Eur. J. Biochem. 89,51 -60 (1978)

The Glutamine Synthetase from Azotohacter vinelandii : Purification, Characterization, Regulation and Localization Jiirgen A. KLEINSCHMIDT and Diethelm KLEINER Biochemisches Institut and Chemisches Laboratorium der Universitiit Freiburg im Breisgau (Received March 2, 1978)

The glutamine synthetase (EC 6.3.1.2) from the Nz-fixing bacterium Azotobucter vinelundii was purified to homogeneity by heat treatment, ammonium sulfate precipitation and ion-exchange chromatography. The following molecular parameters were determined : molecular weight 640 000, subunit molecular weight 53000, partial specific volume 0.710 cm3/g, isoelectric point 4.6, amino acid composition. Most of the molecules are composed of 12 identical subunits but active oligomers of other degrees of polymerization, apparently aggregates with 8, 10 and 24 subunits, were also detected to a lesser extent. The enzymatic activity is regulated via adenylylation-deadenylylation cycles : liberation of AMP was detected upon treatment of the adenylylated form with phosphodiesterase along with a change in the catalytic properties. Adenylylation in vivo is specifically induced by high extracellular ammonia levels. The K , values for the Mg2+-dependent formation of glutamine were independent of the degree of adenylylation for glutamate and ATP, but varied for ammonia. Furthermore the catalytic activity is regulated by several nitrogenous feedback inhibitors. The degree of inhibition in some cases was dependent on the substrate concentrations : the sensitivity towards glycine, alanine and serine decreased with a decreasing ammonia level, while the sensitivity towards ADP or AMP increased with a decreasing ATP concentration. Part of the enzyme (about 30%) seems to be attached to the plasma membrane while the main fraction is found in the cytosol. Assimilation of inorganic nitrogenous compounds by Nz-fixingbacteria can proceed via two routes [l, 21. If high supplies of ammonia are available, it is reductively incorporated into 2-oxoglutarate by the catalytic action of glutamate dehydrogenase, yielding glutamate (‘high ammonia pathway’). If the organisms, however, have to cover their nitrogen demand completely or partially by the nitrogenase-catalyzed reduction of Nz, the resulting low levels of ammonia are assimilated by the two consecutive reactions : Glutamate

+ NH3 + ATP

-+

Glutamine + ADP

+ Pi (la) Glutamine + 2-oxoglutarate + NAD(P)H + H + 2 Glutamate + NAD(P)+ (1 b) -+

(‘low ammonia pathway’). These reactions are catalyzed by the enzymes glutamine synthetase and glutamate synthase respectively. Under condition where the ‘high ammonia pathway’ prevails, nitrogenase formation is usually repressed, while under the conditions of the ‘low ammonia pathway’ no glutamate dehydrogenase is synthesized. Enzymes. Glutamate dehydrogenase (EC 1.4.1.3); nitrogenase (EC 1.7.99.2); glutamine synthetase (EC 6.3.1.2); glutamate synthase (NADPH) (EC 1.4.1.13).

A model has recently been proposed which explains the regulation of the levels of these enzymes, drawing on investigations about the regulation of the nitrogen metabolism in Enterobacteriaceae [3], according to which glutamine synthetase plays a crucial role. In these bacteria the enzyme can exist in two interconvertible forms : an unadenylylated active and an adenylylated less active state [4,5]. Adenylylation is induced by high supplies of ammonia. For Klebsiella pneumoniue, a facultative N2-fixing member of this group, genetic [6- 81 and physiological [9] evidence suggests an inducer function for the nitrogenase synthesis of the unadenylylated glutamine synthetase : thus, when high supplies of ammonia induce its adenylylation, nitrogenase formation ceases. For Nz-fixing members of other bacterial groups only circumstantial evidence for such an involvement of the glutamine synthetase in the induction of nitrogenase synthesis has been obtained. While results from mutants of Spirillum lipoferum support such a regulatory role [lo], investigations with the obligate anaerobe Clostridium pusteurianum (D. Kleiner, unpublished results) and the obligate aerobe Azotobucter vinelandii [ l l ] do not, although some evidence shows that the glutamine synthetase in A . vinelundii, but not in C . pusteuriunum, might be interconvertible [ l l , 121. Although probably not involved in the induction of

Glutamine Synthetase from Azotobacter

52

nitrogenase synthesis, the Azotobacter glutamine synthetase might be connected with another regulation of nitrogenase activity : high extracellular ammonia levels reversibly shut off nitrogen fixation (without affecting the enzyme level), and this inhibition is closely paralleled by a change in the glutamine synthetase activities [l 11. These effects occur irrespective of the reason for the increase in the extracellular ammonia level, i.e. whether by external addition to the culture [ l l ] or by forced excretion from the organism 1131. These observations prompted us to investigate the connections between nitrogenase activity and synthesis, the state of the glutamine synthetase and the extracellular ammonia level. As a first step we wish to report here our investigations on the properties of the glutamine synthetase from A . vinelandii, on the existence of an interconversion by adenylylation which is induced by high extracellular ammonia levels, and some evidence for the localization of the enzyme in the cell. MATERIALS AND METHODS

