Enzymic methylation of foreign sulfhydryl compounds

B1OCHIMICA ET BIOPHYSICA ACTA

217

ENZYMIC METHYLATION OF F O R E I G N S U L F H Y D R Y L COMPOUNDS JON B R E M E R AND DAVID M. G R E E N B E R G

Department o] Biochemistry, University o/Cali]ornia School o/Medicine, San Francisco, Cali]. (U.S.A .) (Received June I7th ,I96O)

SUMMARY

A new transmethylating enzyme system in mammalian tissues, catalyzing methyl transfer from S-adenosylmethionine to sulfhydryl compounds, has been studied. A series of "nonphysiological" sulfhydryl compounds (BAL, mercaptoethanol, O-methylmercaptoethanol, mercaptoacetic acid, fl-mercaptopropionic acid, methylmercaptan and hydrogen sulfide) have been found to act as methyl accepting substrates, whereas "physiological" sulfhydryl compounds (homocysteine, cysteine and glutathione) are inactive. With S-adenosylethionine a "transethylation" is found to take place. The transmethylating enzyme system is present in the microsome fraction of several tissues of the rat and in the liver of 6 mammalian species investigated.

INTRODUCTION

During our studies on the biosynthesis of choline in vitro, it was observed that BAL and mercaptoethanol inhibited the reaction. This inhibition was found to be due to the formation of methylated derivatives of BAL and mercaptoethanol. Further study showed that the observed reactions are catalyzed by an enzyme in the microsome fraction of the cell, and that the reaction products are S-methyl compounds. These reactions, thus, represent a new type of transmethylation from adenosylmethionine in mammalian tissues, where only amines and aromatic hydroxy compounds have earlier been reported as being methyl acceptors. The enzyme has been found to have a low specificity, as all of the following compounds serve as methyl acceptors: BAL, mercaptoethanol, O-methylmercaptoethanol, methylmercaptan, hydrogen sulfide, mercaptoacetic acid, fl-mercaptopropionic acid. The enzyme also catalyzes "transethylation" from adenosylethionine to mercaptoethanol or to O-methylmercaptoethanol. MATERIALS AND METHODS

L-[Me-14C]methionine (4.5/~C/~mole) was obtained from Isotope Specialities, Inc., California. L-[ethyl-14C]ethionine (0.55 /~C/~mole) was kindly supplied for these Abbreviations: BAL, 2,3-dimercaptopropanol; Tris, tris (hydroxymethyl)aminomethane.

Biochim. Biophys. Acta, 46 (1961) 217-224

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J. BREMER, D. M. GREENBERG

studies by Dr. D. GROSS of this laboratory. The labeled compounds were diluted with corresponding unlabeled materials to the required specific activity before use, usually o. I-O.2/~C/~mole. S- EMe-14C]adenosylmethionine and S- [ethyl-laC~adenosylethionine were prepared enzymically from the above mentioned materials as reported elsewhere 1. Preparation of S-methylmercaptoethanol was by the method of FITr AND OWEN2, and its 2,4-dinitrobenzoyl derivative according to CHERONISa (m.p. 77-78°), O-methylmercaptoethanol and its 2,4-dinitrophenyl derivative by the method of CHAPMAN AND OWEN4. All other chemicals used were commercially available. Preparation of miclosomes from liver and other tissues and protein determinations were performed as reported previously 1.

Enzyme assay This was based on the extractability of the methylated and ethylated sulfhydryl compounds in organic solvents and the volatility of these compounds, except for that formed from BAL which was not volatile. The enzymic reaction products, therefore, could be assayed as ether (or toluene) extractable, volatile radioactivity. The following procedure was adopted: Microsomes, Tris-HC1 buffer (5° ~moles, pH 8) and substrates were incubated in a total volume of I ml in tightly stoppered io-ml volumetric flasks in a water-bath at 37 °. After the incubation period the flasks were cooled for a few seconds in ice, opened and o.I ml concentrated HC1 was added. Immediately afterwards ether was added to the Io-ml mark, the flasks were again stoppered, shaken vigorously and left to stand at room temperature to allow complete separation of the phases. Samples of the upper organic layer could then easily be pipetted off for assay of radioactivity.

