active site ligand stabilization of quaternary structures of

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257,,No. 12, Issue of June 25, pp. 7246-7251, Printed LIZ U.S.A.

1982

Active Site Ligand Stabilization of Quaternary Structures of Glutamine Synthetase from Escherichia CoZi*

(Received for publication, February 19, 1982)

Michael R. Maurizi and Ann Ginsburg$ From the Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205

Auto-inactivated Escherichia coli glutamine synthetase contains 1 eq each of L-methionine-S-sulfoximine phosphate and ADP and 2 eq of Mn2+ tightly bound to the active site of each subunit of the dodecameric enzyme (Mauizi, M. R., and Ginsburg, A. (1982) J. Bwl. Chem 257, 4271-4278). Complete dissociation and unfolding in 6 M guanidine HC1 at pH 7.2 and 37 “C requires >4 h for the auto-inactivated enzyme complex ( t l min for uncomplexed enzyme). Release of ligands and dissociation and unfolding of the protein occur in parallel but follow non-first order kinetics, suggesting stable intermediates and multiple pathways for the dissociation reactions. Treatment of partially inactivated glutamine synthetase (2-6 autoinactivated subunits/dodecamer) with EDTA and dithiobisnitrobenzoic acid at pH 8 modifies -2 of the 4 sulfhydryl groups of unliganded subunits and causes dissociation of the enzyme to stable oligomeric intermediates with 4, 6,8, and 10 subunits, containing equal numbers of uncomplexed subunits and autoinactivated subunits. With 970% inactivated enzyme, no dissociation occurs under these conditions. Electron micrographs of oligomers, presented in the appendix (Haschemeyer, R. H., Wall, J. S., Hainfeld, J., and Maurizi, M. R., (1982) J. Biol. C h m . 257,7252-7253) suggest that dissociation of partially liganded dodecamers occurs by cleavage of intra- ring subunit contacts across both hexagonal rings and that these intra-ring subunit interactions are stabilized by active site ligand binding. Isolated tetramers (M, = 200,000; s20,w = 9.5 S ) retain sufficient native structure to express significant enzymatic activity; tetramers reassociate to dodecamers and show a 5-fold increase in activity upon removal of the thionitrobenzoate groups with 2-mercaptoethanol. Thus, the tight binding of ligands to the subunit active site strengthens both intra- and inter-subunit bonding domains in dodecameric glutamine synthetase.

The energetics of ligand binding to protein involves contri- butions from both ligand-protein and protein-protein interactions. Ligand-promoted changes in protein-protein interactions underlie the phenomenon of cooperativity in ligand binding to proteins (1) and, in addition, give rise to the numerous examples of stabilization and destabilization of protein structures by ligands, metal ions, and other inorganic ions (2). A remarkable example of linkage between ligand binding

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + To whom correspondence should be addressed at, National In- stitutes of Health, Building 3, Room 208, Bethesda, MD 20205.

and inter-subunit bonding strength is seen with the avidin- biotin complex, in which the tetrameric protein with bound coenzyme remains associated in 6 M guanidine HCl (3).

Escherichia coli glutamine synthetase forms an extremely tight, inactive complex with L-methionine-S-sulfoximine phosphate, ADP, and 2 Mn2’ bound per subunit ( 4 , 5 ) . The inactive complex is formed on the enzyme by the transfer of the y- phosphoryl group of ATP to the imino nitrogen of L-methionine-S-sulfoximine (6, 7). Recent experiments ( 5 ) have shown that disruption of the inactive complex with recovery of active enzyme requires protonation of protein carboxylate groups and structural perturbations produced by high KC1 concentrations and increased temperature.

The effects of inactive complex formation on the stability of the dodecameric enzyme at neutral pH are reported here. The 12 identical subunits of glutamine synthetase are ar- ranged in 2 superimposed hexagonal rings (8,9). Each subunit contains an active site with 2 metal ion sites essential for catalysis (10) and protein stability (11). Removal of Mn2+ from the high affinity nl site produces a relaxed inactive enzyme of the same molecular weight that has altered hydro- dynamic properties, is more easily denatured, and is more susceptible to covalent modification (11, 12). Ligands (ADP, L-glutamine, arsenate) that increase metal ion binding at the n2 site also stabilize the enzyme under denaturing conditions (13). This paper describes the resistance of the inactive enzyme complex to unfolding and dissociation and the stabilization of bonding domains between adjacent subunits of glutamine synthetase produced by ligand binding at the active site.

