(Received for publication, December 19, 1996, and in revised form, February 20, 1997)
From the Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011
Examined here by directed mutation, circular
dichroism spectroscopy, and kinetics are the relationships of five
residues, Asp13, Glu14,
Lys16, His41, and Arg131, to the
catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no
measurable activity. Mutants E14A and H41N exhibit 1% of the activity
of the wild-type enzyme and 2-7-fold increases in the
Km of substrates. The mutant K16Q has 34% of the
activity of wild-type enzyme and Km values for
substrates virtually unchanged from those of the wild-type system.
Mutation of Arg131 to leucine caused only a 4-fold increase
in the Km for aspartate relative to the wild-type
enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on
kcat are consistent with the putative roles
assigned to Asp13 (catalytic base), His41
(catalytic acid), and Glu14 (structural organization of the
active site). The modest effect of the R131L mutation on the binding of
aspartate is also in harmony with recent crystallographic
investigations, which suggests that Arg131 stabilizes the
conformation of the loop that binds the -carboxylate of aspartate.
The modest effect of the K16Q mutation, however, contrasts with
significant changes brought about by the mutation of the corresponding
lysines in the P-loop of other GTP- and ATP-binding proteins.
Crystallographic structures place Lys16 in a position of
direct interaction with the
-phosphate of GTP. Furthermore, lysine
is present at corresponding positions in all known sequences of
adenylosuccinate synthetase. We suggest that along with a modest role
in stabilizing the transition state of the phosphotransfer reaction,
Lys16 may stabilize the enzyme structurally. In addition,
the modest loss of catalytic activity of the K16Q mutant may confer
such a selective disadvantage to E. coli that this
seemingly innocuous mutation is not tolerated in nature.
Adenylosuccinate synthetase
(AMPSase)1 (see Ref. 1 for review)
catalyzes the following reversible reaction in the presence of
Mg2+ ions: GTP + IMP + aspartate GDP + adenylosuccinate + phosphate (Pi). This reaction is the
first committed step in the formation of AMP from IMP on the pathway
for de novo purine nucleotide biosynthesis and is an
integral part of the purine nucleotide cycle in muscle (2). The
reaction mechanism of AMPSase centers on 6-phosphoryl-IMP, formed
putatively by the nucleophilic attack of the 6-oxyanion of IMP on the
-phosphate of GTP. A second nucleophilic substitution reaction by
the amino group of aspartate on the C-6 of 6-phosphoryl-IMP yields
adenylosuccinate and Pi (3). Two Mg2+ ions are
involved in the reaction mechanism (4). One Mg2+ is in the
active site, associated with the phosphate moiety of the guanine
nucleotide and the N-formyl group of hadacidin, an inactive
analog of aspartate (5). However, crystallographic investigations have
yet to reveal the location of the second Mg2+.
On the basis of preliminary crystal structures of ligated AMPSase,
which are now complete (5, 6), several residues are in positions of
putative significance to catalytic function, ligand binding, and/or
structural organization of the active site. Asp13 of
AMPSase hydrogen bonds with N-1 of IMP and approaches the sixth
coordination site of a pentavalently coordinated
Mg2+. Asp13 is putatively a catalytic
base in the abstraction of the proton from N-1 of IMP (5).
Glu14 hydrogen bonds to the backbone amides 10 and 12 of
the P-loop (residues 8-17 of AMPSase) and to NZ of Lys16.
Glu14 may stabilize the conformation of the P-loop, provide
electrostatic charge balance in the active site, and/or orient NZ of
Lys16 with respect to the -phosphoryl group.
Lys16 of AMPSase corresponds to the essential lysine of the
consensus P-loop sequence GXXXXGK (7, 8). Lys16
interacts with the
-phosphate of GDP and/or anions (nitrate and
phosphate) bound to the
-phosphoryl site (5, 6). Lys16
putatively stabilizes the pentavalent transition state of the
-phosphoryl group during the phosphotransfer reaction.
His41 interacts with phosphate groups located at the
-
and
-phosphoryl binding sites. His41 is a putative
catalytic acid in the phosphotransfer reaction (5). Finally,
Arg131, initially considered a candidate for binding
aspartate (9), may stabilize the closed (ligand-bound) state of the
active site by folding over and perhaps hydrogen bonding with the loop
that recognizes the
-carboxylate of aspartate.
Reported here are mutations of Asp13, Glu14, Lys16, His41, and Arg131, which further probe the roles of each residue in the function of AMPSase.
Escherichia coli strain XL1-Blue came
from Stratagene, a site-directed mutagenesis kit from Amersham Corp.,
and restriction enzymes from Promega. The chemicals used in this study
were obtained from Sigma, and pur A strain
H1238 was a gift from Dr. B. Bachman (Genetic Center, Yale
University).
