N-Acetylglutamate 5-Phosphotransferase of Pseudomonas aeruginosa

Transcription

1 Eur. J. Biohem. 52, (1975) NAetylglutamate 5Phosphotransferase of Pseudomonas aeruginosa Purifiation and LigandDireted AssoiationDissoiation Dieter HAAS and Thomas LESNGER Mikrobiologishes nstitut, Eidgenossishe Tehnishe Hohshule, Zurih (Reeived July 10, 1974) NAetylglutamate 5phosphotransferase (ATP : NaetylLglutamate 5phosphotransferase EC ), the seond enzyme of arginine biosynthesis, was purified over 2000fold from Pseudomonas aeruginosa. The purifiation proedure involved a heat treatment, ammonium sulfate preipitation, and hromatography on DEAEellulose, Sephadex G150, and hydroxyapatite. The purified enzyme was greater than 90 % pure as judged by analytial polyarylamide gel eletrophoresis. A moleular weight of approximately was obtained by gel filtration. Eletrophoresis in sodium dodeyl sulfate gels gave a single band orresponding to a moleular weight of Due to the apaity for selfassoiation, the enzyme an exist in different states of aggregation depending on the nature of ligands and the onentrations of phosphate buffer. As estimated by gel filtration, the moleular weight was about in the presene of NaetylLglutamate. With Larginine, the feedbak inhibitor, and MgATP forms of smaller moleular weight (minimum of approximately ) were found. A onurrent hange in the sedimentation oeffiient as a funtion of ligands was demonstrated by surose gradient entrifugation. The synthesis of Naetylglutamate 5phosphotransferase was not repressed by exogenous Larginine or its preursors. NAetylglutamate 5phosphotransferase, the seond enzyme of arginine biosynthesis, atalyzes the ATPdependent phosphorylation of the yarboxyli group of NaetylLglutamate [l, 21. n several baterial speies inluding Miroous and Pseudomonas, in fungi and in the alga Chlamydomonus the enzyme is subjet to feedbak inhibition by Larginine [3 51. These miroorganisms synthesize ornithine, a preursor of arginine, via a yli aetylation reation Enzymes. NAetylglutarnate 5phosphotransferase or ATP : N aetyllglutamate 5phosphotransferase (EC ) ; Naetylglutamate synthetase or aetylcoa : Lglutamate Naetyltransferase (EC ): ornithine aetyltransferase or NZaetylLornithine : Lglutamate Naetyltransferase (EC ); pyruvate kinase or ATP : pyruvate 20phosphotransferase (EC ); alohol dehydrogenase or alohol : NAD' oxidoredutase (EC ); latate dehydrogenase or Llatate : NAD' oxidoredutase (EC ); atalase or hydrogenperoxide : hydrogenperoxide oxidoredutase (EC ); aldolase or ~frutosel,6bisphosphate ~glyeraldehyde3phosphatelyase (EC ) ; arginase or Larginine amidinohydrolase (EC ). mediated by ornithine aetyltransferase [l, Esherihia oli and Baillus speies, in ontrast, produe ornithine by hydrolyti deaetylation of N'aetylornithine [3,6]. n these organisms Naetylglutamate synthetase, the first enzyme of the pathway, but not Naetylglutamate 5phosphotransferase is inhibited by arginine [3,7]. Previously we have shown that in Pseudomonas aeruginosa endprodut inhibition ats not only on Naetylglutamate 5phosphotransferase [8,9] but also on Naetylglutamate synthetase [lo]. n the present report we desribe the purifiation and some moleular properties of Naetylglutamate 5phosphotransferase from P. aeruginosa. This enzyme undergoes liganddependent assoiationdissoiation reations similar to those of other allosteri phosphotransferases [ll]. The aompanying paper [12] deals with the atalyti and regulatory properties of the enzyme. This work was taken from a dissertation [13].

