Klebsiella Aerogenes JEAN E

Home , Urocanase

JOURNAL OF BACTERIOLOGY, May 1973, p. 666-673 Vol. 114, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Effect of Methionine Sulfoximine and Methionine Sulfone on Glutamate Synthesis in Klebsiella aerogenes JEAN E. BRENCHLEY Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 1 December 1972

At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resistant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthesis via glutamate synthetase enzyme.

Recently it has been found (8) that at least ley, Prival, and Magasanik, manuscript in prep- two pathways are involved in the assimilation of aration). ammonia and the synthesis of glutamate. One Interest in this "low ammonia assimilatory" pathway functions by the glutamate dehydrogen- pathway was furthered when it was found that ase (GltD) (reduced form of nicotinamide-ade- in K. aerogenes catabolite repression of histi- nine dinucleotide [NADPH ]EC 1.4.1.4) reaction dase activity (but not ,B-galactosidase) by glu- converting a-ketoglutarate to glutamate. Am- cose could be overcome during nitrogen limita- monia can be further assimilated by the production. It was also found that this relief from tion of glutamine from glutamate and ammonia repression is correlated with high glutamine by glutamine synthetase (GlnS). synthetase activities and low glutamate dehydrogenase activities. To determine whether the GltD a-ketoglutarate Glutamate (1) absence of glutamate dehydrogenase alone NH,-N could be responsible for increased levels of

GlnSG glutamine synthetase, mutants lacking gluta- Glutamate mate dehydrogenase activity were constructed NH, and characterized. One mutant, MK-275, lacks GltS Glutamine - > Glutamate (2) glutamate dehydrogenase (gltD) activity and a-ketoglutarate yet surprisingly appears to grow as well as the parental strain under all conditions tested The second pathway involves the reaction of (Brenchly and Magasanik, manuscript in prep- a-ketoglutarate and glutamine by glutamine aration). The normal growth and glutamine (amide): 2-oxoglutarate amido-transferase ox- synthetase level show that glutamate dehy- ido-reductase (NADP) (glutamate synthetase, drogenase is not necessary for growth or regula- GltS) to form glutamate. Because the gluta- tion of catabolite repression during nitrogen mine synthetase has a lower Km for ammonia limitation. Thus it appeared that, despite its than glutamate dehydrogenase, it is able to high level of regulation, glutamate dehydrogen- react with ammonia at much lower concentra- ase has no important function in K. aerogenes. tions. In Klebsiella this second pathway is the However, it was felt that it might be possible to major route utilized during growth in media use analogues which would specifically inhibit containing less than 1 mM ammonia (Brench- glutamate synthesis in the gltD strain, and so 66 VOL. 114, 1973 INHIBmON OF GLUTAMATE SYNTHESIS 667 the effects on growth of MK-275 by a number of used as inocula for growth curves in minimal medium analogues were examined. Growth inhibition were grown in the same medium prior to use. Growth occurred with methionine sulfone and methio- temperature was 37 C. Cells to be used for extract nine sulfoximine and these were studied fur- preparation were harvested with rapid cooling and centrifugation when cultures reached a value of 100 5 ther. Since the inhibition by methionine sulfox- Klett units (no. 42 filter, approximately 9 x 108 imine is more pronounced, its effects were cell/ml). The cells were washed twice with cold 0.85% studied in more detail. NaCl and stored as pellets at 0 to 4 C for 8 to 12 h be- Previous work with methionine sulfoximine fore sonic treatment. The cells were suspended to demonstrated its inhibitory effect on Leuconos- 1/100 their original volume in a buffer containing 10 toc mesenteroides (3, 9) and suggested its in- mM imidazole-hydrochloride (pH 7.15), 10 mM volvement in inhibition of glutamine synthesis. MnCl2, and 0.14 ml of