Determination of radioactivity The radioactivity determination was done by liquid scintillation counting under standard conditions. Equal samples of the ether extract were pipetted into two separate counting vials. Scintillation fluid (toluene solution of the fluorescing compounds) and sufficient absolute ethanol to prevent turbidity from the water in the ether extract was added immediately to one vial. The other vial of ether extract was placed under a heating lamp and the ether evaporated. Subsequently the same amount of ether, scintillation fluid and ethanol was added. The difference in radioactivity of the two vials was found to represent the amount of methylated product formed during the incubation. Table I shows a typical experiment, and also gives the results of a test of the efficiency of the ether extraction. The result shows that more than 95 % of the extractable radioactivity was volatile, and that on the second equilibration with HCl--water, 86 % of the volatile radioactivity was extracted into the ether when mercaptoethanol and S-adenosylmethionine were the substrates. Similar checks were performed with O-methylmercaptoethanol, mercaptoacetic and mercaptopropionic acid as substrates. In these cases the extraction of the methylated products into the ether phase was found to be nearly quantitative. With mercaptoethanol as substrate, toluene only partially extracted the methylated product, with O-methylmercaptoethanol, on the other hand, toluene extraction of the methylated reaction product was quantitative. When toluene was used for extraction, a concentrated toluene solution of the scintillator could be added to the extract. By this procedure, Biochim. Biophys. Acta, 46 (I96I) 217-224

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COMPOUNDS

TABLE I TEST OF ASSAY PROCEDURE FOR METHYLATED PRODUCT OF MERCAPTOETHANOL T h e e t h e r e x t r a c t from an i n c u b a t i o n w i t h m e r c a p t o e t h a n o l w a s a s s a y e d for v o l a t i l e a n d nonv o l a t i l e r a d i o a c t i v i t y . The r e s t of t h e e x t r a c t was s u b s e q u e n t l y e q u i l i b r a t e d w i t h o.i i t s v o l u m e of d i l u t e h y d r o c h l o r i c acid ( c o n c e n t r a t e d h y d r o c h l o r i c a c i d - w a t e r s a t u r a t e d w i t h e t h e r (i : io)) a n d a g a i n a s s a y e d for v o l a t i l e a n d n o n v o l a t i l e r a d i o a c t i v i t y . Radioactivity of ether extract before equilibraticn countslmin/ml

Radioactivity cf ether extract after equilibration counts/min/~,l

Total

Nonvolatile

Volatile (by difference)

Total

Nonvolatile

Volatile (by differe, we)

3,405 3,55 °

19o 19o

3,215 3,36 °

3,000 3,045

19o 19o

2,81o 2,855

(2,81o + 2,855) × ioo (3,215 + 3,360)

= 86 % of volatile radioactivity in ether phase

less quenching and a higher counter efficiency was obtained than when ether extraction was used. RESULTS

Identification of reaction product As neither sulfhydryl compounds nor aliphatic hydroxy compounds have been reported as being methyl acceptors from S-adenosylmethionine in animal tissue, it was necessary to determine whether the transmethylation took place on the sulfhydryl or the hydroxyl group of BAL and mercaptoethanol. Mercaptoethanol being the chemically simpler compound, was chosen to establish the methylation site. The two possible methylated products were synthesized and tested as substrates in the reaction. Table I I shows that the free sulfhydryl group was required for the reaction. The following test was also performed with mercaptoethanol as substrate: To part of the dried ether extract (3 ml) containing 17,25o volatile counts/rain was added 25 mg S-methylmercaptoethanol as carrier. Most of the ether was volatilized with a stream of nitrogen. The residue was heated with dinitrobenzoyl chloride and treated with sodium carbonate solution and finally twice recrystallized from methanol-water. The calculated specific activity of the dinitrobenzoyl derivative was 225 counts/min/ mg; found: 234 counts/min/mg after correction for a 23 % quenching effect of the compound in the scintillation counter. A corresponding check was made by adding O-methylmercaptoethanol as carrier and preparing the dinitrophenyl derivative of the sulfhydryl compound. In this case the check for radioactivity could not be done b y scintillation counting as the yellow dinitrophenyl derivative had an almost complete quenching effect in the scintillation counter, but when the compound was checked in a Tracerlab gas flow counter (efficiency 20-25 % of the scintillation counter), the derivative was found to be completely inactive. These experiments therefore, show beyond doubt that an S-methylation had taken place, and as boiled microsomes had nearly no effect, the reaction must be enzymic. Further, the fact that other sulfhydryl compounds also give volatile radioactive products when incubated with rat-liver microsomes and S-adenosylmethionine (Table III) supports this conclusion. B i o c k i m . B i o p h y s . A c t a , 46 ( i 9 6 i ) 217-224

