structural studies of bacillus subtilis glutamine synthetase: further purification, sulfhydryl...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 178, 644-651 (1977)

Structural Studies of Bacillus subtilis Glutamine Synthetase

Further Purification, Sulfhydryl Groups, and the NH,-Terminal Amino Acid Sequence’

ROBERT HSU, SHERWIN J. SINGER, PAMELA KEIM, THOMAS F. DEUEL,2 AND ROBERT L. HEINRIKSON3

Franklin McLean Memorial Research Institute4 and Departments of Medicine and Biochemistry, University of Chicago, Chicago, Illinois 60637

Received May 6, 1976

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxymethylated protein revealed a single NH,-terminal amino acid sequence: HZN-Ala-Lys- Tyr-Thr-Arg5-G1u-Asp-11e-G1n-Lys’”-Leu-Va1-Ser-G1u-Ser~~-CM-Cys-Va1-Thr- Tyr-Ilezo-Ser-Leu-Gly-Phe-Ser2”-Asn-Ser-Leu-Gly- - . The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH, terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5’-dithiobis(2-nitrobenzoic acid).

Glutamine synthetase, which in the presence of a divalent cation catalyzes the synthesis of L-glutamine from ATP, NH3, and L-glutamate, is an important “branch point enzyme” which occupies a pivotal position in cellular metabolism and offers a strategic target for the overall regulation of nitrogen assimilation to the cell (1, 2). Bacillus subtilis glutamine synthetase has a molecular weight of -600,000 and is composed of 12 apparently identical subunits arranged in two superimposed hexagonal

1 This work was supported in part by Grants 74-24 and 75-40 from the American Cancer Society, Illi- nois Division, and Grant HD-07110 from the U. S. Public Health Service.

2 Recipient of Grant CA-13980 from the U. S. Pub- lic Health Service and Faculty Research Award No. 133 from the American Cancer Society.

3Recipient of Grant BMS75-23506 from the Na- tional Science Foundation.

* Operated by the University of Chicago for the U. S. Energy Research and Development Administra- tion under Contract No. E(ll-11-69.

rings (3). This enzyme is remarkably similar in hydrodynamic properties and elec- tron microscopic appearance to glutamine synthetase isolated from Escherichia coli (3). Both enzymes are dodecamers in which the protomer molecular weight is 50,000 (3). However, the two enzymes differ in amino acid composition, in their susceptibility to digestion by carboxypepti- dase A, in their immunological reactivity, and in their respective kinetic parameters (4, 5). Another significant difference be- tween the E. coli and theB. subtilis glutamine synthetases is that the latter does not undergo enzymatic adenylation in response to nitrogen deprivation during growth (3). Fully adenylated E. coli glutamine synthetase differs from nonadenyl- ated enzyme with respect to intrinsic specific activity, divalent cation specificity, and responsiveness to feedback inhibitors, thus providing a unique regulatory mech- anism to the cell. With the B. subtilis enzyme, a single sulfhydryl group is read-

644

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

STRUCTURAL STUDIES OF B. SUBTZZJS GLUTAMINE SYNTHETASE 645

ily alkylated under mild conditions (6) and the resultant changes in catalytic activity are qualitatively similar to those seen in E. coli glutamine synthetase following enzymatic adenylation (6). Before the pri- mary structural features of the enzyme in the vicinity of this modified cysteine residue were investigated, studies were undertaken to characterize the B. subtilis glutamine synthetase preparation more fully in terms of purity and sulfhydryl or disulfide bond content. The present communication describes an additional step in the scheme published earlier (3), which affords an enzyme preparation that is pure by the criterion of automated Edman degradation. The unique NH,-terminal sequence thus obtained provides further sup- port for the existence of identical subunit species in the dodecameric enzyme. Fur- thermore, our studies are consistent with the view that each subunit contains one disulfide bond and two residues of cysteine .