mercaptoethanol per liter. This This inhibitor also has been identified as a buffer is the one used for the glutamine synthetase as- powerful convulsant agent in a number of says, and since this was the enzyme of primary in- animals (4, 11), and extensive biochemical terest it was chosen for all extract preparations. The specific activity of glutamate dehydrogenase was the studies have been reported with glutamine same for extracts prepared with this buffer as for one synthetase purified from sheep brain by Meister prepared in a tris(hydroxymethyl)aminomethane and co-workers (7, 10, 12). They demonstrated buffer. It is not known whether the imidazole buffer that methionine sulfoximine binds glutamine affected the glutamate synthetase stability. synthetase tightly and causes irreversible inhi- The cells resuspended in the imidazole buffer were bition of activity. The results reported here are sonically treated (Bronwill Biosonik Ill sonic oscilla- consistent with methionine sulfoximine inhibit- tor with a needle probe, 25% power setting) three ing the glutamine synthetase from K. aerogenes times for 15 s with 20-s cooling intervals. This fraction was centrifuged for 20 min at 17,000 x g, and the but suggest that the primary growth inhibition supernatant fluids were saved. Extracts were stored in is due to the inability of the glutamate dehy- an ice bath, and the enzymes were assayed routinely drogenase-negative mutant to produce gluta- within 8 h after extract preparation. Protein determi- mate for growth. The effect of methionine nations were made by the method of Lowry et al. (5). sulfoximine and methionine sulfone on the Enzyme assays. Glutamate dehydrogenase and newly reported glutamate synthetase is also glutamate synthetase activities were measured by investigated in these strains. following the rate of NADPH oxidation as described by Meers et al. (8). It was found during these studies that the glutamate synthetase activity is unstable in MATERIALS AND METHODS the reaction mixture unless both glutamine and Strains and media. K. aerogenes mutants were a-ketoglutarate are present. The usual procedure for derived from strains isolated by MacPhee et al. (6). this assay had been to follow the endogenous rate of Strain MK-247 is used as the wild type and has all NADPH oxidation prior to glutamine addition and three enzymes involved in glutamate synthesis. Strain the measurement of the glutamine-dependent gluta- MK-189 lacks glutamate synthetase and cannot as- mate synthetase activity. Most of this activity, how- similate ammonia at concentrations less than 1 mM. ever, is lost within 10 min at 37 C (Fig. 1), but when Strain MK-275 has no glutamate dehydrogenase ac- the reaction mixture contains glutamine and a-keto- tivity and was constructed from a glutamate-requir- glutarate (both are necessary) the activity is stable for ing strain lacking both glutamate dehydrogenase and at least 1 h. Under these conditions the reaction was glutamate synthetase (gItD and gitS). Transductants started by NADPH addition, and the endogenous of this double mutant were selected for their ability to oxidation was determined in separate experiments. grow on limiting ammonia. One transductant, Other experiments demonstrated that about 90% of MK-275, had regained the glutamate synthetase ac- the glutamate synthetase activity is lost when the tivity, but not glutamate dehydrogenase activity, and crude extract is frozen for 18 h. Since the activity is did not require glutamate. Strain MK-8 lacks uroca- more stable in cells stored as a pellet at 4 C (loss of 20 nase, the second enzyme involved in histidine degra- to 25% activity), they were kept in this manner when dation, and cannot form glutamate from histidine (1). the assays could not be completed immediately. Minimal medium contained per liter of distilled Whenever specific activities of different extracts were water (NH4)2SO4, 2 g; Na2HPO4, 6 g; KH2PO4, 3 g; to be compared, the extract preparation conditions NaCl, 3 