EXPERIMENTAL PROCEDURES

Materials-All water was distilled, then deionized and fdtered through a Millipore Corporation Milli Q2 reagent grade system. L- Methionine-SR-sulfoximine, Hepes,’ Tris, and ATP were from Sigma. Sodium dodecyl sulfate, Chelex 100, and urea were from Bio-Rad; ultrapure grade of guanidine hydrochloride was from Schwartz-Mann, and the tetrasodium salt of xylenol orange was obtained from Fisher. [’%]ATP and [y-32]ATP were obtained from New England Nuclear. Unadenylylated glutamine synthetase (GSi) was prepared from E. coli W by the method of Woolfolk et al. (14) and stored in 20 mM Hepes/KOH or 20 mM imidazole/HCl, pH 7.2, containing 100 mM KC1 and 1 mM MnClz at 4 “c.

Assays of Enzyme Activity and Protein-Enzymatic assays for glutamine synthetase and its state of adenylylation (E) were performed using the y-glutamyl transferase assay at pH 7.57 as described (15). The same solutions were used for enzyme activity staining of polyacrylamide gels following electrophoresis under nondenaturing

I The abbreviations used are: Hepes, 4-(2-hydroxyethyl-l-pipera- zineethanesulfonic acid; TNB, dithlobisnitrobenzoic acid; Gdn . HC1, guanidine hydrochloride; GS,, glutamine synthetase with some number (E = 0 - 12) of 5‘-adenylate groups attached/dodecameric enzyme; XO, xylenol orange; HPLC, high pressure liquid chromatography.

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Active Site Ligand Stabilization of Quaternary Structure 7247

conditions. Protein concentrations were determined for the native enzyme by UV absorbance (16) and for the inactive enzyme by the method of Bradford (17) with purified glutamine synthetase as the protein standard.

Preparation of Znactiue Enzyme Complex-Manganese glutamine synthetase (1-10 mg/ml) was combined with 1-5 mM L-methionine sulfoximine in 20 mM Hepes or 20 mM imidazole, pH 7.2, 100 mM KC1, and 5 mM MnC12. ATP (0.2-2 mM) was then added and the solution was incubated at 37 "C for 1-2 h and then overnight at room temperature. Unbound small molecules were removed by dialysis against 3 x IO00 volumes of buffer containing 100 mM KC1. Manganese enzyme retained ~ 0 . 5 % activity after this treatment and had 10.5-11.5 equiv- alents of tightly bound ligandddodecamer. The inactive complex was stable a t 4 "C for >4 months. Inactive enzyme complex containing either [I4C]ADP or L-methionine-S-sulfoximine ["P]phosphate were prepared as above with the addition of [I4C]ATP or [y-"PIATP.

Release of Ligands and Mnz+ from Znactiue Complex-To measure the release of [14C]ADP or L-methionine-S-sulfoximine ["'Plphos- phate from inactive complexes in 6 M Gdn-HC1, the incubation mixtures were diluted 10-20-fold into buffer at pH 7 containing 5-10 mM EDTA, which stops dissociation immediately. Separation of protein-bound and unbound ligands was accomplished by passing the diluted enzyme (<25 pg) through 0.45-p Millipore fiiters (HATF 01300 or Millex HA). The filtrate was counted for radioactivity. When sodium dodecyl sulfate was present, solutions were ultrafiltered in CF 25 ultrafitration cones (Amicon) centrifuged at 900 X g for 20 min.

