The Enzymatic Synthesis of IV-Methylglutamic Acid

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 4, Issue of February 25, 1966

Printed in U.S.A.

The Enzymatic Synthesis of IV-Methylglutamic Acid

(Received for publication, September 15,1965)

W. V. SHAW, L. TSAI, AND E. R. STADTMAN

From the Laboratory of Biochemistry, National Heart Institute, National Institutes of Health, Bethesda, Mary- land 20014

SUMMARY

A species of Pseudomonas which grows on methylamine as the sole source of carbon and nitrogen was isolated from soil enrichment cultures. The lack of pigmentation in the organism and its unusual substrate specificity suggest that it is a species of Pseudomonas distinct from those previously isolated from one carbon substrate enrichments. When grown on methylamine, the new Pseudomonas sp. elaborates an enzyme that catalyzes a reaction between methylamine and L-glutamate to form a hitherto unknown biological intermediate, N-methylglutamate, and ammonia. The enzyme is absent from cells grown on glycerol and ammonia in the absence of methylamine. A number of possible reaction mechanisms to explain N-methylglutamate synthesis have been studied with the conclusion that the reaction is reversible and most likely proceeds by a direct displacement mechanism. Evidence from studies with 15N-labeled substrates are not compatible with a transmethylation mechanism. The possible role of N-methylglutamate synthetase in the one carbon metabolism of this organism is suggested by the results of pulse-labeling experiments showing the appearance of isotope from 14C-methylamine in N-methylglutamate, sarcosine, serine, alanine, and aspartate in the order indicated.

Nonphotosynthetic bacteria utilizing one carbon compounds as their sole source of carbon and energy have been isolated in several laboratories and have prompted studies on the mechanisms involved (l-4). The general aspects of bacterial one carbon metabolism have been reviewed recently (5). Formate, formamide, formaldehyde, methanol, methylamine, and methane have been found to support growth as have polycarbon compounds such as methylurea which must be degraded to one carbon units prior to assimilation (6).

This report describes the isolation of a species of Pseudomonas that utilizes methylamine as the sole source of carbon, nitrogen, and energy. The formation of a new biological compound, N- methylglutamic acid, from methylamine by cell suspensions and cell-free extracts is described. The enzyme system responsible for its synthesis is contrasted with those catalyzing other one carbon transfer reactions, and evidence is presented favoring a

mechanism of direct displacement for the synthesis of N-methylglutamate from methylamine and glutamate.

EXPERIMENTAL PROCEDURE

Isolation and Growth of Organism-Aerobic enrichment cultures prepared from local soil samples in which methylamine was the only added carbon source yielded a number of organisms capable of sustained growth on methylamine in subculture. Two such organisms were isolated in pure culture, one of which resembled in pigmentation and growth characteristics the organism Pseudomonas AM 1 described by Peel and Quayle (3). The other organism was also an aerobic, gram-negative, motile rod with a single polar flagellum conforming to the description of Pseudomonas sp. but was not pigmented and failed to show the clumping noted when Pseudomonas AM 1 is grown on methylamine. The nonpigmented organism was chosen for further study and is the subject of this report.

The growth medium used contained 0.1 M methylamine hydrochloride as the carbon and nitrogen source, 0.02 M potassium phosphate buffer (pH 7.0), and mineral salts. The latter included 5.0 ml of Stock Solution A and 1.0 ml of Stock Solution B per liter of culture medium. Solution A contained per liter: MgS04 .7Hz0, 20 g; CaC12.2H20, 2 g; and FeSO*. 7H20, 2 g. Solution B contained 0.5 g per liter of both MnS04 .4Hz0 and NazMbOl.2H20. With 0.05 M ammonium sulfate as the nitrogen source the nonpigmented Pseudomonas sp. failed to grow on methanol, formate, formaldehyde, or formamide when these substrates were tested at 0.1 o/0 and 0.5% concentrations. Similarly, dimethylamine, trimethylamine, methylaminoethanol, methyl- ethylamine, methylurea, methionine, dimethylglycine, and di- methylaminoethanol failed to support growth. Other ineffective substrates for growth included ethylene glycol, ethanol, glycolate, glyoxylate, oxalate, malate, succinate, fumarate, citrate, glycine, and serine. Good growth was obtained, however, with L-glutamate, L-aspartate, pyruvate, lactate, glycerol, glucose, fructose, ribose, lactose, and sucrose. The organism grows well at 25” and at 30” but not at 37”. The mean generation time at 30” on a rotary shaker is 8 hours with methylamine. No requirements for growth factors have been noted. Storage of the organism on agar slants prepared with the standard methylamine medium has yielded viable cultures for as long as 1 year when the tubes are kept sealed and stored at 3”. Large scale cultivation of the organism was readily accomplished with the standard medium as long as adequate aeration was provided. Cells were generally harvested with the Sharples centrifuge, were washed once with

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936 N-Methylglutamate Synthetase Vol. 241, No. 4

0.02 M potassium phosphate buffer (pH 7.0), and were quickly frozen under liquid nitrogen. Cells prepared in this manner and stored at -40” yielded active extracts for at least 1 year.

Preparation of Cell-free Extracts-Freshly harvested or frozen cells were suspended in a medium containing 20 InM Tris-HCl (pH 8.5) and 5 rnnl 2-mercaptoethanol. Cells were broken by extrusion in an Aminco French pressure cell at 9,000 psi. Un- usually viscous extracts were treated with deoxyribonuclease (5 pg per ml) for 10 min at 30” before centrifugation at 30,000 x 9 for 60 min at 3”. The clear amber supernatant fluid was used without further treatment (crude extract) or was subjected to dialysis against 100 volumes of the mercaptoethanol buffer solution for 8 to 12 hours. All operations were performed at O-3”.

