J. Biochem. 107, 180-183 (1990)
Identification of the Reactive Cysteinyl Residue and ATP Binding Site in Bacillus cereus Glutamine Synthetase by Chemical Modification Yoshio Nakano, Masahiro I ton,1 Eiichi Tanaka, and Kinuko Kimura2 Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo 171 Received for publication, August 15, 1989
Glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming)] [EC 6.3.1.2] is an ammonia assimilatory enzyme which also functions as a key regulator of nitrogen metabolism in many organisms. The enzyme has a molecular weight of ~ 600,000 and is composed of 12 identical subunits (1). Enzymatic activity of glutamine synthetase is regulated by an adenylylation-deadenylylation system in Escherichia coli and several other Gram-negative bacteria (2, 3). In Bacillus species (1, 4-6), no evidence has been obtained to suggest covalent modification of the enzyme. Several amino acid residues at or near the active site of E. coli glutamine synthetase have been reported. Pinkofsky et al reported that an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), reacted with lysine 47 causing a loss of activity (7). Di Ianni and Villafranca reported lysines 383 and 352 reacted with pyridoxal 5'-phosphate, a chemical modification reagent which reacts with lysine residues at a phosphate binding site, and each modification resulted in partial loss of activity (8). To date there have been comparatively few reports pertaining to the active site of Bacillus glutamine synthetase. In a preliminary communication, we reported that modification with an SH reagent, iodoacetamide, inactivated only Mg2*-dependent activity of Bacillus cereus glutamine synthetase, whereas modification with FSBA inactivated only Mn2+-dependent activity (9). Glutamine synthetase of Bacillus subtilis and B. cereus requires both Mn2+ and Mg2* for high activity, and a change in the ratio of Mn2+ and Mg2* concentrations has a marked effect on the activity (10). We suggested that Mn2+ and Mg2* do not share a common binding site. 1
Present address: Department of Bio-Engineering, Tokyo Institute of Technology, Meguro-ku, Tokyo 152. 1 To whom correspondence should be addressed. Abbreviations: IAEDANS, JV-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid; FSBA, 5'-p-fluorosulfonylbenzoylfldenosine; CBS, 4-carboxybenzenesulfonyl; HPLC, high-performance liquid chromatography.
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In this paper, we determined the positions of the amino acid residues modified by a fluorescent SH reagnet, N[ [ (iodoacetyl) amino ] ethyl ] - 5 - naphthylamine -1 - sulfonic acid (IAEDANS) and by FSBA in the primary strucutre of B. cereus glutamine synthetase, which was determined recently (11). MATERIALS AND METHODS Materials—E. coli YMC11 [endAl, thil,hsdRn,supEM, AlacUim, hutC*, 4(glnA-ntrC)2000] (12) harboring pCGSl was grown in M9 medium containing 0.1% glutamate instead of 0.1% NH^Cl. pCGSl is a pBR329 derivative containing the glnA gene of B. cereus IFO3131 (11). Glutamine synthetase was purified and the biosynthetic activity of the enzyme was measured as described previously (10). IAEDANS was purchased from Aldrich Chemical and FSBA was purchased from Sigma Chemical. All other chemicals were of the highest grade commercially available. Chemical Modification with IAEDANS—la order to modify one SH group in glutamine synthetase, the enzyme (0.32 mg/ml) was treated with 3 mM IAEDANS in 100 mM Tris-HCl buffer, pH 9.0, containing 0.5 mM MnCl2, at 37*C. The reaction with IAEDANS was terminated by addition of 20 mM 2-mercaptoethanol. The reaction mixture was applied to 1.5 x 10 cm column of hydroxyapatite equilibrated with 1 mM K-phosphate buffer (pH 6.1). The column was washed with 50 ml of the same buffer and eluted with 50 mM K-phosphate buffer (pH7.6). The absorbance at 280 nm was monitored and the fractions containing the protein were pooled. The modified enzyme solution was lyophilized. Chemical Modification with FSBA—The concentrations of stock FSBA solutions were determined from their absorbance at 232nm (£ = 18.8cm- I -mM-'). A 6-fold excess of FSBA per mol of subunit was added to an aliquot of enzyme (30//M) in 100 mM Tris-HCl buffer, pH7.2, J. Biochem.
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Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, iV-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-l-sulfonic aicd (IAEDANS), or an ATP analog, 5 -p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg}+-dependent activity and activated Mn2+-dependent activity. FSBA inactivated only Mn2+-dependent activity. Mg2* plus Mn2+-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.
