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Mutagenesis and Chemical Rescue Indicate Residues Involved in β-Aspartyl-AMP Formation by Escherichia coli Asparagine Synthetase B

1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês

10.1074/jbc.272.19.12384

ISSN

1083-351X

Autores

Susan K. Boehlein, Ellen S. Walworth, Nigel G. J. Richards, Sheldon M. Schuster,

Tópico(s)

Enzyme Structure and Function

Resumo

Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of β-aspartyl-AMP in the reaction mechanism ofEscherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317–330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation β-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediatesen route to β-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis. Site-directed mutagenesis and kinetic studies have been employed to identify amino acid residues involved in aspartate binding and transition state stabilization during the formation of β-aspartyl-AMP in the reaction mechanism ofEscherichia coli asparagine synthetase B (AS-B). Three conserved amino acids in the segment defined by residues 317–330 appear particularly crucial for enzymatic activity. For example, when Arg-325 is replaced by alanine or lysine, the resulting mutant enzymes possess no detectable asparagine synthetase activity. The catalytic activity of the R325A AS-B mutant can, however, be restored to about 1/6 of that of wild-type AS-B by the addition of guanidinium HCl (GdmHCl). Detailed kinetic analysis of the rescued activity suggests that Arg-325 is involved in stabilization of a pentacovalent intermediate leading to the formation β-aspartyl-AMP. This rescue experiment is the second example in which the function of a critical arginine residue that has been substituted by mutagenesis is restored by GdmHCl. Mutation of Thr-322 and Thr-323 also produces enzymes with altered kinetic properties, suggesting that these threonines are involved in aspartate binding and/or stabilization of intermediatesen route to β-aspartyl-AMP. These experiments are the first to identify residues outside of the N-terminal glutamine amide transfer domain that have any functional role in asparagine synthesis. A great deal of interest in asparagine metabolism has resulted from the finding that certain leukemias can be treated by the administration of l-asparaginase (see review, 1Chakrabarti R. Schuster S.M. Int. J. Pediat. Hematol./Oncol. 1997; Google Scholar). Experiments suggest that the effectiveness of this protocol is dependent upon decreasing the circulating amount of asparagine (2Broome J.D. J. Exp. Med. 1968; 244: 1055-1072Crossref Scopus (159) Google Scholar). Although administration of l-asparaginase is accepted as an essential component of modern therapy, it is fraught with serious side effects and plagued by the appearance of resistant leukemias. An alternative, or adjunct, approach to the use ofl-asparaginase might be to lower circulating asparagine by inhibiting asparagine synthetase (AS), 1The abbreviations used are: AS, asparagine synthetase; AS-B E. coli asparagine synthetase B; GdmHCl, guanidinium hydrochloride; PCR, polymerase chain reaction; CSA, cysteinesulfinic acid; FSBA, 5′-fluorosulfonylbenzoyladenosine; MetRS, methionyl tRNA aminoacyl synthetase; bp, base pair(s); GAT, glutamine amide transfer; I, intermediate. the enzyme responsible for its production. Of several hundred compounds that have been evaluated as AS inhibitors, however, none have exhibited sufficient potency and specificity to warrant clinical consideration (3Cooney D.A. Driscol J.S. Milman H.A. Jayaram H.N. Davis R.D. Cancer Treat. Rep. 1976; 60: 1493-1557PubMed Google Scholar). This failure can be partly explained by the lack of detailed mechanistic information on AS. Two classes of enzymes catalyzing asparagine synthesis have been described that possess no sequence similarity and may consequently have arisen by convergent evolution. Ammonia-dependent asparagine synthetases in prokaryotes such as Klebsiella aerogenes andEscherichia coli (4Reitzer L.J. Magasanik B. J. Bacteriol. 