Assay of Product Formed from 14C-Methylamine by Cell Extracts -Standardized assay conditions were adopted following the tentative identification of the product as N-methylglutamic acid. The reaction was started by the addition of cell-free extract to a 3-ml test tube containing 0.5 to 1.0 ml, final volume. Each incubation contained 100 mM Tris-HCl buffer (pH 8.5) and 5 InM

mercaptoet,hanol in addition to 100 mM r4C-methylamine hydrochloride. Modifications of the standard conditions are described in the presentation of experimental data. The reaction was terminated by the addition of 0.2 volume of 20% trichloroacetic acid to the incubation mixture. After removal of the precipitated protein by centrifugation, an aliquot (0.3 to 0.6 ml) of the supernatant fluid was pipetted onto a small (20 X 5 mm) Dowex 50 (H’) column (100 to 200 mesh). Suitable columns for routine analyses were fashioned from Pasteur pipettes and were dis- carded after each assay. The anionic and neutral products were eluted with water to a final volume of 2.0 ml at which point the effluent pH approached neutrality. The cationic fraction containing amino acids was then eluted with 2.0 ml of 2 N NH40H. An appropriate aliquot of the ammonia eluate was added to 16 ml of a solution prepared by mixing 6 volumes of 95% ethanol and 10 volumes of 0.4% diphenyloxazole in toluene. Radioac- tivity was then measured in a Packard Tri-Carb scintillation spectrometer. The simultaneous determination of radioactivity due to 3H and 14C involved counting samples at 725 volts and again at 1125 volts. At the lower voltage, only carbon was counted, The relative efficiencies for 14C at the two voltages were determined independently so that the contribution of 14C radioactivity at the higher voltage could be calculated and 3H

TABLE I

Identification of N-methylglutamate as product of enzymatic reaction

Two isomers of N-methylglutamate are included for compari- son. Ascending chromatography on Whatman No. 1 paper in solvent systems as follows: A, tert-butyl alcohol-methyl ethyl ketone-formic acid-water (40:30:15:15); B, tert-butyl alcohol- methyl ethyl ketone-concentrated NHdOH-water (40: 30: 10:20) ; C, n-butyl alcohol-acetic acid-water (50:25:25) ; D, methanol- pyridine-water (80:4:20).

I RF in Compound

A

r,-Glutamate......................... 0.53 nn-N-Methylglutamate. 0.65 Radioactive enzymatic product. 0.65 nn-a-Methylglutamate. 0.73 L-Glutamate-r-methyl ester. 0.78

B C -__

0.15 0.43 0.20 0.48 0.20 0.48

D

0.41 0.54 0.54

determined by the difference method. Under the standard assay conditions, no methylamine radioactivity was eluted from the Dowex 50 (H+) columns by 2.~ NH,OH in controls incubated in the absence of enzyme or, alternatively, in the presence of enzyme which had been heated for 5 min at 100”.

The ammonia eluate was lyophilized and redissolved in an appropriate volume of water for ascending chromatography on Whatman No. 1 paper with the solvent systems described by Fink, Cline, and Fink (7). For the isolation of radioactive product on a preparative scale, the incubation mixture was increased %ppropriately, the cation fraction isolated in the usual fashion on a larger column of Dowex 50 (H+), and the eluate was streaked on a large sheet of Whatman No. 3MM paper. After chromatography in Solvent B (Table I), the paper was dried and the radioactive product was localized by autoradiography. The product was eluted and applied to a second sheet of paper for chromatography in Solvent A. The product isolated from the second chromatography step appeared to be homogeneous and free of glutamate and other amino acids as judged by chromatography in additional solvents or by high voltage electrophoresis. Amino acids were detected on chromatograms by spraying with 0.1 y0 ninhydrin in n-butyl alcohol followed by heating at 90” for 5 min. N-Methylglutamate was localized and identified with the use of the spray reagent for secondary amines described by Sweeley and Horning (8). High voltage paper electrophoresis was carried out at 1500 volts and 90 ma in 0.05 M potassium phosphate (pH 7.0).

The experiments designed to detect IbN incorporation in N- methylglutamate from appropriately enriched substrates were processed by the usual Dowes 50 (Hf) procedure. The ammonia eluate was applied to Whatman No. 3MM paper for ascending chromatography in Solvent C (Table I). The presence of tracer r4C-methylamine in the incubation permitted the localization of N-methylglutamate by autoradiography. The radioactive spot was eluted and counted, and the yield of N- methylglutamate was calculated from the known specific activity of r4C-methylamine. The product was then diluted with a known amount of authentic N-methylglutamate and crystallized to constant specific activity. N-Methylglutamate was converted to ammonia by Kjeldahl digestion, and the sample analyzed for r5N content by Gollub Analytical Services, Berkeley Heights, New Jersey.

Chemical Synthesis of N-Methylglutamic Acid-N-Methyl- glutamic acid was prepared by a modification of the method described by Knoop and Oesterlin (9). Dimethyl sulfate, 80 ml, was added with stirring over 1 hour to a solution of 33 g of N-tosylglutamic acid, prepared from toluene sulfonyl chloride and n-glutamic acid, in 350 ml of 20% KOH at 70-80”. After this was stirred for 2 hours, an additional 50 ml of dimethyl sulfate were added slowly. The pH of the mixture was kept above 12 during the course of the reaction by further additions of 20% KOH as needed. The mixture was stirred for 18 hours at 70- 80”, cooled, and acidified with dilute H2S04. After the K2S04 was removed by filtration, the filtrate was extracted six times with 200-ml portions of ether. The combined ether extracts were dried over MgS04, filtered, diluted with 50 ml of toluene, and concentrated to less than 100 ml. On cooling, the crystalline mass was collected by filtration, washed with toluene, and dried under vacuum. The yield of the N-methyl compound (m.p. 130-134”) was 22 g. Another crop of crystals was obtained on concentration of the mother liouor. N-Methvl-N-tosvlglutamic


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acid was purified by recrystallization from ethanol-toluene and from water.

The following procedure was used for the hydrolysis of the above intermediate. N-Methyl-N-tosylglutamic acid, 3.15 g, was dissolved in 10 ml of concentrated HCl and heated in a sealed tube over a steam bath for 6 hours. The tube was cooled, opened, and the contents diluted with 10 ml of water. The mixture was extracted continuously with ether for 12 hours after which the aqueous phase was evaporated to dryness at 50”. The resulting solid material was taken up in a small volume of water, and the solution was passed over a column of Dowex 50 (H+). The column was washed with water followed by 2 N

NH,OH. The ammonia eluate was collected, neutralized with formic acid, and lyophilized. The residue was washed with ethanol and dried in a vacuum, yielding 1.1 g of N-methylglutamic acid, m.p. 153155”. Two recrystallizations from water gave an analytically pure sample, m.p. 158-162°,1 [cr,] 0”. The elemental analysis was performed by Micro-Tech.