MATERIALS AND METHODS

d4uteriaZs. B. subtilis 60029, a strain which lacks alanine dehydrogenase and requires indole, was generously provided by Dr. Ernest Freeze (National Institutes of Health, Bethesda, Md.). Cells grown at 37°C in a minimal salts medium under conditions of limited nitrogen availability were harvested 4 to 6 h after entering the stationary phase (3). The cell paste was frozen immediately in liquid nitrogen and stored at -20°C. Glutamine synthetase was isolated form 4- to &year-old frozen lots of B. subtillis 60029 by a combination of procedures described earlier (3) and in this communication. Step 5 product (37.5 mg) was applied to a hydroxyapatite column equilibrated with imidazole buffer, pH 7.0, and then eluted with linear gradient of potassium phosphate as detailed in the legend of Fig. 1.

Disodium ATP, iodoacetamide, and 5,5’-dithiobis(2nitrobenzoic acid) were obtained from Sigma Chemical Company, St. Louis, MO. Guani- dinium chloride (ultrapure grade) and iodoacetic acid were purchased from Schwarz/Mann Laborato- ries, Orangeburg, N. Y. Beckman Instruments, Palo Alto, Calif., supplied the highly purified phen- ylisothiocyanate, N,N-dimethylallylamine, anhy- drous heptafluorobutyric acid, benzene, ethylace- tate, and butyl chloride used in the automated Ed- man degradation. Hydroiodic acid (57.7% containing hypophosphorus acid as preservative) and sodium dodecyl sulfate were obtained from Fischer Chemi- cal Company, Pittsburg, Pa. Sephadex was obtained from Pharmacia Fine Chemicals, Piscataway, N. J.,

and Bio-Gel beads and hydroxyapatite were the products of Bio-Rad Laboratories, Richmond, Calif. All other chemicals were of the highest grade com- mercially available.

Analytical procedures. Glutamine synthetase activity was assayed by a modification (3) of the procedure developed by Boyer et al. 17). Protein was rou- tinely measured by the Lowry method (8) with crys- talline bovine serum albumin as the reference standard. Amino acid analyses were performed by automated ion exchange chromatography according to the general principles described by Spackman et

al. (9) on a Durrum D-500 analyzer. Samples were hydrolyzed in redistilled constant boiling HCl in uucuo at 110°C for 24 h. Polyacrylamide gel electro- phoresis of the purified enzyme was carried out by a modification of the original procedure of Davis (10) as detailed in a previous paper (3).

Modification of cysteine residues: Denaturation and alkylation. Salt-free, lyophilized protein (8.1 mg) was dissolved in 2 ml of 0.25 M Tris-HCI buffer, pH 8.6, containing 6 M guanidinium chloride and 3 mM EDTA (denaturing solvent). The sample solution was placed in a vial fitted with a magnetic bar and stirred under a constant stream of nitrogen. Freshly prepared iodoacetate neutralized with NaOH was then added (25°C) to a final concentration of 10 mM, and the vessel was sealed and stored in the dark. Alkylation was stopped after 15 min by addition of 20 ~1 of 2-mercaptoethanol followed immediately by 2 ml of glacial acetic acid. The sample was dialyzed first against 50% acetic acid and then against water and was finally lyophilized. A sample (630 pg) of the carboxymethylated protein was subjected to acid hydrolysis and amino acid analysis.

Denaturation, reduction, and alkylation. A sample (40 mg) from the same batch of protein described above was dissolved in 10 ml of denaturing solvent, and this solution was placed in a vial fitted with a magnetic stirring bar and stirred briskly under a stream of nitrogen for 15 min. The deaerated sample was reduced with 1.35 mmol of 2-mercaptoethanol 5O”C, 1 h) and cooled to 25”C, and 1 ml of a solution

containing 270 mg of iodoacetic acid (1.45 mmol) in 1 N NaOH was added. After 15 min of reaction in the dark, alkylation was stopped by addition of a second portion of 2-mercaptoethanol equal to the first, followed by addition of a volume of glacial acetic acid equal to that of the reaction mixture. The sample was dialyzed against 50% acetic acid, then against water, and was finally lyophilized and stored. A sample of this material (610 kg) was subjected to acid hydrolysis and amino acid analysis.

Reaction of sulfhydryls with 5,5’-dithiobis(2&- trobenzoic acid) (DTNB) .5 A sample (4 nmol) of salt-

s Abbreviations used: DTNB, 5,5’-dithiobis(2-nitrobenzoic acid; PTH, phenylthiohydantoin; gc, gas chromatography; tic, thin-layer chromatography; CM, carboxymethyl.