1982; 151: 1299-1313Crossref PubMed Google Scholar, 5Cedar H. Schwartz J.H. J. Biol. Chem. 1969; 244: 4112-4121Abstract Full Text PDF PubMed Google Scholar, 6Cedar H. Schwartz J.H. J. Biol. Chem. 1969; 244: 4122-4127Abstract Full Text PDF PubMed Google Scholar) can employ only ammonia as a nitrogen source (Reaction 1). 6–17141 L­Asp+NH3+ATP→L­Asn+AMP+PPi(REACTION1) L­Asp+L­Gln+ATP→L­Asn+L­Glu+AMP+PPi(REACTION2) L­Gln→L­Glu+NH3a(REACTION3)The second group of asparagine synthetases, on the other hand, is present in both prokaryotes and eukaryotes and employs glutamine as the predominant source of nitrogen in obtaining asparagine from aspartate and ATP (Reaction 2), although ammonia can be employed as an alternative to glutamine (7Humbert R. Simoni R.D. J. Bacteriol. 1980; 142: 212-220Crossref PubMed Google Scholar, 8Mehlhaff P. Luehr C.A. Schuster S.M. Biochemistry. 1985; 24: 1104-1110Crossref PubMed Scopus (12) Google Scholar, 9Van Heeke G. Schuster S.M. J. Biol. Chem. 1989; 264: 5503-5509Abstract Full Text PDF PubMed Google Scholar). In addition, this class of synthetases acts as glutaminases in the absence of aspartate (Reaction 3). E. coli contains two unlinked genes coding for asparagine synthetases (7Humbert R. Simoni R.D. J. Bacteriol. 1980; 142: 212-220Crossref PubMed Google Scholar). Asparagine synthetase A (AS-A), the product of the 990-bp asnA gene for which the complete nucleotide sequence is known, has been isolated and exhibits strictly ammonia-dependent activity (5Cedar H. Schwartz J.H. J. Biol. Chem. 1969; 244: 4112-4121Abstract Full Text PDF PubMed Google Scholar, 6Cedar H. Schwartz J.H. J. Biol. Chem. 1969; 244: 4122-4127Abstract Full Text PDF PubMed Google Scholar, 10Nakamura M. Yamada M. Hirota Y. Sugimoto K. Oka A. Takanami M. Nucleic Acids Res. 1981; 9: 4669-4676Crossref PubMed Scopus (46) Google Scholar). The nucleotide sequence for the 1662-bp asnB gene, encoding asparagine synthetase B (AS-B), has also been cloned and sequenced (11Scofield M.A. Lewis W.S. Schuster S.M. J. Biol. Chem. 1990; 265: 12895-12902Abstract Full Text PDF PubMed Google Scholar). Based on its primary amino acid sequence, AS-B is a purF (class II) amidotransferase, possessing an N-terminal cysteine residue that is essential for glutamine-dependent activity (12Zalkin H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 203-309PubMed Google Scholar). Our recent work describing a number of site-specific AS-B mutants has identified specific amino acids in the N-terminal glutamine amide transfer (GAT) domain that are critical to glutamine-dependent nitrogen transfer (13Boehlein S.K. Richards N.G.J. Schuster S.M. J. Biol. Chem. 1994; 269: 7450-7457Abstract Full Text PDF PubMed Google Scholar, 14Boehlein S.K. Richards N.G.J. Walworth E.S. Schuster S.M. J. Biol. Chem. 1994; 269: 26789-26795Abstract Full Text PDF PubMed Google Scholar). Detailed kinetic analysis of wild-type enzyme and these mutants using alternate substrates and heavy atom isotope effects has also yielded new insights into the mechanistic role of the AS-B GAT domain (15Stoker P.W. O'Leary M.H. Boehlein S.K. Schuster S.M. Richards N.G.J. Biochemistry. 