Vol. 126, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 539-541 Copyright C 1976 American Society for Microbiology
Active Subunits of Escherichia coli Glutamate Synthasel PEKKA MANTSALA AND HOWARD ZALKIN* Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
Received for publication 4 November 1975
The large and small subunits of Escherichia coli glutamate synthase were isolated. The small subunit catalyzes the NH-dependent synthesis of glutamate. The large subunit exhibits glutaminase activity.
Glutamate synthase from Escherichia coli is an iron-sulfur flavoprotein (4) that can utilize glutamine or NH: (P. Mantsala and H. Zalkin, J. Biol. Chem., in press) for synthesis of glutamate. The native enzyme from E. coli is oligomeric and contains nonidentical subunits of molecular weights of approximately 135,000 and 53,000 (4). Recent results (Mantsala and Zalkin, J. Biol. Chem., in press) indicate that flavin and iron are required for glutamine-dependent but not for NH,-dependent glutamate synthesis. The glutamine analogue L-2-amino4-oxo-5-chloropentanoic acid (chloroketone) and iodoacetamide were found to alkylate a cysteine residue in the large subunit of the oligomeric enzyme and specifically inactivate the glutamine-dependent activity without affecting the NH3-dependent activity. These results suggest that glutamine binds to the large subunit. Other experiments have shown that a cysteine residue in the small subunit appears to be required for NH3-dependent glutamate synthase activity. A clear understanding of the function of the nonidentical subunits in catalysis requires their separation in an active form. We report here the isolation of active subunits of E. coli glutamate synthase and show that the small subunit catalyzes the NH3-dependent synthesis of glutamate in the absence of flavin and iron. The small subunit of glutamate synthase does not cross-react with antibodies to E. coli glutamate dehydrogenase. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from E. coli K-12 strain W3110 as described (4; Mantsala and Zalkin, J. Biol. Chem., in press). Anti-glutamate synthase and anti-glutamate dehydrogenase were prepared as described (Mantsala and Zalkin, J. Biol. Chem., in press). The subunits of glutamate synthase were separated (3) on a Bio-Gel A-1.5 m column (2 by 77 cm) at 23 C in buffer solution containing 0.1 M tris(hydroxymethyl)aminomethane ' Journal Paper no. 6045 from the Purdue Agricultural Experiment Station.
(Tris)-acetate, pH 7.7, 0.3% sodium dodecyl sulfate (SDS), 10 mM a-ketoglutarate, 10 mM glutamine, and 10 mM 2-mercaptoethanol. Pooled fractions containing the large and small subunits were dialyzed against 50 mM Trisacetate, pH 7.7, containing 6 M urea and 10 mM 2-mercaptoethanol. Protein fractions were concentrated by ultrafiltration by using an Amicon UM-10 membrane. SDS was removed by chromatography on a Dowex 1 column (1.0 by 2.4 cm) (2, 7). Urea was removed by dialysis against 50 mM Tris-acetate, pH 7.7, and the subunits were stored at -20 C in the same buffer containing 50% glycerol. Glutamine- and NH,-dependent glutamate synthase and affinity labeling by ['4C]chloroketone- were assayed as described (Mantsala and Zalkin, J. Biol. Chem., in press). Glutaminase activity was assayed according to Prusiner and Milner (5). Double antibody precipitation using L1-'4C]carboxamidomethylglutamate synthase (319,000 counts/min per mg of protein) and [1-'4C]carboxamidomethylglutamate dehydrogenase (284,000 counts/min per mg of protein), rabbit antibodies to the two enzymes, and excess goat anti-rabbit immunoglobulin has been described (Mantsala and Zalkin, J. Biol. Chem., in press). The results of subunit separation were similar to those reported by Miller (3). Flavin, iron, and sulfide are removed during denaturation and gel filtration in SDS. SDS gel electrophoresis of the pooled subunit fractions (Fig. 1) shows complete separation without detectable crosscontamination. The final recoveries of the large and small subunits were 24 and 57%, respectively. Losses were encountered during chromatography on Dowex 1 (2). Other less drastic methods of subunit separation were not investigated, but Trotta et al. (6) have reported the separation of apparently inactive subunits of glutamate synthase from Klebsiella aerogenes by using a different technique. NH3-dependent glutamate synthase activity was detected in the small subunit but not in the 539
