0.3 0.6 1.5 ± 0.1

37 2.9 ± 0.5 3.0 ± 0.5 1.0 1.6 ± 0.2
45 5.5 ± 0.7 2.5 ± 0.3 2.2 1.6 ± 0.2
55 9.9 ± 1.5 2.7 ± 0.4 3.7 1.7 ± 0.2
P. abyssi catalytic subunit
55 71.0 ± 4.0 19.7 ± 1.5 3.6 1.0 ± 0.1
Fig. 2. Saturation of Pyrococcus abyssi ATCase by aspartate at
55 °C(5m
M CP, 50 mM Tris ⁄ HCl pH 8.0). (Inset: corresponding
Eadie–Hofstee plot.)
Fig. 3. Saturation of Pyrococcus abyssi ATCase by CP in the pres-
ence of a saturating aspartate concentration (15 m
M aspartate,
50 m
M Tris-acetate pH 8.0). (Inset: corresponding Eadie–Hofstee
plot.)
Aspartate transcarbamylase from Pyrococcus abyssi S. Van Boxstael et al.
2672 FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS
to those of the two substrates [14]. CP and aspartate
saturations were performed in the presence of various
concentrations of PALA. The results, presented as dou-
ble reciprocal plots (Fig. 4A,B), showed that PALA
behaves as a competitive inhibitor toward CP and as a
noncompetitive inhibitor toward aspartate. If the bind-
ing were random, PALA would behave as a linear non-
competitive inhibitor toward both substrates. The
results clearly indicate an ordered binding mechanism,
first CP, then aspartate, and the lack of cooperativity
toward CP at high aspartate concentration cannot be
explained by a random binding mechanism.
Effects of inhibitors

PALA
In the presence of a low aspartate concentration,
PALA stimulates the activity of E. coli ATCase by
promoting transition from the low-affinity T-state to
the high-affinity R-state. This reflects cooperativity:
while the inhibitor blocks the sites which it occupies, it
converts the remaining sites to the R-state, resulting in
an increase in the activity [14–16]. The effect of PALA
on P. abyssi ATCase was tested. Figure 5 shows that
the enzyme is stimulated by PALA at low aspartate
concentrations. The amplitude of the activation decrea-
ses as the aspartate concentration increases: 65 ± 15%
at 0.1 mm aspartate; 20 ± 5% at 0.5 mm aspartate.
At 3 mm aspartate, only direct inhibition by PALA
can be observed. These results confirm the existence of
a cooperative mechanism of aspartate binding.
Phosphonacetate
Native P. abyssi ATCase in crude extracts was repor-
ted to be insensitive to the CP analogues phosphonace-
tate and pyrophosphate [17]. These molecules are
competitive inhibitors of CP in E. coli ATCase [18]. It
was suggested that the P. abyssi CP binding site is
shielded to some extent from the bulk solvent and that
CP may be sequestered by a complex [17]. The isolated
recombinant holoenzyme was tested for sensitivity to
the CP analogue phosphonacetate. The activity was
inhibited almost completely at 90 mm phosphonacetate
(an E
50
value of 18 mm in the presence of 0.05 mm CP

compared with 35 mm in the presence of 0.5 mm CP).
Sensitivity to the inhibitor increased with decreasing
CP concentration, suggesting that phosphonacetate
acts in competition with CP. The observed sensitivity
of the isolated recombinant ATCase to phosphonace-
tate contrasts with the insensitivity of the enzyme in
native P. abyssi ATCase extracts.
Fig. 4. (A) Inhibition by PALA towards CP. Double reciprocal plot:
no PALA (j), 0.25 l
M PALA (d), 0.5 lM PALA (m)and1lM PALA
(.)(37°C, 15 m
M aspartate, 50 mM Tris-acetate pH 8.0). (B) Inhibi-
tion by PALA towards aspartate. Double reciprocal plot: no PALA
(j), 1 l
M PALA (.), 5 lM PALA (d)and10lM PALA (m) (37 °C,
5m
M CP, 50 mM Tris-acetate pH 8.0).
Fig. 5. Saturation of Pyrococcus abyssi ATCase with PALA in the
presence of different aspartate concentrations: (j) 0.1 m
M,(n)
0.5 m
M and (d)3mM (37 °C, 0.5 mM CP, 50 mM Tris ⁄ HCl pH 8.0).
Relative activity defined as A ⁄ A
0
*100; where A is the activity in the
presence of PALA and A
0
the activity in the absence of PALA.
S. Van Boxstael et al. Aspartate transcarbamylase from Pyrococcus abyssi
FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS 2673