1996; 35: 3024-3030Crossref PubMed Scopus (26) Google Scholar, 16Boehlein S.K. Schuster S.M. Richards N.G.J. Biochemistry. 1996; 35: 3031-3037Crossref PubMed Scopus (24) Google Scholar). Identification of key residues in the N-terminal region of AS-B was aided by the availability of sequences for the glutamine-utilizing domains of other purF amidotransferases. On the other hand, both BLAST and FASTP analyses indicate that the primary structure of the C-terminal synthetase domain in asparagine synthetases is unique, with the exception of a small region, termed the P-loop-like motif, that is present in a number of ATP-dependent enzymes that release AMP and PPi as reaction products (17Bork P. Koonin E.V. Proteins Struct. Funct. Genet. 1994; 20: 347-355Crossref PubMed Scopus (112) Google Scholar). The involvement of this motif in ATP utilization during asparagine synthesis, however, has yet to be validated using site-directed AS mutants. While raising interesting evolutionary questions, the observation that sequence similarities between AS-A and AS-B do not exist (11Scofield M.A. Lewis W.S. Schuster S.M. J. Biol. Chem. 1990; 265: 12895-12902Abstract Full Text PDF PubMed Google Scholar) complicates the identification of critical catalytic residues in the C-terminal AS-B synthetase domain. Multiple sequence alignments for several glutamine-dependent asparagine synthetases show 152 conserved residues and 92 positions in which conservative replacements are present. Of the seven regions possessing four, or more, consecutive conserved residues, two of these are in the GAT domain and appear to mediate only nitrogen transfer and glutamine hydrolysis. In this paper, we present the results of random, and site-specific, mutagenesis experiments on two of the other five regions defined by residues 317–330 and 484–500, respectively (Fig. 1). While the latter region does not appear to possess a functional role in catalysis and/or substrate binding, we report evidence supporting the hypothesis that Arg-325, Thr-322, and Thr-323 mediate β-aspartyl-AMP formation, a key intermediate in asparagine biosynthesis (5Cedar H. Schwartz J.H. J. Biol. Chem. 1969; 244: 4112-4121Abstract Full Text PDF PubMed Google Scholar, 18Leuhr C.A. Schuster S.M. Arch. Biochem. Biophys. 1985; 237: 335-346Crossref PubMed Scopus (25) Google Scholar). Restriction and modifying enzymes were purchased from Promega (Madison, WI), Life Technologies, Inc., or New England Biolabs (Beverly, MA). Deoxyadenosine 5′-[α-35S]thiotriphosphate triethylammonium salt (Sp isomer, 1000 Ci/mmol) was purchased from Amersham Corp. All other reagents were the highest possible quality. Oligonucleotide primers were synthesized on an Applied Biosystems 380B DNA synthesizer by the DNA Synthesis Core Facility of the Interdisciplinary Center for Biotechnology Research at the University of Florida. Polymerase chain reactions (PCR) were performed on an Ericomp (San Diego, CA) thermocycler using the GeneAmp DNA Amplification Reagent Kit with AmpliTaq from Perkin-Elmer. Thirty-five cycles consisting of denaturing at 94 °C for 1 min, annealing at 54 °C for 1 min during megaprimer reactions or 52 °C for 1 min during megaproduct reactions, and extension at 72 °C for 1 min were followed by a 10-min completion cycle at 72 °C. Megaprimers were purified by polyethylene glycol precipitation (0.6 volumes of 20% polyethylene glycol 8000 in 2.5 m NaCl were added to the PCR reaction, incubated at 37 °C for 10 min, centrifuged at 10,000 rpm for 10 min, and washed with 2 volumes of 80% EtOH) or agarose gel electrophoresis. After gel electrophoresis, the PCR product was extracted using a Gene CleanII Kit from Bio 101, Inc. (Vista, CA). Double-stranded DNA sequencing was performed using the U. S. Biochemical Corp. Sequenase 2.0 Sequencing kit. Preparation of the template for sequencing was performed by the alkaline lysis method. All strains were derivatives of E. coli K-12: BL21DE3pLys S (F−, ompT, rb−, mb−) generously supplied by Studier and Moffatt (19Studier W.F. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google Scholar), while NM522 (sup E, thi (lac-proAB), hsd5, (r− m−-)/F′ pro AB, lac Iq Z M15) and plasmid pBluescript were obtained from Stratagene (La Jolla, CA). Plasmid pETB was prepared as described previously (13Boehlein S.K. Richards N.G.J. Schuster S.M. J. Biol. Chem. 1994; 269: 7450-7457Abstract Full Text PDF PubMed Google Scholar). E. coli host cells were transformed according to the procedure of Hanahan (20Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8216) Google Scholar). The amino acid sequences of known glutamine-dependent asparagine synthetases were aligned using a simplification of the progressive alignment method of Feng and Doolittle (21Feng D.F. Doolittle R. J. Mol. Evol. 1987; 35: 351-360Crossref Scopus (1541) Google Scholar), as implemented in the program PILEUP in the GCG Sequence Analysis Software Package (22Devereux J.R. Haeberli P. Smithies O. Nucleic Acids Res. 1984; 12: 387-395Crossref PubMed Scopus (11531) Google Scholar). All directed random mutants were constructed using the PCR megaprimer strategy (23Satar G. Sommers S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar) using template pETB, and oligonucleotide primer pairs SS123 and SS82 or SS124 and SS182 (Table I). Primer SS123 and SS124 contained degenerate oligonucleotide sequences for the construction of the mutants and a silent change inserting a restriction site, which was used for screening. The megaproduct was constructed using the megaprimer and SS65, which was 5′ of the coding region of the gene. The final product was cut with the appropriate restriction enzymes, gel-purified, and ligated back into pETB. The identity of the mutated insert was verified by sequencing.Table IOligonucleotides used in construction of site-directed mutants of AS-BMutationOligo numberOligonucleotide sequenceN terminusSS655′ A GCT TCC CAT ATG TGT TCA ATT TTT GGC GTA TTC GAT 3′C terminusSS825′ CGC TTT GTT GGC ACG CGC GCA GTC 3′N terminusSS2245′ GGT CAG CTG TAT GTG GCC TCA G 3′kpn siteSS1485′ AAC CAT CTC GGT ACC GTG CAT CAC 3′Random 1SS1235′ gas acT TaT gaT gTg acc acT aTT cgc gcG TCG ACa ccg aTg TAT TTA 3′Random 2SS1245′ cgc TTC ccg Tac aac acg cca acc agT aaa gAA GCT Tac cTg TaT CGG GAG 3′R325XSS1955′ CAT CGG TGT CGA CGC *** AAT AGT GGT 3′T322YSS1965′ CAT CGG TGT CGA CGC GCG AAT AGT ATA CAC 3′T323XSS1975′ CAT CGG TGT CGA CGC GCG AAT *** GGT 3′T322VSS3515′ CAT CGG TGT CGA CGC GCG AAT AGT GAC CAC 3′T322SSS3525′ CAT CGG TGT CGA CGC GCG AAT AGT GGA CAC 3′T322ASS3535′ CAT CGG TGT CGA CGC CGC GCG AAT AGT GGC CAC 3′T323VSS3545′ CAT CGG TGT CGA CGC GCG AAT AAC GGT C 3′T323SSS3555′ CAT CGG TGT CGA CGC GCG AAT AGA GGT C 3′T323ASS3565′ CAT CGG TGT CGA CGC GCG AAT AGC GGT C 3′R325ASS3575′ CAT CGG TGT CGA CGC GGC AAT AG 3′R325KSS3585′ CAT CGG TGT CGA CGC CTT AAT AG 3′V321ASS3595′ CAT CGG TGT CGA CGC GCG AAT AGT GGT CGC ATC 3′D320ASS3605′ CAT CGG TGT CGA CGC GCG AAT AGT GGT CAC AGC ATA 3′Y319ASS3615′ CAT CGG TGT CGA CGC GCG AAT AGT GGT CAC ATC AGC AGT TT 3′T318ASS3625′ CAT CGG TGT CGA CGC GCG AAT AGT GGT CAC ATC ATA AGC TTC G 3′E317ASS3635′ CAT CGG TGT CGA CGC GCG AAT AGT GGT CAC ATC ATA AGT TGC GAT G 3′E317QSS3705′ CAT CGG TGT CGA CGC GCG AAT AGT GGT CAC ATC