Vol. 171, No. 3, 1990 September
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
28, 1990
Pages
Allosteric
Control of Quaternary States in 23. coli Aspartate Transcarbamylase Raymond Gibbs
Received
1312-1318
August
C. Stevens
and William
N. Lipscombt
Chemical Laboratory, Harvard University, Cambridge, Massachusetts 02138
14,
1990
Changes in the molecular dimensions of ATCase in the unligated T-state are an increase of 0.4 A in the separation of catalytic trimers when 4TP binds. When the R-state is produced by binding of phosphonoacetamide and malonate, addition of CTP or CTP+UTP decreases the separation of catalytic trimers by 0.5 8. In the unliganded Glu239-+Gln mutant, in which the T-state is destabilized so that the enzyme exists in an intermediate quaternary state, ligation of ATP transforms the mutant enzyme to the R-state, whereas CTP converts this enzyme to the T-state. Thus; this mutant is much more sensitive t.o heterotropic allosteric control than is the native enzyme. In this communication we propose a preliminary model based on new crystallographic results that heterotropic regulation occurs partly through control of the quaternary structure by these effecters, thus regulating catalysis. @1990 Academic
Press,
Inc.
Aspartate
transcarbamylase
ferase) from Escherichia
cola is one of the most widely
catalyzes
the first committed
phosphate
and L-aspartate
tivated
by the product
Poised
at the beginning
regulation
to feedback
of the parallel
biosynthesis;
inhibition purine
of the pyrimidine
the flux of metabolites
tein crystallography the catalytic
step in pyrimidine
is not as well understood. on the regulatory
the catalytic
chain produce
How does the binding
opposite
effects on the enzyme’s
whom correspondence
0006-291XBO Copyright All rights
should
be addressed.
$1.50
8 1990 by Academic Press, Inc. of reproduction in any form reserved.
1312
(CTP)
activity?
and ac(ATP;
2).
enzyme Pro-
valuable
into
On the contrary, of ATP
(1).
blocks of DNA.
and CTP
chain at sites more than 60 a from the nearest
controlled?
To
(3).
carbamoyl
this cooperative
and have provided
ATCase
phosphate
5’-triphosphate
pathway,
mutagenesis
cooperativity
between
5’-triphosphate
form the building
and homotropic
enzymes.
the reaction
adenosine
biosynthetic
carbamoyltrans-
and inorganic
by cytidine pathway,
allosteric
that eventually
and site-directed
mechanism
local region
t
studied
to form N-carbamoyl-L-aspartate
The enzyme is susceptible
helps control
(E.C. 2.1.3.2; also called ATC ase, aspartate
insight
heterotropic to the same active site on
And, how is catalysis
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS CTP + UTP complexed T and R-state ATCase. T-state crystals were grown in 100 mM maleic acid, 10 mM Tria HCl, pH 5.7. R-state crystals were grown in 20 mM phosphonoacetamide (PAM), 20 mM malonate, pH 5.8. 1 mM CTP and 1 mM UTP were soaked into the crystals at pH 7.0. 179,967 reflections were collected on the CTP+UTP T-state enzyme. These data were reduced to 41,272 independent reflections to 2.4 di with an Rmrrgc = 7.1%. The crystallographic parameters are: space group P321, a = b - 122.2 A, c = 142.0 A. 213,846 reflections were collected at the Resource for Crystallography at the University of California, San Diego on the CTP+UTP complexed R-st,ate ATCase. These data were reduced to 46,733 independent reflections to 2.3 A with an Lcrgc = 7.1%. The crystallographic parameters are: space group - P321, a = b =: 122.2 A, c = 155.0 A. Both data sets were collected with three different crystals. The program X-PLOR (A. T. Btinger, X-PLOR Manual, version 1.5 (1988)) was used in all refinements. Molecular replacement was used in both structure solutions. Presently, the structures have been refined to 2.4 A resolution and to crystallographic R-values of 0.17 and 0.18 for the T- and R-states, respectively. A complete crystal structure analysis will be published elsewhere. Preliminary crystallographic studies of Gln239 ATCase with effectors ATP or CTP. Screened x-ray precession photographs were taken from crystals