Identification of a Reactive Cysteine Residue at the Glutamine

THE JOWWAL OP EIOLOGICAL CHEMISTRY Vol. 247, No. 19, Issue Of Octo‘Fr 10, pp. 6119-6127, 1972

Printed in C.S.A.

Identification of a Reactive Cysteine Residue at the Glutamine

Binding Site of Carbamyl Phosphate Synthetase”

(Received for publication, May 2, 1972)

LAWRENCE &I. PINKUS$ AND ALTON MEISTER

From the Department of Biochemistry, Cornell University Medical College, New York, New York 10021

SUMMARY

Carbamyl phosphate synthetase from Escherichia coli, which is composed of a light and a heavy subunit, is inhibited with respect to its glutamine-dependent reactions by treatment with ~-2-amino-4-oxo-5-chloro[5-‘~C]pentanoic acid. This glutamine analog reacts with a glutamine- or albizziin- protectable site on the light subunit to yield an enzyme co- valently linked to a 4-oxo[W]norvaline moiety. The molar ratio of bound 14C to the light subunit is close to unity; some binding also occurs on the heavy subunit, but this is noninhibitory. The chloroketone-treated enzyme exhibits increased ATPase activity and a decreased apparent K,,, value for NH&l. Mg++ATP protects the enzyme significantly against chloroketone; allosteric effecters (ornithine, IMP, UMP) offer little or no protection. After reaction of the enzyme with the chloroketone, the enzyme was treated with performic acid and then subjected to acid hydrolysis; S- [‘*C]carboxymethylcysteine was isolated from such hydrolysates of the labeled enzyme and the isolated labeled light subunit. This evidence indicates that the chloroketone reacts with a sulfhydryl group at the glutamine site of the light subunit. This sulfhydryl group does not react with N- ethyhnaleimide at neutral pH which suggests that it is buried. The protective effect of Mg++ATP against chloroketone, the increase in ATPase activity on treatment with chloroketone, and the decreased apparent K, for NH&l may reflect functional intersubunit interactions associated with the binding of glutamine and ATP to the light and heavy subunits, respectively, in the native enzyme.

Carbamyl phosphate synthetase of Escherich.ia coli, which catalyzes Reaction 1 cm utilize either glutamine.

L-Glutamine + 2ATP + HO- K+, Mg++

carbamyl phosphate + 2AL)P + 2H+ + P, + L-glutamate

or ammonia as the nitrogen donor (I) Earlier studies in this laboratory showed that after the enzyme is treated with the glutamine analog, L-Z-amino-4-oxo-5.chloropentanoic acid (Chloro-

* This research was supported in part by the National Institutes of Health, United States Public Health Service.

$ Predoctoral research fellow of the National Institutes of Health, United States Public Health Service.

ketone), it can 110 longer use glutamine as a nitrogen donor, but it can still function with ammonia (2). Roth the chloroketone- treated and the untreated enzyme can catalyze the synthesis of ATP from ADP and carbamyl phosphate, but the chloroketone- treated enzyme exhibits about 3 times as much bicarbonate- dependent ATPase activity as does the untreated enzyme, and the chloroketone-treated enzyme does not catalyze the ATP- and bicarbonate-dependent hydrolysis of y-glutamylhydrox- amate. Inhibition of the glutamine-dependent reaction by chloroketone is prevented by L-glutamine and L-y-glutamylhy- droxamate. These observations led to the conclusion that the chloroketone selectively reacts with the enzyme site that nor- mally accepts glutamine and that it probably alkylates a specific nucleophilic group of the enzyme (2). Recent work in this laboratory in which the enzyme was reversibly dissociated into heavy and light subunits led to the finding that the isolated heavy subunit (mol wt 130,000) can catalyze ammonia-dependent synthesis of carbamyl phosphate, but not the glutamine-dependent synthesis reaction (3). The heavy subunit also catalyzes the bicarbonate-dependent cleavage of ATP and the synthesis of ATP from AD1 and carbamyl phosphate. The data show that the heavy subunit also has the binding sites for the allosteric effecters (UMP, ornithine, ammonia, IMP). The only enzymatic activity associated with the isolated light subunit

(mol wt 42,000) is the hydrolysis of glutamine, indicating that this subunit has a binding site for glutamine. This evidence led to the proposal that in the native enzyme glutamine binds to the light subunit and that its amide nitrogen atom is then trans- ferred to a site on the heavy subunit which contains the functional groups necessary for catalysis of the activation of carbon dioxide and the synthesis of carbamyl phosphate.

In the present work, we synthesized L-2-amino-4-oxo-5.chloro- [5-Wlpentanoic acid and studied the interaction of this compound with the enzyme. The stoichiometry of the reaction, which leads to formation of a labeled enzyme, was measured and a chemical method for modifying the enzyme-bound 4-0~0 [5-14C] norvaline moiety derived from the [Wlchloroketone was applied, which has enabled us to identify cysteine as the amino acid residue of the light subunit that reacts with the chloroketone.

EXPERIMENTAL PROCEDURE

Materials

Carbamyl phosphate synthetase was isolated from E. coli by the procedure of Anderson et (XI. (4). This preparation of the

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enzyme exhibited several small impurities when examined by SDS-gel electrophoresis; a few minor modifications were therefore made in the purification procedure which permitted isolation of a protein which was homogeneous by this criterion. The modified isolation procedure will be described in a subsequent publication from this laboratory. Glutamate dehydrogenase, pyruvate kinase, lactate dehydrogenase, and the nucleotides were obtained from Sigma Chemical Co. The amino acids and dithiothreitol were obtained from Calbiochem. Potassium thiocyanate was obtained from Baker and sodium dodecyl sulfate was obtained from Eastman. Sephadex was purchased from Pharmacia. The organic solvents were redistilled and dried prior to use.

The sulfones were prepared by performic acid oxidation of the corresponding sulfides (5). S-Carboxymethylcysteine was prepared from chloroacetic acid and n-cysteine hydrochloride (6). The l-carboxymethyl-, 3-carboxymethyl-, and 1 ,3-dicarboxy- methylhistidines were kindly provided by Dr. James M. Manning of Rockefeller University. S-Carboxymethylhomocysteine was prepared by Dr. Marian Orlowski. Thiazolidine carboxylic acid was a gift from Dr. Daniel Wellner. n-Albizziin was obtained from Aldrich.

the solution (cooled in ice) for 2 min. A spectrum of the product (chloroketone) exhibited no diazo absorption. The chloroform was removed by flash evaporation and the residual material (the protected chloroketone) was suspended in 20 ml of 6 N HCl and heated at 70-80” for 10 hours. The supernatant solution was decanted and replaced with 20 ml of 6 N HCl and the procedure was repeated until no insoluble material remained. The solutions were combined and the products generated by removal of the protecting groups (benzyl alcohol, toluene) were removed by lyophilization, and the remaining colorless moist powder was dried in Y~CUO over 1’205. The net yield of chloroketone hydrochloride was 49 mg (80%; specific activity, 1.13 Ci per mole). The chloroketone hydrochloride was recrystallized from acetone- water to give the free base.