Sedimentation velocity experiments in the
presence or the absence of PALA
Titration with PALA supports the existence of T- and
R-states with a PALA- or aspartate-induced transition.
However, this does not necessarily reflect the existence
of a major difference in the quaternary structure
between the T- and R-states. In E. coli ATCase, the
quaternary structure transition involves a conforma-
tional change of such amplitude that it can be detected
by sedimentation studies [19–21]. Sedimentation velo-
city experiments by analytical ultracentrifugation were
performed on P. abyssi ATCase both unliganded and
liganded with saturating concentrations of PALA and
the relative difference in sedimentation coefficient
Ds ⁄ s
0
, induced by the binding of PALA was calculated
(Table 3). For comparison, similar experiments were
performed with E. coli ATCase.
PALA binding to P. abyssi ATCase results in a
2.6% relative decrease of the sedimentation coeffi-
cient. As expected, the respective effects of 0.5 and
1mm PALA (both saturating) on the sedimentation
coefficient are identical. The relative difference in
sedimentation coefficient of P. abyssi ATCase is iden-
tical to that observed for E. coli ATCase. Thus,
PALA binding to P. abyssi ATCase induces swelling
of the enzyme indicative of a significant conforma-
tional change.
Allosteric regulation

Influence of the nucleotide effectors on aspartate
saturation
Purified recombinant enzyme is inhibited by CTP and
UTP and activated by ATP. Aspartate saturation of
the P. abyssi holoenzyme in the presence of saturating
amounts of nucleotides was studied at 37 and 55 °C
(Fig. 6A,B). The values of the kinetic parameters are
given in Table 4.
The nucleotides have a pronounced effect on the
affinity for aspartate. ATP decreases the [S
0.5
]
Asp
four-
to fivefold and CTP increases it two- to threefold. The
effect of UTP on the [S
0.5
]
Asp
is less pronounced.
ATP reduces significantly the Hill coefficient. This is
similar to what is observed for E. coli ATCase. How-
ever, an opposite effect of CTP and UTP is not
observed. CTP and UTP do not affect the maximal
activity. Remarkably, ATP provokes an increase of
V
max
of % 35%. In E. coli ATCase no such effect of
ATP on V
max

is observed.
Saturation by the nucleotide effectors
Saturation by the nucleotide effectors was studied at
1.5 mm aspartate, a concentration corresponding to
Table 3. Effect of PALA on the sedimentation coefficients of
Escherichia coli and Pyrococcus abyssi ATCase. N is the number of
measurements, s
0
is the sedimentation coefficient of the unligan-
ded enzyme. Ds is defined as the difference between the sedi-
mentation coefficient of the unliganded enzyme (s
0
) and the
sedimentation coefficient of the PALA-liganded enzyme. SEM
values are given.
PALA (m
M) N
s (sedimentation
coefficient) Ds ⁄ s
0
(%)
P. abyssi ATCase
0 5 10.23 ± 0.05
0.5 2 9.97 ± 0.07 )2.6 ± 0.8
1 2 9.95 ± 0.13 )2.7 ± 1.4
E. coli ATCase
0 6 11.19 ± 0.03
0.3 4 10.89 ± 0.04 )2.7 ± 0.4
Fig. 6. Saturation of Pyrococcus abyssi ATCase by aspartate at (A)
37 °C and (B) 55 °C(5m

M CP, 50 mM Tris ⁄ HCl pH 8.0) in the pres-
ence of nucleotide effectors: no effectors (j), 2 m
M ATP (d),
5m
M UTP (,) and 0.3 mM CTP (m).
Aspartate transcarbamylase from Pyrococcus abyssi S. Van Boxstael et al.
2674 FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS
half the [S
0.5
]
Asp
and in the presence of saturating
CP. These are standard conditions to observe the
maximal amplitude of effector action. Saturations
were performed at 37 and 55 °C. At both tempera-
tures, ATP increased ATCase activity up to 370%,
whereas CTP and UTP inhibited ATCase activity,
respectively, by $ 50 and 40% (Table 5). The con-
centrations of nucleotides at which half the maximal
effect is observed (E
NTP
50
) were slightly lower at
37 °C than at 55 °C, which is in agreement with the
nucleotide interactions with the protein being mainly
ionic or polar. Such interactions are weakened when
the temperature is increased. However, the maximal
amplitudes of the effects were affected little by tem-
perature.
Remarkably, the CTP concentration at which half

the maximal inhibition is observed (E
CTP
50
) is two
orders of magnitude lower than the E
UTP
50
or the
E
ATP
50
. Increasing the CTP concentration to 5 mm
did not increase the amplitude of the inhibition, indi-
cating that if P. abyssi ATCase contains two differ-
ent classes of nucleotide binding sites, like E. coli
ATCase [22,23], the difference in their binding con-
stants is too small to be detected by measurements
of enzyme activity.
Nucleotide effectors competition
In E. coli ATCase, the activator ATP and inhibitor
CTP bind competitively to the same sites on the regu-
latory subunits. UTP, which has little effect by itself, is
a synergistic inhibitor with CTP [24], an effect which
can be mostly ascribed to a positive interaction
between nucleotide binding sites in a regulatory dimer
[22]. Nucleotide competition experiments were per-
formed on P. abyssi ATCase to determine whether
ATP, CTP and UTP have additive, antagonistic or
synergistic effects on the catalytic activity. The princi-
ple of these experiments is to determine if, in the pres-