2 366 Oligomerkation of Pseudomonas NAetylglutamate 5Phosphotransferase MATERALS AND METHODS Chemials Amino aids inluding NaetylLglutami aid, and hymotrypsinogen were obtained from Fluka (Buhs, Switzerland), bovine serum albumin, ovalbumin, arginase, ATP (disodium salt), flaetyllornithine, and Tris from Sigma, pyruvate kinase, alohol dehydrogenase, latate dehydrogenase, atalase, and aldolase from Boehringer (Mannheim, Germany), myoglobin and surose from Shwarz/ Mann (Orangeburg, N.Y.), hydroxyapatite (BiolGel HTP) from Calbiohem, DEAEellulose (DE 52) from Whatman, Diaflo PM30 ultrafiltration membranes from Amion, and the reagents for polyarylamide gel eletrophoresis from Serva (Heidelberg, Germany). Human hemoglobin A was kindly supplied by M. Landolt. Organism and Growth Conditions Pseudomonas aeruginosa PA0 1, a wildtype strain (ATCC 15692), was grown in Fernbah flasks as desribed previously [lo]. The media used were either minimal medium E supplemented with 0.5 % gluose [6] or minimal medium P. Medium P ontained either Lglutamate, NaetylLglutamate, L ornithine, or Larginine, eah at 20 mm, as the only arbon and nitrogen soure [9]. Baterial growth was monitored by measuring the absorbane at 546 nm in an Eppendorf photometer. The ells were harvested early in the exponential phase. For largesale ultivation the organism was grown in an F0300 bioreator (Chemap AG, Mannedorf, Switzerland) equipped with a flatblade turbine and a mehanial foam separator. 2 1 of an overnight ulture grown on medium P plus 20 mm Lglutamate were used to inoulate of medium P plus Lglutamate ontaining all omponents at double onentration. The stirrer speed was 300 rev./min, the temperature 37 "C, and the aeration rate 0.2 (1 air) x (1 working volume)' x (min)'. ph 7.2 was maintained by automati addition of 6 M H,PO,. The mass doubling time was 6270 min. The ells were harvested at the end of the exponential phase by a highspeed ontinuousflow entrifuge (C. Padberg, Lahr, Shwarzwald, Germany) and stored at 20 "C until used. The yield was approximately 1700 g wet weight. Buffers The phosphate buffers used throughout were prepared from a 1 M stok solution of KH2P04/K2HP04 (39:61, v/v). The ph was 7.0 in 0.1 M buffer and 7.2 in 0.02 M buffer. All buffers were supplemented with 2 mm 2meraptoethanol. Where indiated glyerol was inluded at a final onentration of 10% (v/v). Preparation of CellFree Extrats A 15% (w/v) ell suspension in 0.1 M phosphate buffer was sonially disrupted. Cell debris was eliminated by entrifugation at xg for 20 min. The resulting rude extrats were dialyzed overnight against 0.1 M phosphate buffer prior to determination of enzyme ativity. All operations were arried out at 0 4 "C. For enzyme purifiation the ells were broken in a MantonGaulin homogenizer (see Resul t s). Assay of NAetylglutamate 5Phosphotransferase The equilibrium of the enzymati reation strongly favors the substrates, NaetylLglutamate and ATP. At ph 7.0 and 25 "C, in the presene of exess MgZ+, an apparent equilibrium onstant of was found [13]. Hydroxylamine was therefore inluded as a trapping agent in the inubation mixture [1,2]. Either of the produts of the auxiliary reation, NaetylLglutamyl5hydroxamate or inorgani phos phate, was measured. n the standard assay desribed here the hydroxamate was determined by the ferri hloride method [14]. One unit of Naetylglutamate 5phosphotransferase is defined as the amount of enzyme whih atalyzes the formation of 1 pmol of produt per min under assay onditions. The inubation mixture onsisted of: 400 mm NHzOH. HC1,400 mm Tris (base), 40 mm Naetyl Lglutamate (neutralized with KOH), 20 mm MgC12, 10 mm ATP (disodium salt) and up to 0.03 unit of enzyme in a final volume of 1.0 ml. The ph of the inubation mixture ontaining all omponents exept NaetylLglutamate and enzyme was adjusted to 7.2 with NaOH at 25 "C. The reation was started by the addition of NaetylLglutamate. After inubation at 37 "C for 1030 min the reation was terminated by the addition of 1.0 ml of a solution ontaining 5% (w/v) FeCJ 6H20, 8% (w/v) trihloroaeti aid, and 0.3 M HCl. When neessary the preipitate was removed by entrifugation. The volume of all reagents was halved for the assay of purified enzyme. The absorbane of the hydroxamate. Fe3 + omplex was measured at 540 nm [14]. The molar absorption oeffiient of this omplex under the given onditions was M' xm' (mean of 10 determinations k S.E.) as determined by the omplete onversion of a known amount of NaetylLglutamate to the hydroxamate with purified enzyme. Sine the enzyme is ompletely inhibited by an exess of Larginine [9], standard inubation mixtures