C,H60zNCI

Calculated: C 36.3, H 4.84, N 8.45, Cl 21.5 Found : C 36X, H 4.95, N 8.38, Cl 21.4

On paper electrophoresis, the [14C]chloroketone moved toward

L-2-Amino-4-oxo-5-chloropentanoic acid (chloroketone) was prepared on a gram scale essentially as described by Khedouri et al. (2). We encountered substantial loss of product at the step of this procedure in which treatment with acid is used for removal of the protecting carbobenzoxy and benzyl ester groups. We have therefore devised a modification of this method which differs in several significant details from that previously pub- lished (2), which facilitates preparation of [5-i4C]chloroketone in amounts of about 50 mg; the procedure is described below.

the cathode at pH 5.5 and gave a single yellow spot with ninhydrin. The product emerged between proline and glycine 011

the amino acid analyzer (7). Crystalline [14C]chloroketone preparations were found to be stable at 4” for at least 1 year (stored in a vacuum desiccator over P205). No destruction of [l%]chloroketone was observed on storage of frozen dilute (0.1 to 10 mM) aqueous solutions (pH 4 to 5) at -20”.

Methods

Preparation of L-2-Amino-.&oxo-5-chloro[5-14C]pentanoic Acid ([14C]Chloroketone)-N-Benzyloxycarbonyl-L-aspartic acid-a-benzyl ester (Cycle Chemical Co., 108 mg), was dissolved in 1 ml of freshly distilled thionyl chloride (Baker) in a 50-ml round bottom flask equipped with a CaClz drying tube. The Nask contents were heated to 40” on a water bath for 30 min and the thionyl chloride was then removed by flash evaporation under high vacuum at 35”. The remaining colorless oil was dissolved in 0.2 ml of thionyl chloride and this solution was heated for 15 min at 40”. After removal of the thionyl chloride by evaporation under high vacuum, the colorless oily residue was dissolved in 1 ml of dry ethyl ether and used immediately in the reaction with [14C]diazomethane. The flask containing the ethereal solution of the acid chloride was cooled in a Dry Ice-acetone bath; then 7 ml of an ethereal solution of diazomethane (prepared from a mixture of 76 mg of (i4C)Diazald (specific activity, 4.63 Ci per mole; New England Nuclear Corp.) and 228 mg of unlabeled Diazald (Baker) final specific activity, 1.16 Ci per mole) were added rapidly and with swirling; a CaClz drying tube was attached to the flask. The flask was allowed to stand in a bucket containing Dry Ice for 24 hours in a hood, and gradually allowed to return to room temperature. After removal of the ether, the infrared spectrum (in CHCla) exhibited a sharp band at 2105 cm-’ (-(X-N=??) which was of about the same intensity as the carbonyl absorption at 1715 cm-‘. (A band at 1790 cm-l indicates formation of side product N-benzyloxycarbony-or-benzyl aspartate P-methyl ester). The diazoketone was crystal-

Determination of Enzyme Activity-All enzyme assays were carried out at 37”. Carbamyl phosphate synthetase activity was determined essentially as described previously (4). The enzyme (2 to 10 ~1) was incubated in a solution containing sodium ilTP (20 mM), magnesium chloride (20 mM), potassium chloride (100 ITIM), sodium bicarbonate (20 InM), L-glutamine (10 m&r) or ammonium chloride (300 MM), and Tris-HCl buffer (100 1rlM;

pH 8.2 (25”) ; final volume, 0.3 ml. Glutamine-dependent and ammonia-dependent carbamyl phosphate synthetase activities were determined by measuring (a) the ,4DP formed from ATP (l), (6) the [14C]urea formed when [14C]bicarbonate was added to the assay mixture (I), or (c) formation of glutamate as determined with glutamate dehydrogenase (8). Bicarbonate-dependent ATPase activity was determined by measuring the formation of ADP in a reaction mixture identical with that described above but lacking ammonium chloride and glutamine. When the formation of ADP was followed, assays were carried out in the presence and absence of glutamine and correction was made for the activated ATPase activity of the chloroketone-treated enzyme.

Glutaminase activity was determined in reaction mixtures con-

taining L-glutamine (20 mM), Tris-potassium phosphate buffer (0.1 M; pH 7.6 (37”)), and the enzyme; final volume, 0.3 ml. The formation of glutamate was determined with glutamate dehydrogenase.

General Procedure lised for Treatment of Enzyme with [‘“Cl- Chloroketone-Solutions of the enzyme in 150 m&f potassium phosphate buffer (pH 7.8) were treated with 0.01 to 0.25 volume of aqueous 10 mrvr [14C]chloroketone at 37”. The enzymatic activities were determined before and at specific time intervals after addition of chloroketone by removal of small aliquots (2 to 5 ~1) of the reaction mixture and dilution of these into the ap- propriate assay mixtures (volume, 0.3 ml). In the binding experiments the reaction mixtures containing enzyme and [‘“Cl- chloroketone were placed at 0” after incubation and worked up as

ized from dry ether. The white crystalline diazoketone was dissolved in 10 ml of

dry chloroform. Hydrogen chloride gas was bubbled slowly into

1 The abbreviation used is: SI>H, sodium dodecyl sulfate.


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described beIow. The other compounds present in some of these experiments were added just prior to addition of chloroketone at 37”. In all experiments, controls were carried out without chloroketone to establish that the enzyme activity was stable during the experimental conditions employed.

Binding Experiments-After incubation of [‘%]chloroketone with the enzyme, the solution was dialyzed at 4” against three changes of 500 volumes each of 150 mM potassium phosphate buffer (pH 7.8). Alternatively, free [i4C]chloroketone was removed by gel filtration on Sephadex G-25 or G-75; elution was carried out with 150 mM potassium phosphate buffer at 4”. The labeled enzyme was stored at 4” and where necessary, it was con- centrated by ultrafiltration on an Amicon UM-10 XM-50 mem- brane and then used immediately for the determination of protein and radioactivity. Radioactivity was determined by scintillation counting with a Nuclear Chicago counter. Aliquots (10 to 50 ~1 containing 10 to 50 pg of protein) were diluted to 1 ml with water and then counted in 10 ml of Bray’s solution (9). The specific activity of the [i4C]chloroketone was determined in each experiment by counting a [i4C]chloroketone standard; the average specific activity for the [i4C]chloroketone was 1170 cpm per nmole. Protein was determined from its absorbance at 280 nm; a solution containing 1 mg of enzyme per ml exhibits an absorbance of 0.690 (4). Protein was also determined by the procedure of Lowry et al. (10). The binding data are given here as moles per mole of enzyme monomer or subunit with a molecular weight of 170,000 for the enzyme monomer, 130,000 for the molecular weight of the heavy subunit, and 42,000 for the moIec- ular weight of the light subunit (3).

