©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Arginine Residues in the Putative L-Aspartate Binding Site of Escherichiacoli Adenylosuccinate Synthetase (*)

Wenyan Wang , Bradley W. Poland , Richard B. Honzatko , Herbert J. Fromm (§)

From the (1) Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa, 50011

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

Adenylosuccinate synthetase (AMPSase)() 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.

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 -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) .

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 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.


EXPERIMENTAL PROCEDURES

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.


RESULTS

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 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.

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 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.

The K 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.


DISCUSSION

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 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.

Isotope exchange experiments from our laboratory (24) suggested that aspartate prefers binding to the enzyme-substrate ternary complex (GTPIMPenzyme) 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.

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 -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

The enzyme assay solution contained 40 mM Hepes (pH 7.7) and 5 mM MgCl. 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.



FOOTNOTES

*
This research was supported in part by Grants MCB-9218763 and MCB-9316244 from the National Science Foundation and Grant NS10546 from the National Institutes of Health, United States Public Health Service. This article was journal paper J-16180 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project 2575. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 515-294-4971; Fax: 515-294-0453.

The abbreviation used is: AMPSase, adenylosuccinate synthetase.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.