Isolation and Purification of L-Methionine-A-Deamino--Y Mercaptomethane-Lyase (L-Methioninase) from Cbs Tridiu M Sporo Genes'

[CANCER RESEARCH 33, 1862-1865, August 1973]

Isolation and Purification of L-Methionine-a-deamino--y mercaptomethane-Lyase (L-Methioninase) from Cbs tridiu m sporo genes'

Willi Kreis and Catherine Hession

Memorial Sloan-Kettering Cancer Center, New York, New York 10021

SUMMARY the point of high purity, as indicated by a single band in the disc electrophoretic assay. In an attempt irreversibly to deplete biological systems of the essential amino acid L-methionine, L-methionine a-dcam mo- -y-mercaptomethane-lyase was isolated from MATERIALS AND METhODS Clostridium sporogenes (ATCC 7955), highly purified, and characterized. All the investigations undertaken indicate C. sporogenes (ATCC 7955) was grown for 18 hr in a a double function: release of the a-amino- and the â€˜y medium containing Bacto cooked meat medium, 0.35% methanethiol group by a single protein unit. It is possible (Difco Laboratories, Detroit, Mich.); Bacto tryptone, that this bifunctional activity is brought about by gene 1.0% (Difco); proteose peptone, 1.0% (Difco); sodium fusion. thioglycollate, 0. 1% (Matheson Coleman and Bell, East Rutherford, N. J.); and Ucon lubricant LB625, 0.01% (Union Carbide Corp., Pearl River, N. Y.). Batches of INTRODUCTION 4 g of cells were suspended in 3.5 ml of 0. 1 M sodium: potassium phosphate buffer, pH 7.4, containing 0.5 mM This laboratory reported that a methylhydrazin@ de neutralized 2 (Fluka, Buchs, Switzerland) and 5 mM rivative, I-methyl-2-(p-isopropylcarbamoyl) benzylhydra mercaptoethanol (NKPPM buffer) (Sigma Chemical Co., zinc hydrochloride, (NSC 77213), with clinically demon St. Louis, Mo.) were treated in an ultrasonic disinte strated activity in Hodgkin's disease and lymphosarcoma, grator, Model 60W (Measuring and Scientific Equipment showed characteristic disturbance of the normal methyla Ltd., London, England) 3 times for 3 mm over 15 mm tion of RNA, especially tRNA in P815 mouse neoplasms at 20 kc/sec. The cell suspension was kept in an alcohol: (8). This disturbance consisted of a significant in vivo ice mixture throughout the sonic disruptions. All extrac methylation of RNA-guanine at position 7 and a consid tion steps were performed at 0â€”4Â°.Thedisrupted suspen erable reduction of the normal methylation of cytoplasmic sion was centrifuged at 30,000 x g for 30 mm, the pre RNA of these cells. The effect preceded the inhibition of cipitate was washed 2 times with NKPPM buffer, and RNA synthesis and the therapeutic activity of the com the supernatant and the 2 washings of the precipitate pound could be prevented by massive doses of L-methi were then combined. Protamine sulfate (Nutritional Bio onine administered 2 hr before the drug (7). If indeed the chemicals Corp., Cleveland, Ohio), 0.5% in NKPPM undermethylation of RNA (and possibly also DNA) con buffer, was added to a final concentration of 0.25% and tributes to the inhibition of RNA synthesis, it was con the precipitate was discarded after centrifugation. Am cluded that by depletion of the source of methyl groups, monium sulfate was added to 42% saturation and the en i.e., L-methionine, this effect could be achieved more ef zyme-inactive material was removed by filtration through fectively. It was hoped that an L-methionine-degrading glass wool. Additional ammonium sulfate (to 75% satura enzyme would further the studies in this direction, would tion) precipitated all enzyme-active material. After cen help to elaborate on the biological significance and trifugation the precipitate was dissolved in and dialyzed mechanism of DNA and RNA methylation in normal against NKPPM buffer. Two successive gel filtrations and neoplastic tissues, and might be beneficial in tumor on a Sephadex G-200 (Pharmacia Fine Chemicals, Inc., chemotherapy. Piscataway, N. J.) column (50 x 3.0 cm), conditioned and Clostridium sporogenes was used as the source of an cluted with NKPPM buffer, were then performed. One enzyme that degrades 1-methionine to methanethiol, am enzyme-active protein peak was observed. Following monia, and ct-ketobutyric acid. Such an enzyme in crude filtration through the Sephadex G-200 column, the en preparations of C. sporogenes was first described by Wie zyme-active fractions were pooled, precipitated with sendanger and Nisman (21). We carried the isolation to