Polyacrylamide Gel Electrophoresis in SDS-The enzyme was dissociated into its subunits by incubating it in 1% SDS-l% 2-mercaptoethanol in either 100 mM Tris-acetate or 150 mM sodium phosphate buffer (pH 7.0) at 37” for 3 hours (3, 11). 2- Mercaptoethanol was not needed for dissociation of the enzyme. An aliquot of the solution (100 ~1) containing the protein, a few crystals of sucrose, and bromphenol blue dye was layered on a 6% acrylamide-bisacrylamide gel (volume, 1.2 ml). Electro- phoresis was carried out at 25” in a continuous buffer (100 mM Tris-acetate (pH 7.0) containing 0.1% SDS) for 3 to 6 hours until the dye marker emerged. A control gel containing unlabeled enzyme was stained with Coomassie blue (11) to locate the protein. The gel containing 14C-labeled enzyme was sliced into 0.5~cm sections (total gel length, 6 cm), and each section was macerated and homogenized in 0.5 ml of 0.1% SDS solution to extract protein and radioactivity. After standing for 24 hours at 25” each gel section extract was counted by scintillation with Bray’s solution.

Gel Filtration on Sephackx G-200 in I M Potassium Thio- cyan&e-The general procedure of Trotta et al. (3) was followed for separation of subunits. The enzyme was treated with 1 M

potassium thiocyanate in 150 mrvr potassium phosphate buffer (pH 7.6) containing 10 mM dithiothreitol and 10 mM EDTA. The enzyme solution (0.6 ml containing 3 to 4 mg of protein) was added at 4” to the top of a Sephadex G-200 column (23 x 1.9 cm) which was previously equilibrated with the same buffer used for dissociation. The sample was applied at a flow rate of 15 ml per hour and the column was eluted at a flow rate of 6 ml per hour; 0.5.ml fractions were collected. The appearance of protein was followed by determination of the absorbance at 280 nm and the radioactivity was monitored by scintillation counting of 50 ~1 aliquots of each fraction. The presence of heavy and light subunits in the fractions was monitored by gel electrophoresis carried out as described above. After separate pooling of

the heavy and light chain fractions, potassium thiocyanate was removed by dialysis against three changes of 500 volumes each of 150 mM potassium phosphate buffer (pH 7.8) at 4”.

Isolation of Compound X from Hydrolysates of Chloroketone- la6eled Enzyme-In a representative experiment, the enzyme labeled by incubation with [i4C]chloroketone (1.1 mg of protein; 5800 cpm) was oxidized in 3 ml of 0.2% performic acid at 0” for 3 hours (12, 13). The reaction was stopped by dilution with 40 volumes of water at 0”. Acid was removed by lyophilization and the white powder obtained was dissolved in 2 ml of 6 N HCl; hydrolysis was carried out at 108” for 24 hours in vacua. After hydrolysis, the acid was removed by flash evaporation; 4430 cpm were recovered (76%). In some cases 1 mg of carboxymethylcysteine HCI was added at t,his point as a carrier. The solution (pH 2) was then passed through a column of Dowex 50 (H+) to remove phosphate and other negatively charged compounds; the compounds bound to the column were then eluted with 3 N

ammonium hydroxide. The total radioactivity present in the ammonium hydroxide eluate was 87% of the radioactivity added to the column. The eluate was evaporated repeatedly to remove ammonia and the residue was then subjected to electrophoresis or chromatography.

Paper Electrophoresis and Paper Chromatography-Paper electrophoresis was carried out on Whatman No. 3MM paper. The studies at pH 5.5 were carried out on strips, 60 x 2.54 cm, in 40 mM sodium acetate-acetic acid buffer (pH 5.5) with a Savant apparatus, lo”, 50 volts per cm, 30 min. Paper electrophoresis at pH 3.0 was carried out on strips, 30 X 2.54 cm, with 0.58 M

acetic acid adjusted to pH 3.0 by addition of pyridine (14) with a Durrum apparatus, 25” 12.5 volts per cm, 120 min. A picric acid marker was used as an internal reference on each strip. As- cending paper chromatography was carried out on Whatman No. 3MM paper (27 X 30 cm) in 80% phenol for 12 hours. After electrophoresis or chromat,ography, the paper was dried and cut into 0.5-cm sections; each section was soaked in 1 ml of 0.3 M

ammonium hydroxide to elute the radioactivity, which was determined by scintillation counting as described above. The amino acid standards were subjected to electrophoresis or chromatography on separate strips and also on the same strip that con tained the radioactive sample; the amino acids were located with ninhydrin.

RESULTS

E$ect of Chloroketone on Carbamyl Phosphate Synthetase and ATPase Activities Exhibited by Enzyme-When the enzyme was incubated with 0.1 mM chloroketone there was a progressive activation of ATPase (Fig, 1A. Curve 1) and inactivation of glutamine-dependent carbamyl phosphate synthetase activity (Fig. lA, Curve 5). When incubation was carried out in the presence of 100 MM L-glutamine, there was no effect on ATPase activity (not shown in Fig. 1) or on carbamyl phosphate synthetase (Fig. IA, Curve S, open circles). When 20 mM Mg*ATP was included in the incubation mixture, there was less activation of ATPase (Fig. lA, Curve .2) and also less inhibition of glutamine-dependent carbamyl phosphate synthetase (Fig. IA, Curve 4). As indicated in Fig. lA, Curve S (closed circles), incubation of the enzyme with 0.1 MM chloroketone had no significant effect on the ammonia-dependent carbamyl phosphate synthetase. It is of interest that the apparent K, value for NH&l is reduced by treatment of the enzyme with chloroketone. Thus, the untreated and chloroketone-treated preparations exhibited apparent K, values at pH 8.2 for NH&l of 80 to 96 and 40 to 60 rnM,


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0’ ‘- I I , I I I / 20 40 60 00 100 120