g; Na.SO4, 11 mg; MgCl2, 0.2 g; CaCl2, 27 were kept as similar as possible to prevent differential mg; and FeCl, .6H20, 0.2 mg, pH 7.0. Carbon sources loss of activity from influencing the results. were added at 0.4%. When histidine was used as a The glutamine synthetase assay was the non-bio- nitrogen source, the (NH4)2SO4 was omitted ard 0.2% synthetic transferase reaction described by Stadtman histidine was substituted. et al. (14). Blanks without adenosine diphosphate and Complex medium was Luria broth (LB) and con- arsenate were included for every assay condition, and tained per liter of distilled water: tryptone, 10 g; yeast the values were subtracted from those obtained with extract, 5 g; NaCl, 10 g; and glucose, 1 g. Agar was the reaction mixture. added at 1.5% to make either minimal or LB agar. Materials. L-Methionine-DL-sulfoximine, DL-methi- Culture conditions and extract preparation. onine sulfoxide, L-methionine sulfone, and DL-a- Stock cultures were kept on LB agar slants. Cells to be methyl glutamic acid were obtained from Sigma 668 BRENCHLEY J. BACTERIOL. ' ' 'A^MK-275 could be due to the inability of the cell to synthesize adequate quantities of glutamine >. loC >A' Seither to supply glutamine itself for protein A synthesis or supply glutamate made from glutamine the reaction. < 80 by glutamate synthetase

010~~ ~ ~ ~ ~ ~ ~ ~ ~ 2 60 8~~~~~~~~~~~~~0A

5 10 15 60 Preincubation Time (Min.) FIG. 1.20(-/Stability of glutamate synthetase activity. ~~~~~~~2 _ B - 0, Reaction started by glutamine addition. Preincu- 808v bation mixture included a-ketoglutarate and V NADPH. Av, Reaction started by NADPH addition. 40 - Preincubation mixture included a-ketoglutarate and glutamine. 520 Chemical Co. (St. Louis, Mo.). All other chemicals / t are reagent grade and commercially available. Methi- 10 onine sulfoximine was shown by paper chromatogra- phy to have no detectable contamination by gluta- 20-8 B mate. Contamination by glutamate(>1%)could haveand been detected.A, 80Rsr b RESULTS Xl7 Effect methionineof sulfoximineon y c o growth. Severaldeacompoundsc with structures resembling glutamate or glutamine were testedhave for their growth inhibitory effects on lawns of /5^ wild-type gltS and gltD cultures. Some differ- 10 ences were observed with L-methionine-DL-SUI- AfA &^ foximine and methionine sulfone, and their effects were examined further. Figure 2 shows the growth response of these three strains when 80 D cultures were inoculated into glucose minimal 2 media containing 1, 10, or 100 mM L-methio-o X nine-DL-Sld foximine. Theg ltD strainMK-275 is 0 highly inhibited by the compound, and their duration of the inhibition depends on the con- 20 centration of analogue added. This inhibition is D not specific inpreventingcells from initiating 10 growth after a lag since it also inhibits growing cells. MK-189(um tS) grows well but may be growthafateraylagesineinhibited slightly moreaciitythancasinbitsS the wildgroingyttype. I.2 rwhghblgnb2 4 6 8ehoiesloi10 12 Since glutamate dehydrogenase is essential for (hrs2)Time the growth of MK-189 (due to the absence ofs glutamate synthetase activity), its ability to mine (MS). Growth of the strains was followed in grow in the presence of L-methionine-DL-sulfoxi- glucose minimal media at 37 C. Symbols: 0, MK-247; mine shows that glutamate dehydrogenase ac- A, MK-275; 0, MK-189. A, No MS; B, 1 mMMS; C, tivity is not inhibited. The inhibition of 10 mM MS; D, 100 mM MS. VOL. 114, 1973 INHIBITION OF GLUTAMATE SYNTHESIS 669 Inhibition by methionine sulfone was similar to those shown in Fig. 2 except that the inhibiton period was not as long. The ability of both glutamine and glutamate to overcome the inhibition by 10 mM methionine sulfoximine in strain MK-275 is shown in Fig. 3. Glutamine can serve as both a glutamine 0 and glutamate source presumably due to the action of glutaminase converting the large quantities of glutamine to glutamate. (Gluta- .S- mate-requiring strains grow on either glutamate y or