1 This work was supported in part by Grant CA 08748 from the â€˜The abbreviations used are: PPH, pyridoxal phosphate; NKPPM, National Cancer Institute. sodium : potassium phosphate buffer, pH 7.4, containing 0.5 m@i neu Received December 27, 1972; accepted April 18, 1973. tralized PPH and 5 mM mercaptoethanol.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1973 American Association for Cancer Research. L-MethiOninase ammonium sulfate (5.2 g/lO ml), and dissolved in and Radioactive methanethiol released from L-methionine la dialyzed against NKPPM buffer. This pooled sample beled with either 35S or â€˜4Cin the methyl group (Amer was filtered a 2nd time through the Sephadex G-200 sham/Searle) showed good quantitative agreement (33.6 column. The enzyme-active fractions from the 2nd Seph and 33.8 nmoles). No radioactive material was trapped adex G-200 were pooled, and the precipitation and when the substrate was labeled with â€˜4Cin positions I or dialysis steps were repeated. The resulting enzyme-ac 1, 2, 3, and 4 of L-methionine (Amersham/Searle). The tive sample was applied to a DEAE-Sephadex A-SO 3rd reaction product, a-ketobutyric acid, was identified as (Pharmacia) column (45 x 3. 1 cm). The proteins were the 2 ,4-dinitrophenylhydrazonc derivative by paper eluted with a linear gradient of 0 to 0.4 M KCI in NKPPM [Whatman No. 1, system, 1-butanol :ethanol :water without PPH. The 1st of 2 major peaks contained all the (4 : I : 5)] and thin-layer chromatography (silica gel, enzyme activity. After precipitation with ammonium sul system, 75% phenol) and by its melting point and melting fate (5.2 g/ 10 ml), the protein was dissolved in I mM p0- point of mixture with the derivative of authentic a-keto tassium phosphate buffer, pH 6.5, containing 5 mM mer butyric acid. Minor amounts of other reaction products captoethanol. Final purification was achieved on a hy have not yet been analyzed. droxylapatite (Bio-Gel HI) (Bio-Rad Laboratories, Rich Methanethiol in the incubation medium was identified mond, Calif.) column (9 x 3 cm), using a linear gra by a color reaction for mercaptans ( I5) which consisted dient of I mM to 0.2 M phosphate buffer, pH 6.5, which of the addition of 0. 1 ml of sodium nitroprussiate solu contained 5 mM 2-mercaptoethanol. For protection of the tion [1.5 g sodium nitrosopentacyanofcrratc(III) dis enzyme activity in the last 2 columns, 2 ml of 0.5 M solved in 5 ml of 2 N HC1 and followed by the addition of phosphate buffer containing 2.5 mM PPH were added to 95 ml methanol and 10 ml concentrated ammonia] to the all the tubes used for the collection of lO-ml fractions. reaction product after incubation as above and immediate When methanethiol and ammonia were assayed si spectrophotometric determination of the developed multaneously in each individual fraction of the first purple color at 530 nm. The test is, at best, semiquan and last column, there was good qualtitative and quanti titative. tative coincidence. Acceptable coincidence was also Disc electrophoresis was performed on polyacrylamide found for the ratio of methanethiol to ammonia in all the gel at 5Â°according to the methods of Ornstein (13) and combined fractions during the isolation procedure (Table Davis (4). Reagents and conditions were as described I). The overall purification was 120-fold (Table I). earlier (9). About 98% of the enzyme activity was lost during the The molecular weight, as determined by gel filtration process. Disc electrophoretic monitoring of the pro on Sephadex G-200 and by ultracentrifugation and sedi teins present at several steps of the extraction procedure mentation analysis with Rayleigh interferometry (16), was gave evidence of the stepwise purification (Chart I). The about 150,000. enzymatically active fractions of the last column Activity of the enzyme was optimal in the pH range of (Step 6) produced only I band in the disc electrophoresis. 7_S to 8.5 for both functions and absent below pH 6.0. The @ For the assay of methanethiol and ammonia, 50 of Km values under the standard conditions of the enzyme L-methionine-methyl- â€˜@C(1.33 zmoles; specific activ assay were similar for the 2 functions: 90 mM for the re ity, 0.0379 mCi/mmole; Amersham/Scarle, Arlington lease of methanethiol and 78 mM for the release of am Heights, Ill.); 50 j.zl of sodium PPH (0.472 ,umole); SO monia. Maximal enzyme activity was dependent upon the to 90 @lof sodium : potassium phosphate buffer, pH 7.4 presence of PPH. The approximate Km value for the (0.5 M); and 10 to 50 zl of enzyme were mixed in pre latter was 10 @MPPH when measured in the methane cooled Pyrex ignition tubes (10 x 70 mm) each containing thiol assay, indicating that L-methionine is bound loosely 1 small glass bead. Each tube was then â€œpluggedâ€•in and the cofactor more strongly to the enzyme. Dialysis dividually with a fluted paper disc (Whatman No. 1, of the enzyme against 10 mM hydroxylaminc in NKPPM 26-mm diameter) which was moistened with 2 drops of a buffer decreased the activity exponentially to 5% within 3 saturated mercuric chloride solution. The tubes were hr and to less than 2% within 18 hr. Dialysis against plain incubated in a shaking water bath for 15 mm at 37Â°,and buffer reduced the enzyme activity within the same time the reaction was stopped by cooling in ice. For the comple intervals to SO and 5%, respectively. No reactivation was tion of the release of methanethiol-'4C from the reaction observed after adding PPH. The isoelectric point de mixture, the tubes were heated to boiling over a Bunsen termined by electrofocusing on semipurified preparations burner. The paper discs were then removed, and their (LKB Instruments, Inc., Rockville, Md.) was about radioactivity was determined in 10 ml Diotol (5) in a pH 4.2. Packard Model 3380 scintillation spectrophotometer. For The stability of a semipurified enzyme preparation the ammonia determination 250 @zlof 10% trichloroacetic (after step 4) kept frozen between analyses showed a steady acid were added after incubation of the tube. Conway loss of activity of about 1.5%/day over a span of 43 dishes and Nessler's reagent were used for trapping and days. Pure enzyme preparations (after Step 6) lost 85% of evaluating the ammonia. One unit of enzyme is defined their initial activity within 14 days. The enzyme activity as the amount of enzyme necessary to release from the (methanethiol test) increased slightly when heated for IS substrate I @tmoleeach of methanethiol-'4C and/or NH3 mm at 50Â°or for 10 mm at 60Â°with rapid loss of activity per mm. Methanethiol was identified as its mercury salt. after further heating at 60Â°.Within 1 mm there was 90%