Recombinant DNA manipulation was
performed using standard procedures (10). The mutagenic
primers2 in this study are
5-TTTACCTTCGGCACCCCATT-3
(Asp-13
Ala),
5
-ACCTTTACCTGCGTCACCCC-3
(Glu-14
Ala),
5
-ATCTTACCTTGACCTTCGTC-3
(Lys-16
Glu),
5
-ACGAGAGTATTGCCTGCGTT-3
(His-41
Asn), and
5
-CCCGATACCAAGACCGGTGG-3
(Arg-131
Leu). (The bases in
bold letters indicate the mutated sites.) Mutagenesis and isolation of
the mutant cell lines were carried out according to the procedure described previously (11). The mutated plasmids were transformed into
an E. coli pur A
strain (H1238), which does
not produce AMPSase, to prevent mutant protein contamination by
wild-type enzyme.
Mutant forms of AMPSase were purified by the procedure
described previously (12), except for H41N. The H41N mutant has low solubility in 20 mM potassium Pi, pH 7. As a
consequence, the eluent from the phenyl-Sepharose column was
concentrated in 100 mM potassium Pi, pH 7, and
then purified by DEAE-HPLC using 100 mM potassium
Pi, pH 7, with a linear salt gradient from 0 to 1 M NaCl. Protein purity was monitored by SDS-polyacrylamide
gel electrophoresis according to Laemmli (13). The concentration of
purified protein was determined using the extinction coefficient for
wild-type AMPSase at 280 nm (280 = 67.85 mM
1 cm
1) where the
concentration refers to monomers. AMPSase activity was determined as
described earlier (14). 3-20 µg/ml enzyme was used in assays,
depending on the activity of each mutant. A GBC model 918 UV/Visible
spectrophotometer equipped with a Peltier-Effect temperature controller
to maintain the temperature at 25 °C was used to monitor absorbance
changes at 290 nm.
Two samples of the K16Q mutant (0.54 mg/ml) in 40 mM Hepes buffer, pH 7.7, were incubated at either 25 or 4 °C for 2 h. At different times, 5-µl aliquots were removed and added to 1 ml of assay solution containing 150 µM GTP, 200 µM IMP, 5 mM aspartate, and 2 mM MgCl2 in Hepes, pH 7.7, and the activity was measured at 25 °C. The absorbance change at 290 nm was recorded. A parallel experiment was carried out with the wild-type enzyme for the purpose of comparison with the mutant.
In another study, urea (0.5 M) was used to evaluate enzyme stability involving the wild-type and K16Q mutant AMPSases. The enzymes were incubated with 0.5 M urea at 25 °C for different periods of time (0, 1, 2, 4, 8, and 16 min) and then added to the assay solution. Initial rates were measured at 280 nm and 25 °C in solutions containing 0.5 M urea, 20 mM Hepes, pH 7.7, 1 mM MgCl2, 5 mM aspartate, 150 µM IMP, and 60 µM GTP.
Circular Dichroism SpectroscopyCircular dichroism spectra were acquired at room temperature on a Jasco spectropolarimeter, model J-710. Samples (50-300 µg/ml in 10 mM potassium Pi, pH 7.0) were placed in a 1-mm cuvette, and data points were obtained from 200 to 260 nm in 0.5-nm increments. Spectra were normalized for a direct comparison.
All transformants of
the pur A cell line (H1238) grew in the LB
medium. D13A and E14A transformants, however, grew at a slow rate,
comparable to that of the original pur A
cell
line, which must draw its entire supply of adenine from the LB
medium.
D13A, E14A, K16Q, and R131L mutants migrate comparably on phenyl-Sepharose and DEAE-HPLC columns to wild-type AMPSase. However, during concentration (Amicon concentrator) of the eluent from the phenyl-Sepharose column, the H41N mutant precipitated. The precipitated protein could be redissolved upon dilution or by increasing the concentration of KPi at pH 7 from 20 to 500 mM. Samples, precipitated and then redissolved, showed no loss of activity, ruling out the possibility of irreversible denaturation. All mutant proteins were more than 95% pure, as judged by SDS-polyacrylamide gel electrophoresis.
Circular Dichroism Spectroscopic Study of the MutantsThe CD
spectra of D13A, E14A, K16Q, and R131L mutants are almost identical to
that of wild-type AMPSase (data not shown). For these mutants, then, no
global conformational change occurs as a consequence of mutation.
However, the H41N mutant differs significantly from the wild-type
protein in its CD spectrum (Fig. 1), indicating a large
structural perturbation. The altered structure of the H41N mutant may
be responsible for its low solubility in low salt buffer. Based upon
x-ray diffraction studies of AMPSase (7), His41 hydrogen
bonds to Asp21, an interaction that may stabilize the loop
42-53 in the absence of ligands. Differences in the CD spectra of
wild-type and the H41N mutant may stem from conformational differences
in this loop structure in the absence of ligands.