MINUTES

O-

400 -

300 -

200 -

IOO-

O-

/

8 2 I

/ I I I, I I / ’ I / 1 / 1

0 IO 20 30 40 50 60 DISTANCE OF MIGRATION (mm)

FIG. 1 (left). Effect of chloroketone on the glutamine-dependent carbamyl phosphate synthetase, ammonia-dependent carbamyl phosphate synthetase, and ATPase activities of the enzyme. A, enzyme (0.096 mM), chloroketone (0.1 mM), and in some cases other compounds (as indicated below) were incubated at 37” in 150 mM potassium phosphate buffer (pH 7.8). Aliquots (2 to 5 ~1) of the reaction mixtures were pipetted into the appropri- ate assay mixture (volume, 0.3 ml) at various intervals after addition of chloroketone to the enzyme. The chloroketone- enzyme reaction was effectively stopped by dilution when the aliquots were added to the assay mixtures. Curve 1, ATPase; Curve 2 ATPase (ATP and Mg++, each 20 mM, were present during incubakon); Curve S, 0, glutamine-dependent synthetase (100 mM glutamine was present during incubation); l , NHB-dependent synthetase (assayed with 100 mM NH&I); Cuurve Q, glutamine- dependent synthetase (ATP and Mg++, each 20 mM, were present during incubation); Curve 5, glutamine-dependent synthetase. H, enzyme (0.006 mu) and chloroketone (2.5 mM) were incubated as in A. Curve 6, ATPase; Curve 7, NH,-dependent synthetase; Curve 8, glutamine-dependent synthetase (100 mM glutamine was present during incubation); Curve 9, glutamine-dependent synthetase.

FIG. 2 (center). Separation of the subunits of the i4C-labeled enzyme by gel electrophoresis in 0.1% SDS-100 mM Tris-acetate (pH 7.0). A, the enzyme was labeled with 0.1 mM chloroketone as described in Table I, Experiment 2. The binding of chloroketone was 1.8 moles per monomer; the enzyme was 8570 inhibited (gliitamine-dependent activity). The inhibited enzyme (130 pg)

resl)ectively.2 Chloroketone treatment in the presence of glu-

tamine did not decrease the apparent K, value for NH&l. When the enzyme was incubated with 2.5 mM chloroketone there was rapid inactivation of the glutamine-dependent carbamyl phosphate synthetase activity (Fig. lB, Curve 9)) which was not fully prevented by inclusion of 100 mM glutamine during incubation (Fig. lB, Curve 8). Under these conditions there was a slower but significant inactivation of both ATPase (Fig. IB, Curve 6) and ammonia-dependent carbamyl phosphate synthe- rise activity (Fig. lB, Curve 7).

The findings described above are in general accord with earlier observations on the effect of chloroketone on the enzyme (2). However, they also indicate that chloroketone treatment of the enzyme decreases the apparent K, value for ammonia and that

2 The observation that chloroketone treatment of the enzyme decreases the apparent Km value for ammonia in the ammonia- dependent carbamyl phosphate synthetase reaction was first made in this laboratory by Dr. Gerald A. Rosenthal.

FRACTION NUMBER

was then applied to the gel in 50 ~1 of 100 mM sodium phosphate buffer (pH 7.0), and electrophoresis was carried out for 2; hours at 4 ma per gel (volume, 1.2 ml) and 75 volts until the dye marker emerged. The total radioactivity applied to the gel was 1570 cpm; 1240 cpm (79%) were recovered from the gel. 1050 cpm were in the regions which stained for protein, i.e. those marked H and L. Distribution of protein bound radioactivity: light subunit (L), 877,; heavy subunit (H), 137,. R, the enzyme was labeled in the presence of ATP, Mg++, and HCOS- as described in Table I, Experiment 14. The binding of chloroketone was 0.9 mole per monomer; the enzyme was 55y0 inhibited. The conditions of electrophoresis were the same as in A. The total radioactivity applied to the gel was 1060 cpm; 765 cpm (72%) were recovered. Distribution of protein bound radioactivity: light subunit (L), 88%; heavy subunit (H), 1270.

FIG. 3 (right). Separation of the subunits of 1%.labeled enzyme by gel filtration. The enzyme was incubated with [14C]chloroketone as described in Table I, Experiment 2. The labeled enzyme was dissociated into subunits by incubating it in a solution containing 1 M potassium thiocyanate, 150 mM potassium phosphate buffer (pH 7.6), 10 mM dithiothreitol, and 10 mM EDTA for 10 min at 4’. The solution was then applied to a column of Sephadex G-200 (23 X 1.9 cm), and the column was eluted with the same buffer at 4”. The fractions obtained were analyzed for protein and radioactivity as described under “Methods.” The total radioactivity applied to the column was 30,000 cpm; the recovery was 17,000 cpm (577,) in fractions corresponding to the light and heavy subunits.

incubation with a relatively high concentration of chloroketone leads to a decrease in ATPase- and ammonia-dependent synthe-

tase activities as well as glutamine-dependent synthetase activity. As described below, there is evidence that the chloroketone can react with groups on the enzyme which are not

directly associated with the glutamine binding site. Although complete inactivation of the glutamine-dependent

carbamyl phosphate synthetase activity was achieved by incu-

bating the enzyme with 2.5 mM chloroketone, a small amount, i.e. 5 to 10% of the initial glutamine-dependent synthetase activity, remained after the enzyme was incubated with 0.1 mM

chloroketone. In an effort to determine whether this residual activity was due to uninhibited enzyme or whether it was a cat-

alytic property of the chloroketone-inhibited enzyme, the apparent K, value for n-glutamine for the 90% inhibited enzyme was estimated; a value of 0.38 mM was found, which is very close to that for the untreated enzyme. This observation supports

the conclusion that the residual activity observed after chloro-


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TABLE I

Injluence of various compounds on binding of chloroketone and on inhibition of glutamine-dependent carbamyl phosphate synthetase”

Compounds present during incubation T Experi- ment No. (

1 _

- pm& I

?nM

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

10 11 12 13

0.1 0.1 0.1 0.1

14 0.1

15 0.1 16 0.1 17 0.1 18 0.15 19 0.15 20 0.15 21 0.15 22 0.20 23 0.20 24 2.5 25 2.5 26 2.5 27 2.5 28 2.5

Other

Glutamine (100) Albizziin (50) Glutamate (100) Ornithine (10) Ornithine (10) + albizziin

(50) IMP (10) UMP (10) NH&l (100) ATP (l.O), Mg++ (20),

HCO,- (20) ATP (20), Mg++ (20),

HCO, (20) ATP (20), Mg++ (20) HCOs- (20)

Mg++ (20)

Glutamine (100)

Glutamine (100)

Glutamine (100) Glutamine (100) Glut,amine (100) Glutamine (100) Glutamine (100)

Incu- mtion time

min

20 90

115 1020

115 90 90 90 90

90 90 90 90

90

90 90 90 75 75

130 130

75 75 30 60

120 240

1440 -

Chloro- ketone” bound/

mOnOme*

m&s/n%011

0.9 1.8 2.1 2.7 0.35 0.44

1.4 0.3

2.0 1.8 1.9

0.9

1.8 0.4 2.8 0.7 2.0 0.9 1.1 1.8 3.7 5.7

14

Per- cent- age of initial .ctivity

% 50 15

5 0

95 100 90 18

100

8 0

10 10

45

45 10 12 10

100 4

90 4

75 65 55 30

0 0

0 The enzyme (0.006 mM), chloroketone, and other compounds (as indicated) were incubated at 37” in 150 mM potassium phosphate buffer (pH 7.8). Glutamine-dependent carbamyl phosphate synthetase activity was determined initially, and after adding chloroket,one, at the time intervals indicated, by diluting an aliquot (2 to 5 ~1) of the incubation mixture into the standard assay mixture; either the formation of ADP or of glutamate was determined. The binding of chloroketone was determined as described under “Methods.”

b Moles of chloroketone bound per mole of monomer (molecular weight, 170,000).

ketone treatment of the enzyme is due to the presence of uninhibited enzyme since it seems unlikely that the inhibited species would exhibit the same K, value for glutamine as the untreated enzyme.

