THE JOURKAL cm BIOLOGICAL CHEMISTRY Vol. 244, iX;o. 15, Issue of August 10. pp. 4112-4121, 1969

Printed in U.S.A.

The Asparagine Synthetase of Escherichia coli

I. BIOSYNTHETIC ROLE OF THE EXZYJIE, PURIFICATIOr\;, AKD CH,4R.ACTERIZhTIOX OF THE REACTION PRODUCTS *

(Received for publication, February 20, 1969)

HOWARD CEDAR$ ASD J~BIES H. SCHW’ARTZ!

From the Depadment of Microbiology, New York University Medical Center, New I’ork, New York 10016

SUMMARY

Synthesis of L-asparagine was detected in extracts of Escherichia coli K-12 only under conditions where either the amount of asparaginase II was greatly lowered or else its activity was inhibited by S-diazo-4-oxo-L-norvaline (DONV). Asparagine synthetase, the enzyme responsible for the forma- tion of the amino acid, was lacking in extracts of an auxo- trophic mutant which required asparagine. Asparagine repressed the formation of the synthetase, but at a rather high concentration, possibly because asparagine is not readily concentrated by the bacterial cell. A genetic locus for the production of asparagine synthetase (for which we propose the name asn) was found to be between the 73rd and 74th mm of the E. coli chromosome by conjugation and transduction experiments.

Asparagine synthetase was purified 370-fold from a mutant of E. coli impaired in its ability to form asparaginase II. In order to diminish the amount of residual asparaginase fur- ther, extracts were made of this mutant after it was osmoti- cally shocked. The synthetase had a molecular weight of about 80,000; it was stabilized by Z-mercaptoethanol and by 10 % glycerol.

The synthesis of asparagine required aspartate, ATP-Mg2+, and ammonia, and resulted in the stoichiometric production of pyrophosphate and AMP. Glutamine did not serve as a source of amide nitrogen. The pH optimum was 8.4. The enzyme preparation, shown to be free of aspartyl transfer RNA synthetase, in the absence of added ammonia also catalyzed an aspartate-dependent pyrophosphate exchange with ATP, which was inhibited by asparagine. /3-Aspartyl hydroxamate was formed in incubations with hydroxylamine.

The biosynthetic pathways of most of the 20 amino acids which commonly occur in protein are known, and the pathway

* This investigation was supported by ?iational Science Foun- dation Research Grant GB-8034.

$ Predoctoral trainee of the hIedica1 Scientist Training Grant G&IO-1668. The data in this paper are taken from a thesis sub- mitted in partial fulfillment of the requirements for the degree of M.D-Ph.D. in the GradIrate School of Arts and Sciences and the School of ?rledicine of xew York University.

0 Career Development Awardee of the United States Public Health Service (Research Career Program Award (+RI-28,125).

for asparagine is one of the last to be described in Esckericlzia coli. The asparagine synthetases from Lactobacteriaceae catalyze the synthesis of asparagine from aspartic acid and ammonia with the conversion of ATP to AMP and pyrophosphate (1, 2). More recently synthetases from the Novikoff hepatoma (3) and embryo chicken liver (4) have been investigated. These en- zymes differed from t,hc bacterial synthetases by their use of glut,amine in preference to ammonia as the donor of the amide group. In some plants, cyanide provides the carbon and nitro- gen of the carbamyl group, with P-cyanoalanine as a precursor of asparagine (57).

We describe here the purification and properties of the aspara- gine synthetase from E. co&. Since an auxotrophic mutant, whose requirement for asparagine was satisfied by asparagine alone, lacked the enzyme, we are confident that the synthetase activity measured represents the true biosynthetic pathway in E. coli.

EXPERIME3-TAL PROCEDURE

Xaterials

3%Orthophosphate, obtained from Squibb, was converted to 32P-pyrophosphate in a platinum crucible at 400” (8). After it was purified by column chromatography on Dowex 1 (8), the material was 98yc PPi as characterized by its acid lability and by its electrophoretic mobility at pH 4.4.

ATP, labeled with 32P in the o(- and /3-phosphates, was isolated from reaction mixtures after incubation of 32P-pyrophosphate with asparagine synthetase under the conditions described below for the pyrophosphate exchange. After it was eluted from paper electrophoretograms, the ATI’ was radiochemically pure as judged by a second elrctrophoresis, again at pH 4.4. Uniformly labeled l*C-ATP (specific activit!- 500 mCi per mmole) was ob- tained front Kern England ?;uclear Corporation. Its specific activity was low-ercd by the addition of unlabeled ATP as indi- cated.

Uniformly labeled I%-L-aspartate (specific activity 150 mCi per mmole) was obtained from ISew England Nuclear Corpora- tion, and purified by chromatography on a column (1 X 15 cm) of Dowex 1X8 acetate (200 to 400 mesh). /3-Aspartyl hydroxa- mate was synthesized from asparagine, and ol-aspartyl hydroxa- mate from isoasparagine (9). The o(- and P-aspartyl hydroxa- mates could be separated by paper chromat’ography in

4112

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phenol-concentrated ammonia-water, 80: 1: 10.’ Isoasparagine was the gift of Dr. A. Meister. 5-Diazo-4-oxo-norvaline (11) was synthesized by Dr. R. E. Handschumacher of Yale University. Amino acid analysis of 5-diazo-4-oxo-norvaline was performed on a Beckman model 120C amino acid analyzer using procedures described in the manual. Salt-free hydrosylamine was ob- tained from hydroxylamine sulfate after precipitation of the sulfate as the barium salt.

Enzymes-Bovine RNase and DNase, E. coli alkaline phos- phatase, egg white lysozyme, and yeast inorganic pyrophos- phatase were obtained from Worthington.