glutamine, but glutamine-requiring strains can only grow with glutamine.) It appears, therefore, that the growth inhibition of the gltD strain is due to its inability to make glutamine; however, the ability of exogenous glutamine and glutamate to restore growth could be misleading since they might simply be preventing entry of the structurally similar analogue. Thus, the Time (hrs) ability of glutamate to restore growth was FIG. 3. Reversal of methionine sulfoximine inhibi- reexamined by using histidine, which is struc- tion by glutamine and glutamate. Strain MK-275 was turally dissimilar but is degraded to ammonia, inoculated into glucose minimal medium containing glutamate, and foramide and can be used as a 10 mM methionine sulfoximine, and separated into glutamate source (1). The use of histidine as the three cultures. Symbols: 0, no other addition; A, sole nitrogen source in the presence of glucose glutamate addition at 50 ;ig/ml; 0, glutamine addi- also allowed growth of the MK-247 strain under tion at 50 &g/ml. conditions where it phenotypically resembles the gltD mutant. (It has been shown that mM concentrations of either analogue; however, growth in glucose-plus-histidine medium is a at lower concentrations (10 gg/ml) the methio- nitrogen limitation condition during which very nine did not fully reverse the inhibition. The little glutamate dehydrogenase activity is pres- ability of high methionine concentrations to ent.) Figure 4A shows the response of MK-247 restore growth may possibly be due to the growing in glucose-plus-histidine medium. exclusion of the analogues or some interaction MK-247 is more sensitive to the analogue in this between methionine and glutamate synthesis medium than when growing in glucose- regulation. These effects are being investigated. ammonia medium (compare with analogue ad- Inhibition of glutamate synthetase and dition in Fig. 6) as would be expected since glutamine synthetase activities. Previous re- glutamine synthetase is now necessary for am- ports demonstrated methionine sulfoximine in- monia assimilation. The cells readily overcome hibition of glutamine synthetase from sheep the inhibition, presumably because the histi- brain (10); however, its effects on a bacterial dine degradation is providing glutamate. The synthetase had not been explored, nor had its degradation of histidine to glutamate is impor- possible inhibition of the newly described gluta- tant for this resumption of growth (Fig. 4B). mate synthetase reaction been characterized. Strain MK-8 is blocked in the histidine de- To examine these effects, we assayed the ex- gradative pathway (as shown by its inability to tracts for the enzyme activities with and with- grow on histidine as a carbon and nitrogen out analogue addition. MK-275 cultures were source) but contains histidase, is able to obtain used to prepare extracts for the glutamate ammonia as a nitrogen source from the histi- synthetase assays since the results are not dine, and cannot form glutamate. When 10 mM complicated by residual glutamate dehydrogen- methionine sulfoximine is added to this culture, ase activity. To eliminate possible enzyme growth stops, and the culture does not resume instability during preincubation as a cause of full growth even after 22 h of incubation. the decreased activity (see Materials and Meth- Since both methionine sulfoximine and methi- ods), the assays were started by NADPH addi- onine sulfone structurally resemble methionine tion. The amount of inhibition was determined as well as glutamate and glutamine, the ability in two ways: (i) by starting the reaction in the of methionine to overcome the inhibition was absence of analogue, measuring the initial rate, also tested. At high concentrations (2 mg/ml) adding the analogue, and comparing the de- methionine allowed growth in the presence of 10 creased adsorbancy change with that originally 670 BRENCHLEY J. BACTERIOL. activity measured without the analogue. The cause of this high activity is not known, but