Concentrations of free Mn2+ were measured using the dye-metal ion chelator xylenol orange, X 0 (18). Stock solutions of 0.1-1 mM X 0 and 7.4 M Gdn.HC1 at pH 7.2 in 20 mM Hepes/KOH, 100 mM KC1 were passed through small columns of the K' form of Chelex 100 (100-200 mesh) equilibrated in the same buffer. The K+ form of Chelex 100 and the standard MnCL solution were prepared as described previously (19). Mn2+ calibration curves were constructed under the conditions used for dissociation (5.9 M Gdn.HC1, 20 mM Hepes/KOH, 100 mM KC1 at pH 7.2 and 37 "C). In 5.9 M Gdn.HC1 at pH 7.2, the binding of Mn2+ by X 0 produces A€ = 1.1 X lo4 M" cm" at 585 nm; the half-saturation value is 3.7 (IM with a Hill coefficient of 1.8 indicating that there is dye destacking and possibly a 2:l complex formed between X 0 and Mn2+ in 6 M Gdn. HCl. Mn2+ release was recorded continuously on the 0.1 absorbance scale of a Cary model 15 spectrophotometer with a neutral density screen in the reference compartment. Gdn. HCl solution (0.95 ml) was placed in two water- jacketed cells of 10-mm path length and equilibrated at 37 "C; a 50- pl aliquot of the dialyzed, inactive enzyme complex was added to one cell and a 50-4 aliquot of the protein dialysate (20 mM Hepes/KOH, 100 mM KC1, pH 7.2) was added to the other cell as the control. In the experiment of Fig. 1, the increase in AK, nm for each cuvette was recorded with 19.4 p~ X 0 and 5.9 (IM inactive enzyme subunits. Comparable results were obtained when AAfilo nm was measured with 67.6 p~ X 0 and 6.5 p~ inactive enzyme subunits (not shown). After -100 min at 37 "C, the contents of both cells were heated for 2 min at 100 "C (to release all the Mn2+ from the complex) and the AAGI0 nm

was measured at 37 "C. The final AA6,0 ,,,,, of the protein/dye Gdn. HCl solution (corrected for any absorbance change in the control) corresponded to the 2.0 k 0.2 eq of Mn2+ released per enzyme subunit complex initially added (Fig. 1).

Sulfiydryl Determinations-Sulfhydryl groups were measured with dithiobisnitrobenzoic acid by incubating protein at pH 8.1 in 100 mM Tris/HCl, 5 mM EDTA, and 0.4 mg/ml of DTNB. An extinction coefficient of 13,600 M" at 412 nm was used (20). sulfhydryl group exposure in 6 M Gdn HCl was determined by transferring aliquots of enzyme or inactive complex incubated in 6 M Gdn. HCl to a solution of 0.4 mg/ml of DTNB, 5 mM EDTA, 6 M Gdn.HC1, and 100 mM Tris/HCl, pH 8.1. Readings were recorded immediately.

Light Scattering and Fluorescence Measurements-Light scattering was measured at 90" using a Hitachi Perkin-Elmer MPF 2A fluorescence spectrophotometer. Measurements were made with the excitation and emimion monochromaters set a t 360 nm and slits set at 5 nm. Under these conditions, light scattering was proportional to concentration with 50-400 @/ml of protein. Fluorescence was measured in the same spectrofluorometer with excitation at 290 or 300 nm and emission at 340 nm; slits were 4-6 nm. Output was recorded on a Hewlett-Packard 704B X-Y plotter. Cuvettes were housed in a water- jacketed cuvette holder.

Ultracentrifugation-A Beckman Model E ultracentrifuge was used, which was equipped with a rotor temperature and control unit, phase plate and schlieren optics, and absorption optics with photoe- lectric scanner and multiplexer. Instrument calibrations, schlieren

photographs, and viscosity and density measurements were as previously described (12). The observed sedimentation coefficients were corrected to values corresponding to a solvent with the viscosity and density of water a t 20 "C (s20. .,).