646 HSU ET AL.

free, lyophilized enzyme was dissolved in 3 ml of solution containing 2% sodium dodecyl sulfate, 10 mM phosphate buffer, pH 8, and 0.5 mg/ml of EDTA. To this solution was added 0.1 ml of 100 mM sodium phosphate buffer, pH 8, containing 4 mglml of 5&i’- dithiobis(2nitrobenzoic acid). The absorbance at 410 nm was continuously monitored with reference to a reagent blank to determine the net optical density. A similar amount of fully reduced and carboxymethylated enzyme was also subjected to the same reaction conditions (11).

NH,-Terminal sequence analysis. Automated Ed- man degradation (12-14) was carried out in a Beck- man protein-peptide sequencer (Model 890-B) with the Quadrol program (program No. D-XI) supplied by the manufacturer. The partial amino acid sequence of residues at the NH,-terminus of the protomer was derived from degradation of reduced and carboxymethylated B subtilis glutamine synthetase. The phenylthiohydantoin (PTH)-amino acids liberated after each cycle were identified and quantitated as such or as the trimethylsilyl derivatives by gas chromatography (1516) on a Beckman GC-45 unit. When unambiguous identification of specific residues was impossible, the residues liberated were converted back to the parent amino acid (or to alanine in the cases of serine and carboxymethylcysteine) by hydrolysis in 58% HI in sealed, evacuated tubes (17). The resultant amino acids were identified with the amino acid analyzer. Assignments made by these quantitative procedures were confirmed by thin-layer chromatography of the PTH- amino acids (18) on plates of silica gel (Eastman 13181 with fluorescent indicator). The sensitive spot test for PTH-histidine (19) and the phenanthrene- quinone fluorescent spot method (20) for detection of PTH-arginine were also employed on selected samples.

RESULTS

In an earlie communication describing the isolation of glutamine synthetase from B. subtilis, it was reported that the enzyme had been purified 135fold and 73-fold relative to respective Mn2+ and Mg2+ acti- vated activities (3). Disc gel electrophore- sis of the purified native enzyme at pH 9.0 showed one major band of low mobility and two faint bands of greater mobility (3). Our results with enzyme prepared in this fashion were similar. However, sequential Edman degradation of the reduced and carboxymethylated enzyme showed alanine (80%) and glycine (20%) as the NH,- terminal residues. The multiplicity of NH,-terminal residues in the step 5 gluta-

mine synthetase preparation (3) could be explained by heterogeneity of the sample or the presence of nonidentical subunits. Accordingly, attempts were made to purify the enzyme further.

Enzyme Purification B. subtilis glutamine synthetase was

prepared to step 5 as described earlier (3). It was found that Sephadex G-200 was in- terchangeable with Bio-Gel A 1.5 m for the gel filtration step in the previous purification. A representative scheme for the current preparation, as well as one from an earlier experiment, is given in Table I.

Our stepwise yields of enzyme activity consistently showed higher recovery of the Mn2+ activity relative to the Mg2+ activity reported previously (3). The final yield of Mg2+-dependent activity relative to the original level, as measured in the cell extract, was always significantly lower than the final yield of Mn2+-dependent activity. Some differences in the present and earlier results, however, did occur. These differences centered on a variance in the specific Mg2+ activity which may have reflected differences in the condition of the starting materials. The Mg2+ activity was more variable in our present preparations. The original Mg2+ activity in the cell extract was never as great as found previously, but rose more sharply during the early steps of the procedure. In the final steps, the Mg2+ activity deteriorated more rap- idly and eventually fell below that in the presence of Mn2+.

Further purification of the gel filtration product (step 5) was achieved by column chromatography on hydroxyapatite (Fig. 1). Glutamine synthetase was resolved from inactive material eluted earlier from the column. When assayed for its enzymatic activity, the hydroxyapatite product showed essentially no increase in specific activity as compared to that obtained by gel filtration alone. Contrary to the step 5 product, however, the enzyme further purified gave essentially a single sequence on automated Edman degradation. This re- sult will be discussed in greater detail below.