it occurred only with extracts from cultures with glutamate dehydrogenase activity. The addition of 0.1 mM methionine sulfoximine gave no background activity nor did it inhibit the glutamate dehydrogenase activity. There was no background activity (when the L-methionine- DL-sulfoximine was substituted for the substrate) measured for either the glutamate syn- Time (Hrs) thetase (from a gltD strain) or for the glutamine FIG. 4. Effect of methionine sulfoximine on cul- synthetase assays even when the analogue was tures growing in glucose histidine medium. The arrow added at 100 mM. Methionine sulfone addition indicates the division of a culture and the addition of (10 mM) did not give the high background activ- methionine sulfoximine to give a concentration of 10 the mM. A, Strain MK-247; (0) no addition, A) MS ity nor did it inhibit glutamate dehydro- addition. B, Strain MK-8; (0) no addition, (A) MS genase reaction. addition, (0) culture inoculated into medium con- Effect of methionine sulfoximine addition taining only histidine as sole carbon and nitrogen on enzymes during growth. Since the methio- source. nine sulfoximine affected both the glutamate synthetase and glutamine synthetase activities observed. (ii) The analogue was included in the reaction mixture, and the observed, initial en- 10 4 A zyme activity was compared to the amount of activity found without the analogue. The meth- u 80~~~A ods gave comparable results. 80 Figure 5A shows the inhibition by MS of the glutamate synthetase activity. High concentra- 60 tions (10 mM) are required for complete inhibi- 40 tion, but it still occurs at a much lower concen- C) tration than reported for glutamate inhibition o0I of this enzyme (about 20% activity remaining at 40 150 mM glutamate; reference 8). The glutamate 0 synthetase activity is more sensitive to inhibition by methionine sulfone, with only 10% activity remaining at 1 mM concentration (Fig. 5B). Figure 6A shows the inhibition of gluta- MS Concentration (mM ) MSF Concentration (mM ) mine synthetase activity by methionine sulfoximine addition. Glutamine synthetase is much FIG. 5. Inhibition of glutamate synthetase activ- more sensitive than the glutamate synthetase, ity. A, Methionine sulfoximine (MS) addition; B, with 70% inhibition by the 0.01-mM analogue. methionine sulfone (MSF) addition. Since each of the 12 subunits of glutamine f60- , f 0 I I 1 synthetase can exist in two forms (unadenyly- lated or active form in vivo and the adenylylated form which is considered to be inactive in vivo), the activity in the presence of magnesium (which inhibits the activity of the adenylylated mlethOnin sufn IMFadiin form) was also determined. Analogue inhibition in this assay appeared similar to that observed without magnesium. The glutamine synthetase .E 40 D F l\0 activity was also sensitive to methionine sulfone (Fig. 6B) except that the enzyme was much less sensitive to this analogue and 1 mM was required for 80% inhibition. An effort was made to determine whether the methionine sulfoximine could inhibit glutamate MS Concentration (mM) MSF Concentration (mM) dehydrogenase activity. The addition of the FIG. 6. Inhibition ofglutamine synthetase activity. analogue (10 mM) to the reaction mixture gave A, Methionine sulfoximine (MS) addition; B, methio- background activity that was 50% of the total nine sulfone (MSF) addition. VOL. 114, 1973 INHIBITION OF GLUTAMATE SYNTHESIS 671 in extracts, it was of interest to examine the effect of these enzymes when the analogue was 100- added during cell growth. Equal portions of an exponentially growing culture were inoculated into separate flasks containing the same volume 60- of prewarmed glucose minimal medium. Sam- ples were removed at the times indicated (Fig. :.. ;- 200 / D. 7), and the cells were harvested immediately for 20 enzyme assays. Figure 8 shows the specific 4. activities of these enzymes plotted against the .4 .1 _ Klett reading at the time of sampling. As _,) a Uf) opposed to other reports showing glutamate B dehydrogenase and glutamine synthetase levels Z51 100- 311000 varying with the growth of the culture, these enzymes appear relatively constant during ex- 600 A A ponential growth (between 50 and 100 Klett 60- units). Glutamate synthetase appears to in- .2 _ o.0. crease; however, considering the instability of 20- 200 .1 this activity, the significance of this result is -...... 