540
large subunit (Table 1). Addition of the large subunit did not markedly enhance the activity of the small subunit. Although the NHi-dependent activity of the small subunit was less than 1% that of native glutamate synthase, the assays were highly reproducible. Glutaminase activity and affinity labeling by l'4C]chloroketone were confined to the large subunit (Table 2). The small subunit was inactive and did not appreciably enhance these activities of the large subunit. Prior alkylation of the large subunit with chloroketone inhibited
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TABLE 2. Affinity labeling of glutamate synthase subunits by 1 '4C]chloroketone and glutaminase activity"
Subunit
Incorporation of Glutaminase 4Cchloro(% [ketone the native (mol/ ofactivity enzyme) mol of subunit)
0.13 0.062 Large 0 0 Small 0.15b Large + small 0.066b ND' 0 Large subunit alkylated with ['4C]chloroketone 0 ND Large subunit alkylated with ['4C]chloroketone + small subunit " Incorporation of ['4C]chloroketone was carried out in the presence of 55 AM ['4C]chloroketone and 166 Mig of large subunit protein or 149 Mg of small subunit protein. Incorporation of 0.062 mol of chloroketone corresponds to 110 counts/min. The same amounts of the subunits were assayed for glutaminase activity using 0.24 mM L-[U-'4C]glutamine (specific activity, 57.3 mCi/mmol). Glutaminase activity of 0.13% yields, 2,660 counts/min ['4C]glutamate per 15 min. bCalculated according to the amount of large subunit protein. ND, Not determined. "
o
the glutaminase activity just as for the native enzyme. The large and small subunits of glutamate B synthase react with anti-glutamate synthase and decrease precipitation of ['4C]carboxamidomethylglutamate synthase as shown in Fig. 2A. This suggests at least some restoration of the native tertiary structure. Cross-reactivity of glutamate synthase subunits with anti-glutamate dehydrogenase was not obtained (Fig. 2B). CM OF GEL Previous experiments have established that FIG. 1. Densitometer tracings of SDS gel electro- the NH:-dependent activity of E. coli K-12 gluphoresis of the pooled, large subunit (A) and small tamate synthase is approximately 5 to 7% that subunit (B) fractions. The arrow indicates the posi- of the glutamine-dependent activity (Mantsala tion of the tracking dye. and Zalkin, J. Biol. Chem., in press). Flavin adenine dinucleotide is required for the glutamine-dependent synthesis of glutamate but not TABLE 1. NH3-dependent activity of the separated for NH:-dependent synthesis of glutamate, gluglutamate synthase subunits taminase activity, or affinity labeling by chlo% Activity relative roketone. Apoglutamate synthase, resolved of Subunit to native enzyme" flavin, iron, and sulfide, has enhanced NH10.82b Small dependent activity. The NH:-dependent activ0 Large ity of a glutamine amidotransferase may reflect 0.91 Small + large the primitive aminase activity before modificaaThe specific activity of the native enzyme was tion by association of a glutamine amidotransferase function (8). The present experiments 0.92 U/mg of protein. b Corresponds to absorbance change at 340 nm of identify the small subunit of glutamate syn0.014/10 min using 0.3 absorbance full-scale deflec- thase as responsible for the aminase activity. tion on the recorder. Similarity in molecular weights of the small 0 In 4
NOTES
VOL. 126, 1976
ments we did not detect cross-reactivity between the separated subunits of glutamate synthase and anti-glutamate dehydrogenase. Thus, there is presently no evidence to support an evolutionary relationship between the small subunit of glutamate synthase and glutamate dehydrogenase. In anthranilate synthetase (8) the mechanism of glutamine utilization involves transfer of the amide of glutamine from a site on a glutamine-binding subunit to a site for NH: utilization on a distinct aminase subunit. Further experiments are required to elucidate whether the amide of glutamine that is bound to the large subunit of glutamate synthase is transferred to the NH:1 site of the aminase.
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This research was supported by Public Health Service grant CA 11442 from the National Cancer Institute. P. M. was a Fulbright-Hayes Scholar.