AUGUST 1973 I 863

Table 1 Pursfwation and recovery ofenzyme activity Starting material was 20 g of cells. The enzyme activity of the crude homogenate (after sonic disruption Step 1) varied widely from one batch to the other. These values ranged from 0.005 to 0.08 unit/mg protein.Recoveryof

ofPurificationactivityâ€˜4CH,SHenzymeRatio @StepDescription : step'(%)determinationbIHomogenateof stepduring

18tion2Ammonium after sonic disrup I .01001 .

143Sephadex sulfate precipitation3 . 848 . 51 . filtrationlstcolumn6.724.00.9342ndcolumn10.613.20.955DEAE-SephadexG-200 Gel

22.67.41.09matography6Hydroxylapatitecolumn chro

12O.0'@2.11.13matographycolumn chro

a Evaluation by the assay of methanethiol. b In some steps during the extraction procedure, dialysis for removal of ammonium sulfate had to be restricted for loss of enzyme activity.

C At this point of purification, the enzyme is extremely unstable and this value is the best of several experiments.

Table 2 Enzyme activity on different substrates intheFor conditions for enzyme assay see text. All substrates were used saturatedsolutionsame concentration (1 .33 zmoles) except 1-cystine, where a was used. Purified enzyme was used for these assays. a-Keto-y mercaptomethylbutyric acid was synthesized from i-methionine-methyl â€œCaccording to the method of Meister (II).N

reactionreleased, Color mercaptoSubstrate for compounds1-Methionine (nmoles)

+D-Methionine 29.4 0DL-Methionine 0 0L-MethiOnine sulfoxide 17.6 0a-Keto-y-mercaptomethyl-sulfone 14.7 0butyric 0 acida-Aminobutyric 0L-Ethionine acid 81.0DL-Homocysteine 206.01-Cysteine 01-Cystine 38 . 28 .0 0

a A positive color reaction was noticed indicating the release of mer captoethanol. b DL-Homocysteine and 1-cysteine both gave a positive color reaction by themselves.