Other Methods-High pressure gel filtration was carried out with a Hewlett-Packard 1084B Liquid Chromatograph equipped with a Toyo Soda TSK G3000 SW gel filtration column (30 m l ) equilibrated in either 50 mM potassium phosphate, pH 7.2, or 50 mM Tris/acetate, 1 mM EDTA, pH 7.8. Native gel electrophoresis was carried out using the buffer system of Davis (21) and employing a slab gel (15 X 20 cm) composed of 6% acrylamide. All scintillation counting was done with 10 ml Aquasol and 0.50-1.0 ml aqueous sample in a Beckman LS250 liquid scintillation counter.

RESULTS AND DISCUSSION

Disruption of the Inactive Complex under Denaturing Con- ditions-The inactive enzyme complex of glutamine synthetase with L-methionine-S-sulfoximine phosphate, ADP, and 2 Mn2+ bound per subunit displays a remarkable resistance to disruption under denaturing conditions. Treatment with 2% sodium dodecyl sulfate or 8 M urea caused release of only 0- 30% of [I4C]ADP or L-methionine-S-sulfoximine [32P]phosphate even after incubation at 25 or 37 "C for 24 h. When incubated in 2 M NaSCN at 25 "C, native glutamine synthetase dissociated to monomers which slowly reassociated to form large aggregates ( M , > lo6) whereas the inactive enzyme complex had not dissociated after 2 h in 2 M NaSCN. Complete release of ADP and Mn2+ from the inactive complex and the dissociation and unfolding of the protein required -4 h at 37 "C in 6 M guanidine HC1. In contrast, complete dissociation and unfolding of active glutamine synthetase in 6 M guanidine HC1 occurred in less than 1 min (Fig. 1, line to asterisk).

For the data in Fig. 1, [I4C]ADP release was measured at the times indicated and Mn2+ release in 6 M guanidine HCl was recorded continuously by spectrophotometric measurements in the presence of xylenol orange. The quenching of protein tryptophanyl fluorescence upon exposure of tryptophanyl residues to solvent and the increased reactivity of protein sulfhydryl groups with DTNB were used to monitor the extent of unfolding of the subunit. Dissociation of the dodecamer was measured by the decrease in light scattering. As shown in Fig. 1, ligand release and protein dissociation and unfolding in 6 M guanidine HC1 followed the same slow time course.

If rapid disaggregation and unfolding of the protein were dependent on the rate-limiting dissociation of the components of the inactive complex, the reactions in 6 M guanidine HC1 should have followed first order kinetics. However, the kinetics of these changes were non-fist order. Upon addition of 6 M guanidine HC1, 15-25% of the ADP and Mn2+ were released from the inactive complex and 1-2 of the 4 sulfhydryl groups/ subunit reacted with DTNB within 2 min; the remaining reactions occurred slowly with non-fist order kinetics. Heat- ing to 100 "C completed all reactions (Fig. 1). Although the molecular weights of the species present during dissociation of the inactive complex in 6 M guanidine HC1 were not determined, the decrease in light scattering immediately following addition of guanidine HCl (after correcting for solvent effects (22)) was 38%, suggesting that partial dissociation of the dodecamer occurred rapidly; the overall kinetics suggested that dissociation occurred in a stepwise manner with multiple intermediates. When 8 M urea was used instead of guanidine HCl, stepwise dissociation of the enzyme was evident from the multiple species observed upon polyacrylamide gel analysis.

Effects of Active and Inactive Subunits within the Same Dodecamer-Partially inactive enzyme was used to observe the influence of inactivated subunits on the structure and activity of unliganded subunits in the same dodecamer. Glu- tamine synthetase containing less than 12 inactive subunits/


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7248 Active Site Ligand Stabilization of Quaternary Structure