ATA AGT TTG GAT G 3′The codes for oligonucleotides synthesis are as follows: a, 91% A, 3% T, 3% G, 3% C; c, 91% C, 3% T, 3% G, 3% A; g, 91% G, 3% T, 3% C, 3% A; *, 25% A, 25% T, 25% G, 25% C. Open table in a new tab The codes for oligonucleotides synthesis are as follows: a, 91% A, 3% T, 3% G, 3% C; c, 91% C, 3% T, 3% G, 3% A; g, 91% G, 3% T, 3% C, 3% A; *, 25% A, 25% T, 25% G, 25% C. Site-directed mutants with single amino acid changes were obtained as follows. A mutant in which Thr-322 was replaced by serine (T322S), produced in the directed random mutagenesis experiments, was used as the template to construct a cloning cassette. This template was chosen because a unique SalI site, 3′ of the mutagenic area, was created during the original mutagenesis. Megaprimer strategy was again utilized with primers SS196 and SS148 (Table I) creating a uniqueKpnI site 5′ of the mutagenic area. The megaproduct reaction utilized the megaprimer and SS224 (Table I). This 534-bp product was digested with PvuII and SalI and cloned into template T322S, thus creating a new template pETB-KS, containing a 126-bp cassette between the KpnI and SalI sites. pETB-KS was then used as the template to construct single random mutants of Thr-323 and Arg-325. SS148, containing the KpnI site and the corrected sequence for Thr-322, along with SS197 (Thr-323) or SS195 (Arg-325) were used to generate a 126-bp product which was cut by SalI and KpnI and cloned into pETB-KS digested with the same enzymes. Additional site-specific mutations were generated utilizing pETB-KS as a template and SS224 and SS351-SS363 and SS370 (Table I) to produce T322V, T322S, T322A, T323V, T323S, T323A, R325A, R325K, V321A, D320A, Y319A, T318A, E317A, and E317Q. The 534-bp product was digested withSalI and KpnI and cloned into pETB-KS digested with the same enzymes. The identity of mutated inserts generated by PCR and the adjoining cloning sites was confirmed by sequencing before protein expression. Several preliminary assays were performed to determine which AS-B mutants warranted further investigation. First, the solubility of the mutant protein was evaluated by SDS-polyacrylamide gel electrophoresis. Second, the soluble cellular fractions containing crude overexpressed AS-B or AS-B mutant enzymes were assayed by measuring the conversion of aspartate to asparagine as monitored by high performance liquid chromatography amino acid analysis on an Applied Biosystems 420A derivatizer and 130A separation system. Standard assay conditions were as follows: 100 mm NH4OAc or 10 mmglutamine, 10 mm ATP, 10 mml-aspartate, 17 mm Mg(OAc)2, and 50 mm Tris-HCl, pH 7.5. Reactions were initiated with 10 μl of crude extract and incubated at 37 °C for 15 min before being terminated by the addition of trichloroacetic acid, to a 4% final concentration, and filtered through a 0.2-μm syringe filter. All reactions were performed in duplicate. Procedures for the purification and expression of pETB and mutant enzymes have been described elsewhere (13Boehlein S.K. Richards N.G.J. Schuster S.M. J. Biol. Chem. 1994; 269: 7450-7457Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined with an assay kit supplied by Bio-Rad using γ-globulin to construct a standard curve. Apparent affinity constants (Km(app)) for AS-B substrates were determined by incubating purified wild-type or mutant AS-B in reaction mixtures (total volume 160 μl) in which all but one of the substrates were saturating, i.e. at approximately 10 times theirKm(app) value, unless otherwise noted. The highest aspartate concentration used in these experiments was 100 mm, so AS-B mutants for