3 D. Hads and T. Leisinger 361 ontaining 10 mm Larginine were used for blank values. n this way nonspeifi reations an be eliminated. t was observed that small impurities in ommerial NaetylLglutamate are a ause for relatively high blank values in the assay of rude extrats. The enzyme ativity was a linear funtion of both inubation time (up to about 30min) and protein onentration provided that not more than approximately 0.03 enzyme unit per ml of reation mixture was used. Protein Determination Protein was estimated by the method of Lowry et al. [15] with bovine serum albumin as a standard. Misellaneous Determinations LArginine, Lornithine, N'aetylLornithine, and NaetylLglutamate, used as supplements in mediume, were assayed as follows. After harvesting the ells aliquots of the medium were sterilized by filtration. LArginine was degraded to Lornithine by means of arginase. LOrnithine was determined by the ninhydrin method [6]. N'AetylLornithine was deteted qualitatively by its ability to allow growth of E. oli W 39A23R3, a mutant with a blok in the Naetylglutamate synthetase reation [16], in minimal medium E. NAetylLglutamate was onverted to the hydroxamate with purified Naetylglutamate 5phosphotransferase and determined olorimetrially as desribed for the enzyme assay. Polyarylamide Gel Eletrophoresis Dis gel eletrophoresis was performed aording to Davis [17] in 7% arylamide gels (length 6 m). The upper gels were omitted. Protein samples ontaining 0.05 % bromophenol blue as a traking dye and about 10 % surose were layered on top of the gels. Eletrophoresis was arried out at 2 "C and a urrent of 2 ma per gel until the dye had almost reahed the bottom of the gel. The position of the dye was marked with a short piee of opper wire. The gels were stained with Coomassie brilliant blue R250 and destained by diffusion as desribed by Weber et al. [18]. NAetylglutamate 5phosphotransferase ativity was loalized in gels by staining with a reagent speifi for inorgani phosphate [19]. Sine the phosphate ions present in the sample buffer moved with the traking dye, the bottom of the gel arrying the dye was ut off after the eletrophoreti run. The gels were then inubated in the standard inubation mixture at 37 "C for 5 min, quikly rinsed with distilled water and immersed in a reagent onsisting of 0.15 M HC104, 6 mm (NH4)6M H20, and 7 mm triethylamine hydrohloride [19]. After about 10 min a yellowish band formed in the gel indiating the position of the enzyme. Sodium dodeyl sulfate gel eletrophoresis was arried out as desribed by Weber et al. [ls]. The following moleular weight markers were used : bovine serum albumin (mol. wt 68000), atalase from bovine liver (mol. wt ), ovalbumin (mol. wt 43000), aldokase from rabbit musle (mol. wt 40000), hymotrypsinogen from bovine panreas (mol. wt 25700), and sperm whale myoglobin (mol. wt 17200) [18]. NAetylglutamate 5phosphotransferase and the referene proteins were denatured by the standard proedure [18]. The samples were applied in a total volume of 2030 pl to 7.5% arylamide gels (10 m in length). A urrent of 8 ma per gel was used. Marking of the traking dye position, staining, and destaining of the gels were as desribed above. Estimation of Moleular Weight by Gel Filtration The influene of ligands on the apparent moleular weight of Naetylglutamate 5phosphotransferase was studied by hromatography on a Sephadex G150 olumn (1.6 x 44 m) [20]. Rabbit musle pyruvate kinase (mol. wt [21]), yeast alohol dehydrogenase (rnol. wt [22]), and bovine serum albumin (mol. wt [18]) were used for alibration of the olumn. NAetylglutamate 5phosphotransferase and the three protein markers were applied to the olumn in a volume of 1.2ml. The flow rate was 4 ml/h. The fration volume ranged from 0.9 to 1.1 ml in different runs. The void volume Vo was determined with blue dextran 2000 in a separate run sine pyruvate kinase [20] and Naetylglutamate 5phosphotransferase bind to blue dextran. Pyruvate kinase and alohol dehydrogenase were measured by standard methods [23]; the absorbane of bovine serum albumin was monitored at 280 nm. Surose Gradient Centr fugation The sedimentation behaviour of Naetylglutamate 5phosphotransferase was analyzed by the tehnique of Martin and Ames [24]. Rabbit musle pyruvate kinase ( s ' ~, = ~ 10 S [21]), rabbit musle latate dehydrogenase ( s ' ~ =, ~ 7.6 S [25]), and human hemoglobin A = 4.5 S [26]) were used as markers. The samples (0.1 ml) were layered on 520% (w/v) surose gradients prepared in 0.1 M phosphate buffer. Centrifugation was onduted at 3 "C in a Spino L265B ultraentrifuge equipped with a SW50 rotor. After 15 h of entrifugation at rev./min, fra