Binding of [14C]Chloroketone to Enzyme-A series of experiments was carried out in which the enzyme was incubated with [‘“Cl- chloroketone; the binding of radioactivity to the enzyme and the glutamine-dependent carbamyl phosphate synthetase activity were determined (Table I). When the enzyme was incubated was low concentrations (0.1 to 0.15 mM) of [14C]chloroketone, the glutamine-dependent carbamyl phosphate synthetase activity was progressively inhibited (Table I, Experiments 1 to 4) ; in these studies the binding of chloroketone to the enzyme increased progressively reaching a value of 2.1 moles of chloroketone per

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mole of monomer after 115 min. When glutamine was included in this reaction mixture (experiment 5), there was very little loss of glutamine-dependent activity and only 0.35 mole of chloroketone was bound per mole of monomer. The glutamine analog albizziin (Experiments 6 and 9), like glutamine (Experiment 5) and glutamate (Experiment 7) also protected the enzyme against inhibition by chloroketone. The presence of the allosteric acti- vators IMP and ammonia, and the allosteric inhibitor U?tIP did not significantly influence the extent of inactivation by chloroketone or the binding of chloroketone (Experiments 10 to 12). However, there may be a slight protective effect of ornithine against binding of chloroketone (compare Experiment 2 with Experiment 8). The presence of ATP, magnesium ions, and bicarbonate (each 20 mM) afforded some protection against chloroketone after 90 min (Experiment 14) but this effect was not seen when 1 InM ATP was used (Experiment 13). Increasing ATP concentration above 20 mM did not increase protection. Although addition of ATP and magnesium ions alone protected the enzyme (Experiment 15)) it seems likely that dissolved carbon dioxide was present in the reaction mixture. It is notable that only 0.9 mole of chloroketone was bound per mole of monomer after 90 min in the presence of 20 mM ATP, magnesium ions, and bicarbonate (Experiment 14) ; under these conditions the enzyme exhibited 45% of the initial glutamine-dependent carbamyl phosphate synthetase activity. However, when the enzyme was incubated with [14C]chloroketone in the presence of 20 mM ATP, magnesium ions, and bicarbonate for 600 min, about 2 moles of chloroketone were bound per mole of monomer and the enzyme was more than 90% inhibited.

When the enzyme was incubated with 2.5 ITIM [14C]chloroketone, there was marked inhibition and substantial binding even in the presence of glutamine (Experiments 24 to 27). Incubation with 2.5 mM [14C]chloroketone in the absence of glutamine for 1440 min led to the binding of 14 moles of chloroketone per mole of monomer (Experiment 28) ; this value is close to the total number of sulfhydryl groups available for reaction with 5,5’-dithiobis- (2-nitrobenzoic acid) in the enzyme (15).

Localization of Enzyme-bound Chloroketone-After incubation of the enzyme with 0.1 mM [14C]chloroketone as described in Table I, Experiment 2, the inhibited labeled enzyme was subjected to SDS-gel electrophoresis as described in Fig. 211. Under these conditions the light subunit migrated about twice as far as did the heavy subunit and a good separation was obtained. About 87% of the proteill-bound radioactivity was associated with the light subunit. In an experiment in which the enzyme was incubated with 0.1 mM chloroketone in the presence of ATP, magnesium ions, and bicarbonate (Table I, Experiment 14), a similar distribution of radioactivity was observed, i.e. 88% of the protein-bound radioactivity was associated with the light subunit (Fig. 2B).

The heavy and light submits of the [Wlchloroketone labeled enzyme were also separated on a large scale by gel filtration on Sephadex G-200 (Fig. 3). In this experiment, the enzyme was treated as described in Table I, Experiment 2, and then dissociated into subunits by incubation in a solution containing 1 M potassium thiocyanate. The solution was then subjected to gel filtration and the fractions obtained were analyzed for protein and radioactivity. The elution diagram (Fig. 3) shows that most of the radioactivity was associated with the light subunit. In three experiments of this type in which 1.3, 1.5, and 1.9 moles of chloroketone were bound per mole of enzyme, the corresponding values for the binding of chloroketone to the isolated heavy subunit were, respectively, 0.2, 0.36, and 0.4. These


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OXIDIZED ENZYME

:

4H , 2

c=o

b COOH tOOH

i ENZYME

4

y2

YHNH,

COOH

\ 2

OXIDIZED

ENzlYME COOH X

-I- COOH

-% ;f;$j + H 14CH0 + ;H, I

A=0 CHNH,

AH, LOOH

I

YHNH2 COOH

FIG. 4. Possible pathways for the reaction of the chloroketone- treated enzyme with performic acid (see the text).

findings correlate well with the noninhibitory extraneous binding to the enzyme in the presence of glutamine and suggests that such binding is restricted to the heavy subunit. When an experiment of this type was carried out in the presence of 100 mM glutamine, no enzymatic activity was lost and the binding of chloroketone was 0.4 mole per monomer; in this experiment more than 75% of the radioactivity recovered from the Sephadex column was associated with the heavy subunit.

IdentiJcation of Cysteine as Amino Acid Residue that Reacts with Chloroketone-The findings described above indicate that the interaction of chloroketone with the enzyme leads to a cova- lent linkage; thus, neither inhibition of enzyme activity nor the binding of chloroketone is reversed at neutral pH by dilution, SDS-gel electrophoresis, dissociation with 1 M potassium thiocyanate, or gel filtration. Furthermore, when the labeled enzyme was dialyzed for 24 hours at 4” or 25”, 90% of the radioactivity was retained in protein-bound form. However, when the chloroketone-treated enzyme was subjected to acid hydrolysis (6 N HCl, lOSo, 24 hours), analysis on an automated amino acid analyzer (7) revealed the presence of a number of uniden- tified radioactive peaks which either failed to bind to the column or were eluted before aspartic acid. This suggests that the derivatives sought had been degraded during acid hydrolysis. However, the data obtained were consistent with the possibility that the chloroketone reacts with a cysteinyl residue of the enzyme. Thus, the values obtained (after performic acid oxidation) for cysteic acid were invariably lower for the chloroketone- treated enzyme than for the control. However, there was some variability in the values for several other amino acids, and we were therefore unable to establish conclusively by this approach the nature of the amino acid residue (or residues) involved in the binding of chloroketone.