Standard dssuys for Asparagine S?lnthetase-Optimally, about 1 unit (defined below) of enzyme was incubated in a volume of 40 ~1 at the bottom of a conical centrifuge tube with 100 mM Tris-HCl (pH 7.8), 5 mM 2-mercaptoethanol, 10 m&I magnesium acetate, 3 rnhl ATP, 20 mM NH&!l, and 1.5 mM L-aspartate which when radioactive had a specific activity of 40 mCi per mmole. Samples of 10 ~1 were removed during a 15.min incubation, applied to Whatman No. 3hIM paper, and dried by a stream of hot air within 30 set of application. Asparagine was separated from aspartic acid by electrophoresis for 30 min at pH 4.7 in pyridine-acetic acid-water, 25 : 25 : 950, at a potential gradient of 30 volt’s per cm in a tank (Savant Instruments) under Varsol kept at s-15” (12). The location of asparagine, which remained close to the origin, was routinely determined by reference to a marker stained with ninhydrin and occasionally by radioautog- raphy (12). A square of paper (2 x 2 cm) bearing the radio- active product was cut out and counted directly in 5 ml of a toluene-based scintillator.

When asparagine synthetase act,ivity was followed by the formation of l1\IP, enzyme was incubated under standard assay conditions but with the addition of 14C-*\l’l at a concentration of 0.75 m&f (specific radioactivity 67 mCi per mmole). Samples were subjected to electrophoresis at pH 4.4 in 0.05 nr sodium citrate buffer for 90 min at a potential gradient of 33 volts per cm (13). This separated AMP from ATP and ADP as well as from Pl’i. .1MP was located routinely on the paper by its ult,raviolet absorption and also by radioautography. Radio- activity on the paper was again determined by liquid scintilla- tion counting.

hsparagine synthetase activity was also followed by forma- tion of 321’-pyrophosphate with ATP at, a concentration of 0.75 m&f labeled with 32P in the (Y- and P-phosphates (specific activity, 18 mCi per mmole). Electrophoresis at pH 4.4 was used to separate l’l’i from .iI’I’; the 1’I’i was located on the clcctro- phoretogram by radioautography, cut out, and counted by scintillation.

To assay the synthetase by the formation of P-aspartyl hy- drosamate, enzyme was incubated with 50 m&f hydroxylamine under standard conditions but with the omission of NH&l. After the electrophoresis at pH 4.7, the strip bearing radioactive product was cut out and sewn into another paper for electro- phoresis in a second dimension at pH 1.9 (acetic acid-90yc formic acid-water, 87 : 25: 888) (12). Hydroxamate formation was also measured by reaction with FeC13 (14).

Incorporation of 32P-labeled PPi into ,4TP catalyzed by aspara- gine synthetase was carried out under the standard assay condi- t,ions in the absence of NH&l but with the addition of azP

1 Personal communication from Mr. Robert Anthony of The Rockefeller IJniversity, who modified a procedlxe previously described (10).

pyrophosphate (specific activity, 18 mCi per mmole). No special precautions were taken to remove ammonia that might have been present as a contaminant in the components of the reaction mixture. Radioactivity in ATP was absorbed by charcoal, which was washed on glass fiber pads and counted in a gas flow counter (15).

L1 unit is that amount of enzyme which catalyzes the produc- tion of 1 nmole of asparagine per min at 3i”.

Other Enzyme L)elert,li~ations-~lspartyl-tR?;h synthetase was assayed in 100 mar Tris-maleate buffer (pH 7.0), 3 ml\1 ATP, 5 mar 2-mercaptoethanol, 10 nibi magnesium acetate, 1.5 PM 14C-aspartate (specific activity, 150 mCi per mmole), and 0.1 mg of E. coli K-12 tRXA1 (Schwarz I<ioResearch Company) in a volume of 0.1 ml. Eefore use, the tRNA was extracted with phenol and precipitated from the aqueous layer with ethanol. Aspartyl-t.RN-i was counted on glass fiber pads after precipita- tion with cold 5% trichloracetic acid (15).

E. coli asparaginase I and II (16) and alkaline phosphntase (17) were assayed as previously described. Inorganic pyro- phosphatase activity ~vas assayed (18) with the use of high volt- age electrophoresis at pH 4.4 to separate Pi from the radioactive substrate, 321’-l~rrol,hosl,hate.

Bactel-iul Strains and Genetic Experiments-AIll E. coli \vere derived from strain E-12. These were: one of the mutants we isolated from the wild type which was unable to grow under anaerobic conditions (19) (1TC(’ 25288) an ausotroph, ER, (,1sll-, thi-) (ITCC 2528i) the amino acid growth rcquiremcnt of which was sati&,: by L-asparagine only, which was isolated after ultraviolet irr:tdi:Ltion bJ- l>r. 13. Reich of The Rockefeller Unircrsity; Hfr strains .JC12, .J4(1’10), and .11$313 were kindl? supplied by rh. 1:. Low, Yale Univcrsitl-. Strain Xl{1450 bgl+ (20) was obtaitletl from Dr. \V. K. 1Ia:as of this I?cpartment. Descriptions of genetic espcriments follow the notation outlined by Taylor and Trotter (21). ILLcterial matings with Hfr strains and the auxotroph ER, the lxeparation of transducing bacterio- ])hagc, and the transduction espcrimcnts were done as described by XIrFall (22).

Growth of Bacteria, Harvesting, and Preparation of Osn,otically Shocked Bacteria-For the studies on bhe formation of aspara- gine synthetase, cells were gron-n, harvested, and lysed with toluene as we have previou.qly described (19). Cell density was determined turbidimetrically; 1 dsio unit corresponded to 0.93 mg of protein per ml (19). The minimal medium used is that described by 1)avis and hlingioli (23) without citrate.