Glutamine

+ NH20H

y-Glutamylhydroxamate + NHJ (transferase reaction). -+

(2c)

All reactions are dependent on the presence of M$+ or Mn2+ in the reaction medium; reaction 2c additionally requires arsenate and ADP as cofactors. These reactions were used alternatively for the determination of the enzymic activity in crude extracts or purified preparations under the assay conditions of Shapiro and Stadtman [16] for the biosynthetic (phosphate determination) and transferase activity (alterations: for the routine assay the final buffer content was 0.15 mM imidazole, pH 7.1 ; for the determination of the pH profiles the final buffer content was 50mM each of imidazole, 2-methyl imidazole and 2,4-dimethyl imidazole). For the synthetic assay the method of Kohlhaw et al. [17] was used (alterations: cysteine was omitted, and the stop mixture of Shapiro and Stadtman [16] was employed). Double amounts of the stop mixture were used for the determination of the pH profile (to prevent a pH-dependent change in the absorption of the iron . hydroxamate complex). All assays were carried out at 30 "C.

Growth of the Organisms and Preparation of Crude Extracts Azotobacter vinelandii strain OP was kindly supplied by R. H. Burris (University of Wisconsin, Madison, U.S.A.). The organisms were grown aerobically in batch cultures of various sizes (1 - 100 1) in a nitrogen-free medium as described by Strandberg and Wilson [14] at 30 "C and pH 7.0. They were harvested in the logarithmic phase (a) either without further treatment or (b) 10 min after increasing the extracellular ammonia concentration to 10 mM by the addition of diammonium citrate. The glutamine synthetase preparations obtained from these organisms will be designated as GS(N2) and GS(NH3) respectively. The cells were either used immediately or stored at - 15 "C. Crude extracts were prepared from suspensions in 10 mM imidazole buffer (pH 7.0) containing 5 mM MnClz or 60 mM MgCl2 by one of the three methods: (a) passage through a French pressure cell, (b) sonication or (c) the osmotic shock method as described by Shah et al. [15]. Assays for Glutamine Synthetase Activities

Bacterial glutamine synthetases catalyze a variety of reactions [5], inter alia Glutamate + NH3 + ATP -+ Glutamine + ADP + Pi (2a) (biosynthetic reaction) Glutamate NHzOH ATP + y-Glutamylhydroxamate ADP + Pi (2b) (synthetic reaction)

+

+

+

Analytical Procedures

The protein contents were determined by the microbiuret method [18]. Immunological precipitations were carried out by the double-diffusion technique of Ochterlony as outlined by Tronick et al. [12]. Antisera were prepared according to Tronick et al. 1121 with the exceptions that 2 mg protein were used for the primary injection (subcutaneously into the back) and 0.2 mg for boosting. AMP was measured by the luciferin/luciferase method [19] as follows: a 0.1-ml sample was mixed with the same amount of 35 % (w/v) HC104 and dropwise neutralized with 0.4 m12 M KHC03. The mixture was incubated for 15 min at 0 "C and was occasionally stirred vigorously to remove all C02 bubbles. After centrifugation at 4 "C, 0.2 ml of the supernatant was placed into a cuvette together with 0.2 ml 2 pM ATP and 0.005 ml of a myokinase suspension containing 0.01 mg protein. After incubation for 20 min at room temperature, 0.2 ml of a luciferin/luciferase mixture were added, and the bioluminescence was recorded with a bioluminescence detector XP 2000 (Skan, Basel, Switzerland). Disc gel electrophoresis was carried out in 11 x 0.6 cm gels with 4.5 % polyacrylamide and the gel system 6 from Maurer [20]. For the detection of protein bands the gels were stained overnight with an aqueous solution of 7.5 % acetic acid, 40 methanol