Reactive SH Group of B. cereus Glutamine Synthetase
300 A (19x150 mm, Waters) equilibrated with 0.1% trifluoroacetic acid in H 2 0. The adenosyl moiety of the modified peptide was lost under these conditions and CBS-peptide was purified. Tryptic Digestion—The enzyme modified by IAEDANS or purified CBS-peptide was digested with trypsin in 100 mM Tris-HCl (pH 8.0) containing 2 M guanidine-HCl at 37'C. TPCK-trypsin [1 : 100 (w/w)] was added three times at 24 h intervals. After 72 h, the reaction mixtures were applied to a column of TSK-ODS 120T (4.6x150 mm, Tosoh) equilibrated with 0.1% trifluoroacetic acid in H 2 0. HPLC was carried out using a Waters 600 apparatus and fluorescence was detected with a JASCO FP-210 fluorometer. Sequence Analysis—Automated Edman degradation of the purified peptides and determination of phenylthiohydantoin derivatives were performed with an Applied Biosystems sequencer system. RESULTS
240
2S0
320
3BO
400 240 2O0
200
300
320
Wavelength (run)
Fig. 1. A: UV-absorption spectrum of glutamine synthetase modified by IAEDANS (0.35 mg/ml). B: UV-absorption spectra of native (dotted line) and FSBA-labeled enzyme (solid line).
Chemical Modification with IAEDANS—The purified glutamine synthetase was treated with 3 mM IAEDANS for 45 min. Mg2+-dependent activity was inactivated to 50% of the initial activity, in contrast to the activation of Mn2+-dependent activity to 140%. Inactivation of Mg2"1" plus Mn2+-dependent activity was similar to that of Mg2*-dependent activity. The number of reactive sulfhydryl groups was determined to be 12 per molecule of the enzyme, or 1 per subunit, from the ratio of the absorbances at 337 and 280 nm of the enzymne modified by IAEDANS (Fig. 1A). The extinctions of IAEDANS at 337 and 280 nm were taken as 6.8x10' and 1.06x10'M-'-cnr 1 , respectively (13). After inactivation of Mg2*-dependent activity with IAEDANS, the enzyme was digested by trypsin. The peptides were then subjected to HPLC. Elution profiles are shown in Fig. 2, detecting absorption at 210 nm and fluorescence. Figure 2B shows a single peak exhibiting fluoresTABLE I. Amino acid sequence analysis of the AEDANSlabeled peptide. The yields of phenylthiohydantoin amino acid residues in picomoles are indicated. The analysis was started with 666 pmol of AEDANS-labeled peptide. The actual glutamine synthetase sequence in this region from position 298-315 is given. The site of labeling is denoted by X (no phenylthiohydantoin derivative was detected).
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Tlme(mfn) Fig. 2. Separation of peptides obtained by trypsin digestion of SH-labeled glutamine synthetase. The digest was applied to a column (TSK-ODS 120T, 4.6x150 mm, Tosoh) equilibrated with 0.1% trifluoroacetic acid. Peptides were eluted with an increasing gradient of acetonitrile (2%/min) from 0 to 80%. Theflowrate was 1.0 ml per min. Column eluates were monitored by measurement of UV absorption (210 nm) and fluorescence (excitation 337 nm, emission 490 nm). Vol. 107, No. 2, 1990
Cycle
Amino acid
pmol
Glutamine synthetase 298-315
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Leu Val Pro Gly Tyr Glu Ala Pro X Tyr Val Ala Trp
459 498 237 272 225 166 469 258 — 212 326 373 135 52 241 112 110 77
Leu Val Pro Gly Tyr Glu Ala Pro Cys Tyr Val Ala Trp Ser Ala Gin Asn Arg
Ser Ala Gin Asn Arg
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containing 0.5 mM MnCl2 and 50 mM L-glutamate, at 3TC. The reaction with FSBA was terminated by adding of 20 mM 2-mercaptoethanol. Solid ammonium sulfate was added to 70% saturation and the precipitated glutamine synthetase was collected by centrifugation. The precipitate was dissolved in 70% formic acid and was dialyzed against 70% formic acid for 12 h. CNBr Cleavage—The enzyme modified by FSBA was dissolved in 0.7 ml of 70% HCOOH and cyanogen bromide was added in a 50-fold excess over CNBr/methionyl residues. The reaction mixture was incubated at room temperature for 24 h in the dark, and then diluted with 9 volumes of distilled water. The mixture was evaporated and the residue was dissolved in 6 M guanidine HC1. The solution was applied to a column of //-Bondasphere C18-
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TABLE n. Amino acid sequence analysis of CBS-labeled peptide. The yields of phenylthiohydantoin amino acid residues in picomoles are indicated. The analysis was started with 750 pmol of CBS-labeled peptide. The actual glutamine synthetase sequence in this region from position 33-48 is given. The site of labeling is denoted by X (no phenylthiohydantoin derivative was detected). Glutamine synthetase Cycle Amino acid pmol 33-48 1 Asn 165 Asn 2 712 Val Val 3 Glu 626 Glu 4 882 lie lie* 5 Pro 566 Pro 6 Val 383 Val 7 Ser 153 Ser 8 Gin 331 Gin 9 392 Leu Leu 10 Thr 106 Thr 11 — Lys X 12 Ala 303 Ala 13 Leu 286 Leu 14 Asp 150 Asp 104 15 Asn Asn 16 Lys 149 Lys