STRUCTURAL STUDIES OF B SUBTILJS GLUTAMINE SYNTHETASE 647

TABLE I

PURIFICATION OF B.subtiLiis GLUTAMINE SYNTHETASE IN CURRENT AND EARLIER F'REPARATION~ Total protein Specific activity” Percentage recovery

Mn*+ Mgz+ Mn2+ Mg*+

Preparation I (200 g of wet cells) Crude extract 22.3 69.6 36.4 100 100 Streptomycin precipitate 19.7 66.3 70.6 84 171 Ammonium sulfate precipitate 2.70 260.3 276.8 45 92 65”C, pH 5.3 supernatant 0.77 1975.3 803.3 94 73 Bio-Gel A 1.5 m effluent 0.159 3387.0 820.8 35 16

Preparation II (210 g of wet cellsy Crude extract 23.92 54 244 100 100 Streptomycin precipitate 17.78 80 373 109 113 Ammonium sulfate precipitate 3.80 400 1347 116 87 65”C, pH 5.3 supernatant 1.19 1095 4381 100 89 Bio-Gel A 1.5 m effluent 0.13 7333 17773 73 39

a Assays were performed according to the standard biosynthetic assay (3), using either Mn2+ or Mg2+ as indicated.

b As reported by Deuel et al. (3).

Cysteine and Cystine Content ofB. subtilis Glutamine Synthetase

In contrast to the E. coli enzyme, the cysteinyl residues in native B. subtilis glutamine synthetase react readily with al- kylating reagents (6). Furthermore, B. subtilis glutamine synthetase requires 2 mercaptoethanol for stability during purification and storage. With the alkylation of one cysteine residue, an increase in Mn2+ activity, a sharp decrease in Mg2+ activity, and an alteration of the response of the enzyme to feedback inhibitors were previously reported (6). In view of the fact that cysteine appears to be involved in some way with the catalytic activity of glutamine synthetase, it was of importance to determine the number of free sulfhydryl groups and disulfide bonds in the native enzyme.

Amino acid analysis of B. subtilis glutamine synthetase following reduction and S-carboxymethylation under denaturing conditions indicates that there is a total of four cysteine residues per subunit (Table II), in agreement with results published earlier (6). However, only 1.5 carboxymethylcysteine residues were found by analysis of the enzyme denatured and carboxymethylated without prior reduction (Table II).

Corroboration of these findings was provided by Ellman titration. When a sample

4 A,, 0.6 -

t

-7

b ::: 0.5 c

i lb I‘s io is zfo TUBE++-

FIG. 1. Chromatography of the step 5 material from Bio-Gel A 1.5 m on a column (1.5 x 5.9 cm) of hydroxyapatite. The protein, dissolved in 50 mM imidazole buffer, 2 mM EDTA, 2 mM 2-mercaptoeth- ano1, pH 7.0, was adsorbed to a column previously equilibrated with the same buffer. The sample was eluted with a linear gradient of increasing phosphate concentration. The mixing chamber originally contained 35 ml of the starting buffer, as described above, and the reservoir contained an equal volume of the same buffer made 0.4 M in potassium phosphate. Since the phosphate present in the gradient would interfere with the determination of enzyme activity using the biosynthetic method, the y-gluta- myltransferase assay was performed in this case according to the procedure described previously (5). The shaded area indicates enzyme activity.

of denatured enzyme (4 nmol) was allowed to react with DTNB, the enzyme showed a rapid uptake of 2.3 mol of reagent/m01 of subunit after about 13 min. This was followed by a slow steady increase to 2.4 equivalents after 25 min (Fig. 2). An ex- tinction coefficient of 13,600 M-’ cm-’ was

648 HSU ET AL.

used to determine the amount of 2-nitrod- thiobenzoate released (11) and hence the number of cysteinyl groups titrated (Table II). In a control experiment with the reduced and carboxymethylated enzyme, no reaction of DTNB with enzyme was observed. These results, together with studies of carboxymethylation, suggest the presence of one disulfide linkage and two free sullhydryl groups per subunit in the nativeB. subtilis glutamine synthetase.

