From the
Three arginine residues in the putative aspartate binding site
of Escherichia coli adenylosuccinate synthetase were changed
to leucines by site-directed mutagenesis. The mutant enzymes R303L,
R304L, and R305L were purified to homogeneity on the basis of sodium
dodecyl sulfate polyacrylamide gel electrophoresis and characterized by
CD spectrometry and initial rate kinetics. CD spectral analysis
indicated no differences in secondary structure between the mutants and
the wild-type enzyme in the absence of substrates. The
K
Adenylosuccinate synthetase (AMPSase)
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Adenylosuccinate is then cleaved by adenylosuccinate lyase to
form AMP and fumarate. AMPSase also plays a role in the salvage and
nucleotide interconversion pathways
(1) .
AMPSase has been
purified and characterized from many sources
(1) . The enzyme
from E. coli was first purified to homogeneity in
1976
(2) . The purA gene that codes for AMPSase was
cloned in 1987
(3) , and the crystal structure of the enzyme was
recently determined to a resolution of 2.8 Å
(4) . The
enzyme is a homodimer encoded by a single gene, purA, with a
polypeptide molecular weight of 48,000. Three different enzyme
mechanisms have been proposed for AMPase
(1) . The most widely
accepted mechanism, proposed by Lieberman
(5) and
Fromm
(6) , involves a 6-phosphoryl-IMP intermediate formed by
the nucleophilic attack of the 6-oxo group of IMP on the
X-ray crystallography studies
(4) of wild-type AMPSase showed
that much of the enzyme structure involved in GTP binding is closely
related to other GTP binding proteins. A number of studies involving
modification of putative GTP binding residues by site-directed
mutagenesis based upon the crystal structure of the enzyme provided
evidence fully consistent with the three-dimensional structure of the
enzyme
(9, 10) . However, little is known about the other
two substrate binding sites. X-ray diffraction studies of the unligated
enzyme
(4) indicate that residues 298-304 are poorly
ordered. Preliminary x-ray diffraction studies of the enzyme ligated
with MgGDP
Comparison of the Sequence 301-310 in E. coli AMPSase with
the Synthetases from Other Sources-Arg-303 is conserved in all
eight AMPSase enzymes studied thus far. Arg-305 is conserved in all
AMPSase enzymes except the synthetase in Bacillus subtilis;
and Arg-304 is not conserved ().
In the x-ray crystal
structure of the unligated enzyme
(4) , Arg-303-Arg-305 are
located in a poorly organized loop, which defines one edge of the
active site. Preliminary x-ray results of the synthetase ligated with
MgGDP
All the mutant enzymes
were purified by using procedures similar to those for wild-type
AMPSase. The purity of the enzymes was evaluated by SDS-polyacrylamide
gel electrophoresis. All of the enzymes exhibited greater than 95%
purity when electrophoresis is used as the criterion of purity (data
not shown). The molecular mass of the single polypeptide band for each
enzyme form was 48,000 Da.
E. coli purA
The K
Comparison of the cloned AMPSase cDNA sequences from eight
different sources showed that Arg-303 is highly conserved in all
AMPSases, whereas Arg-305 is conserved in seven of the eight known
sequences, and position 304 is a positively charged residue in five out
of eight sequences. These data suggest that there must be some
structural similarity among the AMPSases in this region of the
polypeptide chain.
The conserved Arg-303, and Arg-305 residues
clearly play important roles in the enzyme-catalyzed reaction. The
substitution of leucine at position 303 led to a 250-fold increase of
K
Isotope exchange experiments from our laboratory
(24) suggested that aspartate prefers binding to the
enzyme-substrate ternary complex (GTP
Knowing the
position of bound IMP and that the amino group of aspartate must
approach C-6 of IMP along a direction perpendicular to the plane of the
base permits the generation of a model for the binding of aspartate
(Fig. 1). The model places the
The enzyme assay solution contained 40 mM Hepes (pH 7.7) and 5 mM MgCl
values for GTP and IMP for the mutants
and the wild-type enzyme were comparable. However, the mutant enzymes
exhibited 50-200-fold increases in their values of
K
for the substrate aspartate relative to
the wild-type enzyme. Although the k
values for
the mutant enzymes decreased, the changes were not as dramatic as those
observed for the K
of aspartate. The
modeling of aspartate in the crystal structure of the complex of
adenylosuccinate synthetase with IMP and MgGDP
is
consistent with the results of mutagenesis, placing the
- and
- carboxylates of aspartate near the side chains of Arg-131, -303,
and -305.