Chart I . Disc electrophoresis of protein at different purification steps. DISCUSSION Disc I, supernatant after sonic disruption and centrifugation of cells (105 14 of protein on gel); Disc 2, combined enzyme-active fractions after The data indicate the occurrence of a single protein with 2nd G-200 column [54 zg of protein on gel (Table I Step 4)]. Disc 3, dual enzymatic functions. All the simultaneously per combined enzyme-active fractions after DEAE-Sephadex column [26 @g formed evaluations of methanethiol and ammonia of the of protein on gel (Table I, Step 5)]; Disc 4, combined enzyme-active crude extracts and the individual fractions from all column fractions after hydroxylapatite column [37 @.tgofprotein on gel (Table I, chromatograms up to the final purified product revealed Step 6,)]. Cathode at top: I left, 4 right. For procedure see text. close quantitative ratios within the limits of the assay inactivation at 70Â°. The purified enzyme did not act cx procedure used. Also Km values, loss of enzyme activity clusively on L-methionine but, as demonstrated in Table 2, over 6 weeks, and pH optima indicated dual functions deaminated DL-methionine sulfoxide, L-methionine sul of a single enzyme. Such a phenomenon has been ob fone, L-ethionine, L-cysteine, L-cystine, and DL-homo served previously. An enzyme has been described (18) cysteine. that degrades D-mcthionine but not L-methionine into

