THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 24, Issue of December 25, pp. 85114517, 1973

Printed in U.S.A.

Glutaminase A of Escherichia coli

SUBUNIT STRUCTURE AND COOPERATIVE BEHAVIOR*

(Received for publication, May 2, 1973)

STANDISH C. HARTMAN AND ELEANOR M. STOCHAJ

From the Department of Chemistry, Boston. Un.iversity, Boston, Massachusetts 02215

SUMMARY

Glutaminase A of Escherichia coli is a tetrameric protein consisting of subunits of molecular weight 28,000. The subunits are apparently identical as judged by the observa- tion of a single component on polyacrylamide gel electro- phoresis in the presence of sodium dodecyl sulfate, and by the stoichiometry of binding of 6-diazo-5-oxo[14C]nor- leucine (DON) to the intact enzyme (3.7 eq per molecule). A highly cooperative conformational transition accompanies dissociation of protons from the enzyme with a midpoint at about pH 5.5. This transition can be observed by its effects both upon the kinetic behavior of the enzyme (i.e. in substrate saturation curves) and upon chemical reactivity, as seen in susceptibility to photooxidation. The confor- mational change has a half-time on the order of minutes under appropriate conditions. Substrates and protons bind only to one state of the enzyme so that hyperbolic sub- strate saturation kinetics are observed below the transition pH while sigmoid curves are seen above this point. Thus, protons act as positive heterotropic effecters. At nonsat- urating levels the substrate analogue, DON, acts as a pseudo- homotropic activator. The presence of covalently bound DON in some of the protomeric subunits of the tetramer stabilize the remaining protomers in the active (protonated) state at pH values above the normal transition point. As a consequence, substrate saturation curves are shifted to hy- perbolic form and reaction of [14C]DON with the enzyme is promoted at pH 6, at which pH such reaction does not occur in the native enzyme.

Previous experiments with glutaminase A (glutamine amido- hydrolase, EC 3.5.1.2) isolated from stationary phase cells of Escherichia coli indicated that the kinetic parameter, V,,,/K,, is influenced by a highly cooperative protonic equilibrium in the free enzyme (2). Results reported in this paper show that the enzyme is a tetramer of identical subunits which exhibit homo- tropic and heterotropic cooperativity under appropriate condi- tions. In terms of a model assuming two conformational states,

*This work was supported by United States Public Health Service Grant CA 11161 from the National Cancer Institute. The preceding paper in this series is Reference 1.

substrates and protons bind only to one state, that which exists below a transition point at pH - 5.5. As a consequence, there is a precipitous decrease in the ability to bind substrate above this pH. Taking this behavior as the basis for a possible reg- ulatory role of glutaminase A, we discuss a likely function of this enzyme in the metabolic adaptation which occurs in the t,r~nsit.ion lvtwwn logarithmic and stationary phases of growth.

EXPERIMENTAL PROCEDURES

Purification of Enzyme

Glutaminase was isolated from E. coli B cells, harvested in the statiouary phase, by a modification of the previously pub- lished procedure (3). All operations are carried out at 4” unless otherwise noted.

Step 1: E&action-Five pounds of frozen cell paste are thawed and suspended in 8 liters of cold-distilled water with the aid of a Waring .Blendor, and the suspension is passed twice through a Manton-Gaulin 15M-8TA laboratory homogenizer at 8,000 p.s.i. operating pressure. The suspension is stirred for 10 min at room temperature with about 2 mg of pancreatic deoxy- ribonuclease to fragment highly polymerized DNA. Glacial acetic acid is added dropwise with thorough stirring until the pH of the suspension reaches 4.9 =t 0.1, after which the insoluble material is removed by centrifugation at 20,000 x 9 for 15 min.

Step 2: Heat Step-The extract is heated rapidly to 50” in a water bath, maintained at that temperature for 2 min, and then cooled quickly with cold water. Denatured protein is allowed to precipitate overnight in the cold room after which the super- natant phase is decanted. The residue is centrifuged briefly to recover the rest of the enzyme solution, and the combined solutions are adjusted to pH 7.4 with 10 N KOH.