90-

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80 - 0

$ 7 0 - U

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- w U

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20 -

10 -

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I I I 1 1 I

0 Sulfhydryl Exposure

0 Fluorescence Change

V Light Scattermg Change

1 i

1 I 1 1 I I 1 I 20 40 60 80 100 120 140 160

TIME iminl

FIG. 1. Dissociation of inactive complex and denaturation of glutamine synthetase in 6 M guanidine HCl. Inactive complex of manganese enzyme was incubated at 37 "C in 20 mM Hepes, 100 m~ KC1, and 6 M guanidine HC1 and changes in various parameters were measured. The final fluorescence and light scattering and the number of reactive sulhydryl groups for native and inactivated enzyme (after 2 min at 100 "C) were the same. The per cent ADP released was calculated from the actual amount of [I4C]ADP bound to protein and per cent Mn2+ release was based on the calculated amount of 2 MnZ+ bound/subunit (19). A, [14C]ADP release; 0, Mn2+ release; 0, exposure of sulfhydryl groups; 0, change in protein fluorescence; 0, change in light scattering; *, change in fluorescence, light scattering, and sulfhydryl exposure when active enzyme was used.

dodecamer was prepared by treating the enzyme with substoichiometric amounts of either ATP of L-methionine-S-sulfoximine in the presence of an excess Mn2+ and the other reactant. Fig. 2 shows that the loss of enzymatic activity was directly proportional to the amount of limiting L-methionine- S-sulfoximine used in the inactivation reaction. The increase in fluorescence upon formation of the inactive complex par- alleled the extent of inactivation (Fig. 2) indicating that the inactive complex on one subunit does not perturb the environ- ment of tryptophan residues on adjacent subunits. Treatment of the inactive complex with EDTA removed Mn2+ from active subunits within partially active dodecamers. Fig. 2 shows that removal of Mn2+ from these subunits promotes the same protein conformational change (monitored by b l z ~ . ~ ) observed with native enzyme (11); however, the rate of this conformational change was -2-fold slower than with native enzyme. Treatment with EDTA and DTNB, which leads to modification of all 4 sulfhydryl groups/subunit of native enzyme (23), resulted in modification of less than half the sulhydryl groups of the active subunits in partially inactive enzyme (Fig. 2). Since removal of the metal ions and modification of sulfhydryl groups cause dissociation of native glutamine synthetase (12), the effect of these treatments on partially inactivated enzyme was examined.

Formation of Stable Oligomers of Glutamine Synthetase- Fig. 3 shows that when 15% inactivated glutamine synthetase was treated with EDTA and DTNB, partial dissociation of the dodecamer occurred. Six major species were resolved by high pressure gel filtration (Fig. 3A) and native gel electrophoresis (Fig. 3B). Identical patterns of oligomers were observed when either unadenylylated or adenylylated enzyme

was used, but the following studies were all performed with unadenylylated glutamine synthetase. The molecular weights estimated by gel filtration or by the method of Ferguson (24) using gel electrophoresis indicated that the various species correspond to oligomers with 4, 6, 8, 10, and 12 subunits as well as aggregates of M , > lo6. Individual oligomers remained intact upon repeated gel filtration, after exhaustive dialysis, and during storage for >1 month at 4 "C. Identical oligomers with similar stabilities were obtained in either 50 mM potassium phosphate, pH 7.2, or 50 mM Tris/acetate, pH 7.8, with 1 mM EDTA. Thus, the profiles seen in Fig. 3 do not represent a simple equilibrium distribution of aggregated species but rather stable oligomers of defined composition.

The distribution of oligomers found is a function of the per cent inactive enzyme complex in the enzyme treated with EDTA and DTNB (Fig. 4). At low extents of inactivation (<20%), mostly tetramers were found, but, as the per cent inactivation was increased, the amount of tetramer obtained decreased and higher molecular weight oligomers were fa- vored in turn. Above 70% inactivated enzyme, no dissociation occurred and only intact dodecamers were found. With the native enzyme (0% inactivated enzyme in Fig. 4) oligomers were not detected and only large aggregates (Mr > lo6) were seen. Occasionally, small amounts of monomers and dimers were observed, but these species were too unstable to be isolated. The species with M, > lo6 (Fig. 3A) represent mostly unliganded subunits (monomers or dimers) that dissociated from the partially inactivated dodecamer upon modification with DTNB and then reaggregated to form the large inactive aggregates; this process was also seen with native enzyme treated with EDTA and DTNB (Fig. 4).