J. A. Kleinschmidt and D. Kleiner

and 0.25% Coomassie blue, and then decolorized by several changes of the same mixture without the dye. Staining for activity was achieved by either (a) incubation the whole gels for 1 h in the MnZe/transferase assay mixture and then placing the gels into the FeCI3 stop mixture [16] until the zones of y-glutamylhydroxamate formation showed up as red bands, or (b) slicing the gels into discs of 0.2-cm width, incubating them overnight in 0.2 ml Mn*+/transferase or Mg2+/synthetic assay mixture, stopping the reaction with 0.8 ml of the stop mixture and measuring the absorption at 540 nm. Sodium dodecyl sulfate electrophoresis was performed similarly to the method of King and Laemmli [21]. The samples were dialyzed against bidistilled water for 15 h and then 0.15 ml solution (containing 0.03 - 0.08 mg protein) was incubated for 8 min at 95 "C with 0.3 ml of a preheated solubilization mixture containing 6 M urea, 10 % mercaptoethanol and 4 % sodium dodecyl sulfate in 0.125 M Tris-HC1 buffer of pH 6.8. After addition of 0.02 ml glycerol and 0.003 ml bromphenol blue (0.05 %), the samples were put on gels which contained 10% polyacrylamide, and run with 0.1% mercaptoethanol in the electrophoresis buffer (pH 8.3). For the determination of the isoelectric point the LKB model 8100 apparatus with a 110-ml column was loaded with 4mg protein and 1 % ampholines of pH range 3 - 6. Electrolysis was carried out until the current had dropped to a stable value of 0.5 mA, and 1.5-ml fractions were collected, assayed by reaction 2c, and the pH of every fifth fraction was determined. Amino acid analyses were performed with a Biotronik amino acid analyzer, model LC-6000. Protein samples were hydrolyzed in 6 M HCl for 24, 48, 72, 96 and 120 h at 108 "C in evacuated sealed tubes [22]. Cysteine was determined after oxidation to cysteic acid [23]. Tryptophan was determined colorimetrical1~ ~ 4 1 . For the detection of carbohydrate moieties the purified protein was subjected to the method of Segrest and Jackson [25]. Sedimentation equilibrium centrifugation was carried out according to Yphantis [26] at 4000 rev./min in a Beckman ultracentrifuge model E with an AN-Gtype rotor. The partial specific volume was determined according to Kraky et al. [27]. The protein concentrations were measured as described by Kickhofen and Warth [28].

Criteria for Purity

The purity of the enzyme preparations was estimated by immunodiffusion, by electrophoresis with and without detergent, and by sedimentation equilibrium centrifugation as described above.

53

Molecular Weight Determinations

The molecular weight of the glutamine synthetase was determined by sedimentation centrifugation [26] as described, and by disc gel electrophoresis with gels containing 4,5 and 6 % polyacrylamide. The molecular weight was calculated according to Rodbard and Chrambach [29] by using rabbit muscle lactate dehydrogenase (molecular weight 144000), rabbit muscle aldolase (160000), beef liver catalase (232000), ferritin (450000). E. coli /3-galactosidase (520000) and beef thyroglobulin (660 000) as standards. The molecular weight of the subunits was determined by sodium dodecyl sulfate electrophoresis [21] with horse myoglobin (subunit molecular weight 17200),yeast alcohol dehydrogenase (37000), E. coli glutamine synthetase (50000), beef liver glutamate dehydrogenase (53 000), rabbit muscle pyruvate kinase (57 000), beef liver catalase (58000), bovine serum albumin (68 000) and E. coli RNA polymerase (155000 and 165000) as reference proteins.

Source of Enzymes

Phosphodiesterase (sample from May 1973), myokinase, RNA polymerase, catalase, glutamate dehydrogenase, alcohol dehydrogenase, pyruvate kinase, ferritin, P-galactosidase and lactate dehydrogenase were obtained from Boehringer (Mannheim, F.R.G.). Firefly luciferin/luciferase FL 50 and thyroglobulin were obtained from Sigma (St Louis, U.S.A.). Bovine serum albumin was from Behring (Marburg, F.R.G.) and myoglobin was from Serva (Heidelberg, F.R.G.).

RESULTS Evidence for an Interconversion of the Glutamine Synthetase, which is Specijically Induced by High Extracellular Ammonia Levels As reported previously [ 11,131, an increase in the extracellular ammonia level of A . vinelandii cultures causes a rapid increase in the Mn2 -dependent transferase activity of the glutamine synthetase, which is not prevented by the addition of chloramphenicol. Fig. 1 shows how the other catalytic activities of the enzyme (Eqn 2a-c) respond to such a pulse of ammonia added to the culture as sulfate or citrate: while the Mn2+-dependent biosynthetic activity also shows a small increase, both the Mg2''-supported transferase and biosynthetic activitiesdecrease rapidly. The synthetic activities closely follow the biosynthetic ones (not shown). Thus a high supply of ammonia decreases the physiologically meaningful M$+-dependent biosynthetic activity similarly as in E. coli [30]. +

Glutamine Synthetase from Azotohacter

54 8r

1

J

0

20

40 60 Time (rnin)

80

100

Fig. 1. Eflect of an increase in the extracellular ammonia concentration on the transferase and biosynthetic activities of glutamine synthetase. At zero time ammonium sulfate was added to a batch culture to a final concentration of 2.5 mM. ( x x ) Ammonia MnZ+-dependenttransferase activity; in the medium; (0-0) (0--0) MnZ+-dependentbiosynthetic activity; (A- ---A) Mg2+M&+-dependent biodependent transferase activity; (A-A) synthetic activity ~