ence of a fixed amount of effector A, the addition of
increasing concentrations of effector B can suppress
the response to effector A and lead to the response
observed when only effector B is present. First, the
two inhibitors CTP and UTP were tested separately
against the activator ATP, they were then tested
against each other.
CTP versus ATP
Saturation by CTP was performed in the presence or
absence of 0.2 mm ATP (Fig. 7A). In the presence of
ATP, 10 lm CTP is required to reduce the relative
activity from 280 to 100%. A further increase in CTP
concentration reduces the activity to the same inhibited
level as in the absence of ATP. This experiment shows
an antagonistic effect of CTP on the activation of
ATCase by ATP.
In a reverse experiment, ATP saturation was studied
in the presence or absence of CTP (Fig. 7B). In the
presence of 2 lm CTP, the relative activity was 70%;
0.1 mm ATP was required to increase the relative
activity from 70 to 100%. A further increase in ATP
concentration brought the activity to the same activa-
ted level as in the absence of CTP, thus ATP counter-
acts completely the effect of CTP. Taken together,
these results demonstrate that ATP and CTP have ant-
agonistic effects on activity.
UTP versus ATP
At 0.1 mm, ATP elicited a 110% activation of the
ATCase activity. Increasing the UTP concentration
from 0 to 5 mm resulted in a 50% inhibition of the

activity. This is the maximal inhibition level by UTP
Table 4. Effect of nucleotides on the kinetic properties of the
ATCase. V
max
is the maximal velocity; S
0.5
the concentration at half
saturation and n
H
the Hill coefficient. Activities were determined at
5m
M CP, 50 mM Tris ⁄ HCl pH 8.0. Units (U) are mmoles carbamyl
aspartate formed per hour and per mg ATCase.
V
max
(U) S
0.5
(mM) n
H
37 °C
no effector 2.9 ± 0.5 3.0 ± 0.5 1.6 ± 0.2
+2 mm ATP 4.5 ± 0.3 0.7 ± 0.2 1.1 ± 0.1
+0.3 mm CTP 2.6 ± 0.4 7.0 ± 0.3 1.3 ± 0.2
+5 mm UTP 2.4 ± 0.5 5.0 ± 0.4 1.4 ± 0.2
55 °C
no effector 9.9 ± 1.0 2.5 ± 0.5 1.6 ± 0.1
+2 mm ATP 12.5 ± 1.0 0.5 ± 0.3 1.1 ± 0.2
+0.3 mm CTP 9.0 ± 0.8 7.5 ± 0.5 1.5 ± 0.1
+5 m
M UTP 8.5 ± 1.0 4.0 ± 0.5 1.4 ± 0.2

Table 5. Effect of allosteric effectors on the activity of the ATCase
at 37 and 55 °C. Activities were determined at 1.5 m
M aspartate,
50 m
M Tris ⁄ HCl pH 8.0, 5 mM CP. One hundred per cent is the
activity in the absence of nucleotide effectors. Relative activity
defined as A ⁄ A
0
*100; where A is the activity in the presence of
the effector and A
0
the activity in its absence. E
50
is the concentra-
tion of effector at half maximal effect.
Nucleotides
Relative
activity
(%) 37 °C
E
50
(mM)
37 °C
Relative
activity
(%) 55 °C
E
50
(mM)
55 °C

ATP 370 ± 60 0.20 ± 0.10 350 ± 50 0.25 ± 0.10
CTP 55 ± 10 0.002 ± 0.001 50 ± 10 0.003 ± 0.001
UTP 60 ± 10 0.30 ± 0.20 65 ± 10 0.60 ± 0.20
S. Van Boxstael et al. Aspartate transcarbamylase from Pyrococcus abyssi
FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS 2675
alone (Fig. 8). The reverse experiment, saturation by
ATP in the presence of 0.5 mm UTP (not shown)
confirmed that the effects ATP and UTP are also
antagonistic.
CTP versus UTP
At 0.5 lm concentration, CTP elicits 25% inhibition
(Fig. 9). Increasing the UTP concentration from 0 to
5mm resulted in 50% inhibition of the enzyme, the
maximal inhibition caused by UTP alone (Fig. 9),
showing that the effects of UTP and CTP are antag-
onistic. The reverse experiment, saturation by CTP
in the presence of 0.5 mm UTP confirmed the com-
petitive inhibitory effects of CTP and UTP (not
shown).
Taken together, these results show that the effects of
the nucleotide effectors are neither additive nor syner-
gistic but antagonistic. CTP and ATP act in competi-
tion with each other, as do UTP and ATP. This
suggests that they bind competitively to the regulatory
sites. In this case, UTP and CTP are expected to inhi-
bit in competition with each other too, which is what
is observed, thereby confirming that the nucleotides
bind to the same regulatory sites.
Webb’s formalism allows us to distinguish quantita-
tively among antagonism, additivity and synergism of