cence at 32 min. After further purification of the fraction, the amino acid sequence was determined. The sequence of the purified fluorescent peptide is shown in Table I. The sequence corresponds to amino acids 298-315 of B. cereus glutamine synthetase, whose primary structure was deduced from the nucleotide sequence of the gene {11). Since no phenylthiohydantoin derivative was detected at cycle 9, and the derivatives at every other position were consistent with the known sequence of this region, this result indicated that the cysteinyl residue at position 306 was the site of IAEDANS binding. Chemical Modification by FSBA—B. cereus glutamine synthetase was treated with 150 ^M FSBA for 45 min. In contrast to the case of the modification by IAEDANS, Mg*+-dependent activity was not affected by the modification with FSBA (9). In this same period of time Mn2+dependent activity was inactivated to 25% of its initial value. Figure IB shows the UV absorption spectra of native and FSBA-inactivated enzyme. The enzyme modified by FSBA was cleaved by cyanogen bromide in 70% formic
acid, and the peptides were separated by reverse-phase HPLC. Elution profiles are shown in Fig. 3 using detector wavelengths of 243 and 210 nm. It has been observed previously that the adenosyl moiety is lost under the acidic conditions of cyanogen bromide treament (7). The absorbance of the CBS group at 243-246 nm is detectable. Figure 3A shows that a peak at 52 min has high absorption at 243 nm. The spectrum of the peptide at 52 min had an absorption maximum at 243 nm (data not shown). This peptide was purified and its N-terminal amino acid sequence was determined. The sequence of the first 10 amino acid residues was identical to that of the glutamine synthetase subunit (data not shown). The N-terminal peptide was digested by trypsin and was then subjected to HPLC. Figure 3B shows that a single peak at 39 min was observed at 243 nm. The fraction was purified and the amino acid sequence of the peptide was determined. The sequence is shown in Table II. The sequence corresponds to amino acids 33-48 of B. cereus glutamine synthetase. Since no phenylthiohydantoin derivative was detected at cycle 11 and the derivatives at every other position were consistent with the known sequence of this region, this result indicates that the lysine residue at position 43 was the site of CBS binding. DISCUSSION Glutamine synthetases of B. subtilis and B. cereus each have one reactive cysteinyl residue, and modification of the cysteine with iodoacetamide inactivates Mg2+- dependent activity and activates Mn2+-dependent activity (9, 14). Mg*+ plus Mn2+-dependent activity was also inactivated (10). In contrast, the enzymes of E. coli and Clostridium pasteurianum have no reactive cysteinyl residue (14, 15). Glutamine synthetases of B. cereus and B. subtilis have five cysteinyl residues and three of these cysteines (at positions 167, 207, 306) are unique to Bacillus (11, 16, 17). We determined that one of these cysteines, Cys 306, was modified by iodoacetamide or IAEDANS. The region containing the reactive cysteine is conserved strongly in many bacterial glutamine synthetases (Fig. 4A). The region 339 to 345 of Thiobacillus ferrooxidans glutamine synthetase was shown to be homologous to the sequence J. Biochem.
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2 0 3 0 4 0
Fig. 3. A: Separation of peptides obtained by cyanogen bromide cleavage of glutamine synthetase modified by FSBA. The reaction mixture was applied to a column (//-Bondasphere C18, 19x150 mm, Waters) equilibrated with 0.1% trifluoroacetic acid in H,0. Peptidea were eluted with an increasing gradient (2%/min) of acetonitrile (0-80% in 0.1% trifluoroacetic acid with a flow rate of 1 ml/min). The column eluates were monitored at 243 and at 210 nm. B: Separation of peptides obtained by trypsln digestion of CBS-labeled peptide. Conditions of elution were as described in the legend of Fig. 2. The column eluates were monitored at 243 nm and at 210 nm.
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Reactive SH Group of B. cereus Glutamine Synthetase
a) b) c) d) e)
293 293 292 318 316
NSYKRLVPGYEAPCYVAWSAQNRS 316 NSYKRLVPGYEAPCYVAWSAQNRS| 316 NSYKRLVPGYEAI^^Al NSYKRLVPGYEAFVN NSYKRLVPGYEAPVM 339
B 32 32 32 30 QHLTLYQ: 29 QH0T
Fig. 4. A: Amino acid sequences around the cysteine that was modified by IAEDANS in B. cereus glutamine synthetase. The asterisk indicates Cys modified by IAEDANS. B: Amino acid sequences around the lysine that was modified by FSBA. Asterisks indicate residues labeled by FSBA. Identical sequences are boxed, (a) B. cereus (11), (b) B. subtilis (17), (c) Clostridium acetobutylicum (23), (d) Anabaena (22), (e) E. coli (24).