4 ~~ ~ 368 Oligomerization of Pseudornonas NAetylglutamate 5Phosphotransferase Table 1. Speifi ativity of Naetylglutamate 5phosphotransjerase under various onditions of growth Cells were grown in Fernbah flasks and assayed for enzyme ativity as desribed in Materials and Methods. Mean values of two experiments are given Growth medium Mass doubling time Speifi ativity Medium P + 20 mm Lglutamate + 20 mm NaetylLglutamate + 20 mm Lornithine + 20 mm Larginine Medium E" min units/mg protein a Supplements of NaetylLglutamate, N'aetylLornithine, Lornithine, or Larginine (eah at 2 mm) were without effet on doubling time and speifi ativity tions of 12 drops were olleted from the bottom of the tubes. The marker enzymes were assayed by standard methods [23]; the absorbane of hemoglobin A was measured at 420 nm. RESULTS Control of Enzyme Levels The speifi ativity of Naetylglutamate 5phosphotransferase was determined in rude extrats from P. aeruginosa grown on minimal medium P ontaining Larginine or a preursor thereof as the sole arbon and nitrogen soure. As shown in Table 1, Lglutamate and Larginine allowed good growth ; with NaetylLglutamate and Lornithine slower growth was observed. The speifi ativity of the enzyme, however, showed no gross hange. P. aeruginosa was also ultivated on minimal medium E. Supplements of NaetylLglutamate, N'aetylLornithine, Lornithine, or Larginine had virtually no effet on the enzyme level. At the time of harvest the supplements were not exhausted. This ontrol was inluded beause P. aeruginosa is known to degrade arginine rapidly [8]. n onlusion, the formation of Naetylglutamate 5phosphotransferase appears not to be ontrolled by exogenous arginine or its preursors. Enzyme Purijlation Multiple forms of Naetylglutamate 5phosphotransferase were observed during purifiation. As will be shown, the enzyme undergoes assoiationdissoiation reations whih are influened by effetors of low moleular weight. Crude Extrat. The extration and all subsequent steps were arried out at 04 "C. 415 g of frozen ells (grown on medium P plus Lglutamate, see Materials and Methods) were thawed and suspended homogeneously in 2 1 of 0.1 M phosphate buffer. The ell suspension was passed twie through a Manton Gaulin homogenizer at 500 kg/m2. The homogenate was (entrifuged at x g for 20 min. The supernatant was arefully deanted, a small visous fration above the sediment was disarded of rude extrat were obtained. A small aliquot was dialyzed for the determination of enzyme ativity. Heat Treatment. NAetylLglutamate and L arginine were added to the rude extrat at a final onentration of 20 mm and 5 mm, respetively. The extrat was divided into 60ml portions in large test tubes (2.3~ 19 m). The tubes were inubated first in a 37"C water bath for 2min, then in a 65 C water bath for 4 min and hilled in an ie bath. After standing overnight, the milky suspension was entrifuged at x g for 20 min. The preipitate was disarded and an aliquot of the supernatant retained for dialysis. Ammonium Surfate Preipitation. Solid ammonium sulfate was slowly added to the supernatant, 0.30 g (NH&S04 per ml extrat. The resulting suspension was stirred for 1 h and entrifuged at x g for 30 min. The supernatant was disarded. The preipitate was dissolved in 0.02 M phosphate buffer ontaining 10% glyerol and desalted on a Sephadex G25 olumn (5 x 81 m), equilibrated with the same buffer. Chromatography on DEAECellulose. A DEAEellulose olumn (6 x 34 m) was equilibrated with 0.02 M phosphate buffer ontaining 10 "/, glyerol. The desalted ammonium sulfate fration was applied to the olumn. The flow rate was approx. 210ml/h and 4min frations were olleted. The olumn was washed with of equilibration buffer. Thereby most of the protein and part of the enzyme ativity (peak 1) were eluted. Then a linear 31 gradient from 0 to 0.2 M KC1 (in the equilibration buffer) was applied. The major portion of the enzyme (peak2) was eluted with 0.03 to 0.05 M KCl. Fig. 1 shows the profiles of enzyme ativity and absorbane at 280 nm.