C&N04

Calculated: C 44.63, H 6.86, N 8.66 Found : C 44.93, H 6.83, N 8.76

Radioactive N-methylglutamic acid was prepared by incubat- ing 14C-methylamine of high specific activity (3.3 FC per pmole) with n-glutamate and enzyme under the usual assay conditions. The product was isolated as described, mixed with crystalline (racemic) N-methylglutamic acid, and recrystallized to constant specific activity from a minimum volume of hot water.

Protein was estimated by the biuret method of Gornall, Barda- will, and David (11). Ammonia was determined by the diffusion method of Seligson and Seligson (12). Under the conditions of this procedure, the small amounts of methylamine liberated fail to interfere with the color yield from the reaction of ammonia with Nessler’s reagent. For experiments such as those described in Table IX, the diffusion was allowed to proceed for 12 hours at 37” in the presence of 2 N NaOH rather than Na2C03. Under the latter conditions, the recovery of methylamine was greater than 95%.

Spectrophotometric assays were carried out in the Beckman model DU spectrophotometer. Glutamic dehydrogenase was measured by a modification of the method of Olson and Anfinsen (13). The incubation conditions were similar to those described for N-methylglutamate synthesis. TPNH was the preferred pyridine nucleotide and was used for all such assays.

All amino acids and their derivatives were obtained from Nutritional Biochemicals unless stated otherwise. Glutaconic (trans-pentenedioic) acid was obtained from K and K Labora- tories. The N-formyl and N-acetyl derivatives of L-glutamate were the gift of Dr. Alan Mehler. nn-three-P-Methylaspartate was prepared by Dr. H. A. Barker and P-glutamate was obtained from Dr. Alton Meister. Both 14C- and 3H-labeled methylamine hydrochloride were the products of New England Nuclear as was I%-methyl-S-adenosylmethionine. The 14C-methylcobalamin was prepared by Dr. B. Blaylock by the method of Smith et al. (14). Both lSN+glutamic acid and l5N-methylamine hydrochloride were obtained from Merck Sharp and Dohme of Canada, Ltd. Deoxyribonuclease (pancreatic), n-glutamate decarboxylase (Esch~ichia coli), and alcohol dehydrogenase (horse liver) were obtained from Worthington. Glucose g-phosphate and

1 Melting point reported, 156-158” (10).

glucose g-phosphate dehydrogenase were purchased from Sigma, and pyridine nucleotides were the products of Pabst.

RESULTS

N-Methylglutamate as Product of Methylamine Metabolism-Al- though numerous attempts to demonstrate the oxidation of 14C-methylamine by cell-free extracts were unsuccessful, preliminary experiments indicated that the incubation of crude extract with 14C-methylamine yielded a product which was retained on Dowex 50 (H+) and could be eluted readily with 2 N NH40H. Large scale incubations provided sufficient material for paper chromatographic analysis, which indicated several useful proper- ties of the unknown product. The purified material (see “Ex- perimental Procedure”) gave a weakly positive ninhydrin reaction and failed to cochromatograph with any of the naturally occurring amino acids tested (Fig. I). The ninhydrin color yield was considerably less than that estimated for the expected yield of 1 mole of methylamine per mole of product, and the intensity was not increased by prior treatment of the product with acid or alkali. The radioactive product gave a strongly positive test for a secondary amine (8), and its relative mobility in several solvent systems paralleled that of glutamic acid (Table I). The unknown compound migrated toward the anode in high voltage electrophoresis at pH 7 indicating a net negative charge. These are characteristics expected of N-alkyl dicarboxylic amino acids, and, in view of the source of labeled carbon, N-methylglutamic acid was proposed as the unknown product. The addition of enzymatically prepared radioactive product to authentic N-methylglutamic acid yielded a product with constant specific activity on recrystallization from water (2640, 3090, and 2960 cpm per mg on successive recrystallizations). Chromatography of this material in four solvent systems yielded in each case a

0 GIY C Threo

0 Ser

(I I--t-BuOH/MEK/NH+OH

FIG. 1. Two dimensional chromatogram of the radioactive product formed from ‘4C-methylamine by cell-free extracts. The shaded spot represents the location of the unknown compound by autoradiography. The unknown was later identified as N-methylglutamate as described in the text. Ascending chromatography on Whatman No. 1 paper with Solvents A and B of Table I.


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TABLE II

Effect of dialysis on formation of radioactive product from 14C-methylamine

Each mixture contained crude enzyme (30 mg of protein) or dialyzed enzyme (24 mg of protein) in addition to 100 rnM Tris-HCl (pH 9.0) and 100 mM i4C-methylamine (specific activity, 6000 cpm per pmole). Incubation was at 30” for 30 min in a final volume of 2.0 ml. Boiled extract was prepared by heating the crude extract for 5 min at 100” followed by centrifugation to remove the precipitated protein. The supernatant fluid was either added directly (“untreated”), passed over a cation exchange resin column. or treated with charcoal (20 mg of Norit per ml of boiled extract) before addition.

Enzyme used Boiled extract addition

None Crude enzyme Dialyzed for 4 hrs Dialyzed for 4 hrs Dialyzed for 4 hrs Dialyzed for 4 hrs

Dialyzed for 4 hrs Dialyzed for 4 hrs Dialyzed for 18 hrs Dialyzed for 18 hrs

Untreated None None Untreated Dowex 50 (H+) effluent Dowex 50 (H+) NHIOH elu-

ate Dowex 50 (Kc) effluent Charcoal-treated None Untreated

TABLE III

C-Ivii;vFmine

incorporated into product

patms

0.4 7.9 2.5

6.7 2.9 5.4

9.2 7.1

0.2 2.2

ZdentiJication of L-glutamate as active principle in boiled extract

Incubation was for 20 min at 30” under the standard assay conditions. An aliquot of boiled cell extract (containing 2.9 rmoles of n-glutamate as determined by assay with E. coli glutamic acid decarboxylase) was added to the dialyzed enzyme as indicated. The specific activity of W-methylamine was 6000 cpm per pmole.