(
)
from Escherichia coli (IMP:L-aspartate
ligase (GTP-forming), EC 6.3.4.4.) catalyzes the first committed step
in the de novo synthesis of AMP from IMP.
-phosphorous atom of GTP. Adenylosuccinate is then formed by a
second nucleophilic attack by the amino group of aspartate on the C-6
of 6-phosphoryl-IMP, displacing the phosphate. Hampton and Chu
(7) suggested that the 6-phosphoryl group of IMP could be
important for both IMP binding and catalysis. Initial rate product
inhibition studies suggested that the IMP and aspartate binding sites
are spatially separated but in close proximity
(1, 8) .
and IMP, however, show that Arg-303
approaches, but does not interact with, bound IMP, that Arg-304 is
directed away from the IMP molecule, and that Arg-305 is approximately
5 Å away from both the
-phosphate of MgGDP
and the 6-oxo group of IMP. To understand the roles of Arg-303,
Arg-304, and Arg-305, these residues were changed to Leu to remove the
guanidinium groups. Our results strongly suggest that the three
arginine residues are not part of the GTP or IMP binding sites but
rather are involved in aspartate binding and to some extent in
catalysis as well. This is the first study that implicates specific
residues in the binding of aspartate to AMPSase.
Materials
GTP, IMP, L-aspartate,
phenylmethylsulfonyl fluoride, and bovine serum albumin were obtained
from Sigma. A site-directed mutagenesis kit was obtained from Amersham
Corp. Restriction enzymes were obtained from Promega. E. coli strain XL-1 blue was obtained from Stratagene. E. coli strain purA H1238 was a gift from Dr.
B. Bachman (Genetic Center, Yale University). Unless specified
otherwise, other reagents and chemicals used in the experiments were
obtained from Sigma.
Site-directed Mutagenesis
Recombinant DNA
manipulation was performed using standard procedures
(11) . The
plamid containing a 1.6-kilobase HindIII fragment from PMS204
(3) ligated into PUC118 was used in the mutagenesis step. All of
the mutagenic oligonucleotide primers used in the experiments were
synthesized on a Bioresearch 8570EX automated DNA synthesizer at the
DNA facility at Iowa State University. Mutagenesis was carried out
according to the protocol provided by Amersham Corp. The mutations were
confirmed by DNA sequencing using the chain termination
method
(12) . The 1.6-kilobase HindIII fragment with the
proper mutation was ligated back into PMSN, a plasmid formed by self
ligation of PMS204 after its 1.6-kilobase HindIII fragment was
removed. The newly formed PMS204 plasmids with the correct mutations
were selected and then transformed into XL-1 blue cells. The plasmids
isolated from that strain were used to transform E. coli strain purA H1238, which was then used
for cell culture and protein purification.
Protein Assay
Protein concentration was determined
by the Bradford method
(13) , using bovine serum albumin as the
standard. The concentration refers to monomers.
Purification of Wild-type and Mutant AMPSase
The
wild type and the mutant enzymes were purified by using a
phenyl-Sepharose CL-4B column, a Cibacron blue 3GA column, and a
DEAE-TSK high performance liquid chromatography column. The
experimental details are described elsewhere
(9, 10) .
The purity of the enzyme was checked by SDS-polyacrylamide gel
electrophoresis according to Laemmli
(14) . AMPSase activity was
determined as described earlier
(15) .
