Mutaminase of Escherichia coli

I. PURIFICATIOX- AKIl GENERAL CATALYTIC PROPERTIES*

STANDISH C. HAH’IIIAS~

(J<eceivetl for publication, August 3, 1967)

From the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115

SUMMARY

A glutaminase has been isolated from Escherichia coli in apparently homogeneous form. Two active sites per mole- cule are revealed by titration of the active sites with the specific analogue inhibitor, 6J4C-diazo-5-oxo-L-norleucine, and estimation of molecular weight by gel filtration (mol wt 110,000). The pure enzyme has a specific activity, at pH 5.0 and 25”, of 1,520 pmoles per min per mg of protein, or a turnover number (k& per active site of 1,265 set-1 when glutamine is the substrate. KM and &,,t values are reported for the known substrates, which include amides, esters, and thioesters of glutamic acid. From studies with a large number of structurally related compounds it is concluded that the enzyme will bind only substances having an un- substituted L-glutamyl acyl portion and a substituent in the y position no larger than 2-hydroxyethylamino or 2,2,2- trtiuoroethoxy, and will hydrolyze only those in which the substituent is no larger than methylamino or ethoxy. Trans- fer of the acyl moiety to hydroxylamine or methanol occurs, but at a relatively low rate. It is concluded that the enzyme has a “water site.” Heavy metals inhibit the enzyme, but iodoacetate, iodoacetamide, N-ethylmaleimide, diisopropyl phosphorofluoridate, ethylenediaminetetraacetate, o-phe- nanthroline, and hydrogen peroxide do not. These observa- tions suggest that this enzyme is not related to other well known enzymes with similar catalytic action, such as the serine esterases, thiol acylases, or metal-dependent acylases.

A number of enzymes which catalyze the transfer of the amide nitrogen of glutamine in various biosynthetic reactions have been under investigation in several laboratories (refer- ences cited in Reference 1; 2-5). These enzymes appear to have certain similarities of mechanism and, in addition, are generally susceptible to irreversible inhibition by naturally

*This investigation was supported by IJnited States Public Health Service (irant GM oti591 from the National Institute of General Medical Sciences.

$ Career I)evelopment Awardee, United States Public Health Service.

occurring substrate analogues, O-diazoacetyl-L-serine and 6- diazo-5-oxo-L-norleucine. The latter property is of considerable interest s it not only affords a means of modifying and labeling a group at the substrate-binding site of these enzymes but also may provide insight into aspects of the catalytic process (6). These enzymes may be viewed s effecting (a) the hydrolytic cleavage of glutamine t.o glutamic acid and (b) the transfer of the NH2 group t.o a suitable acceptor. In fact, the simplest reaction of this sort is the hydrolysis of glutaminc, if we regard the proton as the acceptor of the XHz group. We have begun the study of the mechanism of action of a glutaminase (cgluta- mine amidohydrolase, EC 3.5.1.2) as a model for the more complex biosynthetic enzymes of this group with the hope that this work will aid in bridging the gap bet.ween such complex systems aud the well studied hydrolytic amidaye-esterase cn- zymes. A glutaminase from Escherichia coli, reported upon previously by Meister et al. (7), appeared to have several proper- ties of the type we sought, and it was found to be inhibited by covalent reaction with W-6-diazo-5-oxo-cnorlcucine. In this paper we describe the purification and assay of the enzyme, certain of its physical properties, and the kinetic parameters of its known substrates and inhibitors.

EXPERlMEWl-AL PROCEDURE

Purification oj Enzyme

Glutaminase activity (assayed as described below) appears intrarellularly in cultures of I:‘. coli B grown into the stationary phase. Very little enzyme is present in log phase cells. The total activity and the specific activity are essentially independent of the growth medium used, so that the only consideration w&s the yield of cells. Large srale preparations of E. coli U were grown by Grain Processing Corporation, hluscatine, Iowa, in “enriched medium” containing peptone, yeast estract, glucose, and Falts. Cultures were grown at 37” for 16 hours with aera- tion. The cells were shipped &s a frozen paste and stored at -3O”, at which temperature they lost little activity even after 1

year. Purification procedures were carried out at 4”’ unless otherwise noted. Deionized, distilled water was used in all solutions.

E&action--;iny convenient. method for disruption of the cells, such as sonic oscillation or homogenizing with gla.qs beads, may be used. The .\Ianton-Gaulin homogenizer was most

853

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854 Glutaminase of Escherichia coli. I \‘ol. 243, so. 5

convenient for large scale isolation. Ten pounds of frozen cell paste were thawed and suspended 1vit.h the aid of a blender in 8 liters of 0.025 M Tris-chloride, pH 8.0, containing 1 rnlr EDT.1. The cells were ruptured in a Ivlanton-Gaulin laboratory homoge- nizer, model 1511~8T.4 (Manton-Gaulin Manufacturing Com- pany, Everett, hiaqsachusetts). A single passage at. an operat- ing pressure of 8000 psi. released essentially all of the enzyme. A few milligrams of crystalline pancreatic deoxyribonuclease were added t.o t.he homogenate, and the very viscous mixture ~~1s stirred for a few minutes at room temperature until liquc- fact.ion occurred. WYth t.horough stirring, the suspension was carefully brought to pH 4.8 to 4.9 with acetic acid, after which it waq centrifuged in the 3 RA rotor of the Lourdes rent.rifuge at top speed for 30 min. Residual suspended material wan removed from the aupernat.ant, solution by suction filtration through a bed of cellulose powder. The solution was adjusted to pH 7.2 with 10 x SaOH.

Chromatugraphy upon L)B:lB-cell&se-The enzyme solution ws passed t.hrough a column, 5 x 30 cm, of DEAE-cellulose (Cellcx-D, 0.8 meq per g, Bio-Rad Laboratories, Richmond, California) which had been previously equilibrated with 0.025 M pot.a&um phosphate, pH 7.4, containing 1 rnlr EDTA. The column was washed with 2 liters of a solut.ion containing 0.025 M potassium phosphate, pH 7.4, 0.1 M NaCI, and 1 rnM EDTA, then with a solution of similar composition but cont.aining 0.25 M SaCl. The second wash was collected in fractions of 20 ml at a flow rate of 3 to 4 ml per min. The enzyme emerged after an initial dark colored component had been cluted with the 0.25 M NaCl solution, and it.s position was determined by assay of 20-pl aliquots of the fractions.

Ammonium Sulfnte Precipitation-The pooled fractions con- taining the glutaminasc (about 200 ml) were fractionated with solid ammonium su1fat.c bet.ween 357, saturation (20.9 g/100 ml) and 507; saturation (9.4 g/100 ml additional). The precipitate obt.ained after the second treatment with ammonium sulfate was dissolved in t.he minimum amount (about 5 ml) of 0.05 M

potassium phosphate, pH 7.4, containing 1 rnM EDTA. b’ractzonation upon Sephadez-The sample w\‘&s applied to a

column, 3 x !)5 cm, of Sephadcx G-100 (Pharmacia) previously equilibrated with 0.05 M potassium phosphate, pH 7.4, cont.ain- ing 1 mM EDTA, and the column was developed with the same buffer. .I flow rate of 0.5 ml per min was maintained, fractions of 5 ml being collected. The enzyme peak was centered at 330 ml, the bulk of the activity being found in about 30 ml.