NHTTerminal Sequence Analysis of B. subtilis Glutamine Synthetase

A sample of the glutamine synthetase obtained by chromatography on hydroxyapatite (Fig. 1) was reduced and carboxymethylated and 7 mg (140 nmol subunit) of this material was subjected to automated Edman degradation. The following NH,- terminal sequence was derived in this analysis: H,N-Ala-Lys-Tyr-Thr-Argj- Glu-Asp-Ile-Gln-Lyslo-Leu-Val-Ser- Glu-Ser15-CM-Cys-Val-Thr-Tyr-Ile20- Ser-Leu-Gly-Phe-Ser2”-Asn-Ser-Leu- Gly- - . PTH-amino acids were identified

TABLE II FREE SULFI-IYDRYL CONTENT OF REDUCED AND

UNREDUCED B. subtilis GLUTAMINE SYNTHETASP Experimental descriptioti Number of cyst&e

residues per 50,000 g of protein

Found Nearest integer

A. Denatured enzyme, re- 3.9 4 duced and carboxymethylated

B. Denatured enzyme, car- 1.5 2 boxymethylated without reduction

C. Denatured enzyme ti- 2.3 2 trated with DTNB

’ Experimental details are given in Materials and Methods.

b In A and B, the denatured enzyme was carboxymethylated with and without prior reduction. Reac- tion by-products and salts were removed by exhaus- tive dialysis against water and samples were hydrolyzed for 24 h at 110°C in uucuo in 6 N HCI. Cysteine was determined as CM-Cys by amino acid analysis. In C, the cysteine content was determined by release of 2-nitro-5-thiobenzoate; eM = 13,600 M-’ cm-’ during titration of the denatured enzyme with DTNB (cf. Fig. 2).

and quantitated by gas chromatography, amino acid analysis following hydrolytic back conversion (171, and thin-layer chromatography (18). The data so obtained are presented in Table III. Yields of the most easily quantitated derivatives were high from the NH,-terminus through the nine- teenth step, but, as evidenced by gas-chro- matographic analysis, dropped consider- ably from residue 20 through 29. Beyond that point, increased background noise made further identification impossible. Yields of representative PTH-amino acids were: Ala-l, 120 nmol; Ile-8,100 nmol; Val- 12, 75 nmol; Val-17, 35 nmol; and Tyr-19, 25 rmol. The initial yield of Ala-l was about 86% of theoretical, a value higher than that usually encountered with mole- cules of this size (21, 22). Except for residue 1, no multiplicity of PTH-derivatives was observed in gas-liquid chromato- graphs at any cycle and a single sequence was elucidated for the enzyme subunit. Gas chromatography of the material in cycle 1 showed, in addition to alanine, a derivative that appeared to be isoleucine. It was not identified, however, by thin- layer chromatography of cycle 1 and may have been solvent contamination in the first stage of degradation which was removed at this cycle.

Automated Edman degradation of step 5 enzyme (3) described above gave predomi- nant yields of alanine as the NH,-terminal residue, although glycine, leucine, and isoleucine were also observed in lesser quantities. The present analysis indicates that these protein contaminants have been removed by the purification step on hydroxyapatite. The NH,-terminal sequence is presented in Table III together with yields of the PTH-derivatives and a listing of the methods employed in making the identification or quantitation in each case. Gas chromatography failed to give a defi- nite identification of residues 15 and 25; either position could have been serine or carboxymethylcysteine. Hydrolytic back conversion of these residues gave alanine in each case, as would be expected from serine or carboxymethylcysteine (17). The PTH-derivatives of serine and carboxymethylcysteine may be distinguished by