The bacteria used for enzyme purification were grown and osmotically shocked at the New England l<nzyme Center, Boston, Massachusetts. A1 yield of 500 g of shocked cells was obtained from 550 liters of culture medium. The E. coli strain used was our mutant impaired in its ability to grow anaerobically (19). Under all conditions of cultivation this strain produced only 5 to 10yc as much L-asparaginase II as did the wild type. The growth medium, which was designed to minimize the forma- tion of both asparaginase II (19) and aspartokinase (24), con- tained 0.7yc KzHP04, 0.3FG KH&‘04, 0.1% (NH4)$04, 0.02% MgSO+ 0.2% casein hydrolysate (Difco), 0.2y0 glycerol, and 0.02% each of L-methionine, L-lysine, and L-threonine, and, as an antifoaming agent, 2 ml of Polyglycol-2000 (Dow Chemical Company) per 100 liters of medium. A 600.liter fermentor was inoculated with 10 liters of an overnight culture. The bacteria were grown at 37” with forced aeration, and harvesting began when the turbidity of the culture reached 100 Klett units with

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4114 Asparagine Synthetase of E. coli. I Vol. 244, No. 15

the red filter (0.2 11570 unit). The contents of the fermentor were pumped into a DeLaval PX207 centrifuge over a period of 90 min. During this time, growth in the fermentor was allowed to continue with full aeration.

The bacterial pellet was washed and osmotically shocked fol- lowing the procedure of Nossal and Heppel (25) in order to free the cells from asparaginase II (16). This procedure was modi- fied for large amounts of bacteria in that the washes at 0” with 0.01 M Tris-HC1 (pH 7.8), and the sucrose wash, were carried out in the DeLaval centrifuge. Cells became fragile, however, after the sucrose wash. The centrifuge was therefore stopped completely after this wash and the cells gently scraped out of the centrifuge bowl. Immediately following the IO-min wash wit,h cold water the bacterial suspension was made 10 m&r Tris- HCl (pH 7.8), 11 InM magnesium acetate, 1 rnM EDTA, 30 mhr KCI, and 5 mill 2-mercaptoethanol (extract buffer) by the addi- tion of the concentrated components. The shocked bacteria were harvested by a final centrifugation in a Sharples centrifuge. The cells were rapidly frozen in plastic bags by pressing in a thin layer between solid blocks of carbon dioxide.

Purification of Asparagine Synthetase

All procedures were carried out at 4” in a cold room. Cen- trifugation was at 15,000 x g with refrigeration for 10 min unless otherwise specified.

Extraction and Gel Filtration-The frozen bacteria were broken in an Edebo X-press (26) and extracted with extract buffer (defined above) containing 5 Fg per ml of DNase. Unbroken cells and debris were removed by centrifugation. The super- natant was then centrifuged at 30,000 x g for 30 min (12). About 30 ml of the 30,000 x g supernatant (crude extract) were subjected to gel filtration on a column (140 x 4 cm) of Sephadex G-100 (Pharmacia) equilibrated with extract buffer. The flow rate was 30 ml per hour; 15.ml fractions were collected. The fractions containing asparagine synthetase that were uncon- taminated by asparaginase II (see Fig. 1) were collected and

FRACTION NUMBER

FIG. 1. Gel filtration of asparagine synthetase on Sephadex G-100. Blue dextran (Pharmacia, 6 mg) (A), E. coli alkaline phosphatase (10 units) (B), and bovine cytochrome c (Boehringer- Mannheim Corp., 50 mg) (C) were included with the samples as molecular weight markers. The positions of elution of blue dex- tran and cytochrome c were determined spectrophotometrically, the former at 750 nm, the latter at 410 nm. Asparagine synthetase (+a) was measured under standard assay conditions by the appearance of I%-asparagine, and asparaginase II (O----O) was assayed as previously described (16). The amount of protein in the fractions is indicated by their A280 (o- - -0).

combined. The combined Sephadex fractions were brought to 60% saturation with (NH&SO4 by adding a mixture of solid (NH&S04 and NH,HCOB (1O:l) slowly. After 30 min at O”, the precipitate was collected by centrifugation and redissolved in a volume of glycerol buffer (10 m&r Tris-HCl (pH 7.4), 5 mM

2-mercaptoethanol, 10% glycerol) one-fifth that of the original volume and dialyzed 3 times for 6 hours, each time against 100 volumes of the same buffer.

DEAE-Sephadex Chromatography-The dialyzed material was made 0.23 M in NaCl and applied in a volume of 3 to 10 ml to a column (2 X 2.5 cm) of DEAE-Sephadex A-50 (Pharmacia) previously equilibrated with 0.23 ~1 NaCl in the above mentioned glycerol buffer. The column was washed with 5 column volumes of 0.23 M NaCl in glycerol buffer, and then the synthetase was eluted with 5 column volumes of 0.28 M NaCl in glycerol buffer. The flow rate was 10 ml per hour. Fractions containing syn- thetase activity were combined and dialyzed overnight in 1 rn*r Tris-HCl (pH 7.4), 10% glycerol, and 5 rnY 2-mercaptoethanol.

dcid Precipitation-Acetic acid (0.1 ~1) was added dropwise to the dialyzed material from the DEAE-Sephadex column and kept at 0” with stirring until the pH fell to 5.0 as monitored by a pH meter. -1fter 10 min, the precipitate was collected by cen- trifugation and redissolved in glycerol buffer.

Acrylamide Gel Electrophoresis-Electrophoresis was carried out in apparatus from Canal Industrial Corporation, Bethesda, Maryland. Analytical disc gel electrophoresis was done in 5-cm 77; polyacrylamide gel columns at pH 9.5, with stacking gels at pH 8.9, cast with Tris-glycine buffers prepared according to system A of Davis (27). Gels were stained for protein with Coomassie brilliant blue (28). For preparative electrophoresis the “Prep-Disc” apparatus was used at pH 9.5 with Tris buffer as described by Jovin, Chrambach, and Naughton (29), except that no upper gel was used. In addition, the buffers contained 5 mi\r GSH. Following polymerization a preliminary run Iv-as carried out in the absence of sample in order to remove the am- monium persulfate added to catalyze the polymerization of the gel. Sample was applied in 50~~ sucrose, and electrophoresis carried out at 80 volts per cm for 3 hours.