Heat Step-The solution from the Scphadex column was

TARLE I Purificalion of glulaminase

Ten pounds of cell paste were used in this preparjltion. One unit of enzyme catalyzes the hydrolysis of glutamine (at satura- tion) at n rate of 1 pmolc per min at 25” and pII 5.0. -_ .-. .- -

I~raction TOhI

I I T& le:j!C$ 1 “‘:iii 1 Yield activity protem

4. Scphndes, .’ 45,200 5. Heated, dialyzed.. 31,%0 36.4 866 412 6. Elcctrophoresis.. 7,550 4.90,1,540 730

.- -~ ._-.. -̂

.___ %I

100 77 70 58 40 10

-.-

adjusted to pH 5.0 with 1 M acetic acid and hcatcd in a water bat.h at. 50” for 2 min. The suspension was held at 0” for 2 hours, and the precipitated protein was rcmovcd by centrifuga- tion. Concentration of the enzyme solution was achieved by addition of solid ammonium sulfate to 60% saturation (39 g/100 ml), centrifugat.ion, and solution of t.he rrsulting prcc:ipit.ate in 2 ml of 2 111~ potassium succinatc buffer, pH 5.0. Dialysis against the same buffer at 4’ for 24 hours resulted in IJrecipita- tion of considerable am0unt.s of inact.ive protein with little loss of enzyme activity.

The enzyme solution at this stage would withstand storage at -20” and repeated freezing and thawing without losing activity.

.4nalysis by polgacrylamide gel electrophoresis at. pH 5.5 usually showed one major and t.wo minor components after staining with Napht.hol Blue Black dye. Specific activity determination indicated a purity of 50 to 750/;, in Several pX?~~arathllS. A’IOSt. Of the kinetic studies to be described were performed with material at this stage of purity.

Zone Electrophoresti- .The enzyme could be fulther purified by zone electrophoresis on a modified Porath column (8) packed with formalatecl cellulose (Ccllex XF-I, Bio-Rad Laboratories). The column, 2 x 30 cm, wm equilibrat.ed and operated with 2.0 mzr potassium succinatc buffer, pH 5.5, cont.aining 0.2 rnhl EDTA. After application of the sample, clectrophorcsis was carried out for 8 hours at 10 volts per cm, with cooling water in t.he jacket maintained at 6”. Sequential fractions of 2 ml each were then collect.ed from the column by elution with the same buffer. The enzyme migrated about half-way down the column (toward the anode) under these conditions and appeared in about 6 to 8 ml. Concrnt.ration of this solution to 0.5 1111 was accomplished by dialysis under vacuum. This elcctrnphorctic separation resulted in an improvement in specific activity of about 1.5- to 2-fold, wit.h a recovery of about 25yc. III several such runs, the analysis of the product. (50 pg of protein) by polyacrylamide gel electrophorcsis (at pI1 5.5 or 8.0) always showed only one visible band after staining with Naphthol Blue Black. From the results of the analytical gel runs and the constancy of the specific activit.y of the product, WC assume that the cnzvmr at this stage is probably at least 90% pure. Protein determinations were performed by the biuret method and by a micro-Kjeldahl met.hod. The purification procedure is sum- marizcd in Table I. It, should be noted that the initial acid precipitation results in removal of about ‘i5’2 of the total soluble protein, so t.hat purification factors based upon the total soluble protein would he about 4-fold higher than those given.

.I1 ethods of Assay

Titrimetric-The enzymatic hydrolysis of glutamic acid derivatives was usually followed by a titrimctric (pH-stat) assay which is based upon the fact that protons arc generated or con-

sumed dcpcnding upon thr nature of the substrate, as shown in Reaction 1.

gllltalllyl-X + II?0 + glutnmntr + XII,+

glutnmic acid xii

The rate of hydrolysis of the substrate, lil, is related to the rate of addition of standard titrating agent required to maint.ain constant pH, Ii+, by Equation 2.

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~=k- 1 1

1 I l+g)

-l:(Hi) KX

Here (H+) is the hydrogen ion activity of the system, K’o the dissociation constant for the y-carboxyl group of glutamic acid, and K’, the dissociation constant of the conjugate acid of the product, XH. A posit.ive value for H+ indicates that the addi- tion of acid is required to maintain constant pH, while a negative value indicates that alkali must be used as the t.it.rating agent. Values for the dissociation const.ants were determined by the method of half-neutralization at the temperature and ionic strengt.h of the reaction. For the compomlds of chief interest, at 25” and ionic strength 0.2, pK’, was found to be 4.31, PH’~ for hydroxylammonium ion is 6.10, and I>K’~ for methoxyam- monium ion is 4.77. In the pH range of activity of the enzyme (3 to 5.8), t.he second term in the denominator of Equation 2 could be taken &$ zero for derivatives of amines with high pK (such as ammonia), and as unity for derivatives of alcohols and mercaptans.

In the standard determination of enzyme activity, the reaction was carried out in the thermostated microcaell of the Radiometer TTTl pH-stat. The instrument was calibrated against standard buffers at pH values 7.00 and 4.01. The vessel contained glutamine, 30 mM; KU, 0.2 AC; EDTA, 0.2 mhr; and bovine serum albumin, 0.057,. After the solution was adjusted to the desired pH, usually 5.00, the volume was brought to 1.00 ml with water and t.he enzyme was added from a lo- nr 20-~1 pipette. Constant pH was maintained by the automat.ic addition of st.andard IICl, usually 5.00 rnhf, from the 0.5~ml syringe. An amount of enzyme was added to give a rate of between 27, and 15y0 of full scale per min, over which range the rate was linearly related to the enzyme concentration. The react.ion started without lag and proceeded linearly until at least 505$, of t.he contents of the syringe had been delivered. If the serum albu- min wa< omitted, t.he rate of reaction dropped off progressively, probably as a resuli. of surface inact.ivation of the enzyme at the very low protein concentrations present. Inclusion of 10% glycerol by volume in the mixture rather than serum albumin was also effective in maintaining a linear rate. The hydrolysis of other substrates was followed in the same manner, except that standard KOH WL~ used as the tit.rating agent when neces- sary.

One unit of enzyme is defined as that amount which catalyzes the hydrolysis of 1 ~mole of L-glutamine per min at 25.0” and pH 5.0 under the conditions of the above assay. In certain kinetic ,runs at low substrate concentrat.inns, the linear part of thr leacation was rather brief owing to dcl)lrtinn of the substrate and dilution by the titrating agent. In general, a satisfactory cstimat.inn of the initial rate could be obtained in the first minute or so of the reaction.

Formation of y-Methyl Glutamate from W-Clutamic ‘lcid- IJnifnrmly labeled “C-L-glutamic acid (0.2 M, 3250 cpm per pmole) was incubated with methanol (27.8:‘; by volume), 0.2 11 KC1 0.2 mM E;DTA 0.05’;, ’ bovinr serum albumin, and 28.3 unit: of glutaminase,‘in a total volume of 1.8 ml at 25” and pH 5.0. Aliquots ww removed at 5-min intervals, diluted to 5 ml with an aqueous solution containing 50 mg of carrier y-methyl glutamate, and immediately adjusted to pH 7.0 with dilute I\OII. Thr samplrs wcrc individually l)a?;srd through columns,

1 x 10 cm, of Dowex l-acetate to remove the residual labeled glutamic acid. The first 4 ml of eluent were discarded; the next 7 ml were collected. Crystallizat.ion of the methyl glutamate from these fractions was achieved by addition of 3 volumes of acetone and storage at -20”. About 20 mg of product were recovered from each sample after filtration and drying, first with acetone, then in a vacuum desiccator over P,05. “C content was determined by dissolving the weighed methyl glutamate samples in 0.5 ml of Ilyamine 10X (Packard Instrument Cnm- pany) and counting in 10 ml of toluene containing 0.4% 2,5- diphenyloxazole and 0.005% 1,4-bis[2-(5-phenyloxazolyl)]ben- zene in a liquid scintillation counter. The total amount of “C-methyl glutamattt in each aliquot could then be calculated from the fraction of the original 50 mg of carrier recovered.