5 :$, D. Hads and T. Leisinger KC gradient 0.5 E. 1 M, 'g 1.0 : ; 1, 04 L Peak 2. E Y) 2.0._ Z._._ > Y 1.2 E 1.0 = Y 0.6$ g 1.0 E 0.44 W t 8 L > W Peak 1 ; '.. '\ Fig. 1. Chromatography of Naetylglutamate 5phosphotransferase on DEAEellulose. Experimental onditions are stated in the text. The arrow indiates the position where the linear KC gradient Fration number o.a= Fig. 2. Chromatography of Naetylglutamate 5phusphotransferase on Sephadex G150. Conditions are desribed in the text. Most of the 280nmabsorbing material was eluted in the void volume. (0) Enzyme ativity; () absorbane at 280 nm The frations of peak 2 with the highest ativity were ombined (650ml) and onentrated to 51 ml in an Amion ultrafiltration ell using a PM30 membrane. A 1.fold purifiation resulted from the ultrafiltration step. The enzyme ativity of peak 1 was not used for further purifiation. Consequently, the yield of the DEAEellulose step was poor. Control experiments demonstrated that the ourrene of peak 1 was not due to an artifat suh as overloading of the olumn. Rather, tight binding of lowmoleularweight effetors (e.g. NaetylLglutamate), whih were not removed ompletely by Sephadex G25 hromatography, prevented part of the enzyme from adhering to DEAEellulose. Peak 1 was also found, albeit Fration number w i2 begins in the eluate. KC1 onentrations were determined by ondutivity measurements. (0) Enzyme ativity; () absorbane at 280 nm smaller, when desalted rude extrats were hromatographed on DEAEellulose. The enzyme of peak 1 had the same moleular weight as the enzyme of peak 2 as judged by gel filtration with Sephadex G150. The enzyme from both peaks was inhibited normally by Larginine. Chromatography on Sephadex G150. The onentrated solution from the DEAEellulose step was further purified by hromatography on Sephadex G150. A olumn (5 x 89 m) was equilibrated with 0.02 M phosphate buffer ontaining 10 % glyerol. Elution was arried out by upward flow at a rate of 44 ml/h; 15minfrations were olleted. Fig. 2 shows that the enzyme was eluted as a single band. An apparent moleular weight of about was dedued from the position of the peak relative to the void volume of the olumn (f Fig. 6). The frations exhibiting the highest enzyme ativities (total volume 160 ml) were pooled and onentrated to 25 ml by ultrafiltration. Chromatography on Hydroxyapatite. The onentrated pool was applied to a hydroxyapatite olumn (0.9 x 15 m) equilibrated with 0.02 M phosphate buffer ontaining 10% glyerol. The flow rate was 6.4 ml/h, 15min frations were olleted. The olumn was washed first with the buffer employed for equilibration, then with M phosphate ontaining 10% glyerol. The enzyme was eluted with 0.06 M phosphate ontaining 10 % glyerol (Fig. 3). The frations showing the highest ativities were pooled, onentrated immediately after hromatography and stored in small portions at 20 "C. The overall purifiation of the enzyme was greater than 2000fold. Table 2 summarizes the purifiation proedure.

6 370 Oligomerization of Pseudomonas NAetylglutamate 5Phosphotransferase M 0.060M 7 phosphate 0.15 E a. & E Fig, 3. Chromatography of Naetylglutamate 5phosphotransferaue on hydroxyapatite. Details of the experiment are desribed in the text. The arrows mark buffer hanges. (0) Enzymes ativity; Fration number 0 ( ) p rotein onentration, determined by the method of Lowry et al. [51 Table 2. Purifiation of Nueiylglutamute 5phosphotrunsferase Fration Volume Ativity Yield Protein Speifi onentration ativity ml units/ml O/ '0 mdml units/mg Crude extrat Heat treatment Ammonium sulfate DEAEellulose (onentrated pool) Sephadex G150 (onentrated pool) Hydroxyapatite (onentrated pool) Stubility of the Enzyme The enzyme was stable in 0.1 M phosphate buffer at 4 "C. To prevent loss of ativity in more dilute phosphate buffers 10% glyerol was added. The purified enzyme was sensitive to extreme dilution. When stored at 20 "C the purified enzyme lost no ativity over extended periods. Repeated freezing and thawing should, however, be avoided. Purity of the Enzyme The 2000fold purified enzyme gave one major band in analytial dis eletrophoresis (Fig. 4A). Two or three faint bands were judged to ontain less than 10% of the total protein applied to the gel. When the gel was inubated in a omplete reation mixture and then in a reagent speifi for inorgani phosphate (see Materials and Methods), a yellowish preipitate formed at the site of the major protein band. No preipitate was seen when NaetylLglutamate was omitted from the reation mixture. A single strong protein band was found upon polyarylamide gel eletrophoresis in sodium dodeyl sulfate (Fig.4B), suggesting that the enzyme is omposed of subunits of equal moleular size. As in the standard gel, impurities were estimated to amount to less than 10 % of the total protein. Moleular Weight of Subunits The moleular weight of the Naetylglutamate 5phosphotransferase subunits was estimated by omparing their mobility on sodium dodeyl sulfate gel eletrophoresis to the mobilities of six peptides of known moleular weights [18]. Fig.5 shows the alibration urve obtained with the marker proteins. Thus the subunit moleular weight was about Gel Filtration Studies The influene of ligands on the moleular size of Naetylglutamate 5phosphotransferase was investigated in some detail by gel filtration with Sephadex Em. J. Biohem. 52 (1975')