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j . BREMER, D. M. GREENBERG

Whereas methylmercaptan has been found in the urine from patients with severe liver disease, we found it of interest to establish the enzymic formation of this compound from hydrogen sulfide. Table IV shows an experiment with intact and with boiled microsomes where the volatile products from the incubation vessels were trapped according to the method of CHALLANGER5, after the addition of carrier methylmercaptan and dimethylsulfide. The experiment shows that the formation of methylmercaptan is catalyzed by intact microsomes, but also, that there is some methylmercaptan and dimethylsulfide formed non-enzymically. The latter result is in accordance with observation of Dr. J. A. STEKOL (private communication) that a non-enzymic transmethylation from S-adenosylmethionine to sulfhydryl compounds can take place. The only sulfhydryl compound at present known to act as methyl acceptor from S-adenosylmethionine is homocysteine. This transmethylation has been found in yeast and other microorganismse, 7. No corresponding enzyme has been found in animal tissues, although methylation of homocysteine with dimethylthetinS,9 and betaine9 as the methyl group donor has been demonstrated with horse and pigeon-liver methylT A B L E II MERCAPTOETHANOL AND DERIVATIVES AS METHYL ACCEPTORS IN TRANSMETHYLATION FROM ADENOSYLMETHIONINE A p p r o x i m a t e l y I m g (i #1) of t h e listed s u b s t r a t e s w a s i n c u b a t e d w i t h S-[Me-14C]adenosyl m e t h i o n i n e (0.85 #mole, I io,ooo c o u n t s / m i n ) a n d rat-liver m i c r o s o m e s (IO m g protein) for 2 h. Volatile radioactivity in ether extract counts/rain

Substrate

None Mercaptoethanol M e r c a p t o e t h a n o l (Boiled e n z y m e ) S-Methylmercaptoethanol O-Methylmercaptoethanol O-Methylmercaptoethanol (Boiled e n z y m e ) Mercaptoethanol, S-adenosylethionine (o.75 /,mole, 85,ooo c o u n t s / m i n ) Mercaptoethanol, S-adenosylethionine (Boiled enzyme)

o 57,ooo 1,5oo 2,5oo 66,5oo 2,5oo 56,5oo 900

TABLE III VAR1OUS S H COMPOUNDS AS METHYL ACCEPTORS FROM ADENOSYLMETHIONINE T h e listed s u b s t r a t e s (I #1) were i n c u b a t e d w i t h S - a d e n o s y l m e t h i o n i n e ( 0 . 5 / , m o l e , 90,000 c o u n t s / min) a n d rat-liver m i c r o s o m e s (4 m g protein) for i h. Substrate

None Mercaptoacetic acid fl-mercaptopropionic acid P o t a s s i u m sulfide (I mg) Methylmercaptan

Volatile radioactivity ~1 ether extract counts/rain

o 12,4oo 34,4oo 24,75o 56,7oo

B i o c h i m . B i o p h y s . A c t a , 46 (I96I) z i 7 - 2 2 4

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pherases. The enzyme we have found in the microsome fraction of the cell does not seem to be identical with the yeast enzyme. Addition of homocysteine to the incubation mixture did not interfere with choline formation in isolated rat-liver microsomes 1 ; and no increased methionine formation could be detected when carrier methionine was added and reisolated. (The reisolated methionine had some activity, amounting to approx. 5 % of the added S-adenosylmethionine, even from incubations with boiled microsomes, presumably due to non-enzymic degration of S-adenosylmethioninel°.) Neither do other "physiological" sulfhydryl compounds such as glutathione or cysteine act as methyl-accepting substrates in our system, as these compounds do not interfere with the formation of choline in rat-liver microsomes 1. The enzyme apparently is also different from the enzyme system involved in choline biosynthesis. In preliminary experiments where we tried to solubilize the microsome enzymes with n-butanol, it was observed that the butanol treatment always destroyed the choline forming system completely, whereas there still was measurable methylation of BAL. Figs. I and 2 show that a constant reaction rate proportional to protein concentration is obtained at low protein concentrations. Fig. 3 shows the effect of pH on the transmethylation. The reaction rate increased with pH to at least pH 9.5. This indicates that it is the ionized sulfhydryl group which takes palt in the reaction, compatible with a nucleophilic attack on the sulfonium group of S-adenosylmethionine. As the reaction was also easily measurable at a lower pH, later experiments were performed at pH 8, thus decreasing the lability of S-adenosylmethionine which decomposes spontaneously at an alkaline pH.