inhibitor effects [25]. Here the inhibition observed
when both inhibitors are present is smaller than the
sum of the inhibitions elicited by each inhibitor indi-
vidually minus their product (i
1,2
< i
1
+ i
2
) i
1
* i
2
):
0.54 < 0.55 +0.60 ) 0.33 (values taken from Table 5
and Figs 7 and 9). This demonstrates the competition
between the inhibitors.
Fig. 7. Effect of the simultaneous presence of CTP and ATP. (A)
Saturation by CTP alone (s), saturation by CTP in the presence of
0.2 m
M ATP (j) (1.5 mM aspartate, 5 mM CP, 50 mM Tris ⁄ HCl
pH 8.0, 37 °C). (B) Saturation by ATP alone (s), saturation by ATP
in the presence of 2 l
M CTP (j)(1.5mM aspartate, 5 mM CP,
50 m
M Tris ⁄ HCl pH 8.0, 37 °C).
Fig. 8. Effect of simultaneous presence of UTP and ATP. Saturation
by UTP alone (s), saturation by UTP in the presence of 0.1 m
M
ATP (j) (1.5 mM aspartate, 5 mM CP, 50 mM Tris ⁄ HCl pH 8.0,

37 °C).
Fig. 9. Effect of simultaneous presence of UTP and CTP. Saturation
by UTP alone (s), saturation by UTP in the presence of 0.5 l
M CTP
(j)(2m
M aspartate, 5 mM CP, 50 mM Tris ⁄ HCl pH 8.0, 37 °C).
Aspartate transcarbamylase from Pyrococcus abyssi S. Van Boxstael et al.
2676 FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS
Discussion
Pyrococcus abyssi ATCase is a class B ATCase:
evolutionary implications
Calibrated size-exclusion chromatography and sedi-
mentation equilibrium experiments give similar
molecular mass estimates for the holoenzyme of
301 ± 15 and 304 ± 10 kDa. Taken together with the
known trimeric [c
3
] structure of the catalytic subunit
[6], and with the molecular masses for the catalytic
and regulatory polypeptides calculated from the
sequence (34.9 and 17.0 kDa, respectively), these
results are consistent with a [2(c
3
):3(r
2
)] molecular
architecture, typical of class B ATCases, whose proto-
type is E. coli ATCase.
Prokaryotic ATCases fall into three classes, A, B
and C, according to their molecular mass and molecu-

lar organization [26,27]. The limited number of Arch-
aea for which a pyrB (catalytic chain) sequence is
available all have a matching pyrI gene (coding for a
regulatory chain). Thus far, a class B architecture
appears characteristic of archaeal ATCases [28]. On
phylogenetic grounds, two families of ATCases, ATC I
and ATC II, have been recognized, which would both
have been present in the last universal common ances-
tor and later inherited differently in the ancestors of
present-day organisms [29,30]. All class B ATCases
form a clade in the ATC II family [31], and a coevolu-
tion scheme of the pyrB and the pyrI genes, in
response to a need for the conservation of the inter-
actions between their polypeptide products in the holo-
enzyme was proposed. This study supports this
hypothesis and suggests that for archaeal hyperthermo-
philic ATCases, one of the constraints may have been
adaptation to high temperature: indeed, where tested,
the association of the catalytic subunits with regulatory
subunits is a major factor in thermostability [6,32].
The association between catalytic and regulatory
subunits also imposes strong conformational con-
straints on the catalytic sites. At 55 °C, the maximal
observed activity of the holoenzyme is sevenfold lower
than that of the catalytic subunit (Table 2). These con-
straints are, in part, responsible for cooperativity. A
similar phenomenon is observed with Sulfolobus acido-
caldarius ATCase – a threefold difference [32], and
E. coli ATCase – a twofold difference.
Cooperativity towards substrates