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PROTEIN ( 9g)
FIG. 2. Double antibody precipitation. The reaction mixtures contained 13.7 Mg of[I-'4C]carboxamidomethylglutamate synthase or 7.5 pg of [l'4C]carboxamidomethylglutamate dehydrogenase, 2 ,u of antiserum, excess of goat anti-rabbit immunoglobulin, and subunit protein as indicated. The data are corrected for nonspecific precipitation. The experiment measures the capacity of the separated subunits to compete with the radioactive antigens for antibody and decrease precipitation of the radioactive antigen. Symbols: (A) Antiglutamate synthase; (0) large subunit; (A) small subunit; (B) anti-glutamate dehydrogenase. The symbols are the same as in A.
subunit of glutamate synthase with the subunit of E. coli glutamate dehydrogenase has prompted speculation about a relationship between these two proteins (1). We have not detected cross-reactivity between glutamate synthase and anti-glutamate dehydrogenase nor between glutamate dehydrogenase and antiglutamate synthase (Mantsala and Zalkin, J. Biol. Chem., in press). In the present experi-
LITERATURE CITED 1. Blumenthal, K. M., and E. L. Smith. 1975. Alternative substrates for glutamate dehydrogenases. Biochem. Biophys. Res. Commun. 62:78-84. 2. Hennecke, H., and A. Bock. 1975. Altered a subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichia coli. Eur. J. Biochem. 55:431-437. 3. Miller, R. E. 1973. Glutamate synthase from Escherichia coli: an iron-sulfide flavoprotein, p. 183-205. In S. Prusiner and E. R. Stadtman (ed.), The enzymes of glutamine metabolism. Academic Press Inc., New York. 4. Miller, R. E., and E. R. Stadtman. 1972. Glutamate synthase from Escherichia coli. An iron sulfide flavoprotein. J. Biol. Chem. 247:7407-7419. 5. Prusiner, S., and L. Milner. 1970. A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase and asparaginase. Anal. Biochem. 37:429-438. 6. Trotta, P. P., K. E. B. Platzer, R. H. Haschemyer, and A. Meister. 1974. Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. Proc. Natl. Acad. Sci. U.S.A. 71:4607-4611. 7. Weber, K., and D. J. Kuter. 1971. Reversible denaturation of enzymes by sodium dodecyl sulfate. J. Biol. Chem. 246:4504-4509. 8. Zalkin, H. 1973. Anthranilate synthetase, p. 1-39. In A. Meister (ed.), Advances in enzymology. John Wiley & Sons, Inc., New York.
JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 539-541 Copyright C 1976 American Society for Microbiology
Active Subunits of Escherichia coli Glutamate Synthasel PEKKA MANTSALA AND HOWARD ZALKIN* Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
Received for publication 4 November 1975
The large and small subunits of Escherichia coli glutamate synthase were isolated. The small subunit catalyzes the NH-dependent synthesis of glutamate. The large subunit exhibits glutaminase activity.
Glutamate synthase from Escherichia coli is an iron-sulfur flavoprotein (4) that can utilize glutamine or NH: (P. Mantsala and H. Zalkin, J. Biol. Chem., in press) for synthesis of glutamate. The native enzyme from E. coli is oligomeric and contains nonidentical subunits of molecular weights of approximately 135,000 and 53,000 (4). Recent results (Mantsala and Zalkin, J. Biol. Chem., in press) indicate that flavin and iron are required for glutamine-dependent but not for NH,-dependent glutamate synthesis. The glutamine analogue L-2-amino4-oxo-5-chloropentanoic acid (chloroketone) and iodoacetamide were found to alkylate a cysteine residue in the large subunit of the oligomeric enzyme and specifically inactivate the glutamine-dependent activity without affecting the NH3-dependent activity. These results suggest that glutamine binds to the large subunit. Other experiments have shown that a cysteine residue in the small subunit appears to be required for NH3-dependent glutamate synthase activity. A clear understanding of the function of the nonidentical subunits in catalysis requires their separation in an active form. We report here the isolation of active subunits of E. coli glutamate synthase and show that the small subunit catalyzes the NH3-dependent synthesis of glutamate in the absence of flavin and iron. The small subunit of glutamate synthase does not cross-react with antibodies to E. coli glutamate dehydrogenase. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from E. coli K-12 strain W3110 as described (4; Mantsala and Zalkin, J. Biol. Chem., in press). Anti-glutamate synthase and anti-glutamate dehydrogenase were prepared as described (Mantsala and Zalkin, J. Biol. Chem., in press). The subunits of glutamate synthase were separated (3) on a Bio-Gel A-1.5 m column (2 by 77 cm) at 23 C in buffer solution containing 0.1 M tris(hydroxymethyl)aminomethane ' Journal Paper no. 6045 from the Purdue Agricultural Experiment Station.