grown in 45 mM maleic acid, 45 mM Tris Base, pH 5.8 buffer with 10 mM ATP or 2 mM CTP. The crystallographic parameters for the ATP liganded Gln239 enzyme are: a = b = 122 A, c = 156.0 A. The crystallographic parameters for the CTP liganded Gln239 enzyme are: a - b = 122 A, c = 142 A. All enzyme complexes are crystallized in the space group P321. Conformational differences between enzyme with various substrate/effecter complexes. Con&rm&nal changes were determined by first superimposing the a-carbon atoms of one catalytic trimer and then by superpositioning the o-carbon atoms of the other catalytic trimer. The movement of the latter catalytic trimer along the three-fold axis with respect to the first catalytic trimer is the catalytic trimer separation. The unliganded enzyme was used as a reference for the T-state, while the PAM+malonate complex provided a reference for the R-state. These small changes in separation of catalytic trimers are statistically significant because the errors in position of an entire polypeptide chain are much smaller than the errors (o) associated with individual atomic positions. The approximate standard error of the mean for the separation of catalytic trimers is u/h which is about 0.01 A for the n = 1860 C, atoms in two catalytic trimers and for the 0.5 A value of u for an individual C, position based on the Luzzati plot (4). Since the standard error of the catalytic trimer separation also depends on the cell dimension along the c-axis, the unit cell dimensions were determined on at least three different crystals. The average unit-cell dimensions were used in the refinement of the crystal structure.
RESULTSAND DISCUSSION Both
the T and R-states
of ATCase
crystallize
in the space group
unit cell axes are a = b = 122 A, c = 142 A. The R-state 122 A, c = 156 A. Th e separation c-axis cell length When PAM
unliganded
T-state
and malonate
crystals
identical
(a substrate are produced
(c-axis
trimers
structures.
which
can be determined
T-state
and subsaturating
have a c-axis unit-cell
trimers
= 142 A) are soaked in the substrate into its R-state
conformation
dimension 1313
crystals are complexed amounts
of L-aspartate
of 149 A, intermediate
the
are a = b =
by monitoring
the catalytic
of 156 A (5). S oa k’m g an d co-crystallization
When unligated
for the reverse reaction)
In the T-state,
unit cell dimensions
axis along which
the single crystal is transformed
crystal which has a c-axis cell length produce
of catalytic
(th e c-axis is the three-fold
P321.
the move).
analogues as a single experiment5
with phosphate single crystals between
the
Vol.
171, No. 3, 1990
T and R c-axis intermediate
BIOCHEMICAL
cell length
crystal refined
relatively
small fraction
of total scattering
A). The T-state
complexes
triphosphate)
structures
by 0.4 A toward
the R-state.
an 11 A increase in the separation
of catalytic ATP
CTP decreases the separation
binding
bisubstrate analogues
trimers, effect
in both the unliganded
the crystal
that CTP and UTP
structure,
very slightly. with
CTP
In contrast.
Transformation X-ray
tion of Glu239 the T-state
ATP.
Glu239
scattering
occurs with with
In the pAR5
mutant, with
scattering
residues
the neutral
is intermediate
residue
chains
involved
is Asp236
enzyme
(16).
This
region
chain.
1314
destabilizing
enzyme
yielded
a
of 147 A for with
the c-axis length by
of the carboxylate
of
to serine an intermediate enzyme
of the regulatory
occurs
near
the
interacts
interactions Asp236
chain have
cl...r4
interface.
the structure
In the presence
to that shown by the wild-type
interface
of the catalytic
in
muta-
Tyr165.
the T and R forms,
similar
in several
(~4) as found
that in the absence of ligands,
between
Glu239
have been confirmed
of the wild-type
at the C terminus
curves demonstrate
between
of 156 A when complexed
is mutated
Glu239
only
when complexed
thereby
of t,his Gln239
to the mutant
Tyr165
(11). In
trimers
here that the c-axis length
the uncharged
145-153
enzyme is in the R-state,
A specific
chain
One of the interactions
(15); probably,
six new residues
mutant
(14).