In an effort to stabilize the hypothetical enzyme-bound 4-0x0- norvaline moiety, performic acid oxidation was carried out as described under “Methods.” The performic acid-treated protein was then subjected to acid hydrolysis. The hydrolysate

TABLE II

Identification of Compound X (from hydrolysate of performic acid- treated chloroketone-inhibited enzyme) by paper electrophoresis

and paper chromatography

Paper electrophoresis at pH 3.0 and 5.5 and chromatography in 80% phenol was carried out as described under “Methods.” Samples of Compound X containing between 1500 and 2400 cpm were applied to the papers; the values given in parentheses indicate the percentage recovery from the paper after chromatography or electrophoresis.

Compound

Compound X (Experiment I).

Compound X (Experiment 2)......

Compound X (Experiment 3).......................

S-Carboxymethylcysteine S-Carboxymethylcysteine

sulfone. 1,3-Dicarboxymethylhis-

tidine................... I-Carboxymethylhist,idine 3-Carboxymethylhistidine . S-Carboxymethylhomo-

cysteine Thiazolidine carboxylic

acid. Thiazolidine carboxylic

acid sulfone.

- Electrophoresis and migration

pH 5.5

cm

pH 3.0

12 (84%)

12 (91%)

12 O-l (840/;) o-1

12 6

8.5 6 6.5

11

5.5

13.5

4 o-1 o-1

1.5

o-1

7

c Chromatography in 80% phenol,

RF

0.40 (91%)

0.38

0.18

0.47

0.61

0.45

0.79

0.16

was adsorbed and eluted from Dowex 50 (H+) and then examined by paper electrophoresis and chromatography (see “illethods”). According to the pathways indicated in Fig. 4, a Baeyer-Villiger (16, 17) type of rearrangement followed by acid hydrolysis might be expected to yield a [14C]carboxymethyl amino acid and unlabeled serine (Fig. 4, Scheme 1) or a [14C]hydroxymethyl amino acid and aspartate (Fig. 4, Scheme 2). The hydroxymethyl amino acid would be expected to decompose during conditions of hydrolysis releasing the radioactivity in a volatile form ([‘“Cl- formaldehyde), but the postulated carboxymethyl amino acid would be expected to be stable. If the amino acid residue of the enzyme attached to the carbon chain of 4-oxonorvaline were a cysteinyl residue it might be expected that a S-[14C]carboxymethylcysteine derivative could be isolated.

Paper chromatography and paper electrophoresis of the material adsorbed on Dowex 50 (Hi) and eluted with ammonium hydroxide (after acid hydrolysis of the performic acid-treated labeled enzyme preparation) revealed the presence of a highly acidic radioactive compound (Compound X) Table II. Com- pound X mixed with authentic S-carboxymethylcysteine on paper chromatography and paper electrophoresis.3 Thus, the migration of the isolated radioactive product on electrophoresis at pH 5.5 and at pH 3.0 was the same as that of authentic S- carboxymethylcysteine; under these conditions of electrophoresis a number of other possible products were excluded. Thus, sig-

3 Further identification of Compound X as carboxymethylcysteine was obtained by chromatography on the automated amino acid analyzer; Compound X and authentic carboxymethylcysteine eluted together just before aspartic acid.


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TABLE III Inability of glutamine to protect enzyme against p-hydroxymercuri-

benzoate (pMB) and N-ethylmaleimide (NEM)

A solution containing the enzyme (0.006 mM), potassium phosphate buffer (pH 7.8; 150 mM), and (as indicated) glutamine, p- hydroxymercuribenzoate, N-ethylmaleimide, volume, 0.3 ml, was placed at 0”. Aliquots (2 to 10 ~1) were withdrawn 20 set later, and the solutions were then placed at 37”; aliquots were removed for glutamine-dependent carbamyl phosphate synthetase activity determinations 10 and 15 min later.

Inhibitor Glutamine (100 maa)

rnM

pMB (0.1). ......... pMB (0.1). ....... pMB (0.002). pMB (0.002). ....... NEM (0.2) ......... NEM (0.2) .........

Present

Present

Present

Percentage of initial activity

20 set

% %

0 0 0 0

66 44 76 55 78 20 77 25

10 min 15 min

%

0 0

0 0

nificantly different mobilities were observed with S-carboxymethylcysteine sulfone (at pH 3), 1,3-dicarboxymethylhistidine, 1-carboxymethylhistidine, and 3-carboxymethylhistidine (at pH 5.5), S-carboxymethylhomocysteine, thiazolidine carboxylate, and thiazolidine carboxylate sulfone. The thiazolidine derivatives could conceivably have been formed by reaction of [‘“Cl- formaldehyde with cysteine but these were not found. These observations are supported by the results obtained on paper chromatography in 80 ye phenol (Table II).

The experimental conditions employed render extremely unlikely several other possible products. For example, E-N- carboxymethyllysine would be expected to migrate toward the cathode at pH 3. Esters of glutamate or aspartate would be expected to break down during acid hydrolysis to yield neutral radioactive amino acids which would also migrate toward the cathode at pH 3. S-Carboxymethylmethionine would be expected, under the conditions of protein hydrolysis employed, to decompose to homoserine lactone (with loss of radioactivity) or to X-carboxymethylhomocysteine (excluded by the data given in Table II). Other conceivable possibilities include O-carboxy- methylserine, 0-carboxymethyltyrosine, and oc-N-carboxy- methylamino acids; however, formation of such products seems unlikely in view of the previously reported reactivity of chloro- methylketones with proteins (18) .4

4 It should be emphasized that the formation of S-carboxymethylcysteine was observed in these studies after oxidation of the labeled enzyme with 0.2yo performic acid under the conditions given. Under these conditions, virtually all of the protein-bound radioactivity remains in a nondialyzable form after-the performic acid treatment and binds to Dowex 50 (H+) after acid hvdrolvsis. We have subsequently found that when oxidation is carried” out with 2% performic acid at 4” most of the radioactivity originally bound to the enzyme is isolated not in S-carboxymethylcysteine or in the corresponding sulfone, which are stable during acid hydrolysis (in vacua), but in a neutral amino acid, only traces of which are formed when oxidation is done with 0.2% oerformic acid. The identity of this compound is not yet known:- *The formation of S-carboxymethylcysteine implies that Scheme 1 is the predomi- nant pathway when 0.2% performic acid is used. Scheme 2 may occur under other conditions of oxidation. The concentration of performic acid employed, length of oxidation, and the percentage of water in the formic acid solvent may influence the reaction. The nature of per-acid modification of the 4-oxo-norvaline moiety and other protein-bound ketones is worthy of further study.