Additions ‘GMethylamine carbon incorporated into product

None ................................... Boiled extract. ......................... Boiled extract treated with glutamic de-

carboxylase ........................... L-Glutamate only (3 pmoles) ...........

palms

0.6 2.3

1.1 2.4

coincidence of the single radioautograph density with a blue

spot that appeared on the chromatogram after treatment of the

latter with the secondary amine reagent (Table I). Glutamate Requirement-After dialysis, the crude extract

showed a diminished ability to catalyze the formation of N- methylglutamate unless boiled cell extract was added to the incubation. The ability of boiled extract to stimulate formation of

N-methylglutamate was abolished by treatment of the boiled extract with Dowex 50 (Hf) but not with Dowex 50 (K+) or charcoal

(Table II). Furthermore, the factor retained on Dowex 50 (Hf) was readily eluted with dilute NH,OH. After removal of the excess ammonia, this eluate replaced the boiled cell extract. The identification of L-glutamate as the reactive component of the boiled preparation was made possible by the two observations summarized in Table III. The prior incubation of

boiled cell extract with glutamic decarboxylase (E. coli) destroyed

its ability to stimulate N-methylglutamate formation by dialyzed enzyme. Furthermore, the stimulation by L-glutamate was com- parable in degree to that noted with the boiled cell extract.

The discovery that L-glutamate is required for %-methylamine utilization suggested that N-methylglutamate might be formed by Reaction 1, in which ammonia would also be a product.

L-Glutamate + methylamine + N-methylglutamate + ammonia (1)

This conclusion is supported by the data of Fig. 2, which show that stoichiometric amounts of ammonia and N-methylglutamate are produced concurrently and that the total amount ultimately produced is approximately equal to the amount of L-glutamate present initially.

Specificity of Reaction-In order to determine whether the reaction with methylamine is specific with respect to L-glutamate, we examined a number of other amino acids for their ability to stimulate the incorporation of radioactivity from i4C-methylamine into the amino acid fraction. Table IV summarizes the results of two such experiments, wherein it can be seen that the reaction is highly specific with respect to L-glutamate. The sole apparent exceptions are n-glutamine and L-glutamate-y-methyl ester; it is notable that they are both labile glutamate derivatives. Paper chromatography of the reaction products in each instance indicated that the sole labeled product was N-methylglutamate and that extensive hydrolysis of both glutamine and y-methylglutamate had occurred.

Optimum Conditions for N-Methylglutamate Synthesis-The pH optimum for the reaction as determined in pyrophosphate buffer is 8.3 (Fig. 3). Essentially similar profiles have been obtained with phosphate, Tris, and borate buffers over the range of pH 7 to 9. Although preliminary experiments were carried out without the addition of a mercaptan to the reaction mixture subsequent studies suggested a more rapid aging of frozen ex,

6 ---_------_--------~

aNHJ

. N- ‘4 CHJ - Gfutamafe

15 30 60 MINUTES

FIG. 2. Time course and stoichiometry of the reaction. In addition to i4C-methylamine (specific activity, 6000 cpm per pmole) the reaction mixture contained 6 rmoles of L-glutamate and dialyzed enzyme (20 mg of protein) in a final volume of 1.0 ml. Incubation was at 30” in the presence of Tris-HCl, pH 8.5, 100 mM, and 2-mercaptoethanol, 5 mM.


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TABLE IV

SpeciJicity of reaction

TABLE V

Effect of sulfhydryl inhibitors

Each tube contained: Tris-HCl, pH 8.5, 100 mrvr; 2-mercapto- Crude enzyme prepared in the presence of 2-mercaptoethanol ethanol, 5 mM; r4C-methylamine-HCl, 100 mM; dialyzed enzyme, (5 mM) was passed”over a Sephadex G-25 column immediately 25 mg of protein; and additions, 10 mM, as indicated, in a final before use to remove thiol compounds. Incubation was in a volume of 1.0 ml. Reaction was at 30” for 20 min. The specific final volume of 0.5 ml for 20 min at 30” under standard conditions. activity of i4C-methylamine was 24,500 cpm per pmole. The specific activity of methylamine was 9000 cpm per pmole.

Addition

Experiment 1 None...... L-Glutamate. L-Alanine L-Phenylalanine. r-Aminobutyrate. Glycine. L-Histidine L-Leucine. L-Valine. :. Ethanolamine

Experiment 2 None ...................... L-Glutamate ............... n-Glutamate ............... n-Pyroglutamate ........... nn-threo-P-Methylaspartate. @Glutamate. ............... LlGlutamine ................ L-Glutamate-r-methyl ester. N-Formylglutamate ......... N-Acetylglutamate .......... nn-p-Hydroxyglutamate. .... on-a:-Methylglutamate. .....

W-Methylamine carbon incorporated into amino

acid fraction

0.2 4.9 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2

0.4 5.2 0.4 0.4 0.4 0.4 4.2 3.8 0.4 0.4 0.4 0.4

Inhibitor

None p-Chloromercuriphenylsulfonate

N-Ethylmaleimide

Iodoacetate

Arsenite

‘inal concentration of inhibitor

-Methylglutamate synthesized

pmoles

3.4 0.2 0.1 0.2 0.4 0.5 0.2 2.4 0.9

tracts in the absence of 2-mercaptoethanol. Inhibitor studies with sulfhydryl reagents were more conclusive in this regard

(Table V). The rather high concentration of arsenite required

for inhibition suggests that its action is not due to binding of

vicinal dithiols. In the presence of saturating levels of methyl-

m‘+i

0.2 2.0 0.2 2.0 0.2 2.0 0.2 2.0

I I I I I I I 7.0 7.4 7.8 8.2 8.6 9.0

PH

FIG. 3. pH profile for the enzymatic synthesis of N-methylglutamate from methylamine and L-glutamate under the standard conditions described in “Experimental Procedure.” Buffer was potassium pyrophosphate (100 mM).

I I I t 0 I 2 3 4 5

I/p.] x 102

FIG. 4. A, L-glutamate concentration curve in the presence of 100 mrvr methylamine under the standard assay conditions. The initial velocity was determined by stopping the reaction after 10 min had elapsed. B, determination of the Km for L-glutamate by double reciprocal plot. Km calculated from the slope was 6.8 mM.

amine and L-glutamate, the reaction rate is linear for 15 min at

30”. Saturation kinetics were obtained for L-glutamate as seen in Fig. 4. The apparent K,,, for glutamate was calculated to be

6.8 mM by the method of Lineweaver and Burk (15). Similar


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940 N-Methylglutamate Xynthetase Vol. 241, No. 4

kinetics were observed for methylamine with a calculated K, of 92 rnM.

Fig. 5 depicts the enzyme concentration curve usually obtained with extracts of this organism. The departure from linearity at low enzyme concentrations has been studied in some detail and has been found to be unchanged by the addition of boiled extract, alterations in pH or ionic strength, the addition of bovine serum albumin, or prior incubation of the enzyme with either substrate. Preliminary attempts at purification of the crude extract have thus far failed to show more than one protein component .