For an investigation of the specificity of this effect for ammonia a number of other nitrogen sources or nitrogenous metabolites was tested in the same manner. As a diagnostic indication of any changes in the catalytic behavior of the glutamine synthetase we used the ratio of Mn2'-dependent transferase to the Mg+-dependent biosynthetic activity, because both assays reacted most strongly and in reverse directions towards alterations in the extracellular ammonia level. As indicated in Table 1, only ammonia produced strong changes in this ratio. These results suggest that, as in E. coli, the glutamine synthetase can exist in two interconvertible forms. For a definite proof, however, purification of the two states GS(N2) and GS(NH3) and interconversion in a definite reaction medium was desirable. Pur$cation of the Glutamine Synthetase

The purification schemes for the GS(N2) and GS(NH3) enzyme were identical. 100 g cells were suspended in 300 ml of 10 mM imidazole buffer (pH 7.0) containing 5 mM MnC12. The suspension was passed twice through a French press at 20000 lb/ in2 (13.8 MPa). The crude extract was centrifuged at 45 000 x g for 30 min, and the supernatant was aerobically heated to 63 "C for 45 min. The aggregated proteins were sedimented at 27000 x g for 10 min and discarded. To the supernatant, solid ammonium sulfate was added to a final concentration of 60% while the pH was kept constant, and the suspension was stirred for 4 h at 4 "C. After centrifugation

Table 1. EJfect of various nitrogenous compounds ('ml concentrations2 m M ) A. vinelandii cultures on the ratio of the Mn2+-dependent transferase to the M$ + -dependent biosynthetic activity ojglutamine synthetase Compound

Activity ratio

None Ammonia Guanidine Methy lamine Creatine Amino acids: isoleucine, proline aspartic acid, glutamic acid glycine, cysteine, arginine, serine, tyrosine glutamine phenylalanine alanine methionine histidine lysine asparagine

0.14 6.10 0.27

0.28 0.12 0.12 0.13 0.14 0.18 0.20 0.22 0.29 0.33 0.35 0.43

(27000 x g , 10 min) the supernatant was discarded. The residue was resuspended twice with 120ml of 50 ammonium sulfate in 10 mM imidazole and 5 mM MnCh (pH 7.0) for 20 min at 4 "C. After centrifugation (27000 x g, 10 min) the pooled supernatants were partially desalted by adding 200 ml buffer three times and concentrating by ultrafiltration (Amicon XM-100) to 120 ml after each addition; the solutions were then put on a column of DEAESephadex A-25 ( 5 x 25 cm) which had been preequilibrated with 10 mM imidazole (pH 7.0) containing 5 mM MnC12until the gel suspension had a pH of 6.0. After loading with protein the column was washed with 500- 700 ml 10 mM imidazole containing 300 mM NaCl and 5 mM MnClz (pH 7.0) and subsequently with the same buffer (500-700 ml) containing 360 mM NaCl with a flow rate of 60 ml/h. Then the proteins were eluted by a linear gradient prepared by mixing 1000 ml of 360 mM NaCl and 1000 ml 600 mM NaCl in the MnZ+/imidazolebuffer with the same flow rate. The temperature was maintained at 4-6 "C during the chromatography. The glutamine synthetase activities were eluted between 400 and 500 mM NaCl usually in at least two peaks (Fig. 2). The rise in the 280-nm absorbance at the end of the elution is due to nucleic acids. When, as shown in Fig. 2, the last peak was contaminated with nucleic acids, the sample was treated with deoxyribonuclease, ultrafiltrated and re-chromatographed. The immunological test, the sedimentation equilibrium centrifugation and the sodium dodecyl sulfate electrophoresis (Fig. 3 A, B) indicated complete purity of the first peak eluate and more than 95 purity of the second peak eluate for both the GS(N2) and the GS(NH3) preparation. After disc gel electrophoresis,

J. A. Kleinschmidt and D. Kleiner

55

1.00

'

0.60 ' 0.40 '

020.

ct" a10

.

0.06

*

OD4

.