7 D. Haas and T. Leisinger 371 Fig. 4. Polvarylamide gel eletrophoresis of purijied Naetylgluturnate 5phosphotrunsfernse. (A) Dis gel eletrophoresis : 8 pg of the purified enzyme (hydroxyapatite fration, Table 2) were subjeted to eletrophoresis as desribed in Materials and Methods. (B) Sodium dodeyl sulfate gel eletrophoresis: 8 Fg of protein from the hydroxyapatite fration (Table 2) were denatured and applied to the gel. For details, see Materials and Methods L a6 a8 1.o Relative moblllty Fig. 5. Estimation of the moleular weight of Nuetylglutamate 5phosphotrunsfernse subunits by polyarylamide gel eletrophoresis in sodium dodeyl sulfate. The moleular weights of marker proteins are plotted on a logarithmi sale against their mobilities relative to the traking dye [18]. Mobilities are average values from three experiments. (1) Bovine serum albumin; (2) atalase; (3) Ovalbumin ; (4) aldolase; (5) Naetylglutamate 5phosphotransferase; (6) hymotrypsinogen ;(7) myoglobin G150. nitially it was observed that rude extrats prepared in 0.1 M phosphate buffer always yielded two peaks of enzyme ativity (Fig.6E). Both peaks were of about the same size and also ourred after the heat treatment used as the first purifiation step. However, when extrats were transferred from 0.1 M phosphate buffer into 0.02 M phosphate buffer ontaining 10 % glyerol by passage through Sephadex G25, only the highmoleularweight omponent (peak ) was found. The question was whether the two peaks deteted in rude extrats represented isoenzymes or different aggregates of one enzyme. The fat that in 0.02 M phosphate buffer only one omponent was found did not support the isoenzyme hypothesis, but did not rule it out, sine the smallmoleularweight omponent (peak 11) might have been lost due to inativation. To settle this question a rude extrat, partially purified by the heat treatment, was hromatographed in 0.1 M phosphate buffer. The profile of enzymati ativity was analogous to the one shown in Fig. 6E. Both peaks were isolated, onentrated by vauum dialysis and rehromatographed in the same buffer. Peak remained peak. Peak 11, however, was split into peak and peak 1 in a 1 : 1 ratio. This demonstrated that peak ould arise from peak 11, presumably by an aggregation reation. Attempts to dissoiate peak by 1 M phosphate buffer were unsuessful. The aggregation behaviour of the 2000fold purified enzyme was studied in the presene of the substrates, NaetylLglutamate and MgATP, and the feedbak inhibitor, Larginine (Fig. 6A D). n the absene of ligands (Fig. 6A) as well as in the presene of 10 mm NaetylLglutamate (Fig. 6C) symmetrial peaks were found, whih were eluted almost onomitantly with pyruvate kinase. n the presene of 10mM MgATP the orresponding peak was redued to a shoulder and the main ativity ourred as a broad band (Fig.6D). With 10mM Larginine as a ligand, the omponent of high moleular weight was ompletely absent; a skewed peak, probably onsisting of two omponents was obtained (Fig. 6B). Pyruvate kinase, yeast alohol dehydrogenase, and bovine serum albumin were inluded as standards in the experiments of Fig. 6AD. The peaks of these referene proteins were symmetrial and their elution volumes were not affeted measurably by the ligands. n Fig.7 the ratio of the elution volume to the void volume (VJ Vo) is plotted against the logarithms of the moleular weight of the protein markers [20]. Aording to this plot the apparent moleular weight of Naetylglutamate 5phosphotransferase was about in the absene of effetors, in the presene of NaetylLglutamate and in rude extrats (peak ). The predominant omponent found in the presene of MgATP presumably orresponds to peak 1 in

8 372 Oligomerization of Pseudomonas NAetylglutamate 5Phosphotransferase P : h 0.02 E _ 3._ > r 0 %_m 0.1: L,m K o.l( r Q UJ 0.0: a, m E Ol 2? 