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1973 American Association for Cancer Research. L-MethiOfliflase similar products. Rechler and Bruni (14) reported that an REFERENCES enzyme in Salmonella typhimurium had a histidinol de hydrogenase and imidazolyl acetol phosphate amino 1. Binkley, F. A Note on the Specificity of the Enzymatic Cleavage of transferase activity in the same protein molecule. Similar Thio-esters. J. Biol. Chem., 192: 209â€”211, 1951. observations were reported by Yourno et a!. (22). This 2. Biochemical Society Symposium. Two Genes, One Polypeptide dual-function enzyme was thought to be the product of Chain. Federation Proc., 31: 176â€”209,1972. gene fusion resulting in the covalent linkage of the de 3. Chen, S. S., Walgate, J. H., and Duerre, J. A. Oxidative Deamina tion of Sulfur Amino Acids by Bacterial and Snake Venom L-Amino hydrogenase and the aminotransferase (14). Currently, gene Acid Oxidase. Arch. Biochem. Biophys., 146: 54â€”63,1971. fusions are of wide interest (2). In our case, it is likely that 4. Davis, B. J. Disc Electrophoresis II. Method and Application to this enzyme represents another example of the same Human Serum Proteins. Ann. N. Y. Acad. Sci., 121: 404-427, phenomenon. 1964. Less likely is the 2-step mechanism for the double ef 5. Herberg, R. J. Determination ofCarbon-l4 and Tritium in Blood and fect of the enzyme. The deamination of L-methionine Other Whole Tissues. Liquid Scintillation Counting of Tissues. leads to ct-keto- -y-mercaptomethylbutyric acid. This Anal. Chem., 32: 42-46, 1960. product was synthesized according to the method of Meis 6. Kallio, R. E., and Larson, A. D. Methionine Degradation by a ter (I 1). Under the conditions of our assay method, it did Species of Pseudomonas. In: W. D. McElroy and H. B. Glass (eds.), not show any spontaneous decomposition or release of A Symposium on Amino Acid Metabolism, pp. 616-631. Baltimore: Johns Hopkins Press, 1955. methanethiol, which was contrary to the findings of 7. Kreis, W. Mechanism of Action of Procarbazine. In: S. K. Carter Waelsch and Borek (20) for far more vigorous treatment. (ed), Proceedings of the Chemotherapy Conference on Procarba Furthermore, the commercially available Crotalus ada zinc, pp. 35â€”44.Bethesda; National Cancer Institute, 1970. manteus ct-amino acid oxidase (Sigma), when reacted 8. Kreis, W. Metabolism of an Antineoplastic Methyl-hydrazine Dc under the identical conditions of our enzyme prepara rivative in a P815 Mouse Neoplasm. Cancer Res., 30: 82â€”89, 1970. tion, showed deamination of L-methionine exclusively. 9. Kreis, W., Drahovsky, D., and Borberg, H. Characterization of The lack of specificity of the reported purified enzyme Protein and DNA in P815 Cells Sensitive and Resistant to l-@9-D- (Table 2) is expressed in the release of ammonia also from Arabinofuranosylcytosine. Cancer Res., 32: 696-701, 1972. DL-homocysteine, L-cysteine, L-cysteine, L-ethionine, 10. Kreis, W., and Hession, C. Biological Effects of Enzymatic Depriva tion of L-MethiOnine in Cell Culture and an Experimental Tumor. DL-methionine sulfoxide, and L-methionine sulfone, Cancer Res., 33: 1866â€”1869, 1973. whereas D-methionine and a-aminobutyric acid did not 11. Meister, A. Enzymatic Preparation of a-Keto Acids. J. Biol. Chem., act as substrates for the deaminase function of the enzyme. 197: 309â€”317,1952. L-Ethionine did act as substrate for both deamination and 12. Onitake, J. On the Formation of Methylmercaptan from i-Cystine demercaptoethylation. and 1-Methionine by Bacteria. J. Osaka Med. Assoc., 37: 263â€”270, Other microorganisms have been reported to catabolize 1938. methionine such as Proteus rettgeri (3), Escherichia co/i 13. Ornstein, L. Disc Electrophoresis I. Background and Theory. Ann. (12), Pseudomonas (6), and various fungi (17) by de N. Y. Acad. Sci., 121: 321-349, 1964. amination and/or demercaptomethylation. Not all of 14. Rechler, M. M., and Bruni, C. B. Properties of a Fused Protein these microorganisms release methanethiol from methio Formed by Genetic Manipulations. J. Biol. Chem., 246: 1806â€”1813, 1971. nine (19) or a-keto-7-mercaptomethylbutyric acid. Equi 15. Reid, E. E. Organic Chemistry of Bivalent Sulfur, p. 158. New York: molar release of ammonia and methanethiol from D Chemical Publishing Corp., 1958. methionine was reported only with the purified fungal 16. Richards, E. G., and Schachman, H. K. Ultracentrifuge Studies enzyme (17). Rat liver tissue has also been found to re with Rayleigh Interference Optics. I. General Applications. J. lease methanethiol and ammonia from L-methionine (1). Phys.Chem., 63: 1578-1591,1959. However, our experiments did not indicate the presence of 17. Ruiz-Herrera, J:, and Starkey, R. L. Dissimilation of Methionine by an enzyme in homogenized rat liver that catalyzes these Fungi. J. Bacteriol., 99: 544-551, 1969. reactions. 18. Ruiz-Herrera, J., and Starkey, R. L. Dissimilation of Methionine The enzyme described might be useful for studies in by a Demethiolase of Aspergillus Species. J. Bacteriol., 99: 764-770, which a complete removal of 1-methionine is required. It 1969. might also be applied for the quantitative assay of L 19. Segal, W., and Starkey, R. L. Microbial Decomposition of Methio nine and Identity of the Resulting Sulfur Products. J. Bacteriol., methionine in biological samples. The successful use of 98: 908-913, 1969. the enzyme for the inhibition of growth of a tissue culture 20. Waelsch, H., and Borek, E. The Stability of the Keto Acid from and an experimental tumor has been described (10). Methionine. J. Am. Chem. Soc., 61: 2252, 1939. 21. Wiesendanger, S., and Nisman, B. La L-Methionine De-mercapto ACKNOWLEDGM ENTS desaminase: un Nouvel Enzyme A Pyridoxal-phosphate. Compt. Rend, 237: 764-765, 1953. The authors acknowledge the interest and help of Dr. Dorris J. Hutchi 22. Yourno, J., Kohno, T., and Roth, J. R. Enzyme Evolution: Genera son, Dr. R. Barclay, Dr. J. Gill, and J. Begel and the skillful technical tion of a Bifunctional Enzyme by Fusion of Adjacent Genes. Na assistance of Patsy Stroble. ture, 228: 820-824, 1970.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1973 American Association for Cancer Research. Isolation and Purification of l-Methionine-α-deamino-γ -mercaptomethane-Lyase (l-Methioninase) from Clostridium sporogenes

Willi Kreis and Catherine Hession

Cancer Res 1973;33:1862-1865.

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