Specificity of enzyme system The different sulfhydryl compounds (shown in Table III) all gave volatile, radioactive products during incubation with S-adenosylmethionine and rat-liver microsomes (Table III). Furthermore, Table II and V show that adenosylethionine was about as efficient as S-adenosylmethionine as a substrate, both with mercaptoethanol and with O-methylmercaptoethanol as methyl or ethyl acceptors. In cooperation with Dr. H. TARVER and Mr. Y. NATORI of this laboratory, we have also tested adenosylselenomethionine as a methyl donor and hydrogen selenide and methylselenide as methyl acceptors from S-adenosylmethionine. In both cases transmethylation was found to take place. The results with the selenium compounds will be reported separately.

Distribution of enzyme system Microsome fractions from different organs of the rat (protein content not determined) were prepared and incubated in the usual way with BAL as substrate. The results indicated that kidney and lung microsomes have an activity similar to that of liver microsomes, while testis, spleen and intestine microsomes have an activity of between I/IO and 1/3 that of liver. The activity of heart microsomes was insignificant. Tests showed that the extractable activity was not due to choline formation, and incubations done without the addition of BAL, gave negligible activity. Table VI shows the activity obtained with liver microsomes from different species. Guinea pig liver was found to be the best source of the enzyme, while chicken liver exhibited little or no activity. Biochim. Biophys. Acta, 46 (1961) 217-224

j. BREMER, D. M. GREENBERG

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TABLE IV FORMATION

OF M E T H Y L M E R C A F T A N

AND

DIMETHYLSULFIDE

FROM HYDROGEN

SULFIDE

Sodium sulfide (I rag) was incubated with S-adenosylmethionine (o. 7/,mole, 4o5,ooo counts/rain) and rat-liver microsomes (4° mg protein) in Tris-HC1 buffer (IOO /*moles, pH 8) for 2 h. Total volume 1.9 ml. Reaction was stopped, without opening the incubation vessels, by adding sulfuric acid followed by an alcoholic solution containing approx. IOO mg methylmercaptan and approx. IOO mg dimethylsulfide. The methylmercaptan and the dimethylsulfide were then trapped by sucking a stream of air first through the incubation vessel, then through a 4-% solution of mercury cyanide and finally through a 3-% solution of mercury chloride. The mecury methylsulfide precipitated in the mercury cyanide trap was recrystallized from ethyl acetate (m.p. 175°), the dimethylsulfide-mercury chloride precipitated in the mercury chloride trap was recrystallized from benzene and the radioactivity measured. Results are given as counts/min in recrystallized precipitates without correction for presumably small losses of radioactive products during the isolation. Mercury dimethylmercaptide isolated (Hg(SCH~) ~) rag

Radioactivity in recrystallized mercury dimethylmercaptide counts/min

Dimethylsulfidemercury chloride isolated (2(CH~)2S.3HgCI~) mg

Radioactivity in recrystallizeddimethylsulfide-mercury chloride counts/rain

Intact microsomes

395

59,200

512

7,360

Boiled microsomes

325

8,750

471

1,65 °

I ~b ._0 c

.o c "lO.-

~E

2'o

do

s'o

Mg Protein

Time in Minutes Fig. I.

Fig. 2.

Fig. I. Time course of mercaptoethanol methylation from S-adenosylmethionine. Mercaptoethanol (i /*1) incubated with S-adenosylmethionine (o. 5/,mole, 90,00o counts/min) and rat-liver microsomes (3.7 mg protein). Fig. 2. Effect of microsome concentration on the methylation of mercaptoethanol. Mercaptoethanol (i /,1) incubated with S-adenosylmethionine (0. 5/,mole, 90,0o0 counts/min) and rat-liver microsomes for 15 min. Fig. 3. Effect of pH on the methylation of mercaptoethanol. Mercaptoethanol (i/,1) incubated with S-adenosylmethionine (o.5/,mole, 9o,ooo counts/min) and rat-liver microsomes (3.5 mg protein) for 15 min. A - - A , Tris-HC1 buffer, © - - O , Glycine-KOH buffer.

o

pH Fig. 3.