Step 3: Adsorption and El&ion from DEAE-cellulose-DEAEL cellulose (Cellex D, Bio-Rad Laboratories, 0.75 meq per g) is washed with 0.1 M KOH and water several times, then adjusted to pH 7.4 with HCl. A thick slurry of the cellulose is obtained after settling and decantation. Glutaminase is adsorbed to the ion exchanger by stirring 250 ml of the thick cellulose slurry with the enzyme solution for 15 min and allowing the cellulose to settle out. If enzyme assay on the supernatant phase indi- cates significant unadsorbed activity, further small portions of the cellulose slurry are added as before. Following adsorption of the enzyme, the supernatant liquid is decanted and the DEAE- cellulose is suspended in 1 liter of a solution containing 25 mu

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potassium phosphate, pH 7.4, 1 mM EDTA, and 0.1 M NaCI. This slurry is poured into a column 5 cm in diameter, on top of a previously packed bed of DEAE-cellulose 100 ml in total volume. The solvent is passed through the column while the exchanger settles, after which the bed is washed with an addi- tional liter of the same buffer solution. When about 200 ml of liquid remain above the bed, eluting buffer (same as the above except containing 0.25 M NaCl) is added dropwise from a res- ervoir at a rate of 5 ml per min. The glutaminase appears shortly after a peak of colored protein is eluted by the 0.25 M NaCl wash, and is located by enzyme assays.

Solid ammonium sulfate is added to the pooled fractions from the DEAE-cellulose column to 55% saturation (35 g/100 ml). After stirring the suspension for 15 min the precipitate is col- lected by centrifugation, then redissolved in water to give a total volume of 25 ml.

Step 4: Second Heat Step-Acetic acid, 1 M, is cautiously added to the enzyme solution to achieve a pH of 5.0, after which it is heated to 50” for 2 min. Denatured protein is removed by centrifugation after keeping the suspension at 0” for 1 hour. The enzyme solution is concentrated to about 6 ml by vacuum ultrafiltration against 0.02 M sodium acetate buffer at pH 5.0. During concentration and subsequent storage for several days at 4” a copious precipitate of extraneous protein forms, which is discarded.

Enzyme at this stage of purification is completely stable for many weeks at 4”. Unless otherwise noted the kinetic studies reported here were performed with this fraction.

Step 6: Second DEAE-cellulose Fractionation-This separation makes use of the fact, documented later in this paper, that a cooperative protonation of about 4 residues in the glutaminase molecule occurs over a narrow pH range near pH 5.5, a process which affects significantly its net charge and ion exchange be- havior. The whole procedure is carried out at 20-25”. A suspension of DEAE-cellulose is adjusted to pH 6.0 with acetic acid and packed under gravity flow into a column to give a settled bed volume of 20 ml. The bed is washed with several volumes of 1 mM EDTA. The enzyme solution from Step 4 is adjusted to pH 6.0 by careful addition of 0.1 M NaOH and applied to the column. A buffer solution at pH 5.60 containing 0.05 M sodium acetate, 0.12 M NaCl, and 1 mu EDTA is passed through the column until the pH of the eluate is 5.60. Little or no enzyme is washed from the column by this solution. The enzyme is eluted with the same buffer adjusted to pH 5.30 with acetic acid. It appears in the fractions coincident with a drop in pH to 5.3 and is located by assay. The principal fractions are pooled and concentrated by ultrafiltration. Table I sum- marizes the purification procedure.

Determination of Subunit Molecular Weight by Sodium Dodecyl Sulfate Gel Electrophoresis

The procedure of Weber and Osborn (4) was followed using polyacrylamide gels containing 10 y0 acrylamide and 0.27 % methylenebisacrylamide. Glutaminase (Step 5), after denatura- tion with sodium dodecyl sulfate and mercaptoethanol, was subjected to electrophoresis along with marker proteins derived from catalase (mol wt = SO,OOO), ovalbumin (mol wt = 43,000), carboxypeptidase A (mol wt = 34,600), trypsinogen (mol wt = 24,500), and sperm whale myoglobin (mol wt = 17,200). Coomassie brilliant blue was used to stain the proteins. In another run, glutaminase labeled with [6-14C]DONl (1) was

1 The abbreviation used is: DON, 6-diazo-5-oxonorleucine.

TABLE I Purification of glutaminase

Five pounds of frozen E. coli were used. One enzyme unit catalyzes the hydrolysis of 1 rmole of L-glutamine per min at 25” and pH 5.0.