liganded
The site specific
these interactions,
length
of CTP
of the T-state.
interactions
value of 142 A. These results
Lys164 than with
Solution
regulatory
CTP is added
When
is not found
in the R-state
strurture
trimers
here refine-
in ATCase
of catalytic
the T-state.
to the R-state
from solutions
Tyr165.
been replaced
the mutant
inhibitor
catalysis
of an adjacent
to disrupt
trimers
dimer about
concentrations
separation
stabilize
crystal
enzyme is increased
form of the enzyme
of the pAR5
was expected
147 A to the T-state
X-ray
X-ray
and Tyr165
involves
of catalytic
0.6 A in the direction
the T and R states (13). We report
the allosteric
more strongly
Lys164
(PALA)
and malonate
and PAM-malonate
inhibit trimers
analogue
(10). We report
The strong non-covalent
The th ree-dimensional
Gln239
decreases from low-angle
enzymes. with
to glutamine
(12).
When
of Patalytic
increases
of the catalytic
equimolar
T-state
the T-state
studies from this laboratory
between
the mutant
perturbs
the separation
chain (cl)
with
synergistically
decreases by approximately of mutant
diffraction
structure
CTP+IJTP
and UTP
of one catalytic
complexed
trimers
PAM
the separation
on
(8), and PAM
of the regulatory
the T-state
and UTP (uridine
triphosphate)
resolu-
This transformation
a 12” rotation
by 0.5 A toward
enzyme
It is known
for
liganded
substrate
ment to 2.4 A on the wild-type R-state.
are known
at medium
of catalytic
conformation.
has little
the
(7).
determined
about the 3-fold axis, and 15” rotations
the a-fold axes. In the R-state, while
of the tight
the R-state
neglecting
that have been refined are the unliganded,
(9) or weak binding
(5) in the absence of effecters induces to one another
transcarbamylase
the
form has been
structures
with ATP, the separation
Ligation
transforms
reflections
Three-dimensional
CTP ( c yt i d ine triphosphate)
(5). Up on ligation
N-phosphonoacetyl-L-aspartate
relative
in the superlattice
of ATCase
liganded,
L-aspartate
of the intermediate
of 0.25 to 5.5 & resolution
enzyme.
forms of E. COC aspartate
(adenosine
RESEARCH COMMUNICATIONS
with
structure
R-factor
of the wild-type
several enzymatic
liganded
the enzyme
The crystal
to a crystallographic
Transformation
ATP
Saturating
to its R form.
partially
tion (2.6-2.8
(6).
AND EIOPHYSICAL
between makes
of PALA,
enzyme the catalytic
several
(16). and
c...r contacts
Vol.
171, No. 3, 1990
including
a cl...r4
BIOCHEMICAL
interaction
Asp236
has been mutated
mutant
enzyme
pAR5
mutants
and confirms
conducted
the mutant
conclusion
with
confirm
conformation
a tendency
to attribute
amino
An example
contributions
Controversy
in ATCase.
One model
qua.ternary
perturbing
structure
t,he T-+R
an alteration
in substrate
transition
or high affinity sulfinate
mutations
(3) and other
affinity for substrate.
ATCase Our struc-
enzymes
can be very
there are thermodynamic
the effecters
analyses effectors
(19). regulate
cause an alteration
the activity
substrate
affinity
effects in ATCase
is cross-linked
or no homotropic
in the
of the enzyme in turn controls
by ATP
state.
substrate
analogues
by the
cooperativity
through
site-directed
into either
but at least part of the
substrate
analogues
L-cysteine
enzyme do not elicit homotropic and inhibited
The uncoupling (24).
by CTP; through
If th e wild-type
and heterotropic use of site-directed
enzyme
remains
in the alter the
affinity by perturbing
effects on ATCase
and hybridization
cooper-
here, the dominant
of homotropic
that the effecters alter the substrate
mutagenic
the low
experiments.