DRAGASIV, which is thought to be the glutamate-binding site of bovine and chicken glutamate dehydrogenase (18). Our earlier experiments indicated that substrates protected the enzyme against iodoacetamide modification (9). When B. cereus glutamine synthetase was incubated with 50 mM iodoacetamide plus 5mM ATP and 50 mM Lglutamate in buffer containing 100 mM Tris-HCl (pH 9.0) and 0.5 mM MnCl2 for 120 min, the enzyme had 81% of the activity remaining. These results suggested that the Cys 306 is at the active site of the enzyme. The enzyme of B. subtilis, strain DRD2 with glnAlOO mutation in the glutamine synthetase structural gene lacks the Mg2"1"-dependent activity almost entirely, whereas the Mn2+-dependent activity is nearly normal (19). The altered Mg2*-dependent activity of glnAlOO enzyme resembles that of the enzyme modified by iodoacetamide. However Zhang et aL reported that Gly 243 was changed to Ser in the glutamine synthetase of the glnAlOO mutant and none of the sequenced mutations involved cysteines (20). The B. subtilis glnAlOO mutant requires glutamine for growth, although glutamine synthetase of the mutant has nearly normal Mn2+-dependent activity (19). Since its enzyme lacks Mg*+ plus Mn2+-dependent activity, we think that the binding of both Mg2* and Mn2+ to each subunit is required for normal activity of glutamine synthetase in vivo. The cysteinyl residue at position 306 may have a role in the regulation of activity and expression. We also found that the lysine at position 43 was modified by an ATP analog, FSBA. In E. coli glutamine synthetase the lysine at position 47 is modified by FSBA (7). Although the enzyme of B. cereus has a lysine residue (Lys 48) at the same position, no CBS-derivative of lysine at this position was detected. It appears that the ATP binding sites of the Bacillus and E. coli enzymes are similar, although the amino acid sequences have little homology in this region (Fig. 4B) and perhaps their conformations differ. This result supports the atomic model, which places the active
Vol. 107, No. 2, 1990
We thank Dr. K. Horikoshi for permitting us to use the automated protein sequencer, and Mr. W. Bellamy for reading the manuscript and for many useful discussions. REFERENCES 1. Deuel, T.F., Ginsburg, A., Yeh, J., Shelton, E., & Stadtman, E.R. (1970) J. Biol. Chan. 245, 5195-5205 2. Stadtman, E.R. & Ginsburg, A. (1974) in The Enzymes, 3rd ed. (Boyer, P.D., ed.) Vol. 10, pp. 755-807, Academic Press, New York 3. Tyler, B. (1978) Anna. Rev. Biochem. 47, 1127-1162 4. Hachimori, A., Matsunaga, A., Shimizu, M., Samejima, T., & Nosoh, Y. (1974) Biochim. Biophys. Acta 350, 461-474 5. Wedler, F.C. & Hoffmann, F.M. (1974) Biochemistry 13, 32073214 6. Donohue, T.J. & Bemlohr, R.W. (1981) J. Bacterial 147, 589601 7. Pinkofsky, H.B., Ginsburg, A., Reardon, I., & Heinrikson, R.L. (1984) J. Biol. Chan. 259, 9616-9622 8. Di Ianni, C.L. & ViUafranca, J.J. (1989) J. BioL Chem. 264, 8686-8691 9. Nakano, Y. & Kimura, K. (1987) Biochem. Biophys. Res. Common. 142, 475-482 10. Matsuoka, K., Kurebayashi, T., & Kimura, K. (1985) J. Biochem. 98, 1211-1219 11. Nakano, Y., Kato, C, Tanaka, E., Kimura, K., & Horikoshi, K. (1989) J. Biochem. 106, 206-215 12. Backman, K., Chen, Y.-M., & Magasanik, B. (1981) Proc. Natl Acad. Sci. U.S. 78, 3743-3747 13. Hudson, E.N. & Weber,-G. (1973) Biochemistry 12, 4154-4161 14. Deuel, T.F. (1971) J. BioL Chem. 248, 599-605 15. Krishnan, I.S. & Dua, R.D. (1985) FEBS Lett. 185, 267-271 16. Strauch, M.A., Aronson, A.I., Brown, S.W., Schreier, H.J., & Sonenshein, A.L. (1988) Gene 71, 257-265 17. Nakano, Y., Tanaka, E., Kato, C, Kimura, K., & Horikoshi, K. (1989) FEMS Micwbiol. Lett. 57, 81-86 18. Rawlings, D.E., Jones, W.A., O'Neill, E.G., & Woods, D.R. (1987) Gene 63, 211-217 19. Dean, D.R., Hoch, J.A., & Aronson, A.I. (1977) J. Bacteriol. 131, 981-987 . 20. Zhang, J., Strauch, M., & Aronson, A.I. (1989) J. Bacteriol. 171, 3572-3574 21. Almassy, R.J., Janson, C.A., Hamlin, R., Xuong, N.-X., & Eisenberg, D. (1986) Nature 323, 304-309 22. Turner, N.E., Robinson, S.J., & Haselkorn, R. (1983) Nature 306, 337-342 23. Janssen, P.J., Jones, W.A., Jones, D.T., & Woods, D.R. (1988) J. Bacteriol 170, 400-408 24. Colombo, G. & Villafranca, J.J. (1986) J. Biol. Chem. 261, 10587-10591
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a) b) c) d) e)
sites at the subunit interfaces, presented by Almassy et al. (21) and suggests that the difference between the enzymes of E. coli and Bacillus in this region may account for some of the differences in regulation of their activity. Anabaena glutamine synthetase, however, has no lysyl residue in this region (22). A lysine may not be essential for binding of ATP to the active site in glutamine synthetase. These results can not eliminate the possibility that another residue was modified at the same time. Further studies on the active site of Bacillus glutamine synthetase by chemical modification and site-directed mutagenesis are in progress.