5 z 0.1: 0.1( 0.0: 1.2 1, L Moleular wdght Fig.. Estimation of the moleular weight of Naretylglutamate 5phosphotransferase by gel filtration. The VJV, values of the protein markers are averages taken from Fig.6 (AD), those of Naetylglutamate 5phosphotransferase are averages of three experiments similar to Fig. 6 (E). (1) NAetylglutamate 5phosphotransferase (peak 11); (2) bovine serum albumin; (3) alohol dehydrogenase; (4) Naetylglutamate 5phosphotransferase (peak ); (5) pyruvate kinase rude extrats. This enzyme speies had an apparent moleular weight of about n the presene of Larginine some intermediate form(s) appeared. These results show that the enzyme is apable of aggregation with itself and that the state of aggregation depends on lowmoleularweight effetors. t should be pointed out that, with the exeption of the experiment shown in Fig. 6E, the gel filtration studies were onduted at low protein onentrations. The initial onentration of Naetylglutamate 5phosphotransferase applied to the Sephadex olumn was of the order of 20 pg protein per ml. The enzyme was deteted by assaying for its ativity. The reovery of ativity from the olumns ranged from 30% (with 10 mm Larginine) to 90 %. Thus it annot be exluded ( o: 0.0: 0. = vo : 1 j Fration number Fig. 6 Fig. 6. Ge1.filtration of Naetylglutamate 5phosphoiransjerase in the presene and absene ojligands. A Sephadex G150 olumn was run as desribed in Materials and Methods. The void volume is marked by V,,. (AD) 1233pg of purified enzyme from the hydroxyapatite fration (Table 2) and three protein markers (200 pg pyruvate kinase = 1, 300 pg alohol dehydrogenase = 2, 6 mg bovine serum albumin = 3) were applied to the olumn. The olumn was equilibrated and eluted with 0.1 M phosphate buffer ontaining: (A) no ligand; (B) 10mM Larginine; (C) 10mM NaetylLglutamate; (D) 10 mm MgATP. NAetylglutamate 5phosphotransferase was dialyzed overnight against these buffers prior to the hromatographi run. The enzyme was measured by the standard assay exept for (B) where it was assayed at ph 8.9. (At this ph the enzyme is insensitiv to inhibition by Larginine [12].) (E) A rude extrat (16 mg protein) was hromatographed in 0.1 M phosphate buffer

9 D. Haas and T. Leisinger 373 i Fig. 8. Sedimentation of Naetylglutamate 5phosphotransferase in surose gradients. Purified enzyme (7 pg of the hydroxyapatite fration) was entrifuged in surose gradients as desribed in Materials and Methods. The marker proteins are plotted in Fration number arbitrary units. (0) Naetylglutamate 5phosphotransferase; (A) pyruvate kinase; (W) latate dehydrogenase; (0) hemoglobin A; (O)S~~,~ that some omponents of the enzyme were not deteted beause of inativation during hromatography. Density Gradient Cen tr ijiuga t ion Studies The purified enzyme was subjeted to entrifugation in surose gradients. As illustrated by Fig. 8 the enzyme sedimented as a rather broad asymmetrial band whereas the marker proteins formed symmetrial peaks. This behaviour suggests that Naetylglutamate 5phosphotransferase was present in more than a single speies under experimental onditions (low protein onentration, in 0.1 M phosphate buffer). Maximum ativity sedimented at 7.1 S orresponding roughly to an apparent moleular weight of The enzyme was also entrifuged in surose gradients ontaining NaetylLglutamate, Larginine, or MgATP. Table 3 shows that these ligands had an influene on the apparent sedimentation oeffiient of the enzyme. n all ases broad and asymmetrial peaks of ativity were obtained. The highest s0, value was found in the presene of NaetylLglutamate, the lowest in the presene of MgATP. An intermediate value resulted from the addition of Larginine. Thus there is a qualitative orrelation between these results and the gel filtration data. Aording to both methods NaetylLglutamate seems to promote an assoiation and MgATP a dissoiation of the enzyme. On a quantitative basis, there are disrepanies between the moleular weight values obtained with the gel filtration method and those estimated from the sedimentation oeffiients. Several fats, however, Table 3. Effet of ligands on the sedimentation oeffiient of Naeiylglutamate 5phosphotransferase Sedimentation oeffiients were obtained from surose density gradient entrifugation in 0.1 M phosphate buffer with or without addition of ligands. Experimental onditions are given in Materials and Methods. Sedimentation oeffiients are mean values (number of experiments in parentheses). The moleular weight was roughly estimated by using the empirial relation of Paetkau and Lardy [27] M, = [s2,,,11.66 where M, is the moleular weight and s20,w the sedimentation oeffiient in Svedberg units Addition s20.w Moleular weight None 7.1 & 0.2"(4) mm NaetylLglutamate 8.0 (1) mm Larginine 6.6 (2) mm MgATP 5.2 (2) a S.D. S should be borne in mind. First, both methods allow only rude estimates of the moleular weight. Seond, the ourrene of different enzyme speies was learly seen in the gel filtration experiments (Fig.6B, D, E). The broad peaks of ativity observed in surose gradients probably refleted multiple speies whih were in equilibrium with eah other. The s20,w values, therefore, may represent averages of two or more speies. Third, enzyme ativity rather than protein onentration was measured. Possible differenes in speifi ativity of different enzyme speies and loss of ativity during the separation experiments may have influened the piture. Fourth, the protein on