Of some interest is the observation that both ammonia and hydroxylamine are inhibitors of the enzymatic reaction. In the case of ammonia, the inhibition is clearly competitive with respect to methylamine (Ki = 49 mM). Hydroxylamine inhibition has not been studied in sufficient detail to describe the type of inhibition, but it inhibits the reaction approximately 70% at a concentration of 0.4 mM. Incubation of the crude enzyme with hydroxylamine followed by extensive dialysis yields a preparation which is fully active, suggesting that the inhibitor is not binding irreversibly to an enzyme-bound prosthetic group.

All attempts to demonstrate the involvement of a soluble cofactor in N-methylglutamate synthesis have been unsuccessful. Exhaustive dialysis, gel filtration, and charcoal treatment have failed to resolve the crude extracts. In the latter case, treatment with 4 mg of Norit per mg of extract protein have been ineffective. Equivocal results have been obtained by passing the crude extract over a column of Dowex 1 (Cl-). Under such

Mg PROTEIN

FIG. 5. Enzyme concentration curve. Ordinate, yield of N- methylglutamate per 10 min in the presence of dialyzed enzyme under standard conditions.

TABLE VI Coincident transfer of 3H and IT radioactivity from methylamine

to N-methylglutamate

Methylamine (100 mM) and L-glutamate (10 mM) were incubated with dialyzed enzyme (16 mg of protein per ml) for 10 min at 30” in the presence of tracer quantities of C3H3NHz and 14CH3NH?. Aliquots of the substrate (methylamine) and product (N-methylglutamate) were taken to yield suitable counting rates and were assayed for radioactivity as described in “Experimental Pro- cedure.” Radioactive N-methylglutamate was isolated in the usual manner by Dowex 50 (H+) absorption followed by elution with ammonia. The yield of N-methylglutamate was 1.8 pmoles.

Radioactivity due to

Sample Ratio (A:B)

w (A) ‘C (B)

cPm

Methylamine.............. 9,600 12,600 0.76 N-Methylglutamate. 3,900 5,500 0.71

conditions, a 50% reduction in activity has been noted, but the development of turbidity in such preparations and the failure of boiled cell extract to restore activity make it likely that ir- reversible protein denaturation occurred during the resin treatment. No evidence was obtained to implicate conventional methyl transfer coenzymes. When unlabeled methylamine, glutamate, and enzyme were incubated under standard assay conditions in the presence of 14C-methylcobalamin, no radioactivity appeared in the N-methylglutamate isolated at the end of the reaction. Similar experiments performed in the presence of a pool of 14C-methyl-S-adenosylmethionine also yielded negative results. The addition of intrinsic factor sufficient to bind 0.1 mpmole of vitamin Blz coenzyme had no effect on the enzymatic reaction.

Mechanism of Reaction

Reaction 1 could be visualized as occurring by at least five different mechanisms as follows.

Coupled Oxidation-Reduction and Formyl Transfer-Since cell suspensions of methylamine-adapted cells catalyze the oxidation of methylamine to formate, CO*, and ammonia,2 it was possible that Reaction 1 involves oxidation of methylamine to a formyl derivative followed by transfer of the formyl groups to glutamate with subsequent reduction to N-methylglutamate. To determine whether this or any related mechanism is involved, we devised a double isotope experiment to examine the lability of methyl hydrogen atoms during the synthesis of N-methylglutamate. Table VI summarizes the results of an experiment in which 14C3H3NHt was incubated with L-glutamate and enzyme. The ratio of 3H to 14C in the N-methylglutamate formed was essentially the same as that for the methylamine added. Mecha- nisms involving oxidations of the methyl group are therefore very unlikely.

Reversible Oxiclative Deamination-Oxidative deamination as catalyzed by glutamic dehydrogenase (Reaction 2) followed by substitution of methylamine for ammonia in the reverse reaction (React,ion 3) would result in the synthesis of N-methylglutamate.

2 W. V. Shaw, unpublished experiments.


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Glutamate + DPN (TPN) + HZ0 --+

ol-ketoglutarate + ammonia + DPNH (TPNH) + H+ (2)

or-Ketoglutarate + methylamine + DPNH (TPNH) + H+ +

A-methylglutamate + H20 + DPN (TPN) (3)

Sum: Glutamate + methylamine +

N-methylglutamate -l- ammonia (1)

The oxidation of TPNH in the presence of ammonia and (Y- ketoglutarate (Fig. 6A) and the reduction of TPN in the presence

a NH3 ‘\.

d d a

(10, moles) dl.,

0.2 - a.

A ‘\.

‘\ “A

I I I I 2 3 4 5

MINUTES

0.2.

2 0

2

t

2 0.1.

a

”

0 Y I I I I 2 4 6 8 IO

MINUTES

FIG. 6. A, spectrophotometric determination of TPNH oxidation in the presence of a-ketoglutarate (20 /Imoles), dialyzed enzyme (1.2 mg of protein), and amines as noted. The reaction was carried out at 26” in a final volume of 1.0 ml in the presence of Tris-HCI, pH 8.5, 100 mM; and 2.mercaptoethanol, 5 mrvr. B, glutamic dehydrogenase activity of cell extracts as measured by the reduction of TPN+ in the presence of L-glutamate (o- - -0 ). Racemic N-methylglutamate was inactive (O-O). The amino acids were present at a concentration of 10 mM. Other conditions as in A. Similar results were obtained in the presence of DPN+.

TABLE VII Failure of a-ketoglutarate to catalyze N-methylglz~tamate synthesis from 14C-methyZamine (speci$c activity, 60,000 cpm per rmole) in

presence and absence of reduced pyridine nucleotides

L-Glutamate or a-ketoglutarate was present at a concentration of 20 rnM. The DPNH-generating system contained ethanol (4 mrvr), alcohol dehydrogenase (0.16 unit per ml), and DPNH (0.1 rnM). The TPNH system contained glucose 6-phosphate (4 mrvr), glucose G-phosphate dehydrogenase (0.25 unit per ml), and TPNH (0.1 mM). Incubation was for 20 min at 30” in a volume of 1.0 ml. Fractionation of the reaction products was carried out with Dowex 50 (H+) as described in “Experimental Procedure.” Both the activity appearing in the cation fraction (N-methylglutamate) and that found in the anion and neutral fraction (un- identified) are tabulated.