Hydrozamic :icid I+‘ormation-The rates of hydrnxamic acid formation from various glutamyl derivatives and hydroxylamine were determined by incubating the substrate with 0.2 INY EIITA, 0.05% bovine serum albumin, enzyme, and hydrnxylamine hydrochloride, adjusted to the desired pH, at 25”. KC1 was added to adjust the ionic strength to 0.2 M or, in some expcri- ments at higher hydroxylamine hydrochloride concentration, to 0.5 M. The reaction (total volume, 3 ml) was carried out in the t.hermostatcd cell of the pa-stat with relatively concentrated titrating agent (0.1 M) being used to maintain the ~1-1 without changing the volume significantly. Four 0.5~ml aliqunts were removed at intervals (usually 1 min) and added to 0.5 ml of 5yo FeC13.61-120 in either 0.2 M HCI or 0.5 M HCI (the higher con-

centration of acid was used when the higher level of hydroxyla- mine was present). The opt.ical densities of the samples were measured at 500 rnp and the amounts of hydrnsamic acid were determined by reference to a standard of synt.hetic y-glutamyl hydrnxamic acid (9).

Materi4zLs

The following compounds were synthesized by reported methods: L-glutamic y-methylamide (IO), L-glutamic y-ethyl- amide (11)) L-glutamic y-2-t~yclrox~ethylan~idc (12)) L-glutamic y-anilide (13), L-glutamic y-p-nitroanilide (14), L-glutamyl hydroxamic acid (9), A;-acetyl-L-glutamine (15), glutaramic acid (16), S-carbamoxl-L-cysteine (17), 0-acotyl-L-serine (18), nL-2-amino-4-iodobutyric acid (19), 6-diazn-5-oxo-L-nnrlcucine

(20) , and 6-W-6-diazo-5-nxo-L-norleucine (6). L-Glutamic diamidc (21) was converted to the hydrobromidc salt, m.p. 189-l 90”, from ethauol. Dr. Orrie Friedmau generously prn- vided sample3 of r,-glutamic y-2 chlnroethylamide, ncglutamic y-n-butylamide, and nL-glutamic y-dimcthylamide. SCM syntheses of y-2,2,2-trifluoroethyl L-glutamate, r.-2-amino+ cyanobutgric acid, L-glutamic y-methyoxyamide, y-thiomethyl L-glutamate, and y-thinrthyl L-glutamate are reported.

cu-Henzyl y-2,d,2-TriJluoroethyl iV-Carbobenzozy-L-glutamate- cr-Benzyl N-carbobenznxy-L-glutamic acid (3.70 g) (22) was dissolved in 20 ml of dry, freshly distilled diosane containing 2.40 ml of dry, distilled tributylamine. The solution was mixed at 5”, and 1.00 ml of ethyl chloroformate was added. After 30 min at this trmperuturc, 8 ml of 2.2,2-trifluoroethanol in 10 ml of cold dioxane were added together with an additional 2.4 ml of t.ributylaminr. The solution was protected from mnist.ure and stirred at ronm temperature for 2 days, after which it. was poured into 100 ml of ethyl acet.ate. The ethyl acetate phase was washed ~uccessivcly wit,h 150 ml of 0.5 M IICI (twice), water, ,552 I\HCOn, and water; it was then dried with SaZS04. The

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856 Gl~~taminase of Esch.erichia coli. I \.ol. X3, so. 5

product crystallized upon conccnt.ration of the solut.ion and addition of petroleum ether; yield, 3.80 g, 84%. Fine needles, m.p. 81-82”, were obtained after recrystallization from ethyl acetate-pet.roleum ether.

y-2,% ,2-TriJluoroethyl L-(&tar&e-The blocked ester (3 g) was dissolved in ethanol (60 ml) eontaining 57; acetic acid and hydrogenated at atmospheric pressure in the presence of 1 g of palladium black catalyst until uptake of hydrogen ceased. The suspension was cooled in ice and filtered. The product was extracted from the catalyst with 75 ml of water, and then crystal- lized by the addition of several volumes of acetone. The yield was 1 .O g (65$+), m.p. 164165”, unchanged upw recrystalliza- tion.

CrH,oO,XFa

Calculated: C X.7, 11 4.40, S G.11 Found: C 3G.G, II 4.32, S 6.18

L-2-.lniino-4-cyanobutyric ~~lcz&--L-2-Carbobcnzoxylamino-4- cyanobutyric acid methyl ester (m.1). 44-46”) was prepared from N-carbobenzoxy-L-ghrtamine methyl ester (23) essentially by the route described by &oral and Rudinger for the S-tosyl glutamine methyl ester (24). Saponification of the ester (24) gave an oil which wt~s washed with cold water and dried under vacuum. The oil was dissolved in 95:~ ethanol-5’:;. acetic acid and hy- drogenated as described for the trifluoroethyl ester to remove the blocking group without causing detectable reduction of the nitrile function. The product was isolated in 72(& yield after recrystallization from water-acetone. It was identical in all respects with the material prepared by Ressler and Rat&kin by a somewhat different. path (25).

c~-Benzyl .~r-Carbo6enzoxy-L-glutanlic y-Jfethoxyamide-The mixed anhydride from 3.iO g of a-bcnzyl .Y-carbobcnzoxy-L- glutamate and ethyl chloroformatc was prepared as described above. Separately, 1.25 g of methoxyamine hydrochloride were dissolved in 4.8 ml of tributylamine and 6 ml of dry dioxane with mixing at room temperature. The solution of methosga- mine was then added dropnise to the stirred solution of the anhydride at 5’. After a further reaction period of 1 hour at room temperature, the mixture was poured into 100 ml of et.hyl acetate and the resulting solution was washed and dried as described for the trifluoroethyl compound. Crystallization of the blocked methoxyamide occurred upon t.he addition of ligroin and storage at - 20”. The product (3.5 g, 84%), after recrystal- lization from ethanol-water, had a melting point. of 99 to 101”.

L-Glutumic y-.\fethozyamz&-The product of hydrogenation of 3.2 g of the blocked mcthoxyamide was crystallized from water-ethanol to obtain L-glutamic y-methoxgamide in 75:; yield, m.p. 152-153”.

C,jH&;?O(

Calculated: C 46.8, If 7.32, s 15.96 Found : c 40.9, II 7.19, r\’ 15.76

y-Thiomethyl .+Glufunzate--Amiard, Heymb, and Velluz have found that .V-trit,ylglutamic acid may be condensed with amines in the presence of di~yclohexylcarbodiimide to give esclusively the y-substituted derivatives (26). We have used this approach to prepare y-thioesters of glutamic acid.

Trityl-L-glutamic acid (26)) 2 g, was dissolved in dry dioxane, I2 ml, at 5” and the solution was deoxygenated with a stream of dry nitrogen. Dic~~lol~es~Icarbodii~nide, 1.43 g, was dissolved in this solution with stirring, and then 1 .I I 1111 of methyl mtrcap-

tan were added. The mixture was allowed to stand at room temperat.ure overnight under nitrogen; the dicyc*lohcxylurcn wai then removed by filtrat.ion. To remove the trityl-bloc’king group, the dioxanc solution was cooled in an ice bath, and dry HCI gas was bubbled in slowly to saturate the solution. .Uter 5 min at. 0” several volumes of petrolrum ether wem added to lnecipitate an oil. The oil solidified upon repeated trituration with petroleum ether at 0”, and the product was dried OWI

KaOH in the vacuum desiccator. ‘I’l~e thiocster hydrochloride was dissolved in a small volume of ethanol and titrated with trihutylamine to an apparent pI1 of about 5. Crystallization of the product as platelets occurred at -20” to yield 0.45 g (440&), Ill.],. 148-150”. This was raised to 153-154’ up011 rrcrystalliza- tion fro111 water-acetone (the melting point is lower when the sample is heated slowly).