Isoelectric Focusing--;l gradient was prepared from a less dense solution consisting of 51 ml of water, 4 ml of 1 70 ampholine, pH 3 to 10 (LKB Instruments, Inc.), and 5 ml of enzyme solution (combined eluates from the DEAE-Sephadex column), and a more dense solution consisting of 37 ml of m-ater, 8.5 ml of 87, ampholine (pH 3 to lo), and 25 g of sucrose. Both solutions were made 5 mrvr in 2-mercaptoethanol. Focusing was done at 4’ for 30 hours with a final potential of 600 volts (30) in a 110-ml electrophoresis column (LKB) (31).

RESULTS

Conditions Which A$ect Production and Detection of Asparagine Synthetase-The presence of asparaginase II interfered with the detection of asparagine synthesized in extracts of whole, tolu- enized E. coli. In order to measure asparagine synthetase in these crude extracts we have found it convenient to add 5-diazo- 4-oxo-norvaline (DONV) to inhibit both asparaginase I and II. The asparagine analogue is suitable for this purpose because it did not inhibit the purified synthetase under standard assay conditions, but as seen from Table I and as previously reported (II), it did inhibit asparaginase II, the activity t,hat interfered with detection of the synthetase.

The specific activity of asparagine synthetase in extracts of

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TABLE I

Effect of 5-diazo-4-oxo-L-norvaline on measurement of asparagine synthelase and asparaginase II in toluenized E. coli

E. coliwild type K-12 was grown aerobically in minimal medium supplemented with 1% casein hydrolysate and 0.57, glucose and harvested at As,,, of 0.25. Extracts of cells treated with toluene were assayed for asparagine synthetase and for asparaginase II under standard conditions both in the presence and in the absence of 5 mivi 5-diazo-4-0x0-norvaline.

Enzyme activity +5-diazo-4- oxo-norvaline

-5.diazo-4- oxo-norvaline

nnoles/min/mg $wotein

Asparagine synthetase. 1.55

1

0a Asparaginase II. 0.12 2.09

a <O. 1 nmole per min per mg of protein.

wild type E. coli incubated with 5-diazo-4-oxo-norvaline was 120 nmoles per hour per mg of bacterial protein and was unaf- fected under a variety of growth conditions. Similar amounts were obtained from bacteria grown on minimal medium or mini- mal medium enriched with 1% casein hydrolysate. No dif- ferences were seen when glucose, glycerol, or casein hydrolysate was used as sole source of carbon. The specific synthetase ac- tivity in E. coli was approximately one-tenth that reported for crude extracts of lactobacteria (I, 2). The concentration of the synthetase in E. coli was of the same order of magnitude as that in a number of leukemic mouse tumors resistant to treatment wit,h asparaginase (32, 33) and of Sovikoff hepatomas, (3) and was 10 to 200 times greater than the activity in various normal mouse tissues (33,34), including those which are actively engaged in the synthesis of protein, for example, liver and brain.

Formation of asparagine synthetase was repressed by L-aspara- gine (Fig. 2). Relatively high concentrations of the amino acid in the culture medium, however, were needed to inhibit enzyme synthesis. Repression of the synthetase in Lactobacillus arabino- sus was brought about by 200.fold lower concentrationsof aspara- gine. We have previously presented evidence that usparagine is not concentrated by E. coli (19).

Lack of ;l sparagine Synthesis in d sparagine-requiring Jf utant

and Genetic Locus for Production of Asparagine Synthetase-Ex-

tracts of ER, the auxotroph which requires asparagine for growth, failed to synthesize asparagine. It may therefore be concluded that the asparagine synthetase reaction assayed under our condi- tions is the primary biosynt.hetic pathway in E. coli. Washed mutant cells were lysed with toluene and dialyzed in order to free the extracts from any remaining asparagine. The dialyzed estrncts were assayed for enzyme in the presence of 5-diazo-4- oxo-norvaline under conditions where 0.17; of the wild type’s synthetase activity could have been detected. The bacteria for these determinations were grown under strictly aerobic condi- tions in nutrient medium, and the amount of asparaginase II was the same in both the auxotroph and in the wild type grown in parallel as control. Furthermore, this mutant did not show a requirement for any of the amino acids whose biosynthetic path- ways include an aspartokinase reaction, which suggests that the formation of asparagine did not involve @-asparty phosphate as an intermediate.

With the asparagine auxotroph, ER, we were able to map the genetic locus for enzyme production which we propose to name

determined by finding the time of entry of asn into the F- auxo- troph in conjugation experiments using Hfr strains of E. coli with origins from various parts of the E. coli chromosome. Asn lay between the origins of Hfr strains JC12 (60 min) (35) and 54 (80 min) (21). The approximate location of asn was at 70 min, since it entered 15 min after the origin of JC12 and 10 min after that of J4. Furthermore it entered immediately with Hfr strain AR313 the origin of which is at 72 min (21). Finer genetic lo- calization was obt’ained by transducing the asparagine auxotroph with bacteriophage Pl grown on strain AR1450-bgl+ and charac- terizing asn+ transductants. The data shown in Table II lo- calized asn at an equal distance between ilv and bgl. The total number of ilv- transductnnts was 43.5% and the number of bgl+, 39.1%. In agreement with Schaefler and Maas (20), the co- transduction of ilv and bgl was 19.2%. Thus the asparagine locus is between the i3rd and the 74th min on the chromosomal map. We have not been successful in carrying out the reverse cross using strain AU1450 bgl+ as recipient and bacteriophage grown on the asparagine auxotroph as donor.