STRUCTURAL STUDIES OF B. SUBTILE GLUTAMINE SYNTHETASE 649

TABLE III

THE N-TERMINAL SEQUENCE AND TECHNIQUES NECESSARY TO CONFIRM THE IDENTITY OF THE VARIOUS PTH-AMINO ACIDS ARE LISTED BELOW

Number Residue G;:;:pohmya- T:inol+;?r Back conver- Remarks sion

tw-why

1 Ala x

2 LYS X

3 TYr X

4 Thr X

5 -4% 6 Glu 7 Asp 8 Ile 9 Gln

10 LYS 11 Leu 12 Val 13 Ser 14 Glu 15 Ser 16 CM-Cys

X

X

X X

X

X

X

X

X

17 Val 18 Thr 19 Tyr 20” Ile

X X

X

21 22 23 24 25 26 27 28 29 30

Ser Leu Glx6 Phe Ser Asn Ser Leu

GUY

X

X

X X X X

X 50 nmol of Ile-like peak in gc but not confirmed by tic

X X

Large yield of a-amino butric acid on back conversion

Arginine spot test positive

X X

X

X

X X

X

X

X X

X

X Small yield of Ala on back conversion

Small yield of Ala on back conversion Appears on gc as Ser, tic confirmed as

CM-cysteine

X X

X X

X

X

X

X

X

X Weak signal on gc, small yield on back conversion

Small yield of Ala on back conversion X

X

X Yield Glu on back conversion

X Small yield of Ala on back conversion X Asp on back conversion

X

a Residues 20-29 all gave negative Arg and His spot tests, ruling out the possibility of either in this group. b Gln or Glu.

thin-layer chromatography following staining with ninhydrin (23,24). Both residues 15 and 25 appeared to be serine by this method. However, residue 16 gave a distinctive blue spot corresponding to PTH-CM-Cys and this position was therefore identified as cysteine.

DISCUSSION

mine synthetase subunit causes profound changes in enzyme catalytic activity (6) which are qualitatively similar to those associated with enzymatic adenylation of E. coli glutamine synthetase (6). The present study was undertaken as the first part of our investigation aimed at elucidating the structure of the active site ofB. subtilis glutamine synthetase.

Chemical modification of one of the free By a procedure described earlier (31, the sulfhydryl groups in the B. subtdis gluta- enzyme from B. subtilis was purified to a

650 HSU ET AL.

2 4 6 8 IO 12 14 16 16 20 22 24

MINUTES

FIG. 2. Equivalents of -SH per subunit of 50,000 M, titrated with DTNB. (Details for all procedures given in Materials and Methods.)

nearly homogeneous state with a concomi- tant increase of more than loo-fold in specific activity. Preliminary analyses of this preparation by automated Edman degradation showed multiple NH,-terminal residues and sequences. The heterogeneity thus observed could have been due to mi- nor impurities in the enzyme preparation or to the existence in the dodecamer of nonidentical subunits. Many oligomeric enzymes contain structurally different subunits (251, although multiple criteria applied to the question of subunit identity in B. subtilis glutamine synthetase indicated the presence of 12 identical proto- mers in the dodecameric enzyme (3). In order to evaluate these possibilities, the step 5 B. subtilis glutamine synthetase preparation was further purified by ad- sorption column chromatography on hydroxyapatite. Automated Edman degradation of the resulting enzyme preparation gave a single amino acid sequence and the yields of PTH-amino acids at each step were consistent with the quantity of protomer subjected to analysis. These findings are consistent with the view that the enzyme obtained by hydroxyapatite chromatography is homogeneous. They also pro- vide the strongest evidence thus far available that, as is the case with E. coli glutamine synthetase, the 12 subunits of the B. subtilis enzyme are identical.

Previous studies of the sulfhydryl groups of B. subtilis glutamine synthetase (6) showed that four half-cystines were

present per 50,000 g of the enzyme, as ana- lyzed in the reduced state. No attempt to seek a disulfide linkage was made. Only one residue per subunit of the native enzyme was readily alkylated with iodoacetamide. Free cysteinyl residues in native proteins often exhibited variable reactivity toward chemical reagents due to steric factors and this fact has been used in sev- eral cases to detect confirmational changes accompanying derivatization (26). The difference in susceptibility of disulfide bonds towd reducing agents in native and modified proteins has also been employed (11) in attempts to monitor conformational changes associated with chemical modifi- cations. It is well known that disulfide bonds play an important role in stabilizing the three-dimensional structure of proteins; a knowledge of their number and reactivity in native and denatured proteins is therefore of considerable importance.