CGHI~OZSS

Calculated: C 40.4, Ii 6.25, N i.90, S 18.1 Found : C 40.6, II 6.14, S 7X%, S 18.5

The ultraviolet spectrum in 0.001 M HCI showed a single absorp- tion maximum at 235 mp, with -IX, = 4.68 x l@. Stadtman reports A,,,,, = 232 to 233 mc(, rl &I = 4.5 to 4.6 x 10” for similar aliphatic thioesters (27). Treatment of the thiomethyl ester with 4 M hydrosylaminr at J)H 6.2 results in the ahnost instan- taneous formation of 0.98 eq of hydroxamic acid. While t,hc thiomethyl ester is relat.ively stable in aqueous solution below pH .i, it is completely destroyed at pH 7.5 in less than 1 mm, as mra~ured by the loss of the absorption maxirnum at 235 rnp (A;IXI = 4.4X x 103). This reaction also results in the relrase of 0.96 eq of a thiol as measured by the method of Ellman (28). The rapid and total destruction of the thioestcr at pII 7.5 indi- catrs that the product is essentially all in the form of t.he y- thiocster, since any a-thioestcr would not be subject to intramolecular attack by thr cr-amino group and would he expected t,o be considerably more stable under thcac conditions (29). This c~onchrsion is supported by a titration which showed the presence of less than 2yG of material tit.rating in the range from pH 3.5 to 5.0, where the y-carbosyl dissociation of glutanric acid derivatives is observed.

-y-Thioethyl L-Glutanlnte-This material was synthesized from .l’-tritvl-L-gllltami~ acid and ethyl mercaptan as described for the thiomethyl derivative. The yield was 35’;<, m.p. 152-153”. The structure was confirmed essentially as indicated for the thinmethyl ester.

C,II ,BOJNS

Calculated: C 44.0, II ti.85, N i.28, S 16.7 Found : c 43.6, H 6.62, N 7.38, s 16.3

Other materials used which are not specifically described were the best available commercial lnoducts. Compounds used as substrates were recryst.allized and their purity was verified by melting point, optical rotation, and titrat.ion curves. Hydroxyl-

amine hydrochloride was recrystallized t.wice from hot methanol. This treat.ment was found to be necessary to remove inhibitory impurities from the analytical grade commercial compound.

RESULTS

Molecular Ii-eight of Glutuminase by Sephadex Chromatog- raphy--Owing to the very limited amount of pure enzyme available, extensive physical characterization of the enzyme was not attrmlrted. For the lnrrposes of the present studies the

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parameter of principal interest ia an al)prosimatc molcc~ular weight. The met.hod of Whitaker (30) wa~i used for such an est,imate, based upon the position of ehttion of the enzyme from a gel filtration column in relation to the positions of protritts of known tnolecular weight. h good fit to a straight line is ob- taintd with the proteins of known molecular weight. when the ratio of the elution volume to the void volutnc is plotted against log molecular weight (see Fig. 1). With the assumption that the shape of the glutaminasc molecule is not atypical among this group of marker proteins, a molecular weight of about. 110,000 is calculated for this enzyme.

Reaction of Glutaminase with 6-Diazo-5-oxo-L-norleucine- Initial experiments showed that when 1 unit of glutaminase was t.rcatcd for 5 min at pH 5.0 with 6-diazo-5-oso-L-norleucine at a concentration of 1 my, the enzyme was totally and irreversibly inhibited. Howrvrr, if 30 mht glutamine was present ittitiallj with t.hc 6-diazo-5-oso-L-norleucinc, the hydrolysis of the sub-

5.2

log M

lJt(;. 1. Determination of the molccttlar weight of glutatttinasc by gel filtration. A mixture containing ISlt~(b Dextratt (Phar- ma&), 5 tttg of yeast, alcohol dehydroKettasc (Point I ), 20 trig of hexokittase (Point Z), 5 tttg of E. coli nlkalint: phospltatasc (P&l 3), and 200 units of glutaminase (G) in 2 tnl was separated at 4” on a column, 3 X 95 cm, of ,Sephadex (i-100, in a buffer system of 0.05 11 potassium phosphate, pH i.4, rind 0.1 11 KCI, at a flow rate of 0.5 ml per min. The void volume was determined to be 289 ml from the position of the Blue I)extran, and the enzymes were locut,ed by specific assays. V,/V, is the ratio of elution volttme to void volume. The tnolccular weights (M) of the reference en- zymes were taken as 151,000 for alcohol dehydrogenase (31), !Ni,OOO for hexokittase (32), and 80,ooO for nlkaline phosphatasc (33).

4

I I I

5 IO 15

DON ADDED, umoles x IO2

FIN:. 2. Titration of the active. sites of glutntrtinase with J’C- Ci-diazo-5-oxo-t,-norlertcine (Ml.\.). Samples of the electro- phoretically-petrified glut.umittase (20.7 rg of protein, 31.4 units) were treated with varying amounts of 6-“C-(j-diazo-5-oxo-to- ttorleucine (2.14 X 10’ cpttt per @mole) in 0.2 ml of 3 mM sodium acetate, pH 5.0, at 25”. After 30 tnin (the reaction is complete in this time) nliquots were assayed for residual enzyme activity. To the remaining sample, 1 mg of protein was added (a lyophilized ethanol fraction of liver was used, but the source of the protein is not important) to act IIY a carrier during isolation of the labeled enzyme. Thr protein was itnmodintely precipitated with tri- chloracetic: acid and isolated as previously described ((i). Va111es are expressed for the amount of glutatninase in the init.ial mixtttre. 0, (i-diuzo-5.oxo-L-nortettcittc hound; 0, enzyme activity.

strate procerdrd linearly for over 10 tnin at an uninhibited rate. These results suggest. that (a) the Fubstrate cornpeter; effectively with the analogue inhibitor for the same enzyme site, and (b) the interaction between t.he inhibitor and the enzyme is irrevcrsi- ble? s hw been noted for several other enzymes of glutatninr metabolism. A further indication that the binding of the inhibitor is site specific is the observation that the process does not take plave above pH 6.0, in which range the K.” values for all substrates becaomc infinitely large (34) as: measured either bj inhibition of the enzyme or by covalent attachmrttt of IaC: to the enzyme (see below).

Treatment of glutaminase with tY’-6-diazo-5-oso-t-ttorleucitte at pH 5.0, followed by precipitation of the protein with tri- chloracetic acid and isolatiott as previously dcacrilwd (6), showctl that the labeled inhibitor brromcs covalently attarhed to the enzyme. \Yhcn the atnouttt of hi-diazo-5-oso-L-ttorleucille present wax IesF than t.hat required for complete inhibition, the extent of inhibition paralleled the binding of inhibitor, a< shown in Fig. 2. No further amount of 1% is bound aft.er itthihitiott is complete.

It, seems most. reasonable to ittterpret, the lincar relationship

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between the atnount of G-diazo-5-oxo-L-not-lcucitte bound and fractional inhibition in terms of a one-for-one reaction between inhibitor and catalytic sites. n’ith this assumption, these results provide a method for t.he expression of the catalytic rate in absolute term?, i.e. t.urttover ttutnber per site. Rcferc>nce to estimates of protein concentration, Iturity, or tnolecular weight is not, required. The turnover nutnbers reported below arc based upon such titratiotts wit.h tJC-lab&d inhibitor, from which it ir; found that I unit of enzymc~ contains 1.32 x 10T5 pmole of active sites. If the less likely pol;sibility is admitted that bittding of 1 molecule of 6-diazo-5-oso-t,-norlcucint~ inac- tivates n identical catalytic sites (e.g. by ittducing a ronforma- tional change), the turnover ttumbers per catalytic site would in fact be 1 /n times the valurs report,ed bsed upon the nutnber of inhibitor-binding sites.