D13A Mutant
The D13A mutant shows no activity using the
conventional assay, even with 1 mg/ml protein. Considering the
sensitivity of this technique (approximately 1 × 104
A change/min at 290 nm), the activity must be less
than 0.001% wild-type AMPSase. However, GTP and IMP quench the
intrinsic fluorescence of the mutant, indicating that substrates bind
to the D13A mutant. Crystal structures have revealed a hydrogen bond
between the side chain of Asp13 and N-1 of the IMP (5).
Asp13, then, may abstract the proton from N-1 to generate
the 6-oxyanion of IMP, the putative nucleophile in the attack on the
-phosphorus atom of GTP (Fig. 2). The complete loss
of activity due to the D13A mutation is entirely consistent with an
essential catalytic role for Asp13.
E14A Mutant
The E14A mutant exhibits greatly reduced activity. The kcat of E14A is too low to be measured with confidence at pH 7.7. Thus, assays were performed at pH 7.0, where the kcat of wild-type AMPSase increases by 40% and the Km values decrease (Table I). At pH 7.0 the E14A mutant had a kcat of 0.022/s (approximately 1% that of the wild-type enzyme at pH 7.0), and the Km values for substrates are 3-6-fold higher than those of the wild-type enzyme (Table I). Given that Glu14 makes hydrogen bonds that stabilize the P-loop in E. coli AMPSase (7), the dramatic fall-off in kcat of the E14A mutant may be due to a conformational perturbation on Asp13, which, as noted above, is a putative catalytic base. Alternatively, Glu14 may be essential to the electrostatic charge balance in the active site. In crystal structures, Glu14 makes a salt link with Lys16 (see below).
|
The NZ atom of Lys16 probably
interacts with the - and/or
-phosphate groups of GTP (5, 6). The
consensus P-loop lysine is putatively essential for stabilization of a
pentavalent phosphoryl group in the transition state (8) as is well
documented in p21ras (15) and adenylate kinase (16, 17).
However, for AMPSase, the kinetic parameters of the K16Q mutant (Table
I) are similar to those of the wild-type protein. The corresponding
mutant of p21ras (K16N) drastically reduces the affinity of
nucleotides (15), and the corresponding mutant of E. coli
adenylate kinase (K13Q) significantly lowers catalytic activity with a
modest effect on substrate affinity (16). The possibility that the K16Q
mutant of AMPSase may have reverted to the wild-type protein was
eliminated by confirming the sequence of the mutant plasmid in the
transformed H1238 cell line. Also we detected no endogenous AMPSase in
the H1238 E. coli cell line by Western blot analysis (data
not shown). Thus, apparently NE2 of glutamine can substitute for NZ of
Lys16 in maintaining hydrogen bonds. Furthermore, for
AMPSase, the positive charge of Lys16 may not be essential
for the stabilization of the transition state. In fact,
Lys16 hydrogen bonds to Glu14 in ligated
complexes of AMPSase, resulting in a charge-balanced ion pair. In order
for NE2 of the Gln16 mutant to take up the position of NZ
of Lys16, OE1 of Gln16 must hydrogen bond to
Glu14 (Fig. 3). Thus, the observed
Lys16-Glu14 ion pair in the wild-type
enzyme may be replaced by a neutrally charged
Gln16-Glu14 pair. The net electrostatic charge
of the mutated and wild-type active site, then, may be the same. Hence
we observe little influence on kinetic parameters. Neither adenylate
kinase nor p21ras have a P-loop residue equivalent to
Glu14.
If the mutation of Lys16 has only a modest impact on
catalysis, why then is position 16 always a lysine in all known
sequences of AMPSase? The explanation may rest with the stability of
the mutant. At room temperature, the activity of the K16Q mutant is unchanged for 90 min, but at 4 °C the activity decreases
significantly relative to that of the wild-type protein (Fig.
4). It is known that hydrophobic interactions are
weakened at lower temperatures (18). In addition, the stability of
AMPSase dimers decreases with temperature (19). It was also observed
that when the mutant and wild-type enzymes were exposed to 0.5 M urea for varied periods of time and then assayed for
activity, the K16Q enzyme was significantly less stable than its
wild-type counterpart (data not shown). Taken together, these results
suggest that the K16Q mutant is less stable than the wild-type protein,
provided that one accepts enzyme activity as a criterion of stability.
If minor structural alterations in the P-loop lessen the stability of
AMPSase, as revealed by subunit complementation experiments (19),
glutamine may not be permitted at position 16 in E. coli
AMPSase due to selective pressures of evolution. However, if one
argues that the conditions described in Fig. 4 and with 0.5 M urea may not be applicable in vivo, it becomes
difficult to explain the evolutionary preference for lysine over
glutamine at position 16.