Oligomers of the same size and in approximately the same proportions were obtained whether the partially inactive enzyme was produced by fractional reactivation of fully inactive enzyme complex (5) or by fractional inactivation of the native enzyme. Thus, the inactivation and reactivation reactions produce the same average distribution of inactive subunits within the dodecamer. Since the conditions for inactivation (pH 7.0, 0.1 M KC1, 1 mM MnC12) are quite different than

100

80

60

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0 0 0.25 0.50 0.75

Y

- L-Methionine-S-SulfoximineiGS FIG. 2. Structural and catalytic effects of inactive and active

subunits within the same dodecamer. Unadenylylated enzyme was incubated with substoichiometric amounts of L-methionine-S- sulfoximine in the presence of 2 mM MnCL and 1 mM ATP. After 12 h at 22 "C, the activity in the y-glutamyl transferase assay was measured (0). After removing uncomplexed ligands by dialysis against 20 mM Hepes/KOH, 100 mM KCl, 0.1 mM MnC12, pH 7.2, the fluorescence emission (A), relaxation upon EDTA treatment (V), and the extent of reaction of sulfhydryl groups with DTNB in the presence of EDTA (0) were measured.

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those for reactivation (1 M KCl, pH 4.0), this result suggests that inactive subunits are randomly distributed in partially inactive enzyme.

Glutamine synthetase oligomers sedimented at different rates in the ultracentrifuge (Fig. 5). When 35% inactivated enzyme was treated with EbTA and DTNB, five protein components were sufficiently separated after centrifuging at 51,900 rpm for 32 min to estimate sedimentation coefficients. The resolution of five sedimenting species accounts for the dodecamer and the four lower molecular weight oligomers observed after polyacrylamide gel electrophoresis (Figs. 3B and 4) and confirms the absence of a rapid association-dissociation equilibria among these species (25). The s ~ . ~ , values of the slowest to fastest sedimenting components in Fig. 5 were 9.5 S , 12 S, 14 S , 17 S, and 20 S , respectively. The fastest sedimenting species has the sedimentation coefficient of native dodecameric glutamine synthetase (12). The mass distribution in the schlieren pattern of Fig. 5 is -43% dodecamer and -57% oligomers with the most slowly sedimenting com- ponent representing -17%. This slowest sedimenting species (9.5 S) is the tetramer, since a purified tetramer fraction ( A m ",,,. I em = 0.31) obtained by a second HPLC gel filtration had s z ~ . ~ , = 9.4 S , using absorption optics a t 280 nm and the

1 - i + + - + + - I - I - - + - + L " "

I SUBUNITS 1 PER OLIGOMER -> 12 j O - I

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"TRACKING DYE I FIG. 3. Formation of oligomeric intermediates during dis-

sociation of glutamine synthetase. Enzyme containing 15% inactive subunits (obtained with limiting L-methionine-S-sulfoximine) was incubated at pH 7.2 with 10 mM EDTA. The pH was raised to 8.0 with 0.1 M Tris/HCI and 0.4 mg/ml of DTNB was added at 37 "C for 30 min. A, 100 pl containing -0.5 mg of partially inactive enzyme was loaded on a 30-ml Toyo Soda TSK G3000 SW high pressure gel filtration column equilibrated with 50 mM potassium phosphate, pH 7.2, and run at 0.5 ml/min. Fractions of 500 p1 were collected and 10 pl of each fraction was assayed for enzymatic activity (0). The M, standards were: blue dextran, 10,000,000; E. coli glutamine synthetase, 600,000; E. coli glutamate dehydrogenase, 270,000; liver phosphofruc- tokinase, 19O,ooO, dimer of liver catalase, 115,000, bovine serum al- bumin, 67,000. B, A slab gel of 6% acrylamide was made with the discontinuous buffer system of Davis (21). Samples from HPLC gel filtration column were diluted 1:l with glycerol and bromphenol blue and 20 pl of each was loaded in 0.5-cm wells. Gels were stained with coomassie blue and photographed. The electrophoretic gel samples are shown at the approximate position of elution from the HPLC gel filtration column.