0..... unknown. The dramatic effect of methionine 40 60 80 100 sulfoximine addition is the loss of glutamine Klett Units synthetase activity (presumably due to the FIG. 8. Enzyme activities from MK-247 cultures irreversible binding by the analogue). The essen- growing with or without methionine sulfoximine addi- tially complete disappearance and subsequent tion. Results are expressed as specific activities of acquisition of activity are correlated with the enzyme measured from cells harvested at Klett read- inhibition and resumption of growth. Thus, it ings shown by numbered arrows in Fig. 7. A, Samples appears that the major effect of methionine from the culture to which 10 mM methionine sulfoxi- sulfoximine is its irreversible binding to the mine was added at time indicated by arrow. B, Sam- culture without any addition. Symbols: 0, glutamine synthetase. The reappearance of glu- ples from tamine synthetase activity in samples four and glutamate synthetase activity; A, glutamate dehydrogenase activity; 0, glutamine synthetase activity; *, five could be due to synthesis of new enzyme glutamine synthetase activity determined in presence which is resistant to the analogue or to the of magnesium. removal of free analogue either by its binding to the enzyme or its degradation. Glutamine synthetase was assayed in extracts from cultures Inhibition by other compounds. Other com- grown with and without methionine sulfoximine pounds were surveyed for their ability to inhibit addition. The enzyme activity produced in enzyme activity (Table 2). High concentrations these cultures is still sensitive to methionine of a-methyl glutamate are slightly inhibitory to sulfoximine inhibition in vitro (Table 1). glutamate dehydrogenase activity but not at all to the glutamate synthetase. Methionine sulfox- 15 ide inhibits glutamate synthetase at 10 mM 4 ~~~45 concentration. There was no growth inhibition by 10 mM DL-a-methyl-glutamate or DL-methi- 80 2 onine sulfoxide for either strains MK-247, MK-189, or MK-275 growing in glucose am- 40 monia medium. (This may be due partly to the I) use of the DL isomers since DL-methionine-DL- sulfoximine is much less inhibitory than L- 20/ methionine-DL-sulfoximine.) None of these A B compounds appears to allow growth of glutamate- or strains and may 0 2 0 1 2 glutamine-requiring Time (hrs.) not be incorporated into protein. FIG. 7. Growth of MK-247. A, Methionine sulfoximine added to give 10 mM final concentration at the DISCUSSION indicated the arrow. B, No additions. time by large Traditionally, glutamate synthesis was Samples for enzyme determinations were removed (at the times indicated by the numbered arrows), rapidly thought to occur primarily from a-ketoglutarate cooled, and centrifuged. The results of the enzyme and ammonia by the action of a nonregulated assays for these samples are presented in Fig. 8. glutamate dehydrogenase. Recent investiga- 672 BRENCHLEY J. BACTERIOL. TABLE 1. Inhibition of glutamine synthetase in versibly with the glutamate synthetase in vivo, extracts prepared from cultures grown with or without then its effect might not be seen in this experi- methionine sulfoximine ment. The lack of inhibition of the glutamate Inhibition (%) synthetase-negative strain MK-189 shows that the analogue affects some portion of the second MS concna (mM) Treated Nontreated pathway to glutamate synthesis since gluta- culture culture mate dehydrogenase is necessary for its growth. extractb extractc Furthermore, the ability of glutamate, gluta- 0.1 97 90 mine, and histidine (glutamate source) to over- 0.01 60 50 come