Purification step

1. Extract. . . . . 2. Heat step. . . . 3. DEAE-cellulose. . 4. Second heat step

and dialysis. . 5. Second DEAE-cel-

lulose

-7

Total activity

-

- uni1s mg

127,000 14,800 104,000 27,600 73,000 730

50,500

39,400

112

24

Total protein

-

--

.3

Specific activity

vnits/mg 2.8 3.8

100

450

1620

- Purifi-

-fold

1.4 36

160

580

Yield

% 100

82 57

40

31

similarly treated. After electrophoresis the gel obtained was accurately cut into l-mm slices, each of which was dissolved in 0.5 ml of 30% HsOz and assayed for 14C content by liquid scin- tillation counting in Aquasol (New England Nuclear).

Determination of Reactive Enzyme Sites with [K!]DON

These experiments were based on the fact that the reaction of glutaminase A with [6-lJC]DON yields [14C]methanol and 14C bound to enzyme in a fixed ratio (70 : 1) under all conditions tested (Reference 1 and other experiments described below). The enzyme employed (specific activity 350) containing about 220 units per ml, was dialyzed against 0.02 M sodium acetate, pH 5.0, and then against water to remove all buffering species other than the protein itself. A stock solution of [6J4C]DON, 1 mu, had a specific radioactivity of 8.3 x 106 cpm per pmole.

Control Runs at pH 6.0 and 6.0-One-tenth-milliliter aliquots of enzyme were mixed with 15 ~1 of 0.1 M sodium acetate at either pH 5.0 or 6.0 and 35 ~1 of the [i4C]DON solution were added. After reacting 30 mm at 25”, duplicate enzyme assays on aliquots of the two samples showed 0.2% remaining in the pH 5 sample and 100 f 2% remaining in the sample at pH 6. Formation of 14CH30H was determined by adding 5.00 ml of carrier CHZOH to each sample, distillation of about 2 ml of CH,OH in a microstill (b.p. 669, and counting l.OO-ml aliquots of the distillate (g of the total CHzOH) in a toluene-based liquid scintillation medium. The total amount of 14CH30H produced was 112,000 cpm in the pH 5 sample and 865 cpm in the one at pH 6. Essentially identical values were found if the original exposure to DON were continued for 6 hours. Thus, both inactivation of the enzyme and production of 14CH30H occur readily at pH 5 but virtually not at all at pH 6.

Reaction at pH 6 and 6 after Partial Inactivation by Unlabeled DON-Samples of the stock glutaminase solution, at pH 5 but with no buffer added, were treated with limiting amounts of nonlabeled DON for 30 min at 25” to yield preparations which were inhibited to different extents ranging from 10 to 38a/,. The extent of inhibition was determined in two ways: (a) by duplicate or triplicate assays for residual enzyme activity; and (6) by determining the capacity of aliquots to generate 14CH,0H from [6J4C]DON at pH 5.0 under the conditions described above. The calculated values for residual activity obtained by these two procedures were identical within 3%.

Aliquots (0.1 ml) of the partially inhibited enzymes were then treated at both pH 5.0 and 6.0 with excess [14C]DON (35 ~1) as described above. At 0, 2, 4, and 6 hours, 20-~1 samples were removed, added separately to 5 ml of CHsOH and the

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14CH30I-I content of each found after distillation. 14CH30H production had reached a maximum by 2 hours in the runs at pH 5, while 4 to 6 hours were required at pH 6 for complete reaction.

Partitioning of [6-14C]DON at pH 6-In the previous paper it was found that the relative amounts of hydrolysis of DON to CHIOH and of enzyme alkylation by DON were independent of pH in the range 4.3 to 5.5. It was important to show that this ratio does not change at pH 6. A sample of glutaminase was inhibited 35% by treatment with limiting amounts of unla- beled DON and then exposed at pH 6.0 to excess [6J4C]DON for 2 hours. Twenty-microliter aliquots were removed, placed on discs of 3MM filter paper, and the (labeled) protein pre- cipitated on the discs with 10% trichloroacetic acid solution. After exhaustive washing with 5% trichloroacetic acid and then ethanol the discs were counted in toluene-based scintillation medium to determine the 14C covalently bound to protein. r4CHs0H produced in the reaction was determined as before on the remainder of the reaction mixture. The latter value was 18,000 cpm, while that bound to protein (based on the whole reaction volume) was 323 cpm over the control lacking enzyme, for a ratio of 56:l. A similar experiment following this pro- cedure, in which the reaction was conducted at pH 5.0, yielded 112,000 cpm in CH,OH and 1970 cpm bound to protein (57:l). This agreement verifies the assumption that 14CH80H production can be used as a measure of the content of DON-binding sites in the enzyme. The superior sensitivity and lower backgrounds of the methanol assay were reasons for using this procedure. That the partitioning ratio is slightly lower than that found previously (7O:l) is probably due to the fact that the protein samples in these experiments were counted while adsorbed to paper discs, in which state the counting efficiency is somewhat reduced.