Substrate Omissions or additions

L-Glutamate

a-Ketoglutarate

_-

Omit enzyme Enzyme present Enzyme present Enzyme + DPNH system Enzyme + TPNH system

-

i-

: I

‘4C-Methylamine carbon incorpo- ated into product

Iationic Anionic V-m&h- and neu- ilgluta- tral (uni- mate) dentilied)

/.latoms

0.1 0.0 1.5 0.1 0.2 0.8 0.2 0.8 0.6 0.5

of L-glutamate (Fig. 6B) attest to the presence of glutamic dehydrogenase activity (Reaction 2). However, all efforts to demonstrate Reaction 3 have failed. Thus, neither significant oxidation of TPNH or DPNH is observed in the presence of cy- ketoglutarate and methylamine (Fig. 6.4) nor are the oxidized pyridine nucleotides reduced in the presence of N-methylglutamate (Fig. 6B). Furthermore, the data of Table VII show that the enzyme preparations catalyze little or no N-methylglutamate formation in the presence of a-ketoglutarate, methylamine, and a pyridine nucleotide-generating system. The slight formation of N-methylglutamate observed in the presence of TPNH probably reflects the intermediary production of glutamate from endogenously formed ammonia.

A function of glutamic dehydrogenase in N-methylglutamate synthesis is also contraindicated by the data of Table VIII showing that partial purification of the enzyme catalyzing Reac- tion 1 results in appreciable resolution with respect to glutamic dehydrogenase activity. Furthermore, selective denaturntion of the two activities is obtained by heating. After 6 min at 50”, the glutamic dehydrogenase activity is essentially unchanged, whereas N-methylglutamate synthetase activity is decreased more than 50%. Whereas these results fail to support a role of glutamic dehydrogenase in the N-methylglutamate synthetase reaction, final decision must await complete resolution of the two enzyme activities by further purification. Nevertheless, considered with the data of Fig. 6 and Table VII, the results seem to preclude the oxidative deamination mechanism.

Nonoxidative Deaminalion-N-Methylglutamate could arise by an aspartase-like reaction involving (Y ,/3-elimination to form ammonia and glutaconic acid (Reaction 4) followed by substitution of methylamine in the reverse reaction (Reaction 5).


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TABLE VIII Dissociation of N-methylglutamate synthesis from glutamic

dehydrogenase activity

Fractionation was carried out by the addition of crystalline ammonium sulfate to crude cell free extract at 0” in 20 mM Tris- HCl (pH 8.5). The precipitated protein was collected by centrifugation and resuspended in buffer before dialysis to remove the ammonium sulfate. Heat denaturation was performed with dialyzed enzyme by immersing the extract in a water bath at 50” for the time specified at which point an aliquot was pipetted into an equal volume of ice-cold buffer. Enzyme activities were assayed as described in “Experimental Procedure.”

Specific activity

Treatment

Experiment 1 No ammonium sulfate.. O-40yo saturation with am-

monium sulfate. 40-50% saturation with

ammonium sulfate. 5o-GOy(o saturation with

ammonium sulfate. Experiment 2

6.3 11.4

5.2 2.2

9.6 5.7

3.8 19.6

No heating. 2.3 7.9 2 min at 50”. 1.5 9.6 4 min at 50”. 1.2 8.2 6 min at 50”. 1.1 7.8

N-Meth- ylglutamate Glutamic

synthesis dehydrogenase

?n~nzoles/min/mg protein

Glutamate + glutaconate + ammonia (4)

Ratio of activities

0.6

2.4

1.7

0.2

0.29 0.16 0.15 0.14

Glutaconate + methylamine + N-methylglutamate (5)

Glutamate + methylamine +

N-methylglutamate + ammonia (1)

This mechanism is unlikely since no N-methylglutamate was formed when the enzyme was incubated with methylamine and glutaconate under otherwise standard assay conditions. More- over, in the absence of methylamine, no ammonia is liberated when glutamate is incubated with the enzyme in the absence of pyridine nucleotides.

Transrnethylation versus Direct Displacement Reaction--With elimination of the above mechanisms two possibilities remain. N-Methylglutamate may be formed either by a transmethylation reaction, involving transfer of the methyl group from methylamine to the amino group of glutamate or by a direct displacement of the amino group of glutamate by methylamine. On the assumption that Reaction 1 is freely reversible, it is evident that, if the mechanism involves direct displacement, incubation of i4C-N-methylglutamate with either unlabeled methylamine or ammonia will lead to the release of i4C-methylamine (Reactions 6 and 7).

HR HR WHZN-CH + CH,NHs -+ CH,N-CH

COOH + WHaNHz (6)

COOH

HR R WHSN-CH + NH3 -+ H*N-CH + ‘“CHsNH2 (7)

COOH COOH

R = -CH,CH,COOH

On the other hand, if transmethylation is involved, the presence of ammonia should give rise to 14C-methylamine whereas unlabeled methylamine should be inert. The data of Table IX appear to favor the transmethylation mechanism in that ammonia is more effective than methylamine in causing the release of W-methylamine from W-N-methylglutamate. The slight but definite exchange noted when unlabeled methylamine was added prompted further experiments to decide between the transmethylation and direct displacement mechanisms. In this regard, a more definite approach was the use of ‘Wlabeled substrates for the enzymatic reaction. A direct displacement mechanism ought to yield iWmethylglutamate from r5N- methylamine (Reaction 8), whereas a transmethylation reaction in the presence of l5N-glutamate and unenriched methylamine should lead to the formation of r5N-methylglutamate (Reaction 9).

R H R CHa16NHz + HZN-CH -+ CHz16N-CH + NH, (8)

COOH COOH

CH3NH.r + H,&RdH R

+ CH3i5N-CH + NH, (9) COOH COOH

In each of the experiments described in Table X, conditions were ident.ical except for the i5N content of the substrates. In Experi- ment 1, glutamate was unlabeled, whereas methylamine enriched with ‘5N was present. Experiment 2 was designed with the reciprocal combina.tion of ‘SN-glutamate and unenriched methylamine. In each experiment, l*C-methylamine was included to allow for the det.ermination and isolation of the product, N- methyl-W-glutamate, which was then analyzed for ‘5N enrichment (see “Experimental Procedure”). The results summarized in Table X indicate that significant ‘SN enrichment of N-methylglutamate occurs only in the presence of ‘Wmethylamine, a result expected for a direct displacement mechanism. The apparent slight l&N enrichment of N-methylglutamate in Experi- ment 2 probably reflects the incomplete separation of the product from glutamate by paper chromatography. The relative mobili- ties of glutamate and N-methylglutamate are not sufficiently different to exclude rigorously this possibility.