Purification of rlsparagine Synthetase-The crude extract pre- pared as described under “Experimental Procedure” contained 2.1 units of asparaginase II per mg of protein. Asparaginase II was partially separated from asparagine synthetase during gel filtration on Sephades G-100. Only fractions containing the

I. 8

1.6

E” 1.4

. U-J I- 2 1 2

3

1.0 > c > F 0.8

Y

U I= 0.6

s

z z 0.4

w

2 0.

ASPARAGINE CONCENTRATION (gm/ 100 ml)

FIG. 2. Repression of asparagine synthetase by asparagine. E. coli wild type was grown aerobically in the presence of varying amounts of L-asparagine in minimal medium supplemented with 1% casein hydrolysate and 0.5$& glucose. The cells were har- vested at an A570 of 0.25. Extracts of the toluenized cells grown with variotis concentrations of asparagine were dialyzed exhaus- tively in a common vessel, and then assayed for asparagine syn- thetase under standard conditions (“Experimental Procedure”)

asn. Its approximate position on the chromosome was first in the presence of 5 m&f 5-diazo-4-oxo-norvaline.

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Asparagine Synthetase of E. co&i. I Vol. 244, No. 1E

greatest amounts of asparagine synthetase (indicated in Fig. 1) were collected. Inspection of Fig. 1 would suggest that the re- covery of asparagine synthetase should have been less than the 94.5% actually obtained (Table III). An apparent increase in the factor of purification resulted when asparaginase II was separated from the synthetase.

With the use of the equations of Ackers (36) and data from Fig. 1 the molecular weight of asparagine synthetase was esti- mated to be 82,000 and that of asparaginase II, 103,000. Yellen and Wriston (37) have reported a similar molecular weight for the asparaginase of E. co& B as determined by gel filtration on Sephadex G-200.

Both glycerol and 2-mercaptoethanol stabilized the synthetase. Without 2-mercaptoethanol all activity was lost overnight at

4”. The enzyme was also inactivated by agents which react

TABLE II

Genetic rrlapping of locus for aspalagine synthelase formation

The transduction was carried out as described under “Experi- mental Procedure” using bacteriophage grown on AB1450 bgl+ as donor and ER (asn-) as recipient. Asn+ transductants were selected. The frequency of this transduction was 5 X 10m6. Six hundred twenty-four of these were tested for their ability to grow without isoleucine or valine (ilv) and for their ability to ferment salicin (bgl). The diagram at the top of the table shows an ar- rangement of the chromosome consistent with these results, and sperifies the regions of crossover for the various transductants in the table.

Donor (-4111450 bgl+)

bgl+ I

asn+ /

ilv- I

Regions of cross- I II III IV b&A&

over.

Recipient @IL).

Location on chro- mosomnl map (min)

bf-

73 I

asn- I

ilv+ I

74 I

Regions of crOSSO”er

I + III I + IV

II + III II + IV

Phenotype of transductant

bgl+ ilv+ bgl+ ilv- bgl- ilv+ bgl- ilv-

No. of transductants

12-l 110 229 152

Total transductsnts

%

19.9 19.2 3G.F 24.3

with sulfhydryl groups: p-hydroxymercuribenzoate, N-ethyl maleimide, and iodoacetamide. Glycerol was added when it wa! found that activity was lost in a variable manner especially ij the enzyme was frozen. Enzyme in any of the last three stager of purification has been stored in glycerol buffer without loss 01 activity for over 6 months at -60”.

Chromatography on DEAE-Sephadex separated asparagim synthetase from aspartyl-tRNA synthetase (Fig. 3). Demon strntion that this enzyme did not contaminate the preparation o asparagine synthetase is important since the amino acid activat. ing enzyme carried out a vigorous aspartate-dependent ATP, pyrophosphate exchange in crude extracts. As shown below asparagine synthetase also catalyzed a pyrophosphate exchange reaction. Chromatography on DEAE-Sephadex also freed the synthetase from asparaginase I activity.

In attempting to purify the synthetase further by isoelectric focusing, we found that some of the activity banded at pH 5.i (Fig. 4). Most of the enzyme was lost, however, by precipita tion at the isoelectric point. The precipitation at pH 5 used ir the purification procedure also served to concentrate the enzyme This material was used for the experiments described below. K( detect,ed no aspartase (<0.5 nmole per min per ml) or inorganic pyrophosphatase activity (<O.l nmole per min per ml) in thl synthetase preparation at this stage of purification.

The factor of purification after acid precipitation was 37( (Table III). This material was analyzed by polyacrylamidl

FIG. 3. Column chromatography of asparagine synthetase 01 DEAE-Sephadex. Hetails are given in the text. Fractions o 2.8 ml were collected. Asparagine synthetase (wm) wa: assayed under standard conditions, and the aspartyl-tRNA syn thetase (O---O) as described under “Experimental Procedures” A?so (---) was monitored continuously during the collection o fractions and converted to protein concentration.

T-IDLE III

Purijication of aspalagine synthetase

Fraction ~0hllle Enzyme activity Total enzyme Protein Specific activity Recovery

ml units’“/ml unitsQ mglml units’“jnrg R

Crude extract 30 44 1325 35.7 1.2 Gel filtration. _. . 58 22 1250 0.664 32 94.5 60°]0 ammonium sulfate.. _. _. 11 106 1170 3.32 32 88.6 DEAE-Sephadex 14 60 845 0.256 235 63.6 Acid precipitatiou.. 14 GO s45 0.132 454 63.6

a A unit is that amount of enzyme which catalyzes the formation of 1 nmole of asparagine per min at 37”. -

Purification

28.3 26.0

190 370

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$ 4ClO ;: C 5 -a 3 t = -6

f 2- x

a -4

ii 3 s -2

z 2 I ,

10 20 30

FRACTION NUMBER

FIG. 4. Isoelectric focusing. Each fraction (4 ml) was assayed for asparagine synt,hetase (m) by the standard procedure, and for pH (0).

disc gel electrophoresis. Two major protein bands and one minor band appeared on gel columns after electrophoresis at pH 9.5. Preparative gel electrophoresis resulted in the separation of the synthetase from the slower moving major band; but about 70% of the enzymat’ic activity was lost during these procedures. Fur- ther purification by chromatography on calcium phosphate gels was unsuccessful since once the enzyme was adsorbed to the gel at pH 6.5, the activity was not recovered even in washes of 0.5 RI sodium phosphate, pH 9.0.