which the apparent aspartateKm(app) was greater than 10 mm were not saturated by this substrate. All assays contained 100 mm Tris-HCl, pH 8.0, 8 mmMgCl2, and the appropriate purified enzyme (3–15 μg). A 10-fold variation in substrate, centered aboutKm(app), was used to determine the effect of substrate concentration for both wild-type and mutant enzymes. The initial velocity of each reaction was determined spectrophotometrically by following the production of pyrophosphate during asparagine synthesis (Sigma, Technical Bulletin No. BI-100). Each assay was run two to four times, and the averages are presented. Rate and concentration data were fit to the Michaelis-Menten equation to give Km(app) andVmax using the software program Graph Pad Prism (Graph Pad, San Diego, CA). The glutaminase activity of the AS-B mutants was assayed by measuring the formation of glutamate using the reaction of glutamate dehydrogenase in the presence of NAD+(24Bernt E. Bergmeyer H.U. Bergmeyer H.U. Methods of Enzymatic Analysis. Academic Press, New York1974: 331-358Google Scholar). Reaction mixtures (100 μl total volume) contained 100 mm Tris-HCl, pH 8, and 8.0 mm MgCl2with varying concentrations of glutamine (0.5–10 mm). Reactions were initiated by the addition of purified wild-type AS-B or mutant enzyme (0.83 μg) and were incubated for 18 min before being terminated by the addition of 20 μl of 1 n AcOH. The reaction mixture was then added to 380 μl of the coupling reagent (300 mm glycine, 250 mm hydrazine, pH 9, 1 mm ADP, 1.6 mm NAD+, and 2.2 units of glutamate dehydrogenase) and incubated for 10 min at room temperature. The solution absorbance was measured at 340 nm, the amount of glutamate present being determined from a standard curve. Chemical rescue experiments were performed by incubating purified R325A (10.8 μg), R325K (13.5 μg), or wild type AS-B (3.9 μg) with 50 mm aspartate, 10 mmATP, 10 mm glutamine, 15 mm MgCl2, 100 mm Tris-HCl, pH 8, and varying concentrations of guanidinium HCl (GdmHCl), methylamine, urea, thiourea, tetramethylguanidine, or methylguanidine (0.5–50 mm). Asparagine synthesis was quantified by measuring the amount of pyrophosphate released in the enzyme-catalyzed reaction. Independent high performance liquid chromatography experiments were employed to ensure that pyrophosphate and asparagine were formed in a 1:1 ratio under these reaction conditions. All assays were performed in triplicate. An initial evaluation of the functional importance of amino acid residues located in regions 317–330 and 484–500 was carried out using “directed random mutagenesis.” Oligonucleotides SS123 and SS124 (Table I), having a calculated average of two mismatches per oligonucleotide, were used to create the two sets of mutations. Twelve independent clones containing mutations in region 317–330,and 17 independent clones for region 484–500 were evaluated (Table II). Each of these clones was characterized by sequence determination of the inserts and measurement of glutaminase and asparagine synthetase activities in extracts of the expressed protein. Although random mutagenesis of region 484–500 gave eight double mutants, two triple mutants, and seven single mutants, representing a variety of conservative replacements (T489S, S492T, and F485L) and changes in local charge and potential structural modifications (P486L:T489K, R484D:E500K, T489K:E500K, and R484C:P486A:E500K), all of the associated proteins retained their ability to catalyze asparagine synthesis (Table II). We therefore conclude that residues 484–500 are not involved in direct catalysis and/or substrate binding.Table IIActivity