has been ac-
Replacement
of
chain
activation
an enzyme
some distiuc-
effects are present? then the effecters must somehow
We propose
of the regulatory
provides
chemically
studies of aspartate
a quaternary state, as observed in the wild-type enzyme. The uncoupling of ATP activation and CTP inhibition
produces
and
enzyme.
of mutant
in structural
regulating
their
enzyme,
in the wild-type
parameters
has also been observed in other instances
state and heterotropic
ATP
the earlier
changes in a mutant
study of the Gln239
considered
(24) to th e wild-type
occurs in a T-like
in ATCase
minimal
with
Furthermore,
conformational
where
the altered
(22). Binding
but the enzyme is still activated
complished
are in accord
hemoglobin
that
the enzyme
state one finds little
interactions
Lys56
velocity converts
and wild-type
and heterotropic
When
effects remain
part of catalysis
T-like
sedimentation show that ATP
mutant
thereby
affinity;
of homotropic
(23) and L-alanosine
ativity,
mutations,
(20). A n a It erna.tive model suggests tha.t the effectars ca.use
binding
the models.
heterotropic
the Lys143dAla
(21).
The uncoupling tion between
proposes
are present
and Ala236
exists over the way that heterotropic
of ATCase
equilibrium
which
interactions.
for this behavior
that are not ordinarily
control.
and
that this is an oversimplification.
between
also occurs in mutant
from residues
Allosteric
mutant
changes in kinetic
acid, we believe
studies suggest that comparisons
The
changes the enzyme to the T-state
from the crystallographic
we have demonstrated
in a single
and CTP
of interface global
(17).
of the Gln239
reported pAR5,
Difference
ATP
while CTP
effectors promote
our conclusions
it is tempting
to changes complex.
have recently the Gln239,
effecters
the importance
that the heterotropic
Although
T-R
contacts
conformation.
the allosteric
above regarding
more important,
average
interface
The results of this study of the Ala143
studies reported
catalysis
of cl...r4
and co-workers
enzyme to its R-state
conformation.
tural
to that
chain (18). Resembling
at pH 7.0 (5).
interactions
similar
the importance
Schachman
of the enzyme
conformation
enzyme exists in an intermediate
experiments
ATCase
in the T-form
to test its role in interface
an intermediate
of the regulatory
the Ala143
Lys143
to alanine
displays
only in the T form. mutation
with
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
with
with alanine produces an enzyme with CTP inhibition and chain with alanine (25). R e pl acement of Lys60 of the regulatory ATP
activation
and minimal 1315
CTP
inhibition
(26).
IJnlike
.4T-
Vol. 171, No. 3, 1990
BIOCHEMICAL
Case from E. coli, ATCase to a lesser extent
than
from
ATP).
AND BIOPHYSICAL
S. marcescens
Hybridization
experiments
E. coli is substituted
in place of the regulatory
which
characteristics
has catalytic
marcescens which
ATCase
contribute
T-R transition, ATP
(27).
site through
in reference
It appears
and cl:r4
quaternary
state.
the affinity thereby
likely
that
slightly
and co-workers
and cl:r4 interfaces
to the quaternary
binding.
Control
catalysis
a two-state
exist.
R quaternary Finally,
velocity X-ray
Although the MWC tenets is “Two (at We still do not know
and X-ray
the T*R
particularly
ATP
acces-
Schachman
proteins
and CTP
The
possess limited
in effector
has been altered
inter-
control,
conformation”
changes towards
varying
or
or intermediate
“intermediate
the T or
degrees of stability.
do not violate
induce
in the
two- or three-dimensional
differences
a signal through
and
if there are multiple
conformational
states exist, with
how the effecters transmit that
does
is similiar
the effects (18).