Identification of the Reactive Cysteinyl Residue and ATP Binding Site in Bacillus cereus Glutamine Synthetase by Chemical Modification Yoshio Nakano, Masahiro I ton,1 Eiichi Tanaka, and Kinuko Kimura2 Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo 171 Received for publication, August 15, 1989
Glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming)] [EC 6.3.1.2] is an ammonia assimilatory enzyme which also functions as a key regulator of nitrogen metabolism in many organisms. The enzyme has a molecular weight of ~ 600,000 and is composed of 12 identical subunits (1). Enzymatic activity of glutamine synthetase is regulated by an adenylylation-deadenylylation system in Escherichia coli and several other Gram-negative bacteria (2, 3). In Bacillus species (1, 4-6), no evidence has been obtained to suggest covalent modification of the enzyme. Several amino acid residues at or near the active site of E. coli glutamine synthetase have been reported. Pinkofsky et al reported that an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), reacted with lysine 47 causing a loss of activity (7). Di Ianni and Villafranca reported lysines 383 and 352 reacted with pyridoxal 5'-phosphate, a chemical modification reagent which reacts with lysine residues at a phosphate binding site, and each modification resulted in partial loss of activity (8). To date there have been comparatively few reports pertaining to the active site of Bacillus glutamine synthetase. In a preliminary communication, we reported that modification with an SH reagent, iodoacetamide, inactivated only Mg2*-dependent activity of Bacillus cereus glutamine synthetase, whereas modification with FSBA inactivated only Mn2+-dependent activity (9). Glutamine synthetase of Bacillus subtilis and B. cereus requires both Mn2+ and Mg2* for high activity, and a change in the ratio of Mn2+ and Mg2* concentrations has a marked effect on the activity (10). We suggested that Mn2+ and Mg2* do not share a common binding site. 1
Present address: Department of Bio-Engineering, Tokyo Institute of Technology, Meguro-ku, Tokyo 152. 1 To whom correspondence should be addressed. Abbreviations: IAEDANS, JV-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid; FSBA, 5'-p-fluorosulfonylbenzoylfldenosine; CBS, 4-carboxybenzenesulfonyl; HPLC, high-performance liquid chromatography.
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In this paper, we determined the positions of the amino acid residues modified by a fluorescent SH reagnet, N[ [ (iodoacetyl) amino ] ethyl ] - 5 - naphthylamine -1 - sulfonic acid (IAEDANS) and by FSBA in the primary strucutre of B. cereus glutamine synthetase, which was determined recently (11). MATERIALS AND METHODS Materials—E. coli YMC11 [endAl, thil,hsdRn,supEM, AlacUim, hutC*, 4(glnA-ntrC)2000] (12) harboring pCGSl was grown in M9 medium containing 0.1% glutamate instead of 0.1% NH^Cl. pCGSl is a pBR329 derivative containing the glnA gene of B. cereus IFO3131 (11). Glutamine synthetase was purified and the biosynthetic activity of the enzyme was measured as described previously (10). IAEDANS was purchased from Aldrich Chemical and FSBA was purchased from Sigma Chemical. All other chemicals were of the highest grade commercially available. Chemical Modification with IAEDANS—la order to modify one SH group in glutamine synthetase, the enzyme (0.32 mg/ml) was treated with 3 mM IAEDANS in 100 mM Tris-HCl buffer, pH 9.0, containing 0.5 mM MnCl2, at 37*C. The reaction with IAEDANS was terminated by addition of 20 mM 2-mercaptoethanol. The reaction mixture was applied to 1.5 x 10 cm column of hydroxyapatite equilibrated with 1 mM K-phosphate buffer (pH 6.1). The column was washed with 50 ml of the same buffer and eluted with 50 mM K-phosphate buffer (pH7.6). The absorbance at 280 nm was monitored and the fractions containing the protein were pooled. The modified enzyme solution was lyophilized. Chemical Modification with FSBA—The concentrations of stock FSBA solutions were determined from their absorbance at 232nm (£ = 18.8cm- I -mM-'). A 6-fold excess of FSBA per mol of subunit was added to an aliquot of enzyme (30//M) in 100 mM Tris-HCl buffer, pH7.2, J. Biochem.
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Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, iV-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-l-sulfonic aicd (IAEDANS), or an ATP analog, 5 -p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg}+-dependent activity and activated Mn2+-dependent activity. FSBA inactivated only Mn2+-dependent activity. Mg2* plus Mn2+-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.
Reactive SH Group of B. cereus Glutamine Synthetase
300 A (19x150 mm, Waters) equilibrated with 0.1% trifluoroacetic acid in H 2 0. The adenosyl moiety of the modified peptide was lost under these conditions and CBS-peptide was purified. Tryptic Digestion—The enzyme modified by IAEDANS or purified CBS-peptide was digested with trypsin in 100 mM Tris-HCl (pH 8.0) containing 2 M guanidine-HCl at 37'C. TPCK-trypsin [1 : 100 (w/w)] was added three times at 24 h intervals. After 72 h, the reaction mixtures were applied to a column of TSK-ODS 120T (4.6x150 mm, Tosoh) equilibrated with 0.1% trifluoroacetic acid in H 2 0. HPLC was carried out using a Waters 600 apparatus and fluorescence was detected with a JASCO FP-210 fluorometer. Sequence Analysis—Automated Edman degradation of the purified peptides and determination of phenylthiohydantoin derivatives were performed with an Applied Biosystems sequencer system. RESULTS
240
2S0
320
3BO
400 240 2O0
200
300
320
Wavelength (run)
Fig. 1. A: UV-absorption spectrum of glutamine synthetase modified by IAEDANS (0.35 mg/ml). B: UV-absorption spectra of native (dotted line) and FSBA-labeled enzyme (solid line).