If it is assumed that the cttzyme obtained after the cohmm electrophoresis step (specific activity, 1,520) is pure, then the total amouttt of *~C-6-diazo-5-oxo-L-norleuc.irlct bound in t,he plateau region of Fig. 2 allows calculation of an equivalent weight., per itthibitor binding sitcl. The average: value for three different rnzymr preparations was 50,000 + 5,000. Taken with the rst.imated molec~ular wcGght of llO,&lO, these figures indicate two inhibitor sites, hence two catalytic sites, per mole- cule.

It may be WCII from Fig. 2 that a very large csws of B-tliazo- 5-oxo-L-ttorlcuc:inc is tttc’rssary to inactivate the ettzymc totally. Experiments to be reported separateI! sho!v that tnost of the inhihitor is c~ntalytically cleaved to glutamic: acid and methanol, less than 25; reactittg by irreversible binding to the rnzyntc.

Suhstrute SpeciJicity---That kinetic parameters for all cotnltotmds found to he euhst.ratcs of the glutatnittasr art’ list,etl in Table II. A typical set of data is illustrated in Fig. 3, in this case for y-methyl L-glutamate. The data were generally of this quality and always gave a straight. litte in the Lineweavcr-Burk plot, a result showing adherence to Uichaclis kinetics and indcpcndence of the two active sites. In the case of ghttamic acid, the values were derivrd frotn the kinetics of the eschattgc of IIJW with the oxygen atoms of the y-carbosyl group, as deycribetl in detail in an accompanying paper (35).

TABLE II

Kinetic properties

Substrate” -

Glut.amitie ....................... r-Glutamyl met hylttmidc. ........ r-Glrttatnyl hydrazide. ........... r-Glutamyl hydroxamic acid. ... r-Glutntnyl methoxyamide. ......

SEC-’

1265 a.0

14 212

KMC -

m 1,

0.42 3.3

12 5.1

296 5.0 Glutamic :Icid.. 5080 2 .!)” -,-Methyl glutntttate. 645 A4 -,-Rt.hyl glutamate.. 36 50 y-Thiomethyl glrttatnate.. 12GO IO r-Thioethyl gltttntnate.. / 300 , 23

0 .411 materials were of the L configuration. * k,.,, values arc the turnover numbers per (i-diuzo-5-oxo-t,-

ttorleuc:ittr:-t)inding site at 25” and pH 5.0. Estimated error in

kc,,, ,: *5$( c Estimated error in KM, dSO(j$. d Value frotn the TI8W exchange experiments; kinetics of hy-

droxnmate formation yields KM = 2.0 TIIM.

f substrafes

kcat*

TABLE III

Kinetic properties of inhibitors

Competitive inhibit& --.- -.

-f-Glut.umyl meth~latnidc.. _. y-Gtutamyl ethylamide. y-Glutamyl 2-hydroxyethylamide.. .I Glutatttic acid. -,-Trifluoroethyl glutamate. I

__.

2.1 2.8

52 1.2

G7

(1 All materials were of the L configuration. b Nstimuted error, f30C/,.

I/S, M-’

FIG. :I. Estimation of K.q for -,-met,hxl-t,-glutamate. I<c:tction velocities were tinctured by the titrimetric met hod at pH 5.00 and 25O.

Khctt assayed by the titrimctric method at p1I 5.0, gluta- mittase did ttot cat.alyze the hydrolysis of a number of compounds related to glutatnitte at a measurable rate (limit. of detection, 0.1 set-I). Thcsc materials include L-glutamic y-ethylamide, DL-glutamic y-dimethylamide, r.-glut.amic anilide, L-glutamic y-p-ttitroanilide, t,-ghttamic y-2-hydroxyethylamidc, L-glutatttic y-2-~hlorotthylamide, t)t,-glrtt.amic y-n-butJ-lamide, r.-ghttamic diatnidr, glutaratnic acid, h’-acctyl-L-glutamintl, I)-glutamine, t)- or L-asparagitie (at plI 4.5), I.-2-amino-4-cyanot,utyric acid, y-2,2,2-trifluoroethyl L-ghttamate, L-pyroglutatnic acid, S- carbamoyl-t.-cysteine, and O-acety I-L-aeritte. The enzyme failed t.o clrave the attilide derivat.ive to aniline as ntca+urcd h3 the Eratton-JIarrhall reaction (36). Lack of hydrolysis of t.he nitroattilide ws confirmed by spectral measurement+ at 410 trip. The hydrolysis of glutamine could be followed by c&ritnctric detection of the ammottium ion formed (37), but under similar conditions t,-isoghttaminc Whs completely unrclactive.

Competitive Inhibitors-The possibility was examined that certain compounds might be bomld by the enzytne even though they \yere not hydrolyzed. The effect.s of these materials upon

the rate of hydrolysis of y-methyl L-glutamate were measured with the titrimetric assay at pH 5.0, 29, and ionic strength 0.2 M. In each case, apltartnt, K. N values were determined at foul

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levels of inhibitor in order to calculate fYl (Table 111). L-

Glutamic y-methylamide, a substrate, inhibited the hydrolysis of methyl glutamate, as expected. ‘I’hc hydrolysis of the methylamide which occurs during thcsr runs affects the rate metiurements by Jr?;5 than 1 ye. The inhibition is strictly competitive, with a k’, of 2.1 mM. Similarly, glutamic acid inhibits rompetitivrly, the h’, being 5.1 I~JI for glutamic acid plus glutamate ion, or 1.2 I~JI for the glutamic acid alone. By the criterion of competitive inhibition, L-glutamic y-ethylamide (k’, = 2.8 mM), L-glutamic -&hydroxyethylamide (K, = 0.052 M), and 2,2,2-trifluoroethyl r.-glutamate (k’l = 0.067 11) bind significantly to glutaminase even though they appear not to be substrates. On the other hand, the following materials had no

measurable effect upon the reaction of met.hyl glut.amate: I,-glutamic diamide, S-acet.3-l-L-glutalllillc, glutaramir acid, cglutamic y-anilide, r.-2-amino-4-cysnobutyric acid, S-carbam- oyl-L-cystcine, 0-ac:et.yl-L-serinc, nL-methioninc sulfoxide, DL-

mathionine sulfone, and oI.-met.hionine sulfoximine. I conclude

that the k’, values for these compounds must be greater than 0.2 M. The related compounds, DL-allylglycine and DL-2-amino-4- iodobut.yric acid, did not inhibit. the e~lzyrne at a concentration of 0.05 11 when incubated for 30 min at 25” prior to addition of the substrate. These materials were tested in the hope that they might act as active site-directed, irreversible inhibitors.

Phthalein dyes r;trongly inhibit certain glutaminases (3X, 39), but 5 rnhl hromcresol green had no effect upon the hgdrolyris of methyl glutamate (0.05 M) by the E. coli enzyme.

Formation of y-Ghtamyl ftydrozainic .lcid-Meister et al. noted that t.his glulaminasc slowly caat.alyzed the synt,hesij: of y-glutamyl hydroxamic acid when glutamiue was incubated in the presence of hydroxylamine (7). The reaction with hydrosyl-

amine is a general one for all substrates of the enzyme, including glut.amic acid.