(Tris)-acetate, pH 7.7, 0.3% sodium dodecyl sulfate (SDS), 10 mM a-ketoglutarate, 10 mM glutamine, and 10 mM 2-mercaptoethanol. Pooled fractions containing the large and small subunits were dialyzed against 50 mM Trisacetate, pH 7.7, containing 6 M urea and 10 mM 2-mercaptoethanol. Protein fractions were concentrated by ultrafiltration by using an Amicon UM-10 membrane. SDS was removed by chromatography on a Dowex 1 column (1.0 by 2.4 cm) (2, 7). Urea was removed by dialysis against 50 mM Tris-acetate, pH 7.7, and the subunits were stored at -20 C in the same buffer containing 50% glycerol. Glutamine- and NH,-dependent glutamate synthase and affinity labeling by ['4C]chloroketone- were assayed as described (Mantsala and Zalkin, J. Biol. Chem., in press). Glutaminase activity was assayed according to Prusiner and Milner (5). Double antibody precipitation using L1-'4C]carboxamidomethylglutamate synthase (319,000 counts/min per mg of protein) and [1-'4C]carboxamidomethylglutamate dehydrogenase (284,000 counts/min per mg of protein), rabbit antibodies to the two enzymes, and excess goat anti-rabbit immunoglobulin has been described (Mantsala and Zalkin, J. Biol. Chem., in press). The results of subunit separation were similar to those reported by Miller (3). Flavin, iron, and sulfide are removed during denaturation and gel filtration in SDS. SDS gel electrophoresis of the pooled subunit fractions (Fig. 1) shows complete separation without detectable crosscontamination. The final recoveries of the large and small subunits were 24 and 57%, respectively. Losses were encountered during chromatography on Dowex 1 (2). Other less drastic methods of subunit separation were not investigated, but Trotta et al. (6) have reported the separation of apparently inactive subunits of glutamate synthase from Klebsiella aerogenes by using a different technique. NH3-dependent glutamate synthase activity was detected in the small subunit but not in the 539
540
large subunit (Table 1). Addition of the large subunit did not markedly enhance the activity of the small subunit. Although the NHi-dependent activity of the small subunit was less than 1% that of native glutamate synthase, the assays were highly reproducible. Glutaminase activity and affinity labeling by l'4C]chloroketone were confined to the large subunit (Table 2). The small subunit was inactive and did not appreciably enhance these activities of the large subunit. Prior alkylation of the large subunit with chloroketone inhibited
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TABLE 2. Affinity labeling of glutamate synthase subunits by 1 '4C]chloroketone and glutaminase activity"
Subunit
Incorporation of Glutaminase 4Cchloro(% [ketone the native (mol/ ofactivity enzyme) mol of subunit)
0.13 0.062 Large 0 0 Small 0.15b Large + small 0.066b ND' 0 Large subunit alkylated with ['4C]chloroketone 0 ND Large subunit alkylated with ['4C]chloroketone + small subunit " Incorporation of ['4C]chloroketone was carried out in the presence of 55 AM ['4C]chloroketone and 166 Mig of large subunit protein or 149 Mg of small subunit protein. Incorporation of 0.062 mol of chloroketone corresponds to 110 counts/min. The same amounts of the subunits were assayed for glutaminase activity using 0.24 mM L-[U-'4C]glutamine (specific activity, 57.3 mCi/mmol). Glutaminase activity of 0.13% yields, 2,660 counts/min ['4C]glutamate per 15 min. bCalculated according to the amount of large subunit protein. ND, Not determined. "
o
the glutaminase activity just as for the native enzyme. The large and small subunits of glutamate B synthase react with anti-glutamate synthase and decrease precipitation of ['4C]carboxamidomethylglutamate synthase as shown in Fig. 2A. This suggests at least some restoration of the native tertiary structure. Cross-reactivity of glutamate synthase subunits with anti-glutamate dehydrogenase was not obtained (Fig. 2B). CM OF GEL Previous experiments have established that FIG. 1. Densitometer tracings of SDS gel electro- the NH:-dependent activity of E. coli K-12 gluphoresis of the pooled, large subunit (A) and small tamate synthase is approximately 5 to 7% that subunit (B) fractions. The arrow indicates the posi- of the glutamine-dependent activity (Mantsala tion