the MWC
is often referred to as the “two-state model”, states are reversibly accessible to allosteric
but we have determined
CTP
or CTP
scattering
or possibly
we use the phrase
states of allosteric two)
to
state, it is
of reversibly
of ATP
solution
equilibrium
that the effecters induce
model
the T+R
above,
answered.
upon complexation
diffraction,
In this communication
least
enzyme
(28) wit,h modifications
of the number
can detect such subtle
states; thus intermediate
or
is not observed
This model
and co-workers
state differences,
which
regulates
CTP
are not able to detect and describe
of whether
intermediate
binding
Although
site
separation
where the ‘l? and R states are in equilibrium
shift is so slight
Probably
are the main methods
conformations
chain,
ratio
to resolve small quaternary
based on our observation
Thus,
contacts
state in the wild-type
the T-state).
The question
model
sedimentation
the question
interface
trimer
states has not been unambiguously
enzyme that their methods
to answer
(These
(As we have noted
alter
by Her&
results.
conformational
conformations.
site (27).
to alter the R quaternary
states
does slightly
model proposed
wild-type
mediate
in the
signals from the effector
catalytic
of substrate
is not observed
the two allosteric
and, ATP
propose
of S.
signals from the effector
the enzyme’s
at the active site.
state and ATP
that the [T]/[R]
NMR,
alters
transmit
changes
speculate
ability
and CTP
“minor”
of substrate
of difference
and the effector binding
chain to the cl:rl
our new crystallographic
techniques
interactions,
state by transmitting
they do stabilize
sible quaternary
an enzyme
8).
to the primary/secondary reflecting
from
characteristics
are those that have been implicated
which
alter the R-state
(CTP
dimer
produces
and regulatory
the quaternary
regulating
alter the T quaternary
and CTP
the regulatory
of S. marcescens
interfaces
The
ATP
of the amino acid sequence reveals that the residues
differences
from these studies that ATP
to the cl:rl control
control
the regulatory
are illustrated
transition
A comparison
or cl:r4 interface
and CTP partly
dimer
by both where
of E. coli ATCase
to the regulatory cl:rl
is activated
RESEARCH COMMUNICATIONS
(29).
one of the basic oligomers” (29).
the regulatory
global
model
conformational
polypeptide changes
in the quaternary state which in turn controls substrate binding affinity and the T-+R transition. A number of questions remain to be answered but progress has been made in the understanding
of heterotropic
regulation
in aspartate
1316
transcarbamylase.
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS This
work
fellowship
was supported
area detector
allowed
for rapid
GM06920
refinement
(WNL)
of the University
for data collection
in the T and R states, the Pittsburg which
grant
P ro f essor N. Xuong
(RCS). We thank
for use of an x-ray
by NIH
and an NIH
postdoctoral
of California
at San Diego
of the CTP+UTP
Supercomputing
complexed
enzyme
Center for the use of the Cray Y-MP
of the crystal structures,
and HURAY
for ail comparison
calculations.
REFERENCES 1. Jones, M. E., Spector,
L., and F. Lipmann
(1955)
J. Am. Chem. Sot. 77, 819-820.
2. (a) Gerhart, J. C., and Pardee, A. B. (1962) ,I. Bzol. Chem. 237, 891-896. M. R., Smith, K. E., White, J. S., and Jones, M. E. (1968) Proc. Natl. U.S.A. 00, 1442-1449. 3. (a) Kantrowitz, E. R., and Lipscomb, E. R., and Lipscomb, W. N. (1990)
Acta. Crystallogr.
4. Luzzati,
V. (1952)
5. Gouaux,
J. E., and Lipscomb.
6. Gouaux,
J. E., and Lipscomb,
7. Gouaux,
J. E., and Lipscomb,
8. Stevens,
R. C., Gouaux,
11. Wild,
Biochemistry. 29, 389-402.
W. N. (1990) W. N. (1989)
Prot. Natl. Acad. Sci. U.S.A. 86 845-048.
W. N., unpublished
results.
Biochemistry, in press.