In addressing ourselves to the question of the free sulfhydryl and disulfide bond content of B. subtilis glutamine synthetase, we studied the reactivity of SH groups in the native, denatured, and reduced and denatured protein toward iodoacetate. A companion study was carried out in which the disulfide exchange reaction of the enzyme with DTNB was monitored under denaturing conditions. Our results clearly indicate the presence of two free SH groups and a single disulfide bond in each protomer. Chemical modification of one of the two free sulfhydryl groups has been shown to modulate the activity of the B. subtilis glutamine synthetase. Work is currently underway to determine the sequence in the vicinity of their cysteine residue. The single disulfide bond in the enzyme subunit is probably involved in some way with the maintenance of the conformation necessary for the catalytic function of the dodecameric enzyme.

REFERENCES

1. STADTMAN, E. R., SHAPIRO, B. M., KINGDON, H. S., WOOLFOLK, C. A., AND HUBBARD, J. S. (1968) A&an. Enzyme Regul. 6, 257-289.

2. STADTMAN, E. R. (1971) Harvey Lecture 65, 97- 125.

3. DEUEL, T. F., GINSBURG, A., YEH, J., SHELTON, E., AND STADTMAN, E. R. (1970) J. Biol.

STRUCTURAL STUDIES OF B. SUBTILIS GLUTAMINE SYNTHETASE 651

Chem. 245, 5195-5205. 4. DEUEL, T. F., AND STADTMAN, E. R. (1970) J.

Biol. Chem. 245, 5206-5213. 5. DEUEL, T. F., AND TURNER, D. C. (1972)5. Biol.

Chem. 247, 3039-3047. 6. DEUEL, T. F. (1971)5. Biol. Chem. 246,599-605. 7. BOYER, P. D., MILLS, R. C., AND FROMM, H. J.

(1959)Arch. Biochem. Biophys. 81, 249-263. 8. LOWRY, 0. H., ROSEBROUGH, H. J., FARR, A. L.,

AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275.

9. SPACKMAN, D. H., STEIN, W. J., AND MOORE, S. (1968) Anal. Chem. 30, 1190-1206.

10. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-426.

11. HABEEB, A. F. S. A. (1972)Zn Methods in Enzy- mology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 25, Part B, pp. 457-464, Academic Press, New York.

12. EDMAN, P. (1950)Actu Chem. Stand. 4,277-282. 13. EDMAN, P., AND BEGG, G. (1967) Eur. J. Bio-

them. 1, 404-426. 14. FIETZEK, P. O., AND KUHN, K. (1972) Fortschr.

Chem. Forsch. 29, l-28. 15. PISANO, J. J., AND BRONZERT, T. J. (1969) J.

Biol. Chem. 244, 5597-5607. 16. HERMODSON, M. A. ERICKSON, L. H., TITANI, K.,

NEURATH, H., AND WALSH, K. A. (1972) Bio- chemistry 11, 4493-4502.

17. SMITHIES, O., GIBSON, D., FANNING, E. M., GOODFLIESH, R. M., GILMAN, J. G.,, AND BAL- LANTYNE, D. L. (1971) Biochemzstry 10, 4912- 4921.

18. JEPPSSON, J.-O., AND SJ~QUIS?, J. (1967) Anal. Biochem. 18, 264-269.

19. The Spinco Division of Beckman Instruments, Inc., (1971) Palo Alto, California, in sequence, March, 31-2.

20. YAMADA, S., AND ITANO, H. A. (1966) Biochim. Biophys. Actu 130, 538-540.

21. KINGDON, H. S., NOYES, C., LAHIRI, A., AND HEINRIKSON, R. L. (1972) J. Biol. Chem. 247, 7923-7926.

22. MILLER, J. B., NOYES, C., HEINRIKSON, R. L., KINGDON, H. S., AND YACHNIN, S. (1973) J. Exp. Med. 138, 939-951.

23. SUMMERS, M. R., SMYTHERS, G. W., AND OROSZ- LAN, S. (1973) Anal. Biochem. 53, 624-628.

24. SOLAL, M. C., AND BERNARD, J. L. (1973) J. Chromutogr. 80, 140-143.

25. FRIEDEN, C. (1971)Ann. Rev. Biochem. 40, 653- 696.

26. GUIDOTTI, G. (1965) J. Biol. Chem. 240, 3924- 3927.

structural studies of bacillus subtilis glutamine synthetase: further purification, sulfhydryl...

Documents