Enzyme Assays

The titrametric procedure previously described was em- ployed in all experiments (3), generally with n-glutamine as substrate in routine activity determinations, or with y-methyl glutamate as substrate in the kinetic runs at pH value greater than 5. At pH values >5.6 a constant reaction rate was often not achieved for several minutes owing to the slow conforma- tional transitions occurring upon addition of substrate. Rates were measured after this adjustment was complete.

Photooxidation of Glutaminase

Samples containing 20 units of glutaminase were diluted in 0.5 ml of 0.1 M KCl, 0.1% bovine serum albumin, and 1 mM EDTA (added to prevent nonspecific inactivation) then ad- justed to the desired pH. The solutions were transferred to IO-ml beakers, mixed with 0.05 ml of 0.05% methylene blue, and exposed to a loo-watt tungsten lamp at a distance of 10 cm. Aliquots were removed for enzyme assay at zero time and at l- to 2-min intervals over a lo-min period. Exposure under these conditions at pH values greater than 5.6 led to rapid loss of enzyme activity while in controls kept in the dark, or lacking methylene blue, the enzyme was completely stable.

Materials

DON and [B-14C]DON were prepared as previously described (5).

RESULTS

Subunit Molecular Weight-The glutaminase preparation p&Tied by electrophoresis on polyacrylamide gels showed a

Migration Distance, mm

FIG. 1 (left). Polyacrylamide gel electrophoresis of glutaminase A and its subunit. A, gel electrophoresis of enzyme at Step 5 was carried out in Tris-glycine buffer at pH 8.4. Bromphenol blue tracking dye was run just to the bottom of the gel. B, gel elec- trophoresis pattern obtained after dissociation with sodium do- decyl sulfate and mercaptoethanol, according to the procedure of Weber and Osborn (4).

FIG. 2 (right). Migration rates of glutaminase subunit in so- dium dodecyl sulfate-gel electrophoresis. Glutaminase A was subjected to electrophoresis by the method of Weber and Osborn (4), along with marker proteins of known subunit molecular weight. The gels were then stained with Coomassie brilliant blue. The markers are: 1, catalase; 2, ovalbumin; S, carboxypeptidase A; 4, trypsinogen; and 6, sperm whale myoglobin. The position of the glutaminase subunit observed by staining is marked with G, while the region in which “C-labeled glutaminase was located is marked [14C]G.

single component when rerun on analytical scale gels and stained with Coomassie brilliant blue. Further, only a single com- ponent was observed when such preparations were denatured and separated on polyacrylamide gels in the presence of sodium dodecyl sulfate (Fig. 1). That the observed polypeptide is derived from glutaminase is shown by the fact that protein- bound radioactivity from [14C]DON migrates in coincidence with this zone. A comparison of the migration rates on sodium dodecyl sulfate-polyacrylamide gels with those of markers of known molecular weight yields values of 28,500 (by protein staining) and 27,000 (by 14C content) (Fig. 2). Together with the previously determined molecular weight of the intact glu- taminase, 110,000 (3), these values are indicative of a tetrameric subunit composition.

Equivalent Weight from [14C]DON Binding-A previous deter- mination of the number of DON binding sites per enzyme mole- cule was reported (3), based upon the 14C content of protein samples precipitated with trichloroacetic acid after reaction to total inhibition with [14C]DON. A value of approximately two sites per molecule was obtained. In view of the present results that glutaminase might consist of four identical sub- units, these experiments were repeated, but under conditions avoiding the precipitation with trichloroacetic acid which might result in low values due to incomplete precipitation of protein

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TABLE II

Stoichiometry of [14C]DON binding to glutaminase

Enzyme at step 5 (2900 units, 0.0382 pmole of DON binding sites (3)) was reacted to completion with a 70-fold excess of [6- ‘4C]DON (specific radioactivity, 1.41 X 106 cpm per wale). The labeled protein was isolated on a column of Sephadex G-25 (0.9 X 20 cm) using 1% NaCl as solvent and collecting fractions of 1 ml. Radioactivity and protein content were determined in duplicate on each fraction, the latter by the method of Tombs et al. (6) in which twice crystallized, salt-free trypsinogen (Worthington) was used as a standard.