While the maximum rate of hydrolysis of glutamine is inde- pendent of pH between pH 3.5 and pH 5.6 (34), the rate of hydroxamic acid format,ion from this substrate increases with pH at a fixed total concentration of hyrlroxylamine (conjugate acid plus base). Thus, in an experiment with a total hydroxyla- mine concentration of 0.2 11, t.he initial linear rates (v) of hy- droxamic acid formation iu the presence of saturating amounts of glutamine (0.08 M) at pH 4.50, 4.97, and 5.37 were 0.0225, 0.0647, and 0.140 j4mole per min, respectively, at 25” in the presence of 2.6 units of enzyme. The ratios, v/(?;II&H), obtained by dividing these rates by the calculated concentration of hydroxylamine-free base at each pH value, are 4.61, 4.69, and 4.46. Thr constancy of these values is taken to indicate that the enzymatically react,ive species is the free base of hydroxyla- mine.

Studies up011 the direct reaction between glutamic acid and hydroxylamine are not complicated by the occurrence of a concomitant hydrolytic reaction as is the case with the other substrates of the enzyme. The initial rates arc therefore linear

for a much longer time, and if the reaction is run at a pH midway between the pK’ values of glutamic acid and hydrnxylammonium ion, then the pH automatically stays constant. 111 Fig. 4 it may be seen that the rate of hydroxamic acid formation from glutamic acid is a linear function of the total hydroxylamine concentration up to abouL 0.5 M. Under our experiment,al condit.ions, forma- tion of hydroxamic acid is not detectable in the absence of enzyme.

The KM for glutamic acid was determined from the kinetics of

02 04 06 0.0

TOTAL HYCROXYLAMINE, M

FIG. 4. Formation of hydroxamic acid from glutamic acid. L-

Gllltamic acid (0.2 Y total concentration) was incubated with varying amounts of hydroxylamine in the presence of 2.03 units of glutnminase at pll 5.08 and 25” as described under “Experi- mttnt.al Procedure.” At. this pH value and ionic strength the hydroxylrrm~norlium ion is 9.1(% dissociated.

its reaction with hydroxylamine in experiments illustrated in Fig. 5. The slopes of these plots, obtained by a lemt squares fit, yield a K, value of 20.8 + 1.0 rnhl for total glutamic acid (acid plus anion) at pH 5.0X when the total concentration of hydroxylamine was 0.50 Y, and a value of 19.7 + 2.1 mhl when the hydroxylamine was 0.0467 hl. A similar experiment at, pH

4.50 and 0.50 JI total hydroxylamine yielded a value for k’,~ of 6.5 f 0.4 IIN. If the k’,,, values arc caalculated for the cnncen- tration of the acidic form of glutamic acid (pK’ = 4.12 at ionic strength 0.5 M), t.hese arc found to be 2.0 and I .9 ml1 at l)H 5.08 and pH 4.50, respert.ively. In view of the fact t.hat tho KM

values for all other substrates investigated are cssentjally inde- pendent of pH between about pH 3.5 and pH 5.3 (M), it seems very likely that glutamic acid, and not the anion, is the kinetically important form of t.his substrate.

When the reaction between hydrnxylamine and glutamic acid (or any othrr substrate, since these arc eventually converted to glutamic acid or hydroxamic acid) is allowed to go to completion in the presence of glutaminase, the pH-independent equilibrium constant may be calculated according to Equation 3 (at 25” and ionic strength 0.2 M).

k’ eg

_ (IE-COSHOlI)(H#) .--- = 199 f 20 M-’

(R-COOH)(NII,OH) (3)

The ionic forms arc those showy and the activit.y of water is taken as 1. The value of K,, given is the mean of eight runs with various substrates, including two runs in which the equi- librium w&s approached from t.he reverse direction. To confirm

this result au experiment was performed at the calculated equi- librium position. L-Glutamic acid (total concentration, 44 mM; concentrat.ion of acidic. form, i.48 mhl), hydrosylamine hydro- chloride (0.20 11 total; concentration of basic form, 14.7 mu), and y-L-glutamyl hydrosamic acid (22 rnhl) were incubated at

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V/S, umoles rnln-’ Me’

FIG. 5. Est.im:ttiou of KM forgllttnmic acid from rates of hydrox- amic acid formation. Itatcs of hydroxamic acid formation were determined with a ronstant amount of enzyme and varying amounts of glutamic acid, as described under “kkperimentul Proccdruc.” For Crcrre 1, the total hydroxylamine conccntr:ition was 0.50 or and the pll was 5.08; for Ckrve B, the hydroxylumine was 0.04ti7 Y, the pH 5.08; for CWW 3, the hydroxylumirrc was 0.50 M and the pII was 4.50. The temperat urc of the re:tct.ion was 25” in all casts.

I)H 5.0 a11d 25” in a volumr of 1 ml vvith 20 units of glutarninase. 1)etermination of the concent ration of hydrosamic a&l by the ferric chloride method at IO-min intervals gave results of 22.0 (zero Gme), 21.8, 23.0, and 21.7 mnr, so that I co~mlude that the sysl.eni was at equilibrium. Ehrrnfcltl, Marble, and Mrister (40) have determiiicd the equilibrium constanl for the same reaction rata.lyzerl by an enzyme from :I rof.oba+r agilis at. pH i.2 and 37”. In terms of the conventions used here, their data yield values of K,,, of 203 to 363 K~. These results are ill the range of those found for the nonenzymatic reactions Irctwcen carbosylic acids and hydrosylamine by Jcncks et al. (41).

Synthesis of y-J/ethyl Gltrhrnatc---In view of t.hr fact that many rnzymr.s which catalyze hydrolysis and h?dros?-lamillol~sis of carbosylic acyl derivatives effrct an analogous reaction with alcohols, WC attrmptcd to detect such an acyl transfer with mrt.linnol as the acceptor. ‘This reaction would be expected to occur OII the grounds of microscopic reversibility, since y-methyl glutamate is hydrolyzed by the enzyme.

In lrreliminary csl~crinicnts it \\a-: fouild that inethanol in concentrations of up to 40“; by volume did not. affect. the initial rate of hydrolysis of glutamine in t.hc lnesrnce of glutaminase. The ront,inuous titrimct,ric assay was used, and t,he rates were calculated with the use of the pIi value for glutamic acid deter- mined at the appropriate concentration of methanol (ionic

strength, 0.2 I\I KU, 25”). These pI< values inrrea$cd linearI! with the mole fraction of mrthanol from 4.31 to 5.00 in the range 0 to 0.3 (0 t,o .50(-i by volun~c).

When uniformly labrlctl IV-r.-glutumic acid was incubatrd in a solution containing 27.87; met.hannl by volume (6.9 M) and aliquots were analyzed for *Cy-methyl glutamate by the method described under “Esl~erimcntal Procedure,” ester synthesk occurred al a constant rale of 7.3 X IO-* ~.rnio!e niiti-r for 20 min. ‘I’hi:: rate is equivalent to a turnover number, li cat, of 3.20 se0 for the reaction betnect~ glutamie acid and methanol at 6.9 M, or a second order rat.c constant, /~,,,,.~r,~~~r = /c,,~/((‘H&H), of O.li ~-1 set-1.