P. abyssi ATCase is cooperative toward aspartate. The
cooperativity, expressed by the Hill coefficient, is less
pronounced than in the case of E. coli ATCase (1.7
compared with 2.2). This lower cooperativity might
result from changes imposed by adaptation to tem-
perature, but also from a slightly different folding of
the enzyme in the recombinant E. coli host. Indeed, a
Hill coefficient of 2.2 was calculated in native cell
extracts [17], where an association with the carbamate
kinase-like CP synthetase (CK-like CPSase) involved
in the channelling of CP might assist the folding of
ATCase and ⁄ or affect its cooperative behaviour. How-
ever, the high intrinsic thermostability of the recom-
binant enzyme strongly suggests a correct folding in
the mesophilic host and, besides, cooperativity toward
aspartate was found to vary as much between
ATCases from different mesophilic enterobacterial spe-
cies [27] as between native and recombinant P. abyssi
ATCases.
The cooperativity of P. abyssi ATCase appears little
or not affected by temperature, at least in the range
30–55 °C. The half-saturating aspartate concentration
also showed very little variation, whereas maximal
velocity increased sixfold in this same temperature
range. Thus, increasing temperature increases the rate
of the reaction without much affecting homotropic
interactions.
That cooperativity reflects the existence of T- and
R-states characterized by active sites with different
affinity and ⁄ or catalytic velocity is clearly indicated by

the stimulation of activity by PALA at subsaturating
aspartate concentrations: while this bisubstrate ana-
logue and inhibitor blocks the sites which it occupies,
it converts the remaining sites to the R-state. At aspar-
tate concentrations higher than [S
0.5
]
Asp
(3 mm), PALA
behaved simply as an inhibitor. The concentration
range in which P. abyssi ATCase is activated by PALA
is much broader and lower than that in which E. coli
ATCase is activated by PALA (between 10
)10
and
10
)8
m PALA instead of between 1 and 8 lm for
E. coli ATCase). This is in agreement with the higher
affinity of P. abyssi ATCase for CP compared with
E. coli ATCase (a K
m
of 5 ± 2 lm instead of 600 lm).
Our study makes clear that activation by PALA, and
thus cooperativity exists even in the absence of the
CK-like CPSase.
The question arises whether because of specific con-
straints imposed by adaptation to high temperature,
presumably an increased rigidity, the T- and R-states
deduced from the kinetic data correspond to different

quaternary states and if a global quaternary transition
with an amplitude comparable with that observed in
the case of E. coli ATCase effectively occurs with the
hyperthermophilic enzyme. E. coli ATCase undergoes
major structural rearrangements during the T-to-R
transition which result in a global expansion of the
S. Van Boxstael et al. Aspartate transcarbamylase from Pyrococcus abyssi
FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS 2677
molecule, as documented by X-ray crystallography
studies [5,33], small-angle X-ray scattering [34–36] and
sedimentation studies [20,37]. The sedimentation velo-
city experiments performed in this study on both
P. abyssi and E. coli ATCases showed that the binding
of saturating amounts of PALA induced similar
decreases in their sedimentation coefficients (Ds ⁄ s
o
of
2.7%), showing that P. abyssi ATCase undergoes a
conformational change – an increase in volume – of an
amplitude comparable with that of the E. coli enzyme.
It is worth noting that our results for E. coli ATCase
are in quantitative agreement with those recently pub-
lished by Schachman’s laboratory, a decrease of 2.9
and 2.6% in the presence of saturating PALA concen-
trations [19,21].
The existence of structurally distinct T- and R-states
implies the existence of interactions stabilizing these
states. Alignment of the P. abyssi ATCase amino acid
sequence with that of the E. coli enzyme shows that
the residues involved in polar interactions which are of

major importance to stabilize the T- and R-states of
E. coli ATCase [4] are nearly all conserved, with the
exception of rLys
143
which makes a crucial salt link
with cAsp
236
in the E. coli C4–R1 interface. However,
rLys
143
is replaced by an arginine which makes the for-
mation of a salt link possible. This conservation sug-
gests that similar polar interactions stabilize the T- and
R-state of P. abyssi ATCase.
In contrast with E. coli ATCase, P. abyssi ATCase
shows no cooperativity toward CP. The CP coopera-
tivity of the E. coli enzyme is apparent and reflects the
ordered binding of substrates, first CP, then aspartate,
and the cooperativity toward the second substrate [22].
A shorter 80S loop in P. abyssi ATCase, missing one
of the residues between a serine and a lysine which
make interactions with both substrates, might affect
the mechanism of substrate binding. Competitions with
PALA showed, however, that the shorter loop does
not affect the ordered binding of the substrates. The
cause of the lack of apparent cooperativity toward CP
must therefore be looked for elsewhere.
The lack of cooperativity for CP of the pure
P. abyssi ATCase contrasts significantly with the
cooperativity observed at both low and high aspar-