W. N. (1990)
W. N., Cho, Y. J., and Honzatko,
J. E., Stevens, R. C., and Lipscomb,
J. R., Loughrey-Chen,
(b) Kantrowitz,
5, 802-810.
J. E., and Lipscomb.
9. Ke, H. M., Lipscomb, 196, 853-875. 10. Gouaux,
W. N. (1988) S czelzce 241,669-674. Trends in Biochem. SC;. 15, 53-59.
(b) Bethell, Acad. Sci.
R. B. (1987)
Biochemistry, in press.
W. N. (1990)
S. J., and Corder
Biol.
J. Mol.
T. S. (1989)
Proc. Natl. Acad. Sci.
U.S.A. 86, 46-50. 12. Ladjimi,
M. M., and Kantrowitz,
13. Gouaux,
J. E., Stevens,
E. R. (1988)
Biochemistry
R. C., Ke, H. K., and Lipscomb,
27, 276-283 W. N. (1989)
Proc. Natl.
Acad. Sci. U.S.A. 86, 8212-8216. 14. Taut, P., Va.ch&t.e, in press.
P., Middlpton.
15. Robey, E. A., and Schachman, 16. Cherfils,
J., Vachette,
P., Taut,
S. A., a.nd Kant.rowitz,
H. K. (1984) P., and Janin,
1317
E. R.. {l%?O) .7. knot.
Riol.,
J. Biol. Chem. 259, 11180-11183. J. (1987)
J. EMBO 6, 2843-2846.
Vol.
171,
No.
3, 1990
BIOCHEMICAL
17. Newton, C. J., and Kantrowitz, 2309-2313. 18. Eisenstein, 3731.
E., Markby,
AND
BIOPHYSICAL
E. R. (1990)
Proc.
D. W., and Schachman,
RESEARCH
Natl.
COMMUNICATIONS
Acad.
H. K. (1990)
Sci.
U.S.A.
Biochemistry
87,
29, 3724-
19. Gao, J., KuczeraL: K.: Tidor, B.: and Karplus, M. (19891 Science 244, 1069-1072. Again, mutant enzymes may show behavior quite different from that of the native enzyme. For example, the effects of ATP or CTP on kinetic parameters show that the native enzyme is a K-system (Gerhart, J. C., and Pardee, A. B. (1963) Cold Springs Harbor Symp. @ant. Biol. 28,491-496), while the Glu50-+Gln catalytic chain mutant is a V-system (Ladjimi, M. M., Middleton, S. A., Kelleher, K. S., and Kantrowitz, E. R. (1988) Biochemistry 27, 268-276. 20. Hensley, 3736.
P., and Schachman,
21. (a) Her&, Biol. 185, 4172-4181. 22. Chan,
Proc.
Nat?
G., Moody, M F., Taut. P.. Vachette: 189-199. (b) H suanyu, Y., and Wedler,
W. W.-C.,
and Enns, C. A. (1979)
23. Foote, J., and Lipscomb, 24. Baillon,
J., Taut,
25. Corder,
T. S., and Wild,
26. Zhang,
Y., and Kantrowitz,
27. Beck, D., Kedzie, 28. Taut, P., Leconte, 155, 155-168. 29. Monod,
H. K. (1979)
J., Wyman,
W. N. (1981)
P., and Her&,
G. (1985)
J. Ft. (1989)
E. FL (1989)
K. M., and Wild, C., Kerbiriou,
57, 798-801
Chem. 264, 7425-7430. 28, 7313-7318.
J. Biol.
Chem.
264, 16629-16637.
L., and HervC, G. (1982)
J.-P. (1965)
1318
76, 3732-
24, 7182-7187.
Biochemistry
D., Thiry,
U.S.A.
256, 11428-11433.
Biochemistry
J. R. (1989)
J., and Changeaux,
J. Biochem.
Chem.
J. Biol.
Sci.
P., and Jones, T (1985) ,T. Mol. F. C. (1988) J. Biol. Chem. 263,
Canadian
J. Biol.
Acad.
J. Mol.
Biol.
12,
J. Mol.
88-118.
Biol.