4

I. 0

50

100 Fraction number

150

Fig. 2. Chromatography of a partially purified G S ( N H 3 ) preparation (after heat denaturation and ammonium sulfate fractionation) by elution with an NuCl gradient ,from a column of DEAE-Sephadex A-25. (----) NaCl concentration; (A---A) absorbance at 280 nm; (0-0) Mn2+-dependent transferase activity. Aliquots of the fractions were incubated for 15 min with the transferase test mixture, so that the absorbance at 540 nm did not exceed a value of 0.5; data are standardized for 0.5 ml

Fig. 3. Polyacrylamide electrophoresis of sodium-dodecyl-suljatetreated and native glutamine synthetase. (A, B) Sodium dodecyl sulfate electrophoresis of enzyme from activity peaks 1 (60 pg) and 2 (40 pg), respectively; (C, D) disc electrophoresis of enzyme from activity peak 1 (60 pg) and 2 (40 pg), respectively, stained for protein; (E, F) same preparation, stained for Mn2'-dependent transferase activity. GS(NH3) preparations were used for all runs

however, the gels always showed several protein bands (Fig. 3 C, D), all of which exhibited transferase and synthetic activity (Fig. 3 E, F), as has been reported before [31]. The distribution of the glutamine synthe-

0.02 .

Qo1

0

1

2

3

4

5

6

7

Po Iy a c r y la rn id e (%)

Fig. 4. Electrophoretic mobilities of the multiple forms of glutamine synthetuse (60 pgprotein) at dfjerent polyacrylumide concentrations. The numbers at the lines correspond to the ones from Fig. 3c

tase in these different bands was not the same for the two activity peaks eluted from the chromatography column (Fig. 3C, D). Applying the procedure of Hedrick and Smith [32] we found that these bands belong to size isomers of the enzyme (Fig. 4), probably to states with different numbers of subunits: calculations [29] suggested the presence of molecules with 8, 10, 12 and 24 subunits (for the determination of the subunit molecular weight see below). The main band seen in Fig. 3 C - F should then belong to the dodecamer. It is, however, sometimes split into two very close bands which both show glutamine synthetase activity. Very similar electrophoresis patterns were obtained for both the GS(N2) and the GS(NH3) preparations. By running appropriate controls (runs without bromphenol blue, pre-electrophoresis to remove monomeric acrylamide and peroxidisulfate), an artificial production of these isomers by those compounds was ruled out. We are currently investigating whether the distribution into several oligomeric forms is of any physiological significance. The activity ratios of the MnZ'-dependent transferase to the M$+-dependent biosynthetic assay remained constant for both the GS(N2) and the GS(NH3) preparation during the purification. Table 2 provides a summary of the purification procedure with data on recoveries and purities after each step. For the following studies only the fractions from the first chromatography peak were used.

56

Glutamine Synthetase from Azotobacter

Table 2. Scheme for the purification of the A. vinelandii glutamine synthetase One unit (U) corresponds to lpmol y-glutamylhydroxamate formed per min in the Mn2'-dependent transferase assay Purification step

Crude extract Heat denaturation Ammonium sulfate fractionation Ion-exchange chromatography

Total activity obtained

Protein

Specific activity

Recovery

Purification

U

mg/ml

U/mg protein

%

-fold

4971 5034 3496 2846

27.4 10.5 3.1 0.13

0.39 1.2 6 40

100 101 70 57

1 3 15 103

Conversion of the GS(NH3) Form into the GS(N2) Form in vitro

Both enzyme forms not only differed in the activity ratios for the different assays but also in their pH profiles. Since these features are also prominent for the E. coli enzyme, a similar conversion mechanism was anticipated. Therefore we investigated whether the formation of the GS(NH3) enzyme can be reversed by the action of phosphodiesterase and whether AMP was liberated upon this treatment. To avoid interference of possibly adhering fragments of nucleic acids, this preparation was subjected to treatment with 1 % streptomycin sulfate at the beginning of the purification procedure. To avoid excessive quenching in the test for AMP by imidazole the purified GS(NH3) enzyme was dialyzed against a buffer that contained 60 mM MgClz in 10 mM Tris (pH 7.8). 5 ml of the dialyzed enzyme solution with a protein content of 0.51 mg/ml were treated with 0.12 mg snake venom phosphodiesterase at 30 "C, and samples were withdrawn at various intervals and analyzed for the Mg2+-supported synthetic activity and for free AMP. As shown in Fig. 5 , a resurgence of the synthetic activity coincides with a liberation of AMP from the enzyme up to 10mol/mol protein (data for protein characterization see below). These data unequivocally demonstrate that the glutamine synthetase from A . vinelundii is regulated by interconversion and can exist in an adenylated and an unadenylylated state like the E. coli enzyme. Attempts to achieve adenylylation of glutamine synthetase in crude extracts of A . vinelundii by the method of Ebner et al. [33] have failed so far.