FG3Cth Protein content I’c! Content Equivalent weight

i&ml Gwml pmole/ml wdlrmole 6 252 12400 0.0088 28,600 7 625 29100 0.0206 30,300 8 151 7210 0.0054 29,600

or partial cleavage of the 14C moiety, or both. The most serious errors in the previous determination are likely to arise in con- nection with estimation of protein content, which were done with submilligram amounts of material by a micro Kjeldahl method.

Glutaminase was completely inhibited with [6-14C]DON and separated from low molecular weight radioactive products on a column of Sephadex G-25. From the 14C content of the isolated protein, shown in Table II, an average equivalent weight per DON binding site of 29,500 is obtained, a value indicating about 3.7 residues of DON bound per oligomer of mol wt = 110,000. Given the observation that the protein consists of four subunits of molecular weight about, 28,000, these results strongly suggest, that the subunits each contain a binding site and that they are therefore identical.

Kinetic Evidence for Hmotropic Cooperativity--It was pre- viously reported that the parameter, V,,,/K,, for both ester and amide substrates of glutaminase A was a steep function of pH in the range 5.3 to 5.6, with a midpoint at, about, 5.45 (2). Hill plots of the data gave slopes of 4 to 5 and were interpreted as showing that at, least four acidic groups act cooperatively to stabilize the form of the enzyme to which substrate binds. Since protons and substrate bind to the same form of free enzyme, it follows that at a sufficiently high pH (at which the enzyme exists predominantly in the unprotonated, inactive form) homo- tropic cooperativity should be observable iu the substrate sat- uration kinetics of the system. The ester substrate, y-methyl L-glutamate, was used in these experiments because it has K, values in a convenient range and because, for ester hydrolysis, the sensitivity of the pH-stat assay is good in the pH range of interest. Our previous results indicate that qualitatively similar results would be expected for amide substrates as well.

The results shown in Fig. 3 confirm the prediction that the hyperbolic kinetics observed below the transition point, (pH 5 5.5) become shifted to sigmoid curves above this point. The effect of this shift is to increase drastically the concentration of substrate required to yield a stated degree of saturation and to decrease correspondingly the velocity at any fixed concentration of substrate.

E$ect of DON Binding on Kinetics-The sigmoid saturation curves seen in Fig. 3 at pH > 5.6 are indicative of positive homotropic cooperativity in which binding of substrate to one or more of the sites in an oligomeric protein promotes binding to the remaining sites. (The action of protons as positive

FIG. 3. Substrate saturation curves as a function of pH. The linear rates of hydrolysis of r-methyl glutamate were measured by the pH-stat assay method at various concentrations of sub- strate and PH. Velocity values are expressed as a fraction of the extrapolated V max at pH 5.0, and substrate concentrations are normalized to the K,,, observed at pH 5.0,64 mM.

IOC

75

“0 ‘;

w5c 3

25

0 (

I I /

0.05 0.10 0.1 5

[T-Methyl Glutamate], M

0.20

FIG. 4. Effect of covalently bound DON upon the kinetics of hydrolysis of r-methyl glutamate. The linear rates of hydrolysis of r-methyl glutamate were measured at pH 5.80 as a function of substrate concentration for enzyme samples partially blocked with DON by reaction at pH 5.0. The symbols are: native enzyme, circles; enzyme 37.5% inactivated, squares; enzyme 66.5% inac- tivated, triangles. The velocities are normalized to the same amount of active enzyme. Data from the curve for the 66.5y0 inactivated enzyme are replotted in double reciprocal form in the inset to show the conformity with hyperbolic kinetics.

heterotropic effecters is also apparent.) To the extent that the covalent binding of the substrate analogue, DON, mimics the association of a true substrate this agent should tend to stabilize the enzyme in the “active” (or low pH) conformation, even at, high pH. Such an effect can be observed on the kinetic behavior of glutaminase in which a fraction of the total substrate sites is filled with inhibitor moieties. In the experiments depicted in Fig. 4, two samples of glutaminase were partially inhibited at, pH 5.0 with amounts of DON producing 37.5% and 66.5yo

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FIG. 5. Kinetic behavior of glutaminase 94% inactivated by DON. A sample of glutaminase was treated with DON at pH 5 to achieve a 94% decrease in active enzyme. The kinetics of hydrolysis of r-methyl glutamate by this enzyme was examined at pH 6.15 (upper line) and at pH 5.5 (lower line).