tate concentrations in determinations performed on
native crude extracts [17]. A possible explanation for
this discrepancy could be an interaction of ATCase
with one or more proteins present in the crude
P. abyssi extract, linked to the channelling of CP.
This would correlate with the different response
to the CP analogue phosphonacetate of the pure
ATCase and the ATCase in native extracts. Another,
methodological explanation could be that given the
high affinity for CP, a ‘false’ cooperativity was
observed in the CP saturations performed on crude
extracts: due to CP exhaustion in the lower range of
concentrations, initial velocity conditions would not
have been obtained [38].
Allosteric regulation by nucleotide effectors
P. abyssi ATCase is activated by ATP (270%) and
inhibited by CTP and UTP (50 and 40%, respectively).
The amplitudes of the responses to the different allo-
steric effectors do not change between 37 and 55 °C.
Competition experiments showed that the nucleotide
effectors bind competitively to the same regulatory
sites. Their effects on ATCase activity are neither
additive nor synergistic, but antagonistic. Remarkably,
CTP inhibition is already maximal in the micromolar
range, whereas the effects of ATP and UTP reach their
maximum in the millimolar range. This suggests that
CTP is the major physiological regulator of P. abyssi
ATCase activity. In E. coli ATCase, all nucleotides act
in the millimolar range. Alignment of the P. abyssi
and E. coli regulatory chains shows that only two of

the residues involved in interactions with ATP and
CTP in E. coli ATCase are not conserved in P. abyssi,
whereas 13 are conserved [39]. The two nonconserved
residues are involved specifically in ATP binding. It
can thus be suggested that the high affinity of P. abyssi
ATCase for CTP requires extra interactions with CTP,
which do not occur in E. coli ATCase.
Class B ATCases exhibit a varied pattern of
responses to nucleotide effectors (for a review of
mesophilic enzymes, see Wild and Wales [27]). The few
archaeal ATCases studied so far also show diverse
allosteric regulatory patterns: for instance, the ATCase
of S. acidocaldarius is activated by the four nucleoside
triphosphates ATP, GTP, CTP and UTP [32]. Single-
residue changes or the modification of discrete secon-
dary structure elements can dramatically affect the
allosteric response of ATCase, showing that the latter
depends on very subtle networks of intramolecular
interactions [40–43]. A response similar to that of
P. abyssi ATCase is found in the mesophile Yersinia
intermedia [27]. Clearly, adaptation to high tempera-
ture has little or no impact on the patterns of allosteric
response of ATCases.
Although little is understood about the constraints
which have led to the acquisition and conservation
of specific patterns of regulation, it should be con-
sidered that under physiological conditions, class B
ATCases are liganded by nucleotides, and that,
because ATP is a general activator [27], the compet-
itive binding of other nucleotides, inhibitors or less

Aspartate transcarbamylase from Pyrococcus abyssi S. Van Boxstael et al.
2678 FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS
efficient activators than ATP, appears as a conserved
mechanism of modulation of catalytic activity.
Several models have been proposed to explain the
molecular mechanism of effector action [5,44,45]. Glo-
bally, effectors appear to modulate the stability of
interfaces between domains and between subunits,
thereby facilitating (activators) or hindering (inhibi-
tors) the quaternary structure transition from T- to
R-state.
Different properties of the pure ATCase and
ATCase in native extracts may reflect the
channelling of the thermolabile substrate CP
At the optimal growth temperature of P. abyssi
(96 °C), CP turns out to be very unstable with a
half-life < 2 s [10]. The decomposition of CP leads
to the accumulation of toxic amounts of cyanate, a
powerful and indiscriminate carbamylating agent.
Metabolic channelling, a process by which the prod-
uct of an enzyme is directly transferred to the next
enzyme in the pathway without being released in the
bulk solvent, could provide a means to minimize the
thermal decomposition of CP at high temperature.
Indeed, isotopic dilution experiments and coupled
reaction kinetics with P. abyssi extracts showed the
existence of an imperfect channelling between the
CK-like CPSase and ATCase, although no physical
interaction between the two enzymes could be dem-
onstrated [9]. The lack of sensitivity of native

P. abyssi ATCase in cellular extracts to phosphon-
acetate, a CP analogue and an inhibitor of E. coli
ATCase, was proposed to result from a shielding of
the CP binding site from the bulk solvent by an
interaction with another cellular component, possibly
CPSase [17]. The observation that purified P. abyssi
ATCase is normally sensitive to phosphonacetate
supports this hypothesis. It should be mentioned
that a channelling of CP was also observed between
the CPSase and the ornithine transcarbamylase of
Pyrococcus furiosus [10] and that a physical inter-
action between these enzymes could be demon-
strated [11].
Experimental procedures
Strain and plasmids
The E. coli host strain was C600
ATC
À
(F
)
, supE44, hsdR,
endA, thi, D(lac-proAB), D pyrB) (Microbiology, VUB). The
expression vector was pTrc99A (Amersham Pharmacia Bio-
tech, Ghent, Belgium). pSJS 1240 [46] was a gift from S.J.
Sandler.
Chemicals and enzymes
CP (lithium salt), l-aspartate, CTP (sodium salt), UTP
(sodium salt), IPTG, ammonium sulfate, zinc acetate, anti-
pyrine and diacetylmonoxime were purchased from Sigma
(Bornem, Belgium). Sulfuric acid was purchased from Pan-