the inhibition suggests that it is the 0.001 13 15 prevention of glutamate formation by glutamine deprivation which is the important effect. a L-Methionine-DL-sulfOXimine. This is consistent with previous findings bExtracts were prepared from cells taken in sample (Brenchley and Magasanik, manuscript in no. five (Fig. 7a) from cultures growing in the presence histidine as a of 0.01 M methionine sulfoximine. preparation) that cells grown with c Extracts were prepared from cells taken in sample carbon and nitrogen source have low levels of no. five (Fig. 7b). glutamine synthetase, indicating that high activities of glutamine synthetase are not neces- tions have established that a second pathway sary to provide glutamine for protein synthesis exists and that the glutamate dehydrogenase is if glutamate is provided. The less severe growth regulated. The isolation of a mutant lacking inhibition of MK-275 by methionine sulfone is glutamate dehydrogenase activity demon- consistent with the glutamine synthetase being strated that this enzyme was not essential for more resistant to methionine sulfone than to growth in a number of different conditions methionine sulfoximine (Fig. 6). tested. This investigation with methionine sul- The importance of the inhibition by methio- foximine and methionine sulfone shows that the nine sulfoximine and methionine sulfone comes glutamate dehydrogenase negative strain does from their utility in future investigations of the have a phenotype; it is sensitive to these ana- structurally complex glutamine synthetase. Bi- logues. The wild-type Klebsiella and a mutant ochemical analyses have shown that this en- lacking the glutamate synthetase activity are zyme consists of 12 subunits, has modified both quite resistant to the analogues; however, forms, and that the regulation of the modifica- when they are added to cultures of the gluta- tion processes is itself very complicated (2, 13). mate dehydrogenase-negative strain, long lags Certainly, the physiological and genetic charac- occur before growth resumes. terization of mutants resistant to these ana- Studies with methionine sulfoximine and methionine sulfone inhibition of enzyme activ- TABLE 2. Inhibition ofglutamate dehydrogenase and ity demonstrate that both the glutamate syn- glutamate synthetase activities by analogues thetase and glutamine synthetase are sensitive, Concn En- Inhibi- but the glutamine synthetase is sensitive at CompoundaCompounda (mM) zymeb tion (%) much lower concentrations of methionine sulfoximine. Complete inhibition of the glutamate DL-a-Methyl glutamate 10 GltD 39 synthetase occurs at 10 mM L-methionine-DL- DL-a-Methyl glutamate 10 GltS 0 sulfoximine, whereas 0.01 mM causes about Acetamide 10 GltD 7 of Acetamide 10 GltS 100 70% inhibition the glutamine synthetase Acetamide 1 GltS 20 activity. The apparent 30% residual activity Hydroxylamine 100 GltS 16 possibly could be due to protection by gluta- Hydroxylamine 10 GltS 10 mate or ammonia in the reaction mixture since DL-Methionine sulfoxide 10 GltS 100 Ronzio et al. (10) found that these could prevent L-Methionine sulfone 1 GltS 90 the inactivation of sheep brain glutamine syn- L-Methionine sulfone 10 GltD 0 thetase. Cesium chloride 10 GltD 0 The inhibition at lower concentrations and the correlation between the reappearance of a Analogues were added to reaction mixtures prior to the start of the reaction by substrate addition. glutamine synthetase activity and resumption Solutions of the analogues were neutralized to prevent of growth (Fig. 7 and 8) suggest that it is pH change upon their addition to the reaction mix- primarily the inhibition of the glutamine syn- ture. thetase which interferes with normal growth. If, b GltD, Glutamate dehydrogenase; GltS, glutamate however, the methionine sulfoximine binds re- synthetase. VOL. 114, 1973 INHIBITION OF GLUTAMATE SYNTHESIS 673 logues would contribute to the understanding of of glutamine synthetase adenylylation and deadenyly- these enzymes. lation