of the AS-B mutants obtained using directed random mutagenesisMutantAS activity +/−MutantAS activity +/−Mutants with changes in the region defined by residues 317–330E317V−E317V:P329A−Y319F+Y319S:T322I−T322P−T322A:M330I−T322M−T322N:T323N−T322S+E317V:T322S:T323P−T328S+T318S:D320A:T323A−Mutants with changes in the region defined by residues 484–500F485L+P486L:T489K+F485C+R484D:E500K+P486R+P486L:E500K+Y487C+T489K:E500K+T489S+T489M:E500K+S492T+T491S:E500K+E494Q+R484C:P486A:E500K+F485L:S492C+N488I:T491P:A495F+P486L:N488D+ Open table in a new tab In contrast, of the six mutant enzymes associated with single mutations in region 317–330, three were unable to synthesize asparagine, and of the six mutants containing multiple alterations in their sequence, none had detectable AS activity (Table II). All of these mutants were soluble and retained their ability to catalyze glutamine hydrolysis (data not shown), suggesting that gross conformational changes were not responsible for the concurrent loss of synthetase activity. Three AS-B single mutants retaining synthetase activity (Y319F, T322S, and T328S) were then purified according to standard procedures (13Boehlein S.K. Richards N.G.J. Schuster S.M. J. Biol. Chem. 1994; 269: 7450-7457Abstract Full Text PDF PubMed Google Scholar), and kinetic constants for their glutaminase (Table III) and synthetase (Tables IV and V) activities were determined. The synthetase activity of site-specific mutants was fully characterized only if the mutant AS-B exhibited essentially unaltered kinetic parameters for glutaminase activity, in the presence and absence of ATP, relative to wild-type enzyme. The observation of unchanged ATP-dependent stimulation of glutaminase activity provided additional evidence that the ATP-binding site was likely unchanged by the mutation. In the case of all three AS-B mutants, the kinetic parameters for glutamine hydrolysis were comparable to those of the wild-type enzyme. On the other hand, although three of these site-specific AS-B mutant enzymes had similar specificity constants, measured as kcat/Km, to those of wild-type AS-B for all substrates, a substantial decrease inkcat andKm(app) for ATP was observed in the glutamine synthetase activity of the T322S mutant.Table IIIKinetic constants for the glutaminase activity of wild-type AS-B and AS-B mutantsMutantGlutamine + 5 mmATPGlutamine onlyKmkcatkcat/KmKmkcatkcat/Kmmms −1m −1 s −1mms −1m −1 s −1wt AS1.39 ± 0.071.38 ± 0.029931.94 ± 0.180.80 ± 0.03412E317A6.12 ± 0.450.59 ± 0.01968.44 ± 0.480.64 ± 0.0176E317Q2.60 ± 0.070.18 ± 0.004694.01 ± 0.350.18 ± 0.00445T318A1.04 ± 0.081.30 ± 0.0312501.45 ± 0.171.23 ± 0.05848Y319A1.15 ± 0.101.24 ± 0.0310781.23 ± 0.210.38 ± 0.02309Y319F1.03 ± 0.091.0 ± 0.029681.20 ± 0.100.67 ± 0.02556D320A1.89 ± 0.181.49 ± 0.057883.14 ± 0.331.35 ± 0.06430V321A1.11 ± 0.061.16 ± 0.0210451.51 ± 0.271.06 ± 0.06702T322A1.34 ± 0.081.49 ± 0.0311121.39 ± 0.190.97 ± 0.04698T322S1.23 ± 0.081.60 ± 0.0313011.42 ± 0.161.12 ± 0.04789T322V1.21 ± 0.071.72 ± 0.0314211.78 ± 0.221.34 ± 0.06753T322Y0.90 ± 0.070.57 ± 0.016331.11 ± 0.100.44 ± 0.01396T323A0.98 ± 0.050.86 ± 0.018781.38 ± 0.080.77 ± 0.01558T323I0.71 ± 0.040.74 ± 0.0110420.95 ± 0.070.54 ± 0.01568T323L0.94 ± 0.090.79 ± 0.028401.57 ± 0.200.80 ± 0.03510T323S0.84 ± 0.110.74 ± 0.038811.64 ± 0.160.69 ± 0.02610T323V1.14 ± 0.101.11 ± 0.039741.37 ± 0.190.94 ± 0.04686R325A1.59 ± 0.250.77 ± 0.044842.52 ± 0.160.84 ± 0.02333R325K0.91 ± 0.050.71 ± 0.017801.43 ± 0.140.70 ± 0.02490T328S0.92 ± 0.040.71 ± 0.017722.18 ± 0.260.49 ± 0.02225All initial rates were determined measuring glutamate production in a coupled assay. Open table