Reyuirements for clsparagine Sy?zthesis---~sp:lragiile synthetase activity has previously been measured by following the formation of aspartyl hydroxamate in the presence of hl-dros!-lamine (1). IYe, however, have routinely assayed the conversion of W- aspartate to 14C-asparagine in the presence of arnmonkt (“Experi-

mental Procedure”). The rate of product formation ~-as linear Faith time in the range in which up to 105; of the substrate was convertrd. Formation of asparagine was completely dependent upon 1\4g2+ and XTP (Table IV). Less than 17; of the activity was obtained with ADP, CTP, UTP, or GTP. So stimulation of the reaction occurred when 175 i.u. of inorganic pyrophosl)ha- t:lzc were added per ml of incubation misture. *Addition of

1\InClz did not satisfy the requirement for 1\Ig*+ which distin- guishes the 1~‘. coli enzyme from the one from L. arabinosus (1). Xrsimal s\nthetnse activity was seen betn-een 1)H 8 and 8.4 (Fig. 5).

-Yt the 1)1-I of the standard incubation mixture, formation of nslxxragine appeared to be only partially dependent on KHJl. S\nthesis was 90:; dependent upon added SHdCl, however, when precautions were taken to remove contaminating ammonia from components of the assay mixture by prior treatment with Dowes 50 in the Tris form. Th e requirement for added NH&I

slbo varied with the pH of the incubation mixture. Greater dependence was seen at lower values of pH (Fig. 5), although the nrasimal velocity increased with pH.

This pattern of dependence on pH has previously been seen with enzymes which preferentially use unionized ammonia as substrate rather that NHd+ (38). Glutamine, which is the pre- ferrcd ammonia donor for the synthetase from mammalian sources (3), did not substitute for NH&l \\-ith the bacterial en- zyme either at pH 7.8 (Table IV) or at 6.6.

Stoichiometry of Reaction and Characterization of Products-The products of the reaction were asparagine, AMP, and I’Pi. These xvcre produced in nearly equimolar amounts during the course of

the reaction with 0.45 unit of asparagine synthetase (Fig. 6). The stoichiometry, averaged from the values in Fig. 6, was as- paragine, 1.00; AMP, 1.06; PPi, 1.02. In addition to the iden- tification of these products by electrophoresis, additional charac- terizations were also performed.

The radioactivity in the asparagine produced, after elutionfrom the electrophoretogram, was completely converted to ‘4C-aspar- tatc during a brief incubation with purified E. coli L-asparaginase

TABLE Iv

Requiremenls for synlhesis oj asparayine

The standard assay was considered romplete and consisted of 100 rn%~ Tris (pH 7.8)) 5 mM 2mercaptoethano1, 10 mM magnesium acetate, 3 nibI ATP, 20 mM NH&l, 1.5 m&f aspartatc (specific activity, 40 rnCi per mmole) in a volume of 40 ~1. When MnCls was added its concentration was 10 rnlf; glutamine was added at 20 ml{; ADP, CTP, UTP, or ATP, at 3 rnlf.

Components of the incubation Asparagine produced

Complete................... . . . .._... -Magnesium acetate -Magnesium acet,ate + MnC12 . -ATP., . -ATP + ADP, CTP, LTP, or GTP. -NH&‘........ . .._.___.....__.. -NHIC1 + gl~ltamine _. -Enzyme..

51.2 0” 0a Oa 03

li.9 17.7

0a

n <0.5 nmole of asparngine per min per ml.

FIG. 5. The effect of pII on asparagine synthetase. Asparagine synthetase was assayed lmder the standard conditions using Tris-acetate buffers at the variolw values of PI-T. The dependence of maximal velocity on pH (a---w) is presented as the ratio of the activity at the particular pH to that obtained at pH 8.4, the pII optimum. The dependence of the initial rate of 32P-pyrophos- phate incorporation into ATP was determined as described under “Experimental Procedure. ” This rat,e is also graphed (O---O) as a fraction of the maximal velocity of the forward reaction at pH X.4. The requirement for NI14Cl at the various pH values is also shown (X-- -X). The activity of the enzyme was measured both in the presence of 20 m&f NH&l (standard assay condition) or under standard conditions but with NH&l omitted. The results are presented as the percent dependence on added ?jH,Cl:

100% I - Activity without NH&l

Activity with NH&l

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4118 Asparagine Synthetase of L’. coli. I Vol. 244, No. 15

1

5 10 15

TIME (min)

20

FIG. 6. Stoichiometry of the asparagine synthetase reaction. The prodttction of ‘Gasparagine (o), ‘%-AMP (M), and 32P- pyrophosphat,e (X) was followed in separate incubation tithes. All incubations contained 1.5 rnM aspartate, 0.75 mM ATP, 20 IllM

NHICl, 10 rnM magnesium acetate, 5 m&c 2.mercaptoethanol, and 100 m>r Tris-HCl (pH 7.8). I:ither aspartate or ATP was iso- topically labeled. See text.

T.\lILE v

Ifeqtrirements jqr jormnlion a,[ ;t JIP The standard conditions of assay were modified by the addit ion

of r4Clabeled ATP at a concentration of 0.75 rnM with a specific radioactivity of G7 mCi per mmole, and the replacement of radio- active aspartate by unlabeled aspartate. AMP formation was measured electrophoretically (“Experiment,al Procedure”).

Components of the incubation AbIP produced

Complete. -NH&l... .‘.. -Magnesium acetate. -Aspartate.. --I:nzy-me.

a <0.5 nmole per min per ml.