Chemical Modification with IAEDANS—The purified glutamine synthetase was treated with 3 mM IAEDANS for 45 min. Mg2+-dependent activity was inactivated to 50% of the initial activity, in contrast to the activation of Mn2+-dependent activity to 140%. Inactivation of Mg2"1" plus Mn2+-dependent activity was similar to that of Mg2*-dependent activity. The number of reactive sulfhydryl groups was determined to be 12 per molecule of the enzyme, or 1 per subunit, from the ratio of the absorbances at 337 and 280 nm of the enzymne modified by IAEDANS (Fig. 1A). The extinctions of IAEDANS at 337 and 280 nm were taken as 6.8x10' and 1.06x10'M-'-cnr 1 , respectively (13). After inactivation of Mg2*-dependent activity with IAEDANS, the enzyme was digested by trypsin. The peptides were then subjected to HPLC. Elution profiles are shown in Fig. 2, detecting absorption at 210 nm and fluorescence. Figure 2B shows a single peak exhibiting fluoresTABLE I. Amino acid sequence analysis of the AEDANSlabeled peptide. The yields of phenylthiohydantoin amino acid residues in picomoles are indicated. The analysis was started with 666 pmol of AEDANS-labeled peptide. The actual glutamine synthetase sequence in this region from position 298-315 is given. The site of labeling is denoted by X (no phenylthiohydantoin derivative was detected).
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Tlme(mfn) Fig. 2. Separation of peptides obtained by trypsin digestion of SH-labeled glutamine synthetase. The digest was applied to a column (TSK-ODS 120T, 4.6x150 mm, Tosoh) equilibrated with 0.1% trifluoroacetic acid. Peptides were eluted with an increasing gradient of acetonitrile (2%/min) from 0 to 80%. Theflowrate was 1.0 ml per min. Column eluates were monitored by measurement of UV absorption (210 nm) and fluorescence (excitation 337 nm, emission 490 nm). Vol. 107, No. 2, 1990
Cycle
Amino acid
pmol
Glutamine synthetase 298-315
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Leu Val Pro Gly Tyr Glu Ala Pro X Tyr Val Ala Trp
459 498 237 272 225 166 469 258 — 212 326 373 135 52 241 112 110 77
Leu Val Pro Gly Tyr Glu Ala Pro Cys Tyr Val Ala Trp Ser Ala Gin Asn Arg
Ser Ala Gin Asn Arg
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containing 0.5 mM MnCl2 and 50 mM L-glutamate, at 3TC. The reaction with FSBA was terminated by adding of 20 mM 2-mercaptoethanol. Solid ammonium sulfate was added to 70% saturation and the precipitated glutamine synthetase was collected by centrifugation. The precipitate was dissolved in 70% formic acid and was dialyzed against 70% formic acid for 12 h. CNBr Cleavage—The enzyme modified by FSBA was dissolved in 0.7 ml of 70% HCOOH and cyanogen bromide was added in a 50-fold excess over CNBr/methionyl residues. The reaction mixture was incubated at room temperature for 24 h in the dark, and then diluted with 9 volumes of distilled water. The mixture was evaporated and the residue was dissolved in 6 M guanidine HC1. The solution was applied to a column of //-Bondasphere C18-
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50
Tlme(mln)
TABLE n. Amino acid sequence analysis of CBS-labeled peptide. The yields of phenylthiohydantoin amino acid residues in picomoles are indicated. The analysis was started with 750 pmol of CBS-labeled peptide. The actual glutamine synthetase sequence in this region from position 33-48 is given. The site of labeling is denoted by X (no phenylthiohydantoin derivative was detected). Glutamine synthetase Cycle Amino acid pmol 33-48 1 Asn 165 Asn 2 712 Val Val 3 Glu 626 Glu 4 882 lie lie* 5 Pro 566 Pro 6 Val 383 Val 7 Ser 153 Ser 8 Gin 331 Gin 9 392 Leu Leu 10 Thr 106 Thr 11 — Lys X 12 Ala 303 Ala 13 Leu 286 Leu 14 Asp 150 Asp 104 15 Asn Asn 16 Lys 149 Lys