0 5 10 15 20 25 30 35 40 50 60 70 80 90 100 %

FIG. 4. Distribution of oligomers as a function of the per cent inactive glutamine synthetase used. Unadenylylated glutamine synthetase was inactivated by incubation with 1 mM ATP and various substoichiometric amounts of L-methionine-S-sulfoximine. Each partially inactivated sample was then treated with EDTA and DTNB as described in legend to Fig. 3. The samples (5 pg each) were then run on a 6% polyacrylamide slab gel under nondenaturing conditions (21). Gels were stained with coomassie blue. The numbers refer to the per cent inactive enzyme obtained by partial inactivation, with 0% inactive corresponding to native enzyme. The 4 standards in the left hand channel are (from the top): thyroglobulin (660,000), apoferritin (440,000), catalase (220,000), and lactate dehydrogenase (140,ooO). Since these are nondenaturing gels, mobility is not a simple function of molecular weight.

1 FIG. 5. Sedimentation pattern of glutamine synthetase oli-

gomers after 40 min at 51,900 rpm, using schlieren optics with the phase plate angle at 50". Unadenylylated, manganese glutamine synthetase (GS i) was inactivated to the extent of -35% with ATP and a limited concentration of L-methionine-S-sulfoximine and then was treated with EDTA and DTNB at pH 8 as described in the legend to Fig. 3. The oligomeric mixture was dialyzed for 48 h at 4 "C against 0.10 M KPO, buffer at pH 7.1 before centrifugation. A 2-place AnD rotor equipped with a double sector cell (containing the dialyzed protein mixture and the dialysate) and an absorption counterbalance was used for the sedimentation experiment; the protein concentration at the start of centrifugation was 4.0 mg/ml, and the rotor temperature was maintained at 19.5 "C. Sedimentation is from left to right.


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7250 Active Site Ligand Stabilization of Quaternary Structure

ultracentrifugal conditions of Fig. 5. Using the relationship for globular proteins, sds2 = M?/3/M!i/3, and the Szo. and M, values for the native dodecameric enzyme (12), the 9.4 S species has a calculated M, = 200,000. However, a frictional ratio of 1.4 can be calculated from the szo, ui and M, values for both the tetrameric oligomers and the dodecamers.

The presence of [I4C]ADP or L-methionine sulfoximine [32P]phosphate in all oligomeric species (but not in the aggregated material) was shown by autoradiography following native gel electrophoresis and by scintillation counting of fractions from the HPLC gel fitration column. When purified tetramer was prepared by repeated gel filtration, the amount of L-methionine-S-sulfoximine ["P]phosphate bound indicated that 2 of the 4 subunits were complexed (data not shown).

Fig. 3A shows that fractions from the gel filtration column containing dodecamers and lower molecular weight oligomers contain enzymatically active subunits. Activity staining following polyacrylamide gel electrophoresis under nondenaturing conditions showed bands in the exact positions as those shown by protein staining (Fig. 3B) except that no band of activity was seen in the position of the aggregate. Tetramers and hexamers purified by repeated gel filtration and free of contaminating dodecamer displayed significant enzymatic activity; if 2 of the 4 subunits of purified tetramer represent inactive complexes (see above), the activity expressed by the remaining 2 subunits is about 15-20% of that expected for native subunits. Table I shows that all oligomeric species, including dodecamer, have some enzymatic activity and that the activity is increased by removing thionitrobenzoate groups with 2-mercaptoethanol.

Light scattering measurements showed that the tetrameric oligomers did not reassociate in assay solution; thus, the tetrameric unit is sufficient to allow at least partial expression of enzymatic activity. Treatment of the tetramer with 2-mercaptoethanol in assay solution with Mn" present leads to a rapid increase in light scattering (-3-fold) and an increase in enzymatic activity (-5-fold). Gel electrophoresis of tetramer or hexamer treated with 2-mercaptoethanol showed mostly dodecamer and small amounts of decamer present. The spe- cific activities obtained with the dodecamer reconstituted from tetramers and hexamers are about equal and correspond to 3545% active enzyme, which is consistent with about half of the enzyme containing inactive complex (Table I). The increased activity of the tetramer following treatment with 2- mercaptoethanol may be due to removal of inhibitory thionitrobenzoate groups or to favorable conformational changes accompanying assembly of the tetramer into a dodecamer, or, as is most likely, a combination of both effects.