%m7les/min/9nl

5G.3 Y.2 0" 3.2 00

11. The material produced in the absence of added SH,Cl was also asparagine since it too was hydrolyzed to aspartate in incubations with asparaginase II. The radioactive product vvvas separattd from L-isoasparagine by clectrophoresis at p1-I 3.1 (39). -it this pH, asparagine remained in the neutral region, but iaoasparagine migrated toward the cathode. ALIP was further identified by thin layer chromatography on I>EAE-cellu- lose (40).

Formation of AMP was dependent upon aspnrtate, Mg2+ and ammonia (Table V). The PPi produced was quantitatively converted t,o P; after incubation with inorganic pyrophosphat,ase. A small amount of ADP appeared during the incubation and was presumably a product of a contaminating enzymatic activity. This amounted to about 5% of the AMP produced. The ap-

pearance of .WP did not depend on the addition of aspartate, ammonia, or Mg2+. Radioactive AUP (3 mM, specific activity, 67 mCi per mmole), isolated from these reaction mixtures after electrophoresis at pH 4.4, was not converted to -IMP in incubn- tion with the enzyme under the conditions of the standard assay but with ATP omit,ted.

Recently Wilcox and Nirenberg (41) have reported the amidn- tion of glutamyl-tRNAG1” to form glutaminyl-tRNdC1” by par- tially purified enzymes from several bacteria, but not from E. coli. Incubation of the asparagine synthetase for 30 min at 37” with 200 ~g per ml of RKase A at pH 7.4 did not alter the ac- tivity of the synthetase. -2fter dialysis, t,his preparation had a ratio of -lZso:~1260 of 1.24. Furthermore, the addition of tRNA did not affect the pyrophosphate exchange reaction catalyzed by the enzyme, as noted below. These results argue against in- volvement of RS.1 in the synthesis of asparagine.

Pyrophosphafe kkchange and (3-dspartyl Hydroxamate Forma- tion--.&paragine synthetase catalyzed a pyrophosl)hate es- change with ATI’ whose rate was dependent upon enzyme cow

centrat,ion (Fig. 7A). Ko incorporation of I’Pi into ATP was seen when asp&ate or -\Ig2+ was omitted. The rate of the ex- change approximated linearity when less than 57; of the I’Pi added was incorporated into -Yl’P and was presumed to be the initial rate. The ol)timum of the exchange was pH 7.4, slightIT lower than the optimum for the forward reaction (Fig. 5). A similar exchange reartion has been reported for the asparagine synthetase of L. arahinosus (1).

The apparent Michaelis constant for pyrophosphate in the exchaiige reaction w-as 3 nnr (Fig. TB). Under standard assay conditions, addition of l’l’i at a concentration of 3 mnr has litt,le effect on the initial rate of asparagine synthesis. In the accom- panying paper (42) we will show that 1’I’i is a rather weak non- competitive inhibitor of both A’l’l’ and aspartate.

At 3 m&f l’l’i, the initial rate of the exchange was 45yc that of the forward reaction at t,he same pI1. Although the maximal velocity was not measured experimentally, it nas estimated to be 45.5 nmoles per niin per ml of enzyme by estraliolation from the Lineweaver-l%urk plot (Fig. 7B) ; this was SO’;‘? of the rate of the

maximal forward reaction at 111-I 7.1.

It is unlikely that, the pyrophosphate eschanpe observed re- sulted from any activity other than aspnragine synthetase. IYe have shown our preparation to be fret of asparty1-tRN-1 sy-n- the&e (Fig. 3). Furthermore the addition of tRNA (1.5 mg

per ml) did not affect the exchange reaction, and the addition of either 20 m&l ammonium chloride or 1 I~JI asparagine completely

eliminated the exchange. -1sparagine synthetase catalyzed the formation of @-asparty

hydrosamate (Table VI), indicating the occurrence of carbosyl- activation of aspartate. The hydrosamate was identified by two diniensional electrophoresis at pH 4.7 followed by pH 1.9 using P-aspartyl hydrosamate as an internal standard. It was also measured by- reaction with F&la. This product was sepa-

rated from a-aspartyl hydrosamnte by paper chromatography. At 50 ~JI hydroxylnmine, the rate of hydrosamate formation was approximately equivalent to the rate of asparagine synthesis in the presence of 20 m&r ammonium chloride (Table VI). Concen- trations of hydroxylamine greater than 0.1 RI inhibited the en- zyme, so that less hydroxamate was formed. Even in the pres- ence of NH&l, inhibition of the formation of both asparagine and XRIP could be shown with high concentrations of hgdrosglamine.

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Issue of August 10, 1969 H. Cedar and J. H. Xchwartx 4119

TIME (min)

FIG. 7. Pyrophosphate-ATP exchange reaction catalyzed by asparagine synthetase. A, the initial rate of incorporation of 32P-pyro- phosphate into ATP was measured with the amount of enzyme in units as indicated on the figure. phate used was 3 mM (4 X lo7 cpm per ml).

The conrentration of 32P-pyrophos- B, a Lineweaver-Burk plot of the dependence of the initial rate on the concentration of

PPi. All assays were done in 100 mM Tris-HCl (pH 7.4), 5 mM 2-mercaptoethanol, 3 mM EDTA, and 3 In= aspartate. phate was added as 4 X lo1 cpm per ml and unlabeled PPi was added to obtain the concentration indicated.

32P-Pyrophos-

concentration was made 5 m&I greater than the concentration of PPi. The magnesium acetate

TABLE VI TABLE VII Formation of P-aspartyl hydroxamate by asparagine synthetase at Inhibition of asparagine synthetase at high

various concentrations of hydyoxylamine concentrations of inhibitor

Asparagine synthetase (56.9 units per ml) was incubated under Asparagine synthetase activity was measured in the presence of standard assay conditions with ‘4C-aspartate but without NH&l. the various inhibitors under standard assay conditions but with Salt-free hydroxylamine was added in the concentrations indi- cated. @-Asparty hydroxamate was separated from aspartate by electrophoresis at pH 4.7; hydroxamate was purified further by subsequent electrophoresis at pH 1.9.