/QYect OJ I?leat,y .\Iefals---Experimellt~ to test, qualitatively the effect of heavy metals upon glutamina;r were conducted with the standard titrimetric a5;aay lrrncedurr, but. with EDT.1 omitted. Salts of the metal ions were incubated with the enzyme for 5 min at 25” prior to starting the reaction by addition of the substrate, glutamine. Under these conditions, mercuric: nitrate itihibited 100’,; at 0.1 mhr, silver nitrate inhibited 35$; at 0.1 mlr, lead acetate inhibited SC; at 1 mu, and cupric nitrate inhibited 19’ ; at 1 mhr. ~Iagnrsiun~ chloridr, manganese chloride, zinc sulfate, cadmium acetate, cobalt chloride, ferrous sulfate, and calcium chloride were without effect at 1 mM. p-Hydroxymrr- curibenzoatc inhibited 47(x,, al lo-” M and 89% at 2 x IO -j Y; when the lrrelirninary incubation was rhortened to f mm, 2 x 10PS M mrrcuribcnzoste produced 51 C’, inhibit.ion. Glutsmine at 10 IWI prevented inhibition by t!rc latter cnm~mmtl, since the catalytic react ion continued at the uninhibited rate for several minutes when the substrate was present. with the inhibitor from the beginning.

k$cct of Other .IgerA- Preliminary treatment of glutaminase at, 25” vvith 5 m>r iodoacetate or iotloacetamide for 30 min at eit.hcr 1’11 5.0 or l)H 7.0 produced no detectable inhibil.ion of the enzyme. The activity similarl?- was not affected by .Y- ctt~yln~alt+~ide (4 mu, 20 min) or diisol~rol~yll~hosphoroflu- oridak (2 mM, 30 mm).

Aletal-binding agents such as EI)‘I’.A, o-l)henatlt.hrnline, and dithiothrcitol, at conccntration~ up to 5 m;\r, had no inhibitor! effect on the enzyme, rvcn after prolnngctl storage. III fact., thr! usually produced a small slimulatiou of activity, and for this reason low concentrations of EDT.\ were added to the solutions used for l)rcparatinn and assay of the enzyme.

Glutaminase is very rapidly inactivalrd by l)tioto-osidatioii in the l)re>cncr of methylcnc blue. III thrse rspcrimrnts, a snlu- tion of the enzynic (2 to 3 mm in clel)th), contaitling 0.1 ‘; bovine serum albumin and 0.005(x, mct.hylene blue, was csposed at 25” to the light. of a IOO-watt, tungsten lamp at a distance of 15 cm. l!rrdcr these conditions, 94 ‘;:/; of the activity wad loat druitlg 5 mill of illumitlatiotr, whereas in controls in which cit.hcl the nicthylcrir blue or the illuniination was omitted the ac1ivit.v \vai e+riltially conil~lctcly recovcrctl. Enzyme inuctivatrl in this \vay could not bc reactivated I)y retlucing agents such a< dithiot,hrcit.ol.

l’hc nletabolic funct iorl of this glutumina~e ia not at all clrar. The enzyme is tlrtcc~tablc it1 cell extracts only after rxl)onential growth of the culture ceases. The presence of excess ammonia,

glucose, glutamate, or glutamine duritrg the stationary phase had IIO appreciable effect on the level of enzyme formed. It is alsn l)uzzling that tlli-: enzyme, which is inactive in dro above

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about pII 5.8, appears during a period of growth when the medium, at Icast, is becoming alkaline. It might be imagined that the glutaminase is producctl in conjunction with proteo- lytic enzymes at the changeover in metabolism and rnzyme complement accompanying onset of t.he stationary phase to aid in thr reutilization of glutaminr and glutamic acid. However, I have found that 6. coli I{ contains another glutamine amido- hydrola,sc which is present throughout the exponential as well as the stationary phase of growth.1 This enzyme has thr fol- lowing properties. (Q) It is obtained in soluble form upon disruption of the cells, (b) it is active at pH 7 but not at pH 5, (c) it is inhibited by (i-diazo-j-oxo-L-nol.leucille, and (d) its total activity is only about 255; of that of the acid glutaminase. This activity is appreciable since i4(:-glutamine, which is readily accumulated by 6. coli cells from the medium, almrars intra- crllularly almost cxclusivrly as glutamic acid within 30 sec. of its uptake.” Thus the ncctl for an additional means of hydro- Ivzing glutamine is not aplrarent. .\n intriguing lrossibility is that the acid glutaminasr is in fact a drgratlative remnant of snme other enzyme, conceivably one involved in glutamine metabolism during the exponential phase.

.\lI of the compounds found capable of being specifically bound by the enzyme, either as substrates or as comlrctitivc inhibitors, have the common structural elrmcnts

li(J-- Fill,’ I

coo-

r.-2-.~mino-4-c.?-sliol)iit~ri(, acid, in which t hc y-carbonyl osygrn atom is lacking, and S-~arbamo~l-L-c~~tri~~~, in which a mcthylcne group of glutamine is replaced by a sulfur atom, are not bound to a detcctablr rstent. Similarly, 6-diazo-5-oso-L- norleucine is a potetlt inhibitor, but 0-diazoacrtyl-L-yrrinc, the counterpart containing an osygcn atom in the chain, is com- lrletely rcjectctl. Roth thr L-ar-amino group and the carboxyl group must be prcscnt and unsubstituted, as notrd by the inertness of nglutamine, glutaramic acid, .~-ac,et~l-I.-glutamine, and I.-glutamic diamidc. ‘I‘hr strict requirement for the I.- glut amyl moiety is further shown by the lack of binding of close struct.ural analogues such as methioninc sulfositle and O-acetyl- I.-scrine.

;\Iore tolerance of variation is shown with respect to thr sub-

stituent., X. Coml)ounds containing (I-S, c-‘--O, C’--S, and evrn C--X: bonds are both I~ountl and cleavetl. The paramct er, k,.,,, is affectrd bot.h by thr chemical nature of the group, S, and by its size. .\mong groups of similar size, such a< OH and SH?; SCH3, 0c’H3, T\‘IIC113, and NHN&; or SCBHi, OC& T\TI-IC2HS, the rates of hydrolysis decrease ~qmn goitig from the sulfur to the oxygen to the nitrogen derivative. Wit.hin a class of coml~ounds (amides, rstcrs, or thiorsters), k,.:,r is largrly influcnccd by size, the rate gradually rlropl)in g to zero for compounds containing ethyl or slightly largrr substilirrnls.

1 S. C. llartrn:m, Iurprtblished rcsdts. 2 I)avid S. K’rwcombe, rmpublisheti results.

Thr factor most ob\iouely affecting the KY (or K,) values is the chemical naturr of t,he substitucnt. IMween the extremes of the smallest (OH or SJI?), which have low Kw valur.~, and the very large groups, which bind poorly or not at all, all mcmbrrs of each rhemical class have nearly thr samr Kv or Ii,. The valurs for amidrs are significantly lower than t.hosc of the sulfur or oxygen cnmpounds, a result suggrst.ing that the basicity of the carbonyl oxygen atom may be iml)nrtant for binding to some acidic group on the enzyme. The diffcrcntiation of thr sub- r;titurnt effrcts upon thr kirlrtic pammctrrs rau be seen most clearly in the casts of glutamyl ethylamide and trifluornet hyl glutamate, both of which arc bound as competitive inhibitors but are not. hydrolyzed at a detrctable rate.