of the tracking dye. and Zalkin, J. Biol. Chem., in press). Flavin adenine dinucleotide is required for the glutamine-dependent synthesis of glutamate but not TABLE 1. NH3-dependent activity of the separated for NH:-dependent synthesis of glutamate, gluglutamate synthase subunits taminase activity, or affinity labeling by chlo% Activity relative roketone. Apoglutamate synthase, resolved of Subunit to native enzyme" flavin, iron, and sulfide, has enhanced NH10.82b Small dependent activity. The NH:-dependent activ0 Large ity of a glutamine amidotransferase may reflect 0.91 Small + large the primitive aminase activity before modificaaThe specific activity of the native enzyme was tion by association of a glutamine amidotransferase function (8). The present experiments 0.92 U/mg of protein. b Corresponds to absorbance change at 340 nm of identify the small subunit of glutamate syn0.014/10 min using 0.3 absorbance full-scale deflec- thase as responsible for the aminase activity. tion on the recorder. Similarity in molecular weights of the small 0 In 4
NOTES
VOL. 126, 1976
ments we did not detect cross-reactivity between the separated subunits of glutamate synthase and anti-glutamate dehydrogenase. Thus, there is presently no evidence to support an evolutionary relationship between the small subunit of glutamate synthase and glutamate dehydrogenase. In anthranilate synthetase (8) the mechanism of glutamine utilization involves transfer of the amide of glutamine from a site on a glutamine-binding subunit to a site for NH: utilization on a distinct aminase subunit. Further experiments are required to elucidate whether the amide of glutamine that is bound to the large subunit of glutamate synthase is transferred to the NH:1 site of the aminase.
E 06
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This research was supported by Public Health Service grant CA 11442 from the National Cancer Institute. P. M. was a Fulbright-Hayes Scholar.
000
20
40
60
PROTEIN ( 9g)
FIG. 2. Double antibody precipitation. The reaction mixtures contained 13.7 Mg of[I-'4C]carboxamidomethylglutamate synthase or 7.5 pg of [l'4C]carboxamidomethylglutamate dehydrogenase, 2 ,u of antiserum, excess of goat anti-rabbit immunoglobulin, and subunit protein as indicated. The data are corrected for nonspecific precipitation. The experiment measures the capacity of the separated subunits to compete with the radioactive antigens for antibody and decrease precipitation of the radioactive antigen. Symbols: (A) Antiglutamate synthase; (0) large subunit; (A) small subunit; (B) anti-glutamate dehydrogenase. The symbols are the same as in A.
subunit of glutamate synthase with the subunit of E. coli glutamate dehydrogenase has prompted speculation about a relationship between these two proteins (1). We have not detected cross-reactivity between glutamate synthase and anti-glutamate dehydrogenase nor between glutamate dehydrogenase and antiglutamate synthase (Mantsala and Zalkin, J. Biol. Chem., in press). In the present experi-
LITERATURE CITED 1. Blumenthal, K. M., and E. L. Smith. 1975. Alternative substrates for glutamate dehydrogenases. Biochem. Biophys. Res. Commun. 62:78-84. 2. Hennecke, H., and A. Bock. 1975. Altered a subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichia coli. Eur. J. Biochem. 55:431-437. 3. Miller, R. E. 1973. Glutamate synthase from Escherichia coli: an iron-sulfide flavoprotein, p. 183-205. In S. Prusiner and E. R. Stadtman (ed.), The enzymes of glutamine metabolism. Academic Press Inc., New York. 4. Miller, R. E., and E. R. Stadtman. 1972. Glutamate synthase from Escherichia coli. An iron sulfide flavoprotein. J. Biol. Chem. 247:7407-7419. 5. Prusiner, S., and L. Milner. 1970. A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase and asparaginase. Anal. Biochem. 37:429-438. 6. Trotta, P. P., K. E. B. Platzer, R. H. Haschemyer, and A. Meister. 1974. Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. Proc. Natl. Acad. Sci. U.S.A. 71:4607-4611. 7. Weber, K., and D. J. Kuter. 1971. Reversible denaturation of enzymes by sodium dodecyl sulfate. J. Biol. Chem. 246:4504-4509. 8. Zalkin, H. 1973. Anthranilate synthetase, p. 1-39. In A. Meister (ed.), Advances in enzymology. John Wiley & Sons, Inc., New York.