Concentration of hydroxylamine

I

Rate of @-hydroxamate production

M Wdt-S/&%/?d

0.05 51.7 0.10 44.6 0.25 35.3 0.50 22.1 0.75 20.4

Hydroxylamine at these high concentrations also inhibited the ATP-pyrophosphate exchange reaction.

Inhibitors of Asparagine Synthetase-Two classes of compounds were tested for inhibition of asparagine synthesis. A group of amino acids which were selected because they are effecters which influence aspartate kinase (24), when added separately each at a

L-Lysine ................... L-Threonine ................ L-Methionine ............... L-Isoleucinc ............. I,-Valine. .................

All the above ............ Glycine ............... L-Alanine. ...... ...... L-Serine ............... L-Glutamate ........... L-Glutamine ....... p-I/Zyanoalanine ...... ol-Ketosuccinamic acid. L-Asparagine ......... L-Isoasparagine ........ 5-diazo-4-oxo-norvaline.

llzM

25 25 25 25 25

5 each 25 25 25 25 25 25 25

5 5 5

%

12 20 10 20 25 20 10 40 60 40 40 45 40 98 35 60

-

-

_-

-

concentration of 25 MM, inhibited 10 to 20y0 (Table VII) under acids, each at 5 mM, inhibited the reaction by 20%, the extent assay conditions which were standard except for the lowering of expected from the summation of the concentrations of the in- t’he aspartate concentration to 0.1 mM in order to obtain a ratio dividual effecters. of inhibitor to substrate of 250. A mixture of all of these amino Compounds structurally similar to aspartate were also tried.

0.1 mM aspartate.

Inhibitor Concentration Extent of inhibition

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4120 11 spa~agim Syzthefase of I:‘. coli. I Vol. 244, so. 15

Asparagine itself was a 1)otent inhibitor of the reaction, with an ;~(.li?2o~Zedg,tlenis--n-e thank 1)~ l%rooks Low, Pearl Cooper, affinity for the synthetaae greater than that of aspartate. DC- and Robert Kadner for their help with the genetic experiments, tailed studies of product inhibition will be lwesented in the ac- and Dr. StunleJ- Charm and the staff of the Sew E:ngland En- companying paper (42). Other compounds were poor inhibitors zyme Center whcrc bacteria I\-ere grown and harvested in large (Table VII). Inhibition both with swine and with 5-d&o-4- quantities. oxo-norvaline n-as antagonized by high aspartate concentrations. Since asparagine was eluted approximately 2 min before 5-diazo- 4-oxo-norvaline, amino acid anal\-sis of the 5-diazo-4-oxo-nor- valine used did not exclude the possibility of contamination by a small amount of asparagine. The slight inhibition by all amino acids tested suggests the possibility that the a-amino group is the common inhibitor)- feature; this is strengthened by the ob- servation that XH,Cl is a coml’etitive inhibitor of aspartate (42).

DISCUSYIOX

The pathway of aspnragine biosynthesis in E. cobi nppenrs to involve only one enzyme, asparagine synthetase. Except for a transaminution reaction which results in the conversion of as- paragine to a-ketosuccinamic acid, described originally in live1 (43), asparaginc, in contrast to glutamine, has not been COII- sidered an intermediate in any biosynthetic ljathway other than the synthesis of protein. Thus we might expect that the pro- duction of the enzyme would not be under the control of an3 metabolite other than asparagine. The amount of the synthe- tase in cells grown in enriched media XV:IS the same as that in cells grown in minimal medium. *isparagine, however, both repressed the formation of the synth&se and inhibited its ac- tivity. Many other compounds were tested for inhibition and were found to be without significant effect, for esamplc the effer- tors \yhich influence the activit- of nspartoki~lase in K. coli. Substances structurally related to asparagine, including gluta- mine, were found to be poor inhibitors (Table VI).

Xlthough asparagine was found to repress the formation of the synthetase, rel)ression required relatively- high concentrations of the amino acid in the growth medium (Fig. 2), probably be- cnwe nspamgine is not rcndily taken up by I!‘. coli. These high concentrations of asparagine are likely to be greater than those the organism would be cspectcd to encounter in nature. I’ersis- tence of t,he control mechanism, however, sugge+ that it, has some physiological value. -1 possible explanation for its l’crsis- tellce is that the rcpressiou functions in response to cshanges in the collcrntration of asparagine which arise from wit.hin the organism.

-i mechanism for this enzymatic w&ion has already bee11 proposctl for synthetases from other bacterial sources (1).

Aspartate + ATI + E ti Emasparty1-ARIP + PI’; (1)

E-aspartyl-Ah4P + XII, + :tspnrn;ille + AhIP (‘4

E-aspartyl-AMP + NII,OH --f

Our results support’ this reaction pathway. 11-e have identified the products and have shown the enzyme capable of catalyzing a pyrophosphate exchange (Reaction 1) in the absence of added ammonia as well as the formation of @-aspart,- hJ-droxamate (Reaction 3). The rate of both ~\‘~I’-pSrol)hosp~late exchange and hydrosamate formation approached the rate of the over-all synthesis of asparagine. Furthermore, the exchange reaction is inhibited by ammonia, suggesting that Reaction 1 is probably the rate-limiting step in the over-all pathway.

2.

3

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Issue of August 10, 1969 H. Cedar and J. H. Schwartz 4121

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Howard Cedar and James H. SchwartzREACTION PRODUCTS

THE ENZYME, PURIFICATION, AND CHARACTERIZATION OF THE : I. BIOSYNTHETIC ROLE OFEscherichia coliThe Asparagine Synthetase of

1969, 244:4112-4121.J. Biol. Chem. 

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