Reac. ATP sodium salt was purchased from Boehringer-
Mannheim (Brussels, Belgium). Tris-base was purchased
from Invitrogen (Bruges, Belgium). Leupeptine and amino-
ethylbenzylfluoride (AEBSF) were purchased from ICN.
PALA was obtained from the Drug Synthesis and Chem-
istry Branch, Developmental Therapeutics Program, Divi-
sion of Cancer Treatment and Diagnosis, National Cancer
Institute (Bethesda, MD, USA). Restriction enzymes were
from Amersham Pharmacia Biotech.
Cloning and expression
pyrBI genes from P. abyssi were amplified by PCR using
primers designed to generate a NcoI restriction site coinci-
ding with the ATG initiation codon of pyrB and a BamHI
site after the end of pyrI. The PCR product was cloned in
the pTrc99A vector and transformed into competent cells
of the ATCase-deficient strain C600
ATC
. In order to
improve expression, the cells were cotransformed with the
pSJS1240-vector [46]. The P. abyssi pyrBI genes were
expressed from the IPTG-inducible trc promotor of
pTrc99A. Cells were grown in a 12 L Biolafitte fermentor,
in rich 853 medium [47] containing 50 lgÆmL
)1
ampicilline
and 50 lgÆmL
)1
spectinomycine. IPTG was added to a final
concentration of 2 mm at A
600

¼ 2. Cells were harvested at
A
600
¼ 6 and kept at )80 °C.
Purification of P. abyssi ATCase holoenzyme
Twenty grams of cells were resuspended in 100 mL 100 mm
Tris ⁄ HCl, pH 8.2 containing the protease inhibitors leu-
peptine (1 lgÆmL
)1
) and AEBSF (100 lgÆmL
)1
). Cells were
lysed by cooled sonication in a Heat System Branson soni-
cator (model W-225R; Fisher Block, Kortrijk, Belgium) or
30 min. The cell extract was centrifuged (30 min, 10 000 g).
Heat denaturation
The supernatant was incubated for 20 min at 85 °C after
which it was cooled on ice. In order to remove denatured
proteins, the protein solution was centrifuged (10 min,
10 000 g).
First anion-exchange chromatography
The supernatant was applied on an Amersham Pharmacia
Biotech 26 ⁄ 60 column packed with Source 15Q medium.
The column was equilibrated with 20 mm Tris ⁄ HCl
(pH 8.2), 2 mm b-mercaptoethanol, 0.1 mm zinc acetate.
S. Van Boxstael et al. Aspartate transcarbamylase from Pyrococcus abyssi
FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS 2679
The protein was eluted with a linear 0–0.5 m NaCl gradient
in 1000 mL of buffer at a flow rate of 8 mLÆmin
)1

. The
eluted fractions showing ATCase activity and activation by
the nucleotide ATP, were pooled and dialysed against
20 mm Tris ⁄ HCl (pH 8.2), 2 mm b-mercaptoethanol,
0.1 mm zinc acetate.
Second anion-exchange chromatography
The dialysed protein was applied on a 10 mL MonoQ col-
umn equilibrated with 20 mm Tris ⁄ HCl (pH 8.2), 2 mm
b-mercaptoethanol, 0.1 mm zinc acetate. ATCase was elut-
ed with a 0–0.25 m NaCl gradient in 250 mL of buffer at a
flow rate of 2 mLÆ min
)1
. The eluted fractions showing
ATCase activity and activation by ATP were pooled and
concentrated using a Centriplus 100 column (Amicon,
Millipore, Belgium).
Size-exclusion chromatography
Final purification was achieved by size-exclusion chroma-
tography. This method also allows to separate free ATCase
catalytic subunits from ATCase holoenzyme. Two 16 ⁄ 60
colums in series, packed with Superdex 200 pg (Amersham
Pharmacia Biotech) equilibrated with 20 mm Tris ⁄ HCl
(pH 8.2), 2 mm b-mercaptoethanol, 0.1 mm zinc acetate
were used. Five hundred microlitres of concentrated protein
was injected on the column and eluted at a flow rate of
0.5 mLÆmin
)1
.
The ATCase fractions were concentrated and found to
be > 95% pure as judged by Coomassie Brilliant Blue