cence at 32 min. After further purification of the fraction, the amino acid sequence was determined. The sequence of the purified fluorescent peptide is shown in Table I. The sequence corresponds to amino acids 298-315 of B. cereus glutamine synthetase, whose primary structure was deduced from the nucleotide sequence of the gene {11). Since no phenylthiohydantoin derivative was detected at cycle 9, and the derivatives at every other position were consistent with the known sequence of this region, this result indicated that the cysteinyl residue at position 306 was the site of IAEDANS binding. Chemical Modification by FSBA—B. cereus glutamine synthetase was treated with 150 ^M FSBA for 45 min. In contrast to the case of the modification by IAEDANS, Mg*+-dependent activity was not affected by the modification with FSBA (9). In this same period of time Mn2+dependent activity was inactivated to 25% of its initial value. Figure IB shows the UV absorption spectra of native and FSBA-inactivated enzyme. The enzyme modified by FSBA was cleaved by cyanogen bromide in 70% formic
acid, and the peptides were separated by reverse-phase HPLC. Elution profiles are shown in Fig. 3 using detector wavelengths of 243 and 210 nm. It has been observed previously that the adenosyl moiety is lost under the acidic conditions of cyanogen bromide treament (7). The absorbance of the CBS group at 243-246 nm is detectable. Figure 3A shows that a peak at 52 min has high absorption at 243 nm. The spectrum of the peptide at 52 min had an absorption maximum at 243 nm (data not shown). This peptide was purified and its N-terminal amino acid sequence was determined. The sequence of the first 10 amino acid residues was identical to that of the glutamine synthetase subunit (data not shown). The N-terminal peptide was digested by trypsin and was then subjected to HPLC. Figure 3B shows that a single peak at 39 min was observed at 243 nm. The fraction was purified and the amino acid sequence of the peptide was determined. The sequence is shown in Table II. The sequence corresponds to amino acids 33-48 of B. cereus glutamine synthetase. Since no phenylthiohydantoin derivative was detected at cycle 11 and the derivatives at every other position were consistent with the known sequence of this region, this result indicates that the lysine residue at position 43 was the site of CBS binding. DISCUSSION Glutamine synthetases of B. subtilis and B. cereus each have one reactive cysteinyl residue, and modification of the cysteine with iodoacetamide inactivates Mg2+- dependent activity and activates Mn2+-dependent activity (9, 14). Mg*+ plus Mn2+-dependent activity was also inactivated (10). In contrast, the enzymes of E. coli and Clostridium pasteurianum have no reactive cysteinyl residue (14, 15). Glutamine synthetases of B. cereus and B. subtilis have five cysteinyl residues and three of these cysteines (at positions 167, 207, 306) are unique to Bacillus (11, 16, 17). We determined that one of these cysteines, Cys 306, was modified by iodoacetamide or IAEDANS. The region containing the reactive cysteine is conserved strongly in many bacterial glutamine synthetases (Fig. 4A). The region 339 to 345 of Thiobacillus ferrooxidans glutamine synthetase was shown to be homologous to the sequence J. Biochem.
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2 0 3 0 4 0
Fig. 3. A: Separation of peptides obtained by cyanogen bromide cleavage of glutamine synthetase modified by FSBA. The reaction mixture was applied to a column (//-Bondasphere C18, 19x150 mm, Waters) equilibrated with 0.1% trifluoroacetic acid in H,0. Peptidea were eluted with an increasing gradient (2%/min) of acetonitrile (0-80% in 0.1% trifluoroacetic acid with a flow rate of 1 ml/min). The column eluates were monitored at 243 and at 210 nm. B: Separation of peptides obtained by trypsln digestion of CBS-labeled peptide. Conditions of elution were as described in the legend of Fig. 2. The column eluates were monitored at 243 nm and at 210 nm.
183
Reactive SH Group of B. cereus Glutamine Synthetase
a) b) c) d) e)
293 293 292 318 316
NSYKRLVPGYEAPCYVAWSAQNRS 316 NSYKRLVPGYEAPCYVAWSAQNRS| 316 NSYKRLVPGYEAI^^Al NSYKRLVPGYEAFVN NSYKRLVPGYEAPVM 339
B 32 32 32 30 QHLTLYQ: 29 QH0T
Fig. 4. A: Amino acid sequences around the cysteine that was modified by IAEDANS in B. cereus glutamine synthetase. The asterisk indicates Cys modified by IAEDANS. B: Amino acid sequences around the lysine that was modified by FSBA. Asterisks indicate residues labeled by FSBA. Identical sequences are boxed, (a) B. cereus (11), (b) B. subtilis (17), (c) Clostridium acetobutylicum (23), (d) Anabaena (22), (e) E. coli (24).