is mediated by metabolic transformation of P1,-regulatory protein. Proc. Nat. Acad. Sci. U.S.A. In addition, recent findings have shown a 68:2949-2953. correlation between high levels of glutamine 3. Heathcote, J., and J. Pace. 1950. Inhibition of the growth synthetase and low glutamate dehydrogenase of Leuconostoc mesenteroides by the toxic factor from levels and have suggested the involvement of 'agenized' zein: reversal by L-glutamine. Nature (Lon- don) 166:353-354. glutamine synthetase itself in regulation. If the 4. Hrebicek, J., J. Kolousek, M. Wiederman, and 0. Cha- reappearance of glutamine synthetase activity ramza. 1971. Changes of the incorporation of [7"Sel- represents an increase level of enzyme (inactive methionine and of electrical activity in various brain enzyme bound to inhibitor plus new, active structures of the cat after administration of methionine sulphoximine. Brain Res. 28:109-117. enzyme) and high levels of glutamine synthe- 5. Lowry, O., J. Rosebrough, A. Farr, and R. Randall. 1951. tase (or another product under the same con- Protein measurement with the Folin phenol reagent. J. trol) repress glutamate dehydrogenase produc- Biol. Chem. 193:265-275. tion, then the level of glutamate dehydrogenase 6. MacPhee, D., I. Sutherland, and J. Wilkinson. 1969. Transduction in Klebsiella. Nature (London) activity should decline after L-methionine-DL- 221:475-476. sulfoximine addition. The results in Fig. 8A 7. Manning, J., S. Moore, W. Rowe, and A. Meister. 1969. showing the lower levels found in the fifth Identification of L-methionine-S-sulfoximine as the sample and other preliminary results suggest diastereoisomer of L-methione-SR-sulfoximine that inhibits glutamine synthetase. Biochemistry that this occurs. 8:2681-2685. Two different and genetically (by transduc- 8. Meers, J., D. Tempest, and C. Brown. 1970. Glutamine tion) unlinked mutations have been found in (amide):2-oxoglutarate amino transferase oxido-reduc- Klebsiella, which cause a glutamine require- tase (NADP), an enzyme involved in the synthesis of glutamate by some bacteria. J. Gen. Microbiol. ment. Thus far it has been difficult to demon- 64:187-194. strate that either is the gene encoding for the 9. Pace, J., and E. McDermott. 1952. Methionine sulphoxi- enzyme itself. The possible isolation of mutants mine and some enzyme systems involving glutamine. with a glutamine synthetase resistant to methi- Nature (London) 169:415-416. 10. Ronzio, R., W. Rowe, and A. Meister. 1969. Studies on onine sulfoximine or methionine sulfone would the mechanism of inhibition of glutamine synthetase allow genetic analysis to establish which (if by methionine sulfoximine. Biochemistry 8:1066-1075. either) of these mutations encode the glutamine 11. Rowe, B., and A. Meister. 1970. Identification of L- synthetase. This work is currently in progress. methionine-S-sulfoximine as the convulsant isomer of methionine sulfoximine. Proc. Nat. Acad. Sci. U.S.A. ACKNOWLEDGMENTS 66:500-506. 12. Rowe, B., R Ronzio, and A. Meister. 1969. Inhibition of I thank Boris Magasanik for helpful discussions. glutamine synthetase by methionine sulfoximine. This work was supported by grants RC-Bacteria from Studies on methionine sulfoximine phosphate. Bio- Research Corporation and by Public Health Service grant 1 chemistry 8:2674-2680. R01 GM 19423-01 from the National Institute of General 13. Shapiro, B., and E. Stadtman. 1970. The regulation of Medical Sciences. glutamine synthesis in microorganisms. Annu. Rev. Microbiol. 24:501-524. LITERATURE CITED 14. Stadtman, E., A. Ginsburg, J. Ciardi, J. Yeh, S. Hennig, 1. Brill, W., and B. Magasanik. 1969. Genetic and metabolic and B. Shapiro. 1970. Multiple molecular forms of control of histidase and urocanase in Salmonella ty- glutamine synthetase produced by enzyme catalyzed phimurium strain 15-59. J. Biol. Chem. 244:5392-5402. adenylylation and deadenylylation reactions. Advan. 2. Brown, M., A. Segal, and E. Stadtman. 1971. Modulation Enzyme Regul. 8:99-118.