Kinetic Studies of the Wild-type and Mutant
AMPSases
The concentrations of the stock solutions for GTP and
IMP were determined by using their extinction coefficients at 253 and
248 nm, respectively. For each reaction, the increase at 288 instead of
280 nm was recorded at 23 °C. For mutant enzymes, the
K for aspartate was obtained by holding
GTP and IMP at their saturating concentrations
(10K
), whereas the
K
values for GTP and IMP were obtained by
using an aspartate concentration of about 30 mM. 1-80
µg of the purified enzymes were used in each kinetic assay
reaction, depending on the specific activity of the enzyme.
Circular Dichroism Spectrometry
Circular dichroism
spectra of the wild-type enzyme and the mutant enzymes were acquired on
a JASCO J710 spectropolarimeter equipped with a data processor. Samples
(100 µg/ml) were placed in 1-mm cuvettes, and data points were
collected in 0.1-nm increments. Each spectrum was calibrated to remove
the background of the buffer and smoothed by using the program in the
computer of the spectrometer. The data were analyzed by the JASCO
analysis program or by the computer program PSIPLOT.
and IMP clearly reveal the locations of
Arg-303-Arg-305, the side chains of Arg-303 and Arg-305 project
into the active site, but do not directly bind to either IMP or
MgGDP
. Site-specific mutagenesis studies were
undertaken to better understand the roles of the three arginine
residues in question.
Mutagenesis of E. coli AMPSase purA cDNA and Purification of
the Mutant Enzymes
The oligonucleotide primers used in the
mutagenesis experiments are shown in together with the
sequencing primer for confirming the mutants. In our study, Arg-303,
-304, and -305 were changed to leucines to remove the side chain
guanidinium groups as a means of disrupting salt bridges or hydrogen
bonds while at the same time keeping the size of the side chain
comparable with that of the original residue.
H1238 harboring the mutant PMS204 plasmid grew more slowly than
the bacteria containing the wild-type plasmid. Given the same culture
conditions, the cell yield of bacteria expressing R303L and R304L was
80% of that of the E. coli expressing the wild-type enzyme,
while R305L was only 40%. The mutations at the three arginine sites had
a negative affect on the normal growth of the cells, suggesting that
the mutated enzyme might exhibit some differences in properties from
the wild-type AMPSase.
Secondary Structure Analysis
CD spectrometry was
used to analyze the secondary structures of the mutant AMPSase and the
wild-type enzyme. The CD spectra of the four enzymes were
superimposable (data not shown) from 200 to 260 nm. These observations
indicated that the mutant residues did not disrupt global secondary
structures detectable by CD.
Kinetic Analysis of AMPSase Mutants
The kinetic
parameters for GTP, IMP, and aspartate are summarized in
I.
values for GTP
and IMP were only marginally affected by the mutations compared with
the wild-type enzyme, suggesting that Arg-303, Arg-304, and Arg-305 are
not involved in binding of the substrates GTP or IMP. However, the
K
values for aspartate showed very
significant increases for all three mutants compared with wild-type
AMPSase. The K
values exhibited by R303L,
R304L, and R305L were 250, 290, and 44 times greater than that of
wild-type AMPSase. The changes suggest that all of the residues are
involved in aspartate binding. Another kinetic parameter,
k
is also significantly different in the mutant
enzymes relative to wild-type AMPSase. The k
for
R303L, and R304L were 0.133 and 0.183 s
,
respectively, equivalent to 13.3 and 18.3% of the wild-type activity.
On the other hand, in the case of R305L, a k
value of 0.0082 s
was observed, only 0.8% of
that of the wild-type enzyme. The changes in enzymatic activity suggest
that Arg-303, Arg-304, and Arg-305 may play a role in AMPSase catalysis
as well as binding of aspartate. Comparison of the specificity
constants, k
/K
, is
also available in I. For aspartate, the specificity
constants decreased 5.3
10
, 6.4
10
, and 1.9
10
for R303L,
R304L, and R305L, respectively, relative to wild-type AMPSase. These
findings demonstrate that by changing arginine residues 303-305
to leucine, the affinity of the enzyme for aspartate is drastically
reduced. This may result from the disruption of the salt linkages or
hydrogen bonds between these arginines and the substrate aspartate
and/or between these arginines and other residues important for
maintaining the proper enzyme conformation for aspartate binding and
catalysis.