DRAGASIV, which is thought to be the glutamate-binding site of bovine and chicken glutamate dehydrogenase (18). Our earlier experiments indicated that substrates protected the enzyme against iodoacetamide modification (9). When B. cereus glutamine synthetase was incubated with 50 mM iodoacetamide plus 5mM ATP and 50 mM Lglutamate in buffer containing 100 mM Tris-HCl (pH 9.0) and 0.5 mM MnCl2 for 120 min, the enzyme had 81% of the activity remaining. These results suggested that the Cys 306 is at the active site of the enzyme. The enzyme of B. subtilis, strain DRD2 with glnAlOO mutation in the glutamine synthetase structural gene lacks the Mg2"1"-dependent activity almost entirely, whereas the Mn2+-dependent activity is nearly normal (19). The altered Mg2*-dependent activity of glnAlOO enzyme resembles that of the enzyme modified by iodoacetamide. However Zhang et aL reported that Gly 243 was changed to Ser in the glutamine synthetase of the glnAlOO mutant and none of the sequenced mutations involved cysteines (20). The B. subtilis glnAlOO mutant requires glutamine for growth, although glutamine synthetase of the mutant has nearly normal Mn2+-dependent activity (19). Since its enzyme lacks Mg*+ plus Mn2+-dependent activity, we think that the binding of both Mg2* and Mn2+ to each subunit is required for normal activity of glutamine synthetase in vivo. The cysteinyl residue at position 306 may have a role in the regulation of activity and expression. We also found that the lysine at position 43 was modified by an ATP analog, FSBA. In E. coli glutamine synthetase the lysine at position 47 is modified by FSBA (7). Although the enzyme of B. cereus has a lysine residue (Lys 48) at the same position, no CBS-derivative of lysine at this position was detected. It appears that the ATP binding sites of the Bacillus and E. coli enzymes are similar, although the amino acid sequences have little homology in this region (Fig. 4B) and perhaps their conformations differ. This result supports the atomic model, which places the active
Vol. 107, No. 2, 1990
We thank Dr. K. Horikoshi for permitting us to use the automated protein sequencer, and Mr. W. Bellamy for reading the manuscript and for many useful discussions. REFERENCES 1. Deuel, T.F., Ginsburg, A., Yeh, J., Shelton, E., & Stadtman, E.R. (1970) J. Biol. Chan. 245, 5195-5205 2. Stadtman, E.R. & Ginsburg, A. (1974) in The Enzymes, 3rd ed. (Boyer, P.D., ed.) Vol. 10, pp. 755-807, Academic Press, New York 3. Tyler, B. (1978) Anna. Rev. Biochem. 47, 1127-1162 4. Hachimori, A., Matsunaga, A., Shimizu, M., Samejima, T., & Nosoh, Y. (1974) Biochim. Biophys. Acta 350, 461-474 5. Wedler, F.C. & Hoffmann, F.M. (1974) Biochemistry 13, 32073214 6. Donohue, T.J. & Bemlohr, R.W. (1981) J. Bacterial 147, 589601 7. Pinkofsky, H.B., Ginsburg, A., Reardon, I., & Heinrikson, R.L. (1984) J. Biol. Chan. 259, 9616-9622 8. Di Ianni, C.L. & ViUafranca, J.J. (1989) J. BioL Chem. 264, 8686-8691 9. Nakano, Y. & Kimura, K. (1987) Biochem. Biophys. Res. Common. 142, 475-482 10. Matsuoka, K., Kurebayashi, T., & Kimura, K. (1985) J. Biochem. 98, 1211-1219 11. Nakano, Y., Kato, C, Tanaka, E., Kimura, K., & Horikoshi, K. (1989) J. Biochem. 106, 206-215 12. Backman, K., Chen, Y.-M., & Magasanik, B. (1981) Proc. Natl Acad. Sci. U.S. 78, 3743-3747 13. Hudson, E.N. & Weber,-G. (1973) Biochemistry 12, 4154-4161 14. Deuel, T.F. (1971) J. BioL Chem. 248, 599-605 15. Krishnan, I.S. & Dua, R.D. (1985) FEBS Lett. 185, 267-271 16. Strauch, M.A., Aronson, A.I., Brown, S.W., Schreier, H.J., & Sonenshein, A.L. (1988) Gene 71, 257-265 17. Nakano, Y., Tanaka, E., Kato, C, Kimura, K., & Horikoshi, K. (1989) FEMS Micwbiol. Lett. 57, 81-86 18. Rawlings, D.E., Jones, W.A., O'Neill, E.G., & Woods, D.R. (1987) Gene 63, 211-217 19. Dean, D.R., Hoch, J.A., & Aronson, A.I. (1977) J. Bacteriol. 131, 981-987 . 20. Zhang, J., Strauch, M., & Aronson, A.I. (1989) J. Bacteriol. 171, 3572-3574 21. Almassy, R.J., Janson, C.A., Hamlin, R., Xuong, N.-X., & Eisenberg, D. (1986) Nature 323, 304-309 22. Turner, N.E., Robinson, S.J., & Haselkorn, R. (1983) Nature 306, 337-342 23. Janssen, P.J., Jones, W.A., Jones, D.T., & Woods, D.R. (1988) J. Bacteriol 170, 400-408 24. Colombo, G. & Villafranca, J.J. (1986) J. Biol. Chem. 261, 10587-10591
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a) b) c) d) e)
sites at the subunit interfaces, presented by Almassy et al. (21) and suggests that the difference between the enzymes of E. coli and Bacillus in this region may account for some of the differences in regulation of their activity. Anabaena glutamine synthetase, however, has no lysyl residue in this region (22). A lysine may not be essential for binding of ATP to the active site in glutamine synthetase. These results can not eliminate the possibility that another residue was modified at the same time. Further studies on the active site of Bacillus glutamine synthetase by chemical modification and site-directed mutagenesis are in progress.