in a new tab Table IVKinetic constants for the glutamine-dependent synthetase activity of wild-type AS-B and the AS-B mutantsMutantGlutamineAspartateATPKmkcatkcat/KmKmkcatkcat/KmKmkcatkcat/Kmmms −1m −1 s 1mms −1m −1 s −1mms −1m −1 s −1wt AS-B0.69 ± 0.071.01 ± 0.0514630.68 ± 0.071.05 ± 0.0415440.18 ± 0.011.10 ± 0.036111E317A3.73 ± 0.420.55 ± 0.021474.67 ± 0.680.52 ± 0.031110.77 ± 0.120.62 ± 0.03805E317Q3.67 ± 0.500.15 ± 0.014117.6 ± 2.40.086 ± 0.00450.80 ± 0.080.13 ± 0.004163T318A0.33 ± 0.020.25 ± 0.0037576.60 ± 0.540.33 ± 0.01500.24 ± 0.020.34 ± 0.021416Y319A0.68 ± 0.070.59 ± 0.018681.07 ± 0.070.56 ± 0.015230.17 ± 0.010.69 ± 0.014058Y319F0.43 ± 0.080.52 ± 0.0412090.82 ± 0.080.71 ± 0.028660.15 ± 0.020.71 ± 0.024733D320A0.67 ± 0.090.25 ± 0.013732.60 ± 0.180.28 ± 0.011120.18 ± 0.020.30 ± 0.011667V321A0.57 ± 0.030.74 ± 0.0212983.1 ± 0.20.72 ± 0.012320.48 ± 0.061.2 ± 0.12500T322A0.43 ± 0.080.14 ± 0.013302.1 ± 0.20.24 ± 0.011140.068 ± 0.0060.16 ± 0.012353T322S0.19 ± 0.030.13 ± 0.016841.20 ± 0.090.16 ± 0.011350.049 ± 0.0040.170 ± 0.0043469T322V0.18 ± 0.030.045 ± 0.0022501.28 ± 0.090.041 ± 0.001320.06 ± 0.010.058 ± 0.003935T322Y0.39 ± 0.070.022 ± 0.0015740.7 ± 8.50.044 ± 0.0041.080.17 ± 0.030.030 ± 0.002171T323AaKinetic constants for this mutant were determined using an end point assay.0.42 ± 0.030.054 ± 0.00112832.2 ± 3.00.066 ± 0.0032.050.20 ± 0.020.050 ± 0.001250T323I0.50 ± 0.020.240 ± 0.00448015.6 ± 0.80.270 ± 0.00517.20.37 ± 0.040.29 ± 0.01783T323L0.41 ± 0.030.23 ± 0.0156120.1 ± 1.00.210 ± 0.00110.50.12 ± 0.010.220 ± 0.0041833T323S0.30 ± 0.020.47 ± 0.0115663.50 ± 0.250.49 ± 0.011400.21 ± 0.020.64 ± 0.033047T323V0.30 ± 0.030.35 ± 0.0111178.60 ± 0.410.39 ± 0.01450.11 ± 0.020.41 ± 0.023727T328S0.45 ± 0.070.42 ± 0.029330.55 ± 0.060.39 ± 0.017090.15 ± 0.020.77 ± 0.055133The T323F, R325A, R325K, R325H, R325I, R325L, R325T, and R325Q AS-B mutants had no detectable synthetase activity.a Kinetic constants for this mutant were determined using an end point assay. Open table in a new tab Table VKinetic constants for the ammonia-dependent activity of wild-type AS-B, AS-B mutantsMutantAmmoniaAspartateKmkcatkcat/KmKmkcatkcat/Kmmms −1m −1 s −1mms −1m −1 s −1wt AS-B12.0 ± 0.80.69 ± 0.02581.02 ± 0.060.84 ± 0.02824E317A4.5 ± 0.430.69 ± 0.0215321.3 ± 3.60.78 ± 0.0436E317Q8.5 ± 0.40.12 ± 0.0021473.8 ± 19.60.16 ± 0.022.2T318A10.17 ± 1.050.26 ± 0.012611.6 ± 1.30.23 ± 0.0119.8Y319A17.4 ± 1.40.56 ± 0.02322.08 ± 0.250.45 ± 0.01216Y319F8.14 ± 1.180.72 ± 0.04880.99 ± 0.200.45 ± 0.04455D320A16.6 ± 1.60.20 ± 0.01123.79 ± 0.390.14 ± 0.0137V321A22.6 ± 1.90.85 ± 0.04384.16 ± 0.300.64 ± 0.02154T322A13.4 ± 2.20.080 ± 0.00661.31 ± 0.200.11 ± 0.0184T322S11.1 ± 1.80.16 ± 0.01141.80 ± 0.300.14 ± 0.0178T322V4.50 ± 0.70.040 ± 0.0018.91.83 ± 0.300.039 ± 0.00421T322Y10.6 ± 1.60.020 ± 0.002.0737.5 ± 4.50.021 ± 0.0010.53T323A2.60 ± 0.050.16 ± 0.0012749.0 ± 3.20.110 ± 0.0042.2T323I23.3 ± 1.30.36 ± 0.00815.442.5 ± 1.60.43 ± 0.00710.1T323L12.05 ± 1.700.21 ± 0.0081728.4 ± 4.10.33 ± 0.0212T323S9.13 ± 0.860.56 ± 0.02614.33 ± 0.520.47 ± 0.02109T323V12.08 ± 0.690.38 ± 0.013110.5 ± 1.50.38 ± 0.0236T328S9.4 ± 1.30.47 ± 0.02500.78 ± 0.130.59 ± 0.03756The T323F, R325A, R325K, R325H, R325I, R325L, R325T, and R325Q AS-B mutants had no detectable synthetase activity. Open table in a new tab All initial rates were determined measuring glutamate production in a coupled assay. The T323F, R325A, R325K, R325H, R325I, R325L, R325T, and R325Q AS-B mutants had no detectable synthetase activity. The T323F, R325A, R325K, R325H, R325I, R325L, R325T, and R325Q AS-B mutants had no detec

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