compared with the
wild-type enzyme, as well as a dramatic decrease of
k
/K
by 3 orders of magnitude. On the other hand, the
K
values for GTP and IMP were not greatly
affected by this mutation. The R305L mutant retained only 0.8% of the
catalytic activity of the wild-type enzyme. It exhibited
K
values for GTP and IMP that are similar
to the wild-type enzyme. Although the K
for aspartate increased 45-fold,
k
/K
decreased to 1/5000 of that of the wild-type enzyme. This
significant loss of catalytic activity is consistant with our
observation that the purA
H1238 strain of
E. coli harboring the R305L-PMS204 plamid grew much more
slowly, and yielded only 40% of the cell weight, compared with the
bacteria harboring the wild-type plasmid. This suggests that even
though the vector allows 40-fold overexpression of AMPSase
(3) ,
replacement of Arg-305 with leucine impaired the catalytic activity of
the enzyme, which in turn may have led to a decrease in AMP synthesis.
IMP
enzyme) rather than
to the free enzyme. It is reasonable to suggest that for wild-type
AMPSase, a conformational change is required to facilitate the binding
of aspartate. Arg-305 is within 5 Å of the carbon 6 of IMP and
has the same conformation both in the presence and absence of the
active site ligands, MgGDP
and IMP. Arg-305 is
available for direct interaction with aspartate, consistent with the
decrease in k
and increase in
K
for aspartate, exhibited by the Leu-305
mutant. Very probably the proper orientation of Arg-305 is required for
the precise alignment of substrates in the quarternary complex of
enzyme, MgGTP
, IMP, and aspartate, the breakdown of
which is rate limiting in the AMPSase reaction
(15) . Arg-304
stabilizes the conformation of the loop consisting of residues
297-305 by forming a salt link to Glu-281, but only in the
presence of active site ligands. Hence the effect of the Leu-304 mutant
on the K
of aspartate is probably
indirect and due to the conformational destabilization of the
297-305 loop. Arg-303 hydrogen bonds to a loop consisting of
residues 125-129. The loop folds over the ribose moiety of IMP
but is disordered in the absence of the ligand. Again Arg-303 could
stabilize the conformation of the 297-305 loop and thereby
enhance the affinity of the enzyme for aspartate. However, as Arg-303
approaches the active site, a direct role in the binding of aspartate
cannot be excluded. One other residue, Arg-131, is in position and
available for a direct interaction with aspartate.
-carboxylate of aspartate in
a salt link with Arg-305 and the
-carboxylate in a salt link with
Arg-131. The amino group of aspartate would be fixed in the vicinity of
C-6 of IMP by a hydrogen bond to the carbonyl of residue 38. The model
is speculative, inasmuch as we do not know the structure of the enzyme
in its complex with the putative 6-phosphoryl IMP intermediate. The
presence of the 6-phosphoryl group may trigger additional
conformational changes in the enzyme, particularly in the vicinity of
residues 38-40.
Figure 1:
Stereo view of aspartate modeled in the
active site of AMPSase. The starting point for the model is the
preliminary P3 crystal structure of the complex of the
synthetase with IMP, GDP, and Mg
. Aspartate has been
placed in the active site without energy minimization. All ligands are
drawn with boldface lines.
Table:
Alignment of E. coli AMPSase amino acid sequence
301-310 with the sequences of the AMPSase from other sources
Table:
Oligonucleotides used in site-directed
mutagenesis
Table:
Kinetic parameters of wild-type and mutant
AMPSases from E. coli
. When GTP was
the variable substrate, IMP concentration was fixed at 0.45
mM. Asp was fixed at 5 mM for wild-type AMPSase and
at 30 mM for the mutants. When IMP was the variable substrate,
the GTP concentration was fixed at 0.25 mM, and the Asp
concentration was fixed at 5 mM for the wild-type AMPSase and
30 mM for the mutants. When Asp was the variable substrate,
GTP and IMP concentrations were fixed at 0.25 mM and 0.45
mM, respectively.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.