N-Methylglutamate Synthesis as Adaptive Process-The novel aspects of N-methylglutamate synthesis prompted assays of

TABLE IX

Reversibility of reaction

Each tube contained: Tris-HCl, pH 8.5, 100 mM; 2-mercaptoethanol, 5 mM; N-methyl-‘%-glutamate, 5 mM; and enzyme, 20 mg per ml, in a final volume of 0.5 ml. The total radioactivity added as N-methylglutamate was 13,000 cpm. After 20 min of incubation at 30”, the reaction was stopped by the addition of 0.1 ml of 20% trichloroacetic acid. Volatile amines were collected from an aliquot of the protein-free supernatant as described in “Experimental Procedure,” and were assayed for radioactivity.

Addition Amount Total volatile amine (methylamine) recovered

None ...................... NH&l. ................... NH&l. ................... CH,NH,Cl. ............... CHaNH&l. ...............

pmoles CM

480 10 3,620 50 5,020 10 920 50 750


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TABLE X

Mechanism of reaction

Incorporation of i&N derived from methylamine or glutamate into N-methylglutamate. The standard incubation included: Tris-HCl, pH 8.5, 100 mM; 2-mercaptoethanol, 5 mru; and enzyme protein, 20 mg per ml, plus the reactants as indicated, in a final volume of 1.0 ml. W-Methylamine (specific activity, 3800 cpm per Imole) was present in each experiment to permit the detection and purification of the N-methylglutamate formed. After 30 min of incubation at 30”, the protein was removed by precipita- tion with trichloroacetic acid, and the supernatant was subjected to Dowex 50 (H+) treatment and ammonia elution as described in “Experimental Procedure.” The product, N-methylglutamate, was isolated by paper chromatography and analyzed for *6N content by mass spectrometry (see “Experimental Procedure”).

Experiment

IA 100 48 1B 100 48 2A 100 0 2B 100 0

Reactants

Methylamine

i

L-Glutamate

pmoles ato?n qo ezcess

10 0 10 0 10 56 10 56

Product

N-Methylglutamate

pmozes atom qo ezcess

5.2 49 5.1 55 5.0 7 4.3 7

Mg PROTEIN

FIG. 7. N-Methylglutamate synthesis as an adaptive process. Cell-free extracts were prepared in the French pressure cell in the usual fashion from bacteria grown on methylamine or on glycerol plus ammonia as described in the text. The enzyme activity was compared with dialyzed extracts by the standard radioactive assay and is plotted as the yield of N-methylglutamate from methylamine and L-glutamate per 10 min of incubation.

enzyme activity in extracts of bacterial cells grown on a carbon source other than methylamine. Pseudomonas sp. was grown alternatively on methylamine or on glycerol and ammonia under otherwise identical conditions. The ability of cell-free extracts to catalyze N-methylglutamate synthesis from methylamine and glutamate was comnared with logarithmic nhase cells from

I

FIG. 8. Pulse-labeling of amino acids from ‘4C-methylamine. Resting cell suspensions were incubated in the presence of W- methylamine at zero time. A nonradioactive pool of methylamine was added at 12 set to yield a WOO-fold dilution of the ‘4C material. Aliquots were taken at the times indicated and subjected to hot ethanol extraction and two dimensional paper chromatography. The dashed lines and the ordinate scale on the left refer to radioactivity incorporated into the N-methyl amino acids (sarcosine and N-methylglutamate). The solid lines and the scale on the

ok ~~~ _~~..~~ . . ~~ . .._ right refer to the other amino acids as indicated.

each culture as a source of enzyme. Fig. 7 depicts the results of such an experiment wherein extracts of cells grown in glycerol fail to synthesize N-methylglutamate. Only an additive effect was noted when extracts of glycerol and cells grown in methylamine were mixed and assayed under similar conditions.

Incorporation of Radioactivity from 14C-Methylamine into Amino

Acids by Intact Cells-Because N-methylglutamate synthesis represented an unusual reaction, it was of interest to examine what relat)ionship it might have to other pathways of carbon assimilation in intact cells. Preliminary experiments indicated that radioactive N-methylglutamate and other amino acids were rapidly synthesized from 14C-methylamine by whole cells, but the small amounts of labeled products formed precluded the accurate measurement of specific activities. Thus, an attempt was made to evaluate a possible precursor-product relationship for N-methylglutamate and other amino acids by the use of pulse-labeling techniques.

Cells grown on methylamine were harvested in logarithmic phase, washed, and resuspended in substrate-free growth medium at a concentration of 20 mg per ml (dry weight). After pre- incubation for 5 min at 26” to deplete endogenous substrates, 14C-methylamine (3.3 PC per pmole) was added rapidly to a final concentration of 0.07 mM. Ten seconds after the addition of radioactive substrate, a suitable aliquot was taken for analysis, and the W-methylamine pool was diluted by the addition of unlabeled methylamine to a final concentration of 70 InM. The initial sample and subsequent aliquots over a 30-set period were

SECONDS

1 I!


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subjected to hot ethanol extraction, concentration, and Dowex 50 (Hf) absorption as described for the isolation of Ai-methylglutamate. Labeled amino acids were located by autoradiography after two dimensional chromatography (Solvents B and il of Table I). The radioactive amino acids were eluted and identified as N-methylglut,amate, sarcosine, serine, alanine, and aspartate by cochromatography with the authentic compounds.

The total amount of radioactivity in the major amino acid products isolated is shown in Fig. 8. The labeling of sarcosine is transient, disappearing after addition of the unlabeled methylamine pool, and is consistent with its synthesis de novo from methylamine. An equally plausible interpretation is that sarcosine is an early product of N-methylglutamate labeling, is rapidly degraded, and does not reappear since an endogenous precursor such as glycine is limiting. The late appearance of radioactivity in aspartate is consistent with the observations that whole cells of this organism oxidize 14C-methylamine to 14C02 via formate and that cell-free extracts contain phosphoenol- pyruvate carboxylase as well as an avidin-sensitive pyruvic carboxylase system.2 The hypothesis that radioactive serine and alanine are derived from sarcosine is only suggested by the data. Information on the relative pool sizes of the labeled products and other important intermediates or precursors would be necessary for a final judgment.