0

80

40

120

Time (min)

Fig. 5. Time dependence of A M P liberation and the M?+-dependent synthetic activity as a consequence of the treatment of a GS(NH3) preparation with phosphodiesterase. Protein concentration : 0.51 mg/ AMP liberated per mol protein; (M Mgz+) ml. (A-A) dependent synthetic activity

when the predominantly adenylylated GS(NH3) form is treated with phosphodiesterase to yield the unadenylylated enzyme (Fig. 6 ) . The pH profile of the GS(N2) preparation still indicates a small degree of adenylylation. In contrast to E. coli, the isoactivity pH for the Mn2' -dependent transferase test is at pH 8.6 instead of 7.1, while the isoactivity pH for the Mg2+-dependent transferase activity is at pH 6.8 (Fig. 7). The Mg2+-supported synthetic activities show no isoactivity pH but only broad maxima around pH 7.5 for both forms. Molecular Parameters

p H Proj2es As thoroughly examined for the E. coli glutamine synthetase, the enzyme forms differing in the degree of adenylylation also differ in their pH profiles, especially for the transferase activities. The ratio of the Mn2+-supported to the Mg2+-supported transferase activity at the isoactivity pH has widely been used as an indicator of the degree of adenylylation [34]. A similar shift in the pH profiles can be observed

Table 3 gives a compilation of the molecular parameters of the glutamine synthetase from A . vinelandii. Both the predominantly unadenylylated GS(N2) and the predominantly adenylylated GS(NH3) form gave identical values for molecular weight, subunit and amino acid composition, and isoelectric point within the experimental errors. Electrophoretical runs of mixtures of the glutamine synthetase from A . vinelandii and E. coli always gave to bands indicat-

J. A. Kleinschmidt and D. Kleiner

57

r

Table 3. Molecular characterization of the glutamine synthetase from A. vinelandii Parameter Molecular weight : from sedimentation equilibrium centrifugation from disc gel electrophoresis main band satellite bands

J

I 70

6.2

28

8.6

PH

Fig. 6. pH profiles f o r the Mn2'-dependent transferase activities of dI;fferently adenylylated states of glutamine synthetase which were obtained by varying the incubation time of a GS(NH3) preparation with phosphodiesterase. 0.4 mg/ml of the enzyme were incubated at 30 "C with 0.8 pg/ml phosphodiesterase in 10 mM imidazole buffer of pH 7.0. To complete deadenylylation more phosphodiesterase (25 pg/ml) was added after 100 min. For the transferase assay 0.4-ml samples were diluted 20-fold with 10 mM imidazole/S mM MnClz buffer (pH 7.0), and assayed according to Shapiro and Stadtman [16]. Numbers on the curves give the incubation time (minutes) with phosphodiesterase (----) pH profile for a GS(N2) preparation

12

-.--

*

c

>

"

I-

;;.

8

L

C - 0 L

E'Z

c

CII

--E

:3

TI c-

$

4

73

Number of types of subunits Subunit molecular weight Apparant specific volume Isoelectric point

Value

650000

27000

632000 544000 400000 1 200 000 1 53000 f 3000 0.710 cm3/g 4.6

Table 4. Amino acid composition of the glutamine synthetase from A. vinelandii Results are given as number of residues per subunit of molecular weight 53 000 Amino acid

Amount

Lysine Histidine Arginine Aspartic acid and asparagine Threonine Serine Glutamic acid and glutamine Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phen ylalanine Tryptophan

32 14 17 56 22 27 46

28 41

44 4 28 13 25 32 20 26 3

N

m

z

0

62

%0 Z8 PH Fig. 7. pH projiles for the M 2 ' -dependent transferase activities. Details as in Fig. 6 with the exception that the samples were diluted with 10 mM imidazole/60 mM MgClz buffer (pH 7.0) 5.4

ing small but significant differences in their molecular weight. According to the molecular weight determinations for the dissociated and for the undissociated enzyme we assume that, as in E. coli, the native enzyme is predominantly dodecameric and is composed of identical subunits. However, as pointed out before, the activity is not restricted to the dodecamer, but oligomers of apparently lower and higher degree of polymerization also exhibit marked activities.

The amino acid composition is shown in Table 4. If compared to the compositions of the enzyme from other sources [5,35-401, both prokaryotic and eukaryotic, only the slightly lower arginine content is remarkable. According to an empirical formula derived by Marchalonis and Weltman [41], the amino acid contents of proteins from different organisms can be compared to indicate possible homologies : the individual differences in mo1/100 mol of each of the 17 amino acids (the tryptophan content is usually disregarded because of frequent high errors) are squared and summed, giving a value termed SdQ. When comparing more than 100 proteins of known sequence [41] the authors found that most proteins known to be highly homologous showed S A Q values less than 50. Only 2% of the proteins known to be

58

Glutamine Synthetase from Azotobacter

Table 5. Calculations of S A Q for the glutamine synthetases from various sources SAQ is explained in the text Enzyme source

A . vinelandii E. coli [5] B. stearothermophilus [35] B. subtilis 1361 Soybean nodule cytosol [37] Pea [38] Rat liver [39] Ovine brain [40]