Inability of Glutamine to Protect against Inhibition by N-Eth- ylmaleimide and p-Hydroxymercuribenzoate-N-Ethylmaleimide and p-hydroxymercuribenzoate have been reported to be potent inhibitors of carbamyl phosphate synthetase (15, 19). We therefore sought to learn whether these compounds could interact with the cysteine residue at the glutamine binding site of the enzyme. As indicated in Table III, both of these reagents inhibited the glutamine-dependent carbamyl phosphate synthetase activity of the enzyme. Substantially the same results were obtained in the presence and absence of glutamine. Thus, no significant protection by glutamine against N-ethylmaleimide or p-hydroxymercuribenzoate occurred under conditions where glutamine protects completely against inhibition by chloroketone. N-Ethylmaleimide and p-hydroxymercuribenzoate also inhibited the ammonia-dependent synthetase and ATPase activities of the enzyme to about the same extent as found for the glutamine-dependent carbamyl phosphate synthetase activity.

E$ect of Chloroketone on Glutaminase Activity of Enzyme- Treatment of carbamyl phosphate synthetase with N-ethylmaleimide leads to a dramatic (more than 100-fold) increase in the glutaminase activity of the enzyme, but all of the other activities catalyzed by the enzyme are inhibited (20). The enhanced glutaminase activity of the N-ethylmaleimide-treated enzyme is markedly inhibited by chloroketone. Such inhibition is partially prevented by the presence of 20 mM L-albizziin, a glutamine analog that is not hydrolyzed by the enzyme. Thus, when the N-ethylmaleimide-treated enzyme was incubated in 0.1 M

Tris-phosphate buffer (pH 7.6) containing 0.2 mM chloroketone at 37”, 63, 37, 8, and O%, of the initial glutaminase activity remained after 5, 15, 30, and 60 min, respectively. When this experiment was carried out in the presence of 20 InM n-albizziin the corresponding values were 89, 78, 62, and 52%.

The binding of [14C]chloroketone to the N-ethylmaleimide- enzyme was determined as follows. A solution (volume, 0.7 ml) containing the enzyme (8.25 nmoles), EDTA (0.5 mM), Tris- phosphate buffer (pH 7.6; 0.1 M), and N-ethylmaleimide (50 mnf) was placed at 37” for 20 min and then dialyzed against 1000 volumes of the same buffer at 4” to remove excess N-ethylmaleimide. An aliquot (300 ~1) of the dialyzed solution was incubated with 0.16 mM [14C]chloroketone for 24 hours at 25”; after incubation the preparation did not exhibit glutaminase activity. It was then dialyzed against 2000 volumes of the same buffer at 4” to remove free [i4C]chloroketone. Determinations of radioactivity and protein in three separate experiments indicated binding of 0.80, 0.88, and 1.1 mole of chloroketone per enzyme monomer. SDS-gel electrophoresis was carried out as described in Fig. 2; 94% of the recovered radioactivity was associated with the light subunit and the remainder with the heavy subunit.

When N-ethylmaleimide-enzyme was inactivated by dialysis against 100 mM sodium acetate buffer (pH 4.0), it then bound only 0.08 mole of chloroketone under the conditions stated above. It seems probable that treatment of the enzyme with N-ethylmaleimide blocks enzyme groups responsible for extraneous binding of the chloroketone; this suggests that the binding of chloroketone to the active N-ethylmaleimide enzyme takes place almost exclusively at the glutamine binding site of the light subunit.

DISCUSSION

The present work shows that close to 1 mole of 4-oxonorvaline becomes attached to the light subunit of the enzyme after it is treated with chloroketone and that such binding is prevented by glutamine or albizziin. The data also show that virtually all


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of the material bound at the site protected by glutamine binds to a cysteine residue. Although reaction of the enzyme with chloroketone leads to some binding to the heavy subunit and perhaps slight additional binding to sites on the light subunit not protected by glutamine, this seems to reflect the ability of the chloroketone to function as a general alkylating agent similar to (but less reactive than) iodoacetate. The binding to the heavy subunit which occurs in the presence of glutamine is 0111~

associated with inhibition of the catalytic properties of the enzyme when several moles of chloroketone are bound (at high chloroketone concentrations). Such inhibition seems to be gen- eralized in that it affects several activities of the enzyme and is probably associated with binding of chloroketone at a number of enzyme sites. When the enzyme is incubated with a low (0.1 mM)

concentration of chloroketone the glutamine binding site is labeled under conditions in which there is relatively little extraneous labeling of the enzyme.

The present findings and the previous observation that the isolated light subunit exhibits glutaminase activity and that it confers ability to utilize glutamine when added to the isolated heavy subunit, are in accord with the conclusion that the glutamine and ammonia binding sites are located on different subunits of the enzyme. However, it is evident that association of the two subunits leads to very significant functional inter-rela- tionships. For example, treatment of the native enzyme with chloroketone produces a very substantial increase in the bicarbonate-dependent ATPase activity; this activity is known to be catalyzed by the isolated heavy subunit and not by the isolated light subunit. In addition, the present studies show that the chloroketone-inhibited enzyme exhibits a decreased apparent K, value for ammonium chloride as compared to the untreated native enzyme or the fully active enzyme which has been labeled on the heavy subunit by treatment with chloroketone in the presence of glutamine. This suggests that interaction of the glutamine analog with the glutamine binding site 011 the light subunit effects substantial changes in the functional efficiency of the heavy subunit. I f these effects occur in the native enzyme, it might be supposed that glutamine, in binding to the light subunit, would increase its own rate of utilization by facilitating the formation or utilization of activated carbon dioxide. In addition, binding of glutamine to the light subunit in some manner may increase the affinity of the heavy subunit for ammonia trans- ferred to it from the light subunit.

phate amidotransferase (34), and CTP synthetase (31, 35). That an enzyme sulfhydryl group reacts with a glutamine analog is consistent with the finding of a decrease in the number of sulfhydryl groups available for reaction with 5,5’-dithiobis(2- nitrobenzoate) and other sulfhydryl reagents after treatment with the analog. However, were the reactive sulfhydryl group buried and thus unavailable for reaction with 5,5’-dithiobis(2- nitrobenzoate) it would not be detected in this way. This ap- pears to be the case for anthranilate synthetase (32, 33). That the sulfhydryl group of carbamyl phosphate synthetase which reacts with chloroketone is buried, is evident from the present studies on N-ethvlmaleimide treatment of the enzyme. After extensive treatment of the native enzyme with N-ethylmaleimide about two more sulfhydryl groups may then be reacted when the enzyme is unfolded in 4 M urea.5 Since N-ethylmaleimide does not inhibit the glutaminase activity and fails to react with the chloroketone-reactive sulfhydryl group in the native enzyme this group may be considered to be buried.