reduction of enzyme activity, respectively, as measured at pH 5.0. Velocity as a function of substrate concentration was then determined at pH 5.80, where the untreated control shows highly cooperative kinetics. It is seen that, as an increasing fraction of sites are filled with inhibitor, the kinetic behavior of the un- filled sites approaches hyperbolic form. A double reciprocal plot of the data for the sample with 66.5% filled sites (inset) shows a good fit to a straight line and yields a K, for y-methyl glutamate (67 mM) identical with that obtained for the native enzyme at pH 5 (64 mM) (3). I f it is assumed that the DON molecules, at pH 5.0, react completely randomly with the sites in a tetrameric molecule (see below), the distribution of enzyme forms in the 66.5% inhibited enzyme containing 0, 1, 2, 3, and 4 active (unblocked) protomers is calculated to be 0.01, 0.10, 0.32, 0.39, and 0.19, respectively, while the fraction of the re- maining active sites represented by each of these species is 0.04, 0.21, 0.47, 0.28, and 0. Thus, by far the greatest proportion of the remaining active sites is located in molecules bearing 1 or more DON residues, the presence of which apparently stabilizes those active sites in the correct conformation for binding.

It is interesting to note that, at pH 5.8, covalently bound inhibitor actually effects a net increase in reaction velocity at low substrate concentrations. Such an “activation by in- hibitor” is related to the effect of the reversible competitive inhibitor and aspartate analogue, succinate, in activating as- partate transcarbamylase under certain conditions (7).

When the reaction with DON (at pH 5.0) is increased to the point at which 94yo of the activity is lost, 83% of the remaining active sites are present in tetrameric species having only a single empty protomer. Such species, for which homotropic cooper- ativity is impossible, will dominate the kinetic behavior and thus hyperbolic saturation kinetics should be observed under all circumstances. The linearity of double reciprocal plots ob- tained with enzyme inhibited to this extent, even at pH 6.15 (Fig. 5), is in agreement with this expectation. Data for this enzyme preparation obtained at pH 5.5 are included for com- parison, at which pH the “normal” K,, 70 mu, is found. A much larger K,, estimated to be about 1 M, is seen at pH 6.15, a result indicating that, at a point between pH 5.8 and 6.15, the cooperative heterotropic stabilization by proton binding is lost even though the substrate sites are nearly filled by pseudo- substrate groups (DON). The biding of substrate to the remaining active site of the tetramer therefore must “force”

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TABLE III Promotion of [W]DON reaction with glutaminase at pH 6

by covaleatly bound DON

Samples of enzyme, inhibited at pH 5.0 with limiting amounts of unlabeled DON to various extents, were treated at pH 6.0 with excess [6-W]DON. The reactivity of these samples was deter- mined by measuring the [Wlmethanol produced as described un- der “Experimental Procedures.” The details of each of the as- sumed models are described in the text.

Enzyme sample Model

10% inhibited

30% inhibited

38yo inhibited

- I

I II III IV I II III IV I II III IV

Fraction of remaining active sites promoted”

Predicted

0.27 0.03 0.10 0.10 0.66 0.22 0.30 0.27 0.76 0.32 0.38 0.34

-

_-

-

Experimental

0.04 f 0.01

0.20 f 0.03

0.29 f 0.03

0 [W]Methanol produced from [14C]DON at pH 6 compared to that at pH 5.

the energetically unfavorable association of a proton, with the concomitant increase in K,. It is also seen in Fig. 5 that V,., is the same at pH 6.15 as it is at 5.5, a result in agreement with our previous conclusion that no catalytically significant proton dissociations occur in the ES complex (2).

The effect of DON in these experiments is not a result of dissociation of enzyme into subunits, since glutaminase, reacted to completion with [14C] DON, has an identical position of elution from gel filtration columns as does the untreated protein (3), as well as the same migration rate in polyacrylamide gel elec- trophoresis.

Promotion of DON Binding by DON-A more quantitative way of assessing the effect of bound DON upon the conforma- tional state of glutaminase is to use an “all-or-none” assay based upon the reaction of [14C]DON with the enzyme after a known fraction of the sites have been filled with unlabeled DON. Use is made of the fact that, under the conditions employed, native glutaminase does not react at a measurable rate with [6J4C]DON at pH 6.0 to produce labeled protein or methanol, as it does at pH 5.0. However, when the enzyme is partially inhibited with unlabeled DON at pH 5.0, some fraction of the unblocked sites now exist in enzyme conformers capable of reaction with [‘“Cl- DON even at pH 6. Exposure to excess [14C]DON for a pro- longed period (6 hours, 25’) allows all such ‘Lpromoted” sites to react.2 A quantitative measure of the 14CHs0H produced, relative to that generated by reaction at pH 5.0, gives a value for the fraction of sites promoted by the presence of bound (cold) DON.