SAQ value with enzyme from A. vinelandii

E. coli

0 9 21 22 23 31 20 26

0 23 20 22 31 24 22

B. B. stearosubtilis thermophilus

soybean nodule cytosol

0 22 32 26 51 49

0 53 26 22

heterologous had an SAQ less than 100 and none less than 50. Keeping in mind the purely empirical basis of the formula, SdQ values less than 50 suggest the possibility of an evolution from a common ancestor. If these calculations are applied to the amino acid contents of the glutamine synthetases known so far, the SdQ values shown in Table 5 are obtained. All comparisons show values below or only slightly above 50, suggesting evolution from a common ancestral protein. The closest similarity is shown for the proteins from A . vinelandii and E. coli; these glutamine synthetases are also the only ones listed in the table where regulation by adenylylation-deadenylylation has been proved. Previous SdQ calculations [42,43] indicated that the homology suggested does not only cover the glutamine synthetases but extends over all proteins with ATPase activity, thus pointing towards evolution of all ATP-requiring processes from a common prototype reaction (see also [44]). No carbohydrate moiety was found to be attached to the molecule, if tested by the method of Segrest and Jackson [25].

Michaelis Constants K , values for the Mg2'.-dependent biosynthetic reaction, which is the most significant reaction physiologically, were measured for the substrates ammonia, ATP and glutamate. Two of the substrates were employed at the saturation concentrations indicated, and the concentration of the third reactant was varied. Care was taken to ensure that initial velocities were recorded, and the data were plotted according to Lineweaver and Burk for both the GS(N2) and the GS(NH3) enzyme. Straight lines were always obtained. The results (Table 6) show that, except for ammonia, the Michaelis constants are identical for both forms. The higher K , for ammonia of the GS(NH3) enzyme is explicable in view of the fact that this state prevails

0 47 20 55 43

pea

0 55

rat liver

ovine brain

0 13

57

0

Table 6. K, values for the Md+-dependent biosynthetic reaction Substrate K,,, measured

Concentrations of the other substrates

K,,, of

Glu

ATP

NH4'

GS(N2)

GS(NH3)

7.5 7.5

50 50

0.15

0.23 1.3 0.94

mM NH4' Glutamate ATP

100 100

-

1.2

0.94

under conditions where this substrate is supplied in high amounts.

Feedback Inhibitors Bacterial glutamine synthetases are subject to feedback inhibition by numerous metabolites [5,16]. In E. coli the unadenylylated and the adenylylated enzyme are often inhibited to various degrees by the same compound [16]. Table 7 summarizes the effects of various compounds on the M$+-supported biosynthetic activity of both forms under substratesaturation conditions, while Table 8 shows the results under various substrate-limiting conditions. Under substrate-saturation conditions, both the predominantly unadenylylated and the predominantly adenylylated Azotobacter enzyme are inhibited to about the same degree by the compounds listed, in contrast to the results reported for E . coli [16]. An identical sensitivity of both forms was also found when ammonia was supplied in limiting amounts (0.3 mM). Interesting changes in the inhibitory power can be observed when one of the substrates is supplied in activity-limiting concentrations (Table 8). If the ammonia level is decreased, the enzyme becomes less sensitive towards inhibition by glycine, alanine and serine. The physiological meaning for this decreased

J. A. Kleinschmidt and D. Kleiner

59

Table 7. Effect of various metabolites on the MSZ’-dependent biosynthetic activity catalyzed by the predominantly adenylylated GS(NH3) and the predominantly unadenylylated G S ( N 2 )form of the A. vinelandii glutamine synthetase At zero time the enzyme (25 pg/ml and 6 pg/ml respectively) was added to the assay mixture which contained the inhibitors. Reaction time: 15 min. The substrates were present at saturation levels (see Table 6) Inhibitor

Residual activities at inhibitor concentrations (mM) of

1

2

5

1

2

5

46 55 15 96 93 93 58

29 37 58 93 78 87 36 97

44 53 72 98 90 93 58 88 94

31 33 50 94 76 86 34

94 96

73 69 87 100 95 96 73 100 100

% Glycine 64 Alanine 73 Serine 91 100 Tryptophan 98 CTP 99 AMP ADP 83 100 Carbamoyl phosphate Glucosamine 6-phosphate 100

83

85

95

A limiting amount of glutamate, finally, will reduce its utilization by an increased inhibitory power of alanine and serine. The decreased sensitivity against inhibition by ADP, however, is not easily understandable. Localization

After centrifugation of the crude extract at 45 000 xg, most of the glutamine synthetase activity was found in the supernatant. A significant fraction of the activity, however, was always found in the sediment. The percentage varied according to the extraction method : while after passage through the French press or sonication about 5- 10% were sedimentable, cell disintegration by the more gentle osmotic shock method yielded about 30 % of the activity in the easily sedimentable fraction. These results indicate extended, albeit relatively loose, association with the plasma membrane. The glutamine synthetase from this fraction could not be solubilized by treatment with butan-1-01 or with deoxyribonuclease. Intact cells do not show any glutamine synthetase activity. DISCUSSION