Definitive evidence for the reaction of a glutamine analog with a sulfhydryl group in a glutamine binding site has come from studies on 2-formamido-A-ribosylacetamide 5’.phosphate : L-glutamine amido-ligase (36, 37). Treatment of this enzyme with [Wlazaserine followed by reduction with sodium borohy- dride and enzymatic hydrolysis of the enzyme led to isolation of a peptide containing S-[Wlcarboxymethylcysteine (36). A cysteine residue has also been identified in the active site of guinea pig liver transglutaminase (38). The available data, which indicate that an enzyme sulfhydryl group is intimately involved in the utilization of the amide nitrogen of glutamine by several enzymes, suggest that this functional group may be of general significance in the mechanism of action of the glutamine amido transferases.

REFERENCES

1. ANDERSON, P. M., AND MIGISTER, A. (1966) Biochemistry 6, 3157

2. KHICDOURI, Ii:., ANDERSON, P. M., AND MI~ISWX, A. (1966) Biochemistry 6, 3552

3. TROTT~L, P., HXXHEMEYER, 1~. H., AND MEISTEH, A. (1971) Proc. Nat. Acad. Sri. U. S. A. 68, 2599

4. ANDXRSON, P. M., WELLNTEIC, V. P., ROSENTHAL, G. A., AND MEISTEI~. A. (1970) Methods Enzumol. 17A, 235

5. FINKLE, B.‘J., AND S&H, E. L. (1958) J. Biol. Chem. 230, 669 6. MICHAELIS, L., AND SCHUBERT, M. P. (1934) J. Biol. Chem. 106,

331 The ability of ATP and magnesium ions to partially protect

the native enzyme against inhibition by the chloroketone suggests an additional interaction between the ATP binding sites 0x1 the heavy subunit and the glutamine binding site on the light subunit. ATP may have an allosteric as well as a substrate function in the action of the enzyme; thus, this protective effect ~nay possibly be of a direct steric nature or it may be allosteric.

.I number of enzymes have previously been inhibited through u*e of halomethylketone substrate analogs derived by modification of a substrate carboxyl group. For example, studies have been reported on chymotrypsin (21-23) and trypsin (24, 25), in which a histidine residue was labeled, and papain (26) in which a cysteine residue was labeled.

7. Moom, S., AND STEIN, W. H. (1963) Methods Enzymol. 6, 819 8. BERNT,’ E:, AND BERGMEYER, H. U. (1963) in Methods of

Enzvmatic Analusis (BERGMEYER. H. U.. ed) PD. 384-388, Academic Press,“New: York --

9. BRAY, G. A. (1960) Anal. Biochem. 1, 279 10. LOI~RY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,

R J. (1951) J. Biol. Chem. 193, 265-275 11. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V., JR. (1967)

Biochem. Biophys. Res. Commun. 28, 815 12. SHAW, F,. (1967) Methods Enzymol. 11, 683 13. HIRS, C. H. W. (1956) J. Biol. Chem. 227, 611 14. HEXTER, C. S., AND WESTHISIMER, F. H. (1971) J. Biol. Chem.

246, 3934 15. FOLEY, R., POON, J., AND ANDERSON, P.M. (1971) Biochemistry

10, 4562

The glutamine analogs, L-6-diazo-5-oxonorleucine and L-aza- serine, have been used as inhibitors of several glutamine amido- transferases (27-31). Evidence, based on sulfhydryl group titrations before and after reaction with the analog indicate that

1G. BAEYER, A., AND VILLIGEX, V. (1899) Ber. 32, 3625 17. HASSALL. C. H. (1967) Org. React. 9, 73 18. SHAW, E: (1970) Physiol. Rev. 60, 244 19. ANDERSON, P. M., MATTHEWS, S. L., AND FOLEY, R. E. (1970)

Fed. Proc. 29, 400

there is probably a functional sulfhydryl group in the active site 5 V. P. Wellner, personal communication; unpublished work of anthranilate synthetase (32, 33)) phosphoribosyl pyrophos- carried out in this laboratory.


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20. PINKUS, L. M., WELLI\‘F:R, V. P., AND MEISTER, A. (1972) Fed. Proc. 31, 1459

21. SCHOELLMANN, G., AND SHAH, E. (1963) Biochemistry 2, 252 22. STEVENSON, K. J., AND SMILLIE, L. B. (1965) J. Mol. Biol. 12,

937-941 23. ONG, E. B., SHAW, E., AND SCHOELLMANN, G. (1965) J. Biol.

Chem. 240, 694-698 24. PETRA, P. H., COHEN, W., AND SHAW, E. N. (1965) Biochem.

Biophys. Res. Commun. 21, 612 25. SHAW, E., AND GLOVER, G. (1970) Arch. Biochem. Biophys. 139,

298 26. HUSSAIN, S. S., AND LOWE, G. (1965) Chem. Commun. 16, 345 27. MEISTER; A. (i962) in The’Enzymes,.vol. 6, 2nd Ed, p. 247 28. PREISS. J.. AND HANDLER. P. 11958) J. Biol. Chem. 233. 493 29. TAMIR,‘H.‘, AND SRINIVAS~N, P. R.‘(1969) J. Biol. Chek. 244,

6507-6513

30. HARTMAN, S. C. (1968) J. Biol. Chem. 243, 853-863 31. LONG, C. W., LEVITSKI, A., AND KOSHLAND, D. E., Ja. (1970)

J. biol. Chem. 246, 86-87. 32. NAGANO. H.. ZALKIN. H.. AND HENDERSON. E. J. (19701 J.

Biol. dhed. 246, 38iO-38iO ~

33. HWANG, L. H., AND ZALIUN, H. (1971) J. Biol. Chem. 246, 2338-2345

34. HARTMAN, S. C. (1963) J. Biol. Chem. 238, 303G-3047 35. LEVITZICI, A., STALLCUP, W. B., AND KOSHLAND, I). E., JR.

(1971) Biochemistry 10, 3371 36. DAWID, I. B., FRENCH, T. C., AND BUCHANAN, J. M. (1963)

J. Biol. Chem. 238, 2178-2185 37. LI, H.-C., AND BUCHANAN, J. M. (1971) J. Biol. Chem. 246,

4713-4719 38. FOLIO J. E., AND COLE, P. W. (1966) J. Biol. Chem. 241, 551%

5525


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Lawrence M. Pinkus and Alton MeisterCarbamyl Phosphate Synthetase

Identification of a Reactive Cysteine Residue at the Glutamine Binding Site of

1972, 247:6119-6127.J. Biol. Chem.

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Identification of a Reactive Cysteine Residue at the Glutamine