Table III records the observed “fractions of remaining active sites promoted” by prior reaction with DON to three different degrees of inhibition, together with the value for this fraction predicted by various assumed models for the cooperative behavior

* There is no distinction implied in the term “promoted” be- tween a change induced by ligand binding and stabilization of a pre-existing state by binding (8).

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of the enzyme. The predicted values are based on the fol- lowing assumptions. (a) The two reaction modes of DON with glutaminase (alkylation of enzyme and hydrolysis to meth- anol) occur in the same ratio at pH 6 as they do at pH 5. This assumption was verified by determination of the i4C bound to protein and that converted to 14CHs0H as described under “Ex- perimental Procedures.” (b) The protomers of the native en- zyme are equivalent and independent at pH 5.0, so that the filling of sites by covalent binding of,DON occurs in completely random fashion. Since strictly Michaelis-Menten kinetics are seen with all substrates below pH 5.4 (no homotropic cooper- ativity), this seems a valid assumption.

For each of the partially inhibited samples, the predicted values of Table III were calculated from the expected statistical distribution of enzyme forms (e.g. Aa, AJ, A& AL, and I4 for a tetramer where A indicates an empty site and I, one containing covalently bound DON) and the fraction of active sites in each form, using an Adair-type expression essentially as outlined by Koshland (9). The four models considered are:

I. Four sites per oligomer; the presence of one, two, or three inhibitor moieties promotes the reaction of DON at pH 6 to the remaining sites; i.e. AJ, A&, and AI8 can all react with DON at their A subunits.

II. Four sites per oligomer; the presences of two or three in- hibitor moieties is required for promotion; i.e. only A& and AI, can react at pH 6.

III. Two sites per oligomer; the presence of bound DON at one site promotes reaction at the other.

IV. Four sites per oligomer, in which cooperativity occurs only between pairs.

Of the relatively simple models treated, II is the only one which gives agreement with experimental observations. Models which include various combinations of positive and negative cooperativity give less satisfactory agreement. More complex cases can be constructed in which the cooperative effects depend upon the geometry of subunit interactions (9), but these appear to offer no advantage over model II. In addition, models as- suming numbers of sites greater than four could accommodate the results but the determination of subunit molecular weight suggests that the number is unlikely to be greater than four. In conclusion, the presence of 2 or more DON residues in a tetrameric aggregate provides sufficient conformational sta- bilization to the remaining sites for them to be reactive at pH 6. The assumption of their being only two cooperating binding sites cannot account for the results.

Photooxidation of Glutaminase as Function of pH-Exposure of glutaminase to light in the presence of methylene blue results in rapid loss of enzyme activity if the pH of the solution is held at 5.6 or above, whereas the enzyme is stable to such exposure at pH 5. The rates of decay of activity are first order to at least 90% inactivation. Fig. 6 shows a plot of the first order rate constants for inactivation versus pH, from which it is clear that a highly cooperative transition takes place between states which are totally different in their susceptibility to photooxidation. The midpoint of the plot is at pH 5.53, a value which differs by less than 0.1 unit from that found previously for the pH de- pendence of Vm,,/Km in the catalytic reaction. It seems rea- sonable to conlude that the two states are identical with those which are active and inactive with respect to substrate binding. The Hill coefficient determined from the line in the transition region of Fig. 6 (pH 5.4 to 5.6) is estimated to be 6. Since only three experimental points were available for the determination, each with a substantial uncertainty, it does not seem warranted

a //

I ,, I

0.3 - 0

a 0

0.2 -

T .r E x-

0.1 -

PH

FIG. 6. Rates of photooxidation of glutaminase as a function of pH. The first order rates of enzyme activity loss were determined during exposure of glutaminase to photooxidation in the presence of methylene blue at various pH values.

to treat this figure as implying anything other than a high degree of cooperativity.a

Rate of Phase Transition-The results reported in this paper indicate that at pH values above 5.5 glutaminase A exists pre- dominantly in a state which is unable to bind substrate and that an increase in either substrate or proton concentration promotes a transition to the active state. This transition can be observed to occur over a period of several minutes under appropriate conditions, for example, when substrate is added to a solution of enzyme initially at a pH > 5.6, or when, in the presence of substrate, the enzyme is shifted from a pH > 6 to a point in the transition range. One such example is shown in Fig. 7 in which the rate of hydrolysis of y-methyl glutamate increases from near zero to a final constant value when this substrate is added to glutaminase at pH 5.8 in the pH-stat. The half-time for the transition under these conditions is about 1.5 min. This would appear to be an example of enzyme hysteresis as defined by Frieden (lo), representing a slow change in conformation. As the cooperative properties of the enzyme would imply, these results offer further evidence that the transition between recog- nizable states involves significant structural isomerizations and not merely rapid proton association or dissociation.