staining after denaturing PAGE. Twenty grams of wet cells
yielded 3 mg of pure P. abyssi ATCase holoenzyme.
Determination of ATCase concentration
The concentration of the pure holoenzyme was determined
by absorbance measurements at 280 nm using an extinction
coefficient of 26 985 m
)1
cm
)1
, calculated according to [48].
Estimation of the molecular mass by
size-exclusion chromatography
The molecular mass of ATCase was estimated using two
XK 16 ⁄ 60 columns in series packed with Superdex 200 pg.
The column was equilibrated with 20 mm Tris ⁄ HCl
(pH 8.2), 2 mm b-mercaptoethanol, 0.1 mm zinc acetate,
300 mm NaCl buffer. The elution volumes of the standard
proteins (Amersham Pharmacia Biotech) thyroglobuline
(660 kDa), ferritine (440 kDa), catalase (232 kDa), aldolase
(158 kDa) and bovine serum albumin (67 kDa) were deter-
mined. A standard curve was made with the logarithm of
the molecular mass of the proteins in the y-axis and the elu-
tion volumes (V
e
) in the x-axis. Comparison of the elution
volumes of the P. abyssi and E. coli ATCases with the
standard curve gave an estimate of their molecular mass.
Enzyme assays
ATCase activity was assayed using the colorimetric method
[49]. Absorption was measured at 466 nm using a SHIM-

ADZU UV-1601PC spectrophotometer. Unless specified
otherwise, assays were performed in 50 mm Tris ⁄ HCl,
pH 8.0. Aspartate saturations were performed in the pres-
ence of 5 mm CP at 30, 37, 45 and 55 °C, taking into
account the large DpH ⁄°C (0.28 ⁄ 10 °C) of Tris buffer. For
aspartate saturations at 45 and 55 °C, a blank value was
subtracted for each different aspartate concentration to
account for chemical carbamylation. At 30 and 37 °C the
chemical carbamylation is negligible.
Data analysis
Hyperbolic saturation curves were fitted to the Michaelis–
Menten equation, using origin
TM
software. For sigmoidal
aspartate saturations, the Hill equation was used to deter-
mine the kinetic parameters. Maximal velocity was deter-
mined graphically from the saturation curve, being the
maximal observed specific activity [50,51]. A minimum of
two independent experiments was carried out at each tem-
perature (at least three independent experiments at 37 and
55 °C). Aspartate saturations in the presence of nucleotides
were performed twice at both 37 and 55 °C. The Hill coeffi-
cient was obtained by determination of the slope in the Hill
plot: log(V ⁄ (V
max
) V)) vs. log([aspartate]). All other curves
were fitted manually.
Analytical ultracentrifugation
Sedimentation velocity and sedimentation equilibrium
experiments were performed in a Beckman Optima XL-A

analytical ultracentrifuge, equipped with an AN-60 Ti ana-
lytical rotor (Analis, NV, Belgium). The concentration along
the cell was measured with an optical absorption detection
system at 280 nm. The sedimentation velocity experiments
were carried out at 10 000 r.p.m. at 20 °C. Protein concen-
trations were 1.5 mgÆmL
)1
(P. abyssi ATCase) or
2mgÆ mL
)1
(E. coli ATCase) in 20 mm Tris ⁄ HCl pH 8.0,
150 mm NaCl, 20 lm zinc acetate. Sedimentation coeffi-
cients were determined with Beckman software based on a
nonlinear least squares fit, using the mixedfit program for
Windows [52–54]. The calculated sedimentation coefficients
for both E. coli and P. abyssi ATCase were not corrected
for density, nor for viscosity of the solvent and the change
in buoyant density weight caused by the binding of PALA,
following the calculation procedure used by [19,21]. The
sedimentation equilibrium experiments were carried out at
4 °C, at 6000 r.p.m. Protein samples were at a concentration
Aspartate transcarbamylase from Pyrococcus abyssi S. Van Boxstael et al.
2680 FEBS Journal 272 (2005) 2670–2683 ª 2005 FEBS
of 0.5 mgÆmL
)1
in 20 mm Tris ⁄ HCl pH 8.0, 150 mm NaCl,
2mm b-mercaptoethanol. After 67 h, equilibrium was
reached (determined by comparing scans taken at 5 h inter-
vals). The equilibrated scans were analysed with the same
Beckman software and the mass was determined.

Acknowledgements
This work was supported by the Flemish Science Foun-
dation (FWO, grant G. 0448.99), by the Flanders Inter-
university Institute for Biotechnology (VIB) and by the
Research Council (OZR) of the Vrije Universiteit Brus-
sel. SVB gratefully acknowledges the Flemish Institute
for the Improvement of Scientific and Technological
Research in Industry (IWT) for a specialization grant.
The authors are grateful to Tony Aerts (Biomedical Sci-
ences, University of Antwerp) for performing the AUC
experiments and to the Drug Synthesis and Chemistry
Branch, Developmental Therapeutics Program, Divi-
sion of Cancer Treatment and Diagnosis, National can-
cer Institute (Bethesda, MD) for the gift of PALA.
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