The conservation of lysine at positions equivalent to 16 in all known sequences of AMPSase may stem alternatively from survival disadvantages associated with a 3-fold reduction in kcat. The wild-type activity of AMPSase in E. coli may barely meet the requirement of the cell for adenine nucleotides. Thus, even a 3-fold reduction in activity of the K16Q mutant relative to the wild-type enzyme could be catastrophic. In an attempt to evaluate the amount of AMPSase in E. coli (TG 1 cells), a quantitative Western blot experiment was employed with antibody against E. coli AMPSase. The signal from a crude extract of TG 1 cells was compared with that of purified AMPSase; the amount of AMPSase was approximately 1.5 mg of AMPSase/g of wet TG 1 cells (data not shown). Assuming that the weight percents of total DNA and RNA in E. coli are 1 and 6% (20), respectively, with a quarter of this nucleotide pool in the cell associated with adenine and that the free adenine nucleotide concentration (ATP, ADP, and AMP) is approximately 5 mM, it is possible to calculate the total adenine content of the bacterial cell. With a kcat value of 1.4/s for AMPSase (Table I), the calculated minimum time for a doubling of an E. coli population is approximately 21 min, close to the observed time for population doubling under optimal conditions. Therefore, an E. coli cell containing K16Q AMPSase as the endogenous enzyme should grow at a significantly slower rate than an E. coli cell containing wild-type AMPSase. Such a mutation in a critical enzyme with an extremely low turnover number may not be acceptable for cell survival in a competitive environment.
Based upon the amount of AMPSase in E. coli and its low turnover number, it is tempting to suggest that the AMPSase reaction is the rate-limiting factor in the generation time of the bacterium. Although our calculations seem to support this hypothesis, it is possible that an as yet unrecognized activator of the synthetase may negate this suggestion.
H41N MutantThe H41N mutant has broadly altered kinetic
constants relative to wild-type AMPSase, including a
kcat that is approximately 0.01/s (1% of the
wild-type enzyme) and Km substrate values increased
by 2-6-fold relative to those of the wild-type enzyme (Table I). The
uniform increase in Km values for all substrates
implies that the mutation perturbs the entire active site. A hydrogen
bond between His41 and the - and/or
-phosphoryl
groups of GTP is one of three enzyme-ligand interactions putatively
responsible for the 9-Å conformational change in the loop 42-53 (5).
The loop 42-53 folds over the guanine nucleotide; the 6-fold increase
in Km for GTP may stem from a weakened interaction
between Asn41 and the guanine nucleotide relative to
observed His41-guanine nucleotide interactions in the
wild-type system. Increases in Km for IMP and
aspartate to some extent may stem from the decrease in GTP affinity.
Synergism in the binding of IMP and GTP is suggested by studies of Wang
et al. (21). Furthermore, Mg2+ binds to guanine
nucleotides and putatively to the
-carboxylate of aspartate (5).
Thus, the observed 6-fold increase in the Km for
aspartate may be due entirely to the 6-fold increase in the
Km for GTP and, presumably, bound Mg2+.
The altered CD spectrum of the H41N mutant may not be relevant to its
kinetic properties; the CD spectrum measures the conformation of the
unligated mutant, whereas the kinetics probe the ligated mutant. The
severely depressed kcat of the H41N mutant is in
harmony with crystallographic studies that implicate His41
as a catalytic acid in the phosphotransfer
step.3
The guanidinium group of Arg131 is
close to the loop that binds to the -carboxylate group of aspartate
(Fig. 5), and its removal affects the affinity for
aspartate (Km,Asp increases 4-fold)
without a significant influence on other parameters. Mutation of
Arg303 or Arg305 to leucine increases
Km for aspartate by 100-fold (21); these residues
putatively bind directly to the
- and
-carboxylates, respectively, of aspartate. Thus, the 4-fold increase in
Km of the R131L mutant is consistent with long
range, electrostatic interactions between Arg131 and
aspartate, and the stabilization of the aspartate-bound conformation of
the loop 298-303.
Summary
Mutations at positions 13, 14, 41, and 131 are
consistent, as noted above, with their putative roles in catalysis and
conformational changes in AMPSase from E. coli. The high
activity of K16Q was not anticipated on the basis of recent
crystallographic structures of AMPSase, which clearly show
Lys16 making hydrogen bonds with a nitrate anion in the
-phosphoryl site (5) and with a Pi anion in the
-phosphoryl site and the
-phosphate group of bound GDP (6). On
the basis of crystallographic structures, the role played by
Lys16 appears to be as significant as Arg305,
His41, or Asp13, where mutations cause at least
a 99% reduction in kcat. Thus, the positive
charge on Lys16 does not play a significant role in
stabilizing the transition state. Further mutations at position 16 should reveal whether glutamine is the next best alternative to lysine
or whether other substitutions at position 16 result in mutants with
significant catalytic capacity.