10 374 Oligomerization of Pseudomonus NAetylglutdmate 5Phosphotransferase entration was low (7 28 pg/ml), but not idential in different experiments. The effet of protein onentration on the aggregation state was not investigated. DSCUSSON Previously Naetylglutamate 5phosphotransferase has been partially purified from Esherihia oli [2,28], Baillus hrevis [29], and Chlamydomonas reinhardi [5,30]. n E. oli and B. brevis the enzyme is not subjet to feedbak inhibition by arginine [7,29]. The Chlamydomonas enzyme was studied in some detail by Farag6 and Dknes [ LArginine was shown to at as an allosteri inhibitor and to indue onformational hanges in the enzyme. Gel filtration experiments indiated that Larginine and Naetyl Lglutamate had no influene on the apparent moleular weight of the enzyme. We have purified Naetylglutamate 5phosphotransferase over 2000fold from P. aeruginosa. The enzyme level, whih aounted for less than 0.05 % of the total soluble protein, ould not be inreased by manipulation of the growth medium (Table 1). The enzyme thus appears to be synthesized onstitutively as the majority of the arginine biosyntheti enzymes in P. aeruginosa [8,34]. Essentially the same onlusion was reahed by saa and Holloway [8], who investigated the levels of arginine biosyntheti enzymes in the wildtype and in bradytrophi mutants under onditions of arginine exess and deprivation. t many miroorganisms there are aggregates of enzymes belonging to the same metaboli pathway. Suh aggregates may be regarded as funtional units whih failitate the flux of metabolites ( hanneling ) and inrease the atalyti ativity [35]. NAetylglutamate 5phosphotransferase of P. aeruginosa was deteted in several states of aggregation in rude extrats as well as after purifiation. However, there was no indiation that this enzyme forms aggregates with other enzymes involved in arginine biosynthesis, in partiular with Naetylglutami 5semialdehyde dehydrogenase, the enzyme following in the pathway. The apparent absene of multienzyme omplexes in the arginine pathway does not neessitate that they are absent in vivo. Often aggregates are held together by very weak proteinprotein interations whih are interrupted during frationation proedures [36]. As shown with essentially pure Naetylglutamate 5phosphotransferase, the different aggregation states were due to selfassoiation of the enzyme. At present it is diffiult to derive a model aounting for all moleular weight data. Based on Sephadex G150 hromatography the largest enzyme aggregate (termed peak, Fig.6A, C, E) had an apparent moleular weight of about , whereas the smallest omponent exhibiting enzymati ativity (termed peak 11, Fig. 6D, E) had a moleular weight of approx ntermediary forms were also observed (Fig. 6B, D). The subunit moleular weight was Thus peak 1 presumably onsists of two subunits while peak might be a tetramer of peak 11. Conurrent with the hange in moleular size (estimated by gel filtration) there was a hange in the sedimatation oeffiient as a funtion of ligands (Table 3). The moleular weight estimates obtained from the sedimentation oeffiients do not fit into a simple oligomerization pattern. Rather than representing distint moleular forms they may be indiative of two or more speies in an equilibrium. A wide variety of selfassoiating enzymes is known. A review by Frieden [37] demonstrates that most of these enzymes are allosteri or regulatory, whih is onsistent with the idea that flexible onformations and subunit interations are essential for regulation of enzyme ativity. n fat, it seems that most, if not all, allosteri phosphotransferases are in a monomerpolymer equilibrium [ 111. Welldoumented examplesarephosphofrutokinase [38,40] and aspartokinase [ from various soures. Aspartokinase from P. j7uoresens is subjet to onerted feedbak inhibition by two pairs of endproduts: lysine plus threonine and methionine plus threonine. Dungan and Datta [41] showed that these amino aids aused an inrease of both the Stokes radius and the sedimentation oeffiient of aspartokinase. The substrates, aspartate plus ATP, had little or no effet on the protein struture. There appears to be a orrelation between the degree of inhibition and the oligomerization of the enzyme. t was suggested that the formation of oligomers may be involved in the inhibition mehanism [41]. n the ase of Naetylglutamate 5phosphotransferase of P. aeruginosa the relationship between enzyme struture and ativity is more omplex in that not only the feedbak inhibitor (Larginine) but also the substrates (NaetylLglutamate and ATP) promote strutural hanges in the protein. We wish to thank Dr A. Einsele for help with the largesale ultivation of P. ueruginosa and Vreni Kurer for exellent tehnial assistane. Finanial support ame from the Swiss National Foundation for Sientifi Researh (projet ). REFERENCES 1. Udaka, S. & Kinoshita, S. (1958) J. Gen. Appl. Mirohid Baih, A. &Vogel, H. J. (1962) Biohem. Biophys. Res. Commun. 7, Udaka, S. (1966) J. Bateriol. 91, De Deken, R. H. (1962) Biohem. Biophys. Res. Commun. 8, Denes, G. (1970) Methods Enzymol. 17A, Eur. 3. Biohem. 52 (1975)

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