DISCUSSION

A scheme is presented in Fig. 8 which summarizes the results reported in this paper concerning the cooperative properties of glutaminase A of E. coli. The conclusion that the enzyme is tetrameric is based upon (a) the molecular weight of the single observable component after sodium dodecyl sulfate gel elec- trophoresis; (b) the stoichiometry of DON binding to the pure enzyme; (c) the Hill coefficient of about four in the relationship between V,,,/K, and pH; and (d) the statistical behavior of cooperativity between DON-binding sites. The last two points actually argue that the number of cooperating sites is at least

8 After photooxidation at pH 6, alkaline hydrolysates of the enzyme contained 4.5 residues of methionine per subunit, while controls treated at pH 5 contained 5.8 residues, essentially the same value observed with nonexposed enzyme (6.0 Met residues per subunit). It appears that the pH-dependent conformational change exposes 1 (essential) methionine residue per protomer to photooxidation.

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8517

o-------J 0123456789

Time, minutes

FIG. 7. Slow conformational adjustment upon addition of sub- strate to glutaminase at pH 5.80. T-Methyl glutamate, 0.2 M, was added to a sample of enzyme at pH 5.80 in the pH-stat. The production of glutamate (uptake of standard alkali) was followed until a constant rate was achieved. The reaction is linear from time zero when the experiment is performed at pH 5.0.

Phase Transition at pH-5.5

pH 5.0 (active) pH 5.8 (inactive)

+DON

6’)

i

High 6) +H’

FIG. 8. Scheme summarizing the cooperative conformational changes of glutaminase A.

four, Two significant conformational states are included, con- nected by a single transition in which symmetry is conserved, although there are no data available which allow a clear dis- tinction to be made between this and a sequential transition model.

The action of DON as a pseudohomotropic activator provides yet another example of the close relationship between the be- havior of this affinity label and that of true substrates. There is one significant difference, however; namely, that once bound,

DON remains covalently fixed to the enzyme regardless of con- formational state, and therefore its presence cannot be used as a conformational probe. As a consequence the observation that at least 2 molecules of bound DON must be present to promote reactions of [14C]DON with the remaining sites at pH 6 does not necessarily indicate a sequential mechanism since the form AJI, at pH 6, may exist with all protomers in the inactive (un- protonated) conformation. Apparently this is the case at pH 6.15, even with the form AI,, as judged by the increased K, for methyl glutamate found at this pH (Fig. 5).

The pronounced cooperativity observed with glutaminase in response to substrate and proton concentrations suggests a metabolic regulatory function, and adds further interest to the question of the physiological role of this enzyme. We have previously noted, as has Prusiner (11)) that the activity of the enzyme increases markedly when cultures of E. coli undergo transition from the logarithmic to stationary phases of growth, concomitantly with depletion of a good carbon source. It has been suggested that the enzyme may act to provide either NH4+ for synthesis of other nitrogenous compounds (12) or glutamic acid as a secondary carbon source (13). We have proposed (14) that glutaminase A may have a more far reaching function, namely, to hydrolyze glutamine during a period of metabolic deceleration. By depleting glutamine the biosynthesis of amino acids, nucleotides, and amino sugars dependent upon this substrate will be shut down so that the limited energy supplies available to the cell can be diverted to maintenance rather than to continued proliferation. Clearly, further studies on the induction and activation of glutaminase A in vivo will be of value, in conjunction with the molecular properties of the enzyme described here, in assessing its role in cellular metab- olism.

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248, 8506-8510 2. HARTMAN, S. C. (1968) J. Biol. Chem. 243,870-878 3. HARTMAN, S. C. (1968) J. Biol. Chem. 243,853-863 4. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-

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Standish C. Hartman and Eleanor M. StochajCOOPERATIVE BEHAVIOR

: SUBUNIT STRUCTURE ANDEscherichia coliGlutaminase A of

1973, 248:8511-8517.J. Biol. Chem. 

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