Table 8. Effect of feedback inhibitors on the M?+-dependent hiosynthetic assay catalyzed by the predominantly unadenylylated form GS(N2) under substrate-limiting conditions Effector concentrations: 2 mM. Substrate limitation for NH4’ 0.3 mM, for ATP 1 mM, for glutamate 2 mM. Other assay conditions as in Table 7, except that the reaction time was shortened to 2 min in order to ensure that initial velocities were determined Inhibitor

Residual activity when limiting substrate is ATP

Glycine Alanine Serine Tryptophan CTP AMP ADP Carbamoyl phosphate Glucosamine 6-phosphate a

58 35 66

87 96 68 27 100 100

glutamate

48 35 51 97 91 97 72 100 90

NH4’

80 12 79 92 91 87 55

100” 100”

Errors exceed 10

sensitivity could be as follows: even if these metabolites are still present in sufficient amounts in the cell, they do not interfere with the slower assimilation of the suboptimal levels of the extracellular nitrogen source. In contrast, a reduced intracellular ATP level, signalling lack of energy, enhances the inhibitory powers of several endproducts, especially AMP and ADP. This ensures a direction of the energy flow away from amino acid synthesis to more urgent problems.

The glutamine synthetase from A . vinelundii in many respects resembles the enzyme from E. coli as to amino acid composition, molecular weight, subunit composition, sensitivity toward the same feedback inhibitors, and being also regulated by adenylylationdeadenylylation. This regulatory property is of considerable interest. Gancedo and Holzer [45] compared the behaviour of the glutamine synthetase from nine different bacterial strains towards sudden changes in the extracellular ammonia concentrations and found evidence for an interconversion of the enzyme only for the species belonging to Enterobacteriaceae. Recently Johannson and Gest [46],however, presented evidence that adenylylation-deadenylylation of glutamine synthetase occurs in the phototrophic bacterium Rhodopseudomonas cupsuluta. Our investigations show unambiguously that the activity of this enzyme from A . vinelundii is regulated via such cycles. Thus the suggestion of Tronick et al. [12] seems to hold that all gram-negative bacteria possess interconvertible glutamine synthetase, whereas the grampositive organisms do not. Possibly the ancestral form of the gram-negative bacteria existed in an environment with high fluctuations in the ammonia level, where the evolution of such a ‘life-saving’ regulatory mechanism [30] was of major advantage, in order to prevent a depletion of the ATP pool by an excessive synthesis of glutamine following a strong increase in the ammonia concentration [30]. To the best of our knowledge the occurrence of multiple active forms of a bacterial glutamine synthetase containing different numbers of subunits has

60

J. A. Kleinschmidt and D. Kleiner: Glutamine Synthetase from Azotobacter

not been reported previously. Our previous suggestion [3 11, that the extent of dissociation and re-association is influenced by the state of adenylylation, does not hold in the light of our recent findings, which rather indicate a facilitated dissociation upon prolonged storage of the whole cells at subzero temperatures. The influence of the substrate concentrations on the inhibitory power of certain feedback inhibitors suggests cooperative or antagonistic binding [47] : while binding of ammonia would enhance binding of the amino acids glycine, alanine or serine, binding of ATP would be antagonistic to these amino acids and to AMP and ADP, and binding of glutamate would be antagonistic to alanine, serine and ADP. The validity of these suggestions, however, has still to be proved by binding studies. The centrifugation experiments strongly indicate that a certain amount of the glutamine synthetase from A . vinefundiiis attached to the plasma membrane. Similar results have been obtained with K . pneumoniae (Kleiner, unpublished results). Whether this attachment alters the catalytic properties or whether it points towards another function of the enzyme, e.g. in ammonia transport [ll], has to be elucidated by further investigations. We thank Prof. H. Holzer for stimulating discussions and his continued interest in these studies, H. Henninger for the ultracentrifugation studies, and C. Kuklik for the amino acid determinations. This work was supported by grants from the Gesellschaft fcr Strahlen- und Umweltforschung and from the Deutsche Forschungsgemeinschaft (K129817, KI 29818 and K1298/9).

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J . A. Kleinschmidt, Biochemisches Institut der Albert-Ludwigs-Universitat Freiburg, Hermann-Herder-StraDe 7, D-7800 Freiburg i. Br., Federal Republic of Germany

D. Kleiner, Chemisches Laboratorium der Albert-Ludwigs-Universitat Freiburg, AlbertstraDe 21, D-7800 Freiburg i. Br., Federal Republic of Germany