TABLE I Enzymatic activity of oligomers

Oligomers were prepared by treating 15% inactivated enzyme with EDTA and DTNB as described in the legend to Fig. 3. Oligomers were separated by gel filtration (Fig. 3A) and 10 pl of various fractions were assayed in the standard y-glutamyl transferase assay solution with and without addition of 30 mM 2-mercautoethanol.

Glutamine synthetase activity

~~~~a~~~ Predominant oligomer No addition ethanol

2-Mercapto-

added unitslmg

2 Aggregate 0.40 0.92 5 Dodecamer/decamer 26 35 6 Octamer 21 29 7 Hexamer 16 39 9 Tetramer 8 40 Untreated, native enzyme 99 99

The formation of the oligomers is clearly related to the ability of inactivated subunits to protect some of the sulfhydryl groups of active subunits from reaction with DTNB (Fig. 2). With native glutamine synthetase treated with EDTA, modification of sulfhydryl groups causes dissociation of the enzyme, most likely by preventing the reversal of an inter- mediate unfolding step or by preventing the reassociation of subunits. The modified sulfhydryl groups in the tetramers and hexamers are located in positions that prevent reassociation of the oligomers to form dodecamers, since treatment with 2- mercaptoethanol to remove the thionitrobenzoate groups leads to reaggregation of the oligomers to dodecamers. A possible model for the formation of oligomers is that the inactive liganded subunits in a dodecamer restrict the conformational flexibility of adjacent active subunits by preventing partial unfolding and blocking the reaction of certain sulfhydryl groups with DTNB and thereby preventing disruption of the bonding contacts between subunits at those locations. It appears that, in addition to dodecamers and large aggregates, only oligomers with even numbers of subunits (4,6,8, and 10) are formed; monomers and dimers are not observed because these species have a high tendency to reaggregate into material of M, > lo6. The dissociation of two subunits at a time from the dodecamer was previously observed when glutamine synthetase was treated with EDTA and 1 M urea (26). Oligo- mers formed by the present method, however, are quite stable in buffer and can be isolated as individual species.

Examination of the electron micrographs of glutamine synthetase obtained by Valentine et al. (9) reveal the two superimposed hexagonal rings of the native enzyme as well as structures missing 2, 4, or 6 subunits with the remaining subunits in the same positions as in the original double hexa- gons. These structures were more prevalent in samples not fured with glutaraldehyde and may have arisen during preparation of grids for the micrographs. In the accompanying appendix, electron microscopy of separated oligomeric species confirm the estimates of molecular size of the oligomers presented here and suggest that formation of the oligomers occurs by cleavage through the hexagonal rings producing structures with DP symmetry containing pairs of subunits from each ring. Studies of the oligomers and reconstitution of dodecamers from the oligomers should provide valuable information about the relative strengths of isologous and heterologous subunit interactions within glutamine synthetase dodecamers.

CONCLUSION

The very tight binding of L-methionine-S-sulfoximine phosphate, ADP, and 2 Mn2+ to the subunit active site strengthens intra- and inter-subunit bonding domains in the dodecameric glutamine synthetase from E. coli. Disaggregation and unfolding of the inactive enzyme complex in 6 M guanidine HC1 occurs very slowly by a mechanism involving multiple pathways and stable intermediates. Furthermore, subunits in tetrameric structures partially saturated with inactivating ligands retain some activity, demonstrating that in this case the dodecameric structure is not necessary for the expression of enzymatic activity.

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1. Weber, G. (1975) Adu. Protein Chem. 29, 1-83 2. Klotz, I. M., Darnall, D. W., and Langerman, N. R. (1975) in The

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M R Maurizi and A Ginsburgfrom Escherichia coli.

Active site ligand stabilization of quaternary structures of glutamine synthetase

1982, 257:7246-7251.J. Biol. Chem.

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