DISCUSSION

The present studies were undertaken to investigate the assimilation of one carbon units by nonphotosynthetic bacteria. Whereas the Pseudomonas sp. chosen for study is able to utilize methylamine as the sole source of carbon and nitrogen, its lack of pigmentation and the fact that it fails to grow on methanol, formaldehyde, and formate differentiates it from similar organisms reviewed by Quayle (5) as well as several other pseu- domonads described more recently (4, 16). Although resting cells readily oxidize methylamine to carbon dioxide and ammonia, it has not been possible to demonstrate methylamine oxidation by cell-free extracts. Formate has been identified as an intermediate in the oxidation of methylamine by intact cells and is oxidized to CO2 by cell-free extracts but methanol and formaldehyde are not oxidized by cells or crude extracts.’ In this respect, the organism described here differs from the Pseudomonas sp. discussed by Johnson and Quayle (17) and by Anthony and Zatman (4).

The original goal of tracing the net synthesis of CZ units from C1 compounds in this organism has not been accomplished. However, the formation of N-methylglutamate from methylamine and glutamate by intact cells and cell-free extracts represents the first recognition of this compound in biological systems. The early appearance of methylamine carbon in sarcosine and N-methylglutamate observed in pulse-labeling experiments with intact cells suggests a possible pathway for the utilization of reduced C1 units. However, attempts to demonstrate the direct methylation of glycine or transmethylation between sarcosine and N-methylglutamate in cell-free extracts were unsuccessful. It is noteworthy that similar pulse-labeling experiments with *4C-methylamine in another Pseudomonas sp. failed to show an intermediate suggestive of N-methylglutamate (18). The latter studies indicated that serine was the first amino acid to show labeling from methylamine, a finding similar to

experiences with Pse~~domonas AM 1 when ‘%-methanol or formate was the C1 substrate (19).

That still another pathway for methylamine assimilation may exist is apparent from the data in Table VII. Although (Y- ketoglutarate fails to replace glutamate as an acceptor for the methyl group it is clear that a radioactive product other than N-methylglutamate is formed which is not bound to Dowex 50 (H+). No serious attempt has been made to identify this product; however, it is anionic as judged by its affinity for Dower 1 (OH-) and is stable to extremes of pH.

The mechanism responsible for the enzymatic synthesis of N-methylglutamate remains somewhat unclear. From the results of Table VI it is evident that the methyl group is trans- ferred intact from methylamine to glutamate. Furthermore, the 15N experiments (Table X) favor the view that the entire methylamine molecule is incorporated into the product. Whereas a pyridine nucleotide-mediated reaction analogous to glutamic dehydrogenase appears not to be involved, it is possible that a coupled oxidation-reduction may occur via an enzyme- bound prosthetic group. All efforts to resolve extracts for such a cofactor have been unsuccessful. The experiments summarized in Table IX indicate that the reaction is reversible in that ammonia may displace ‘Gmethylamine from N-methyl-14C-glutamate, but it is not clear as to why unlabeled methylamine fails to exchange with its 14C counterpart if the reaction is a direct displacement as is indicated by the results of the ‘SN experiments. It is plausible that when displacement occurs in the reverse reaction there is a high degree of specificity for ammonia as the dis- placing nucleophile.

Information is currently lacking as regards the optical configuration of the N-methylglutamate formed in the enzymatic reaction. The determination of configuration of the enzymatic product has been impossible since only small amounts of N- methylglutamate have been isolated directly from the reaction products and because the compound is a poor substrate for the enzymatic techniques commonly used for the microdetermination of optical configuration (20). Determination of the presence or absence of inversion at the ac carbon during the course of the reaction would clarify whether displacement by methylamine occurs directly on glutamate or whether a deaminated enzyme- glutaryl complex occurs before the addition of methylamine. The former would be expected to lead to inversion whereas the latter should favor retention of configuration (21). Information on these points is not currently available.

1. LEADBETTER. E. R.. AND FOSTER. J. W.. Arch. Mikrobiol., 30,

2. 91 (1958). ’

KANEDA, T., AND ROXBURGH, J. M., Can. J. Microbial., 6, 87 (1959).

3. PE‘EL, D., AND QUAYLE, J. R., Biochem. J., 81, 465 (1961). 4. ANTHONY, C., AND ZATMAN, L. J., Biochem. J., 92, 609 (1964). 5. QUAYLE, J. R., Ann. Rev. Microbial., 16, 119 (1961). 6. IYER, S. N. AND KALLIO, R. E., Arch. Biochem. Biophys., 76,

295 (1958) . 7.

8.

9.

FINK. K.. CLINE. R. E., AND FINK, R. M.. Anal. Chem., 36, 389, (1963). ’

SWEELEY, C. C., AND HORNING, E. C., J. Am. Chem. Sot., 79. 2620 (1957).

KNOOP, F., AND OESTERLIN, H., Z. Physiol. Chem., 170, 186 (1927).

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10. SUGASAWA, S., J. Pharm. Sot. Japan, No. 550, 1041 (1927); Chem. Abstr., 22, 1573 (1928).

11. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem., 177, 751 (1949).

12. SELIGSON, D., AND SELIGSON, H., J. Lab. Clin. Med., 38, 324 (1951).

13. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem., 197, 67 (1952).

14. SMITH, E. L., MERVYN, L., JOHNSON, A. W., AND SHAW, M., Nature, 194, 1175 (1962).

15. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot., 66, 658 (1934) .

16. STOCKS, P. K., AND MCCLESKEY, C. S., J. Bacterial., 88, 1065 (1964).

17. JOHNSON, P. A., AND QUAYLE, J. R., Biochem. J., 93, 281 (1964).

18. LEADBETTER, E. R., AND GOTTLIEB, J. A., Bacterial. Proc., 104 (1964).

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20. GREENSTEIN. J. P.. AND WINITZ. M., Chemistry of the amino _ . acids, Vol. 5, John Wiley and Sons, Inc., New York, 1961, p. 1753.

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W. V. Shaw, L. Tsai and E. R. Stadtman-Methylglutamic AcidNThe Enzymatic Synthesis of

1966, 241:935-945.J. Biol. Chem.

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The Enzymatic Synthesis of IV-Methylglutamic Acid

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