In contra$t to the behavior of certain other glutaminascs which preferentially catalyze transfer of the glutamyl group to amine acceptors such as hydroxylamine rat.her than to water (14, 40, 42, 43), the B. coli rnzyme is predominantly a hydrnlase. This fact is due in part. t,o the acidic pH rcquiretl for activity, iti which rangr most amines are largely in thr unreactive, protonatcd form. 111 addition to this effect, a marked discrimination against nuclrophiles other than wal,er can be shown to exist. The relative rhrmical rractivity of hydrosylamine compared with water can be est.imatrd from the equilibrium ratios of hytlrosamic acid to carbosylic acid when the activities of the nuclcophiles arc cxpresstrl in the same units. I f t.he activity of water is taken as 55.5 11, the equilibrium ratio of hytlrnsamir acid to glutamic acid given under “Results” becomes 1.1 x 10’. .Jencks el nl. report a value of about 2 x 10J for similar equilibria achirved nonenzymatically (41). The initial rate of the rn- zymatic reaction between glutamic acid and hydrosyluminc, t.aken from the data of Fig. 4 and crlne~scd as a second order rate constant, is

The unalognue rate constant for the virtual hydrolysis of glu- tamic acid (35) is

Thus, hytlrosylamine reacts rnzymatically some 100 times fastrr than watrr. This is ahout two orders of mugtlitudc slower than it would bc expected to rract on thr basis of t.hc equilibrium data, howcvrr. Similarly, if we coml)are thr second order rate constant for rructioti between mctlianol and glutamic acitl (0.4i h1-r set-1) wit.11 that for water, wc sre that water reacts 194 timrs faster than methanol. In view of obsrrvations that mrthanol is generally a somewhat better nucleophilt than water in nonenzymatic reactions and in certain rnzymat.ic processes of acyl clerivatives (44), these results show that a very large dis- crimiiration against thr reaction of met.hanol exists in the case of glutaminasc. Thrsc results anI those with hydroxylamine show definitely that t.he water molecule reacts with some int.crmediate from a more or less sl)ccifir site and not from free solution. ‘I’hercfore the rate constant, kmo, calculatrd above has qucstion- ablr mraning except for the purposes of this comparison. Whether the other two nuclcnphilcs react from t.he solvrnt phase cannot. be decided, although the linearity of the curve in Fig. 4 is consistrnt with this lrossibility in the ca+e of hydroxylamine.

The tlatiire of the “water site” could in lrriticil)le rangr from

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S(j2 Glutaminase of Escherichia coli. I \‘ol. 243, x0. 5

a true binding rite involving imnlobilization of a single molceule of watrr in a rritifal position with rcs;l)clct to the intcrmcdiatc t.0 be hydrolyzed, t.o a solvent-likr aqueous pool from which larger mole~les are restrictrd. I~etwecn (hcsr rst rrmes is the po5si- bility that t,he reactive water exists in UII icr-like structure form- ing an effcct.ivc bridge for rapid proton Vansfrrs (45). At. this stage these are only conjectures, but certainly this question lies at t.he heart of understanding the mechanism of the process.

Enzymes which catalyze hydrolysis of amides are often found to act upon rclat,ed acyl derivatives, such as cst.crs and t.hioesters (46). This is the case with the “scrillc chtcrases,” e.g. chymo- trypcin, trypsin, and subtilisill; thiol acylares, e.g. Jjapain and ficin; a metallornzymc (carl,os~l)cl,titiasr); prpsin (4i); and thi* glutaminase. It would IW of obvious interest to know if glutaminake is related to any of these &her groups of enzymes, or whether it ures a basically different mechanism for hydrolysis of aryl derivatives. The latter is a real possibility, since the masimum rate of amide hydrolysis catalyzed by glutaminase is about. 3 X 10’ greater than that of chymotrypsin acting upon S-acct~l-L-phenS-lalanille amide (,48), about I .5 x lo2 greater than papain acting upon S-hcnzo?-I-L-argillille amide (49), about lo-fold great.cr than the rate of hydrolysis of carbobenzoxyglycyl- cphenylalanine in tho presence of carboxypeptidase A (50) and ahour 5 x lo” greater than prpsin acting upon carbosy- berrzor;yhistidyl-L-pher~ylalanyI-L-tryl~to~~har~yl ethyl ester (51).

1:‘. coli glutaminase and the scrine est.erases, typified 1~) chymotrypsin, differ in scvrral respects (52). First, diiso- prop~Il)hosphorofluoridatr, a potent. and general inhibitor of scrinc esterac;eq 1, has ii0 cffcct up011 glutamina5e. Second, glutaminase is maximally active from below pH 4 t.o above pH 5.5, while thr rerine enzymes characteristically arc dependent. up011 a basic group nith apljarent. pli about 7. Third, while glutaminase can catalyze hydrolysis of amides and esters at similar rates, esters arc hydrolyzed about 103 times faster than corresponding amides by chymot.rypnin and trypsin.

\Yhile it seems unlikely t.hat. the detailed mechanisms of glutaminase and scrine estcrases have much in common esccpt possibly in a formal sense, the rclationshiJ) with a typical thiol acylase, such as Jjapain, may be closer. Glutaminase and papain have qualitatively similar propertics when compared by the three criteria listed above (53). That l)H profiles arc not iden- tical, but rach is charact.crizcd by a broad region of constant masimum velocity in the acidic region. However, it is not at

all certain that glutaminwe is a thiol enzyme. While it is SW-

sitivr to inhibition by heavy metals and organic mcrcurials, more specific sulfhydryl reagrnts (such as inrlnnc*ctic acid, iodoacct.a- mide, and .~-et.h~lnialcimidc) arc without. effect. Glutaminase

is remarkably resistant to thr art.ion of hydrogen peroside under coilditioiis which readily iuact,ivate papain. 111 t.he presence of 0.3 AI hydrogen pcroxidc at J)H 4.6 and 25’, the enzyme has a half-life of about. 3 hours, while papain is inactivated within scc~~~~tls. &idc from the susccl)t ibility towards hravy metals,

which are known to bind to l)rnteins by grouJ)s other than thinls (54), glutaminase dots not exhibit. the behavior expected of sulfhydryl enzymes. Another dissimilarity between glutaminase and papain is their sensitivity t.oward 1)hotoinactivation in the J)rezencc of methylene blue. Glutaminasc is irreversibly in- hibited within minutes, a result SUggeStiUg that imidazole groups ma\- be important for the activity of the CIIZJTIW (56). On the

other hand, papain, even after fi0 min of similar treatment, can

be totally rcartivatcd by rsposurr to rcduring sgeilts such as dithiothrcitol.3

It +ccm+ reasonable to reach the tentativr conclusion t,hat. glutamina~c~ is not rrlatcd to the mrtal-~ontainillg or mctal- activated acy1au.s even though we do not yet have the rnnfirm- ing nicbtal analy.scs. 111 ad:lition to t,hc lack of inhibition of glutuminasc observed with metal-binding agents which &late the csscntiul mc~t.als of carbosypeptidase (56) and leucinc aminoJ)ctptidasc (5)) t,hc latter cnzymcs arc maximally artivc in neutral or alkalinr solution, in which glutami~~asc is inartive. A mruningful coml)arison brtwren glutaminase and prpsin is dif?irult I~ause little is known as yet about thr mrchanistic rnurt;c of tliis l)rnteolyt.ic enzyme. Their pH optima arc vrr? different, mid it tlor* not 7rem likely that thr combination of catalytic, groups which function optimally at pH 2 to 3, as in pcl)~in, could scrvr with the necessary effectivctnrxs in gin- tamina-c: at pII 5.5.

The foregoing arguments point up the likelihood that the action of glutaminase upon carbnxylir drrivativos involves a new mechanistic J)ath\vay-at least, vw not. operative in other well studied acylares. This possibility, c,nupled with the fact thst this ~~IIz~I~: has a significantly higher turnover number than any related cnzymc yet found, makes t.he mechanism of action of glutamina~r of vrry considerable interest. The two accom- pallying l)al~rs deal with studies on this l)roblern (34, 35).

.-lcknol~le~lg,rtr,ct--The author wishes to thank Miss PYeanor 11. Stochaj for espcrt a&tancc.

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Standish C. HartmanCATALYTIC PROPERTIES

: I. PURIFICATION AND GENERALEscherichia coliGlutaminase of

1968, 243:853-863.J. Biol. Chem. 

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