]3iochim. Biophys. Acta, 46 (1961) 217-224

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V

RELATIVE EFFICIENCY OF ADENOSYLMETHIONINE AND OF ADENOSYLETHIONINE AS SUBSTRATES

The labeled substr~tes (o.75 /*mole) were incubated with O-methylmercaptoethanol (i /*l) and rat-liver microsomes ( i o mg protein). Counts/rain incubated

Substrcae

Volatile radioactivity in toluene extract, counts]rain rain

40 rain

36,800 I6,5oo

59,600 28,25o

20

S-adenosylmethionine S-adenosylethionine

135,000 85,000

TABLE

VI

METHYLATION OF O-METHYLMERCAPTOETHANOL IN DIFFERENT SPECIES

O-methylmercaptoethanol (,,~ I # 1 ) i n c u b a t e d with S-adenosylmethionine (o.27 /*mole, i i o , o o o counts]min) and liver microsomes from the listed species (5 mg protein) for 15 min. Species

Volatile radioactivity of toluene extrax~ coums/min

Rat Mouse Rabbit Guinea pig Beef Sheep Chicken

23,700 2o,I oo 25,2 o o 54,8oo I i ,6oo 20,200 I, I o o

DISCUSSION

In the study of the metabolism of BAL by several authors u-is most of the sulfur of this compound was found to be excreted in the urine in organic form. No unchanged BAL could be detected, but some of the excreted compounds gave a positive nitroprusside test, showing that free sulfhydryl groups were still present. GUNINA14 found that most of the radioactive hydrogen sulfide when injected was excreted as sulfate in the urine. On the basis of our results, it is reasonable to conclude that the BAL metabolites are S-methylated products. The fact that the BAL metabolites excreted in the urine, at least in part, give a positive nitroprusside test, indicates that only one of the sulfhydryl groups may be methylated. Further work is required to clarify the exact nature of the reaction products from BAL. Since hydrogen sulfide and methylmercaptan serve as methyl group acceptors from S-adenosylmethionine in our system, methylmercaptan and dimethylsulfide may well be metabolic products of hydrogen sulfide in vivo. GUNINA recovered approx. 60 % of the injected [35S]hydrogen sulfide in the urine after 6 days, mainly as sulfate. The rest of the radioactivity was not accounted for and may well have escaped as the highly volatile methylmercaptan and dimethylsulfide. Methylmercaptan has been isolated from the urine of patients with severe liver Biochim. Biophys. Acta, 46 (i961) 217-224

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disease 15. It has been assumed that this compound represents a pathological degradation product of methionine, but no enzyme splitting methionine to give methylmercaptan, has been reported in animal tissue. On the other hand, cystathionase has been found to attack cysteine, with hydrogen sulfide as one of the reaction products 16. The formation of methylmercaptan in liver disease, therefore, may be explained by a block in the oxidation of cysteine to sulfate and taurine, leading to an increase in hydrogen sulfide formation. Methionine can also give rise to cysteine and hydrogen sulfide via cystathionine, but methionine would not possess enough methyl groups for complete "detoxification" of the hydrogen sulfide formed. The report by WATSON17 on a patient with liver disease who lapsed into coma each time methionine was given accords with this. This theory is also supported by the finding that methylmercaptan is far more toxic to rats than is dimethylsulfide is. It has been reported that the glucoside derivative of BAL (BAL-INTRAV) is far less toxic than BAL itself 19. It would be of interest to try this compound as a methyl acceptor. ACKNOWLEDGEMENT

This investigation was aided by research grants from the National Institutes of Health (H-3o74 and CY-3715), United States Public Health Service and the Cancer Research Funds of the University. REFERENCES 1 j . BRSMER AND D. M. GREENBERG, Biochim. Biophys. Acta, 46 (196o) 2o 5. 8 p. S. FITT AND L. N. OWEN, J. Chem. Soc., (1957) 2250. 8 N. D. CHERONIS, Technique o[ Organic Chemistry, Vol. V I (Micro and Semi-micro Methods) I n t e r science Publishers, New York, 1954, p. 491. J. H. CHAPMAN AND L. N. OWEN, J . Chem. Soc., (195 o) 579. 5 F. CHALLANGER,Aspects o] the Organic Chemistry o] Sulphur, B u t t e r w o r t h s Scientific Publications, London, 1959, p. 18. e S. K. SHAPIRO, Biochim. Biophys. Acta, 29 (1958) 4o5 . S. K. SHAPIRO AND D. A. YPHANTIS, Biochim. Biophys. Acta, 36 (1959) 241. 8 j. DURELL, D. G. ANDERSON AND G. L. CANTONI, Biochim. Biophys. Acta, 26 (1957) 27 o. 9 N. H. SLOANE AND E. M. BOGGIAI~IO,Arch. Biochem. Biophys., 87 (196o) 217. 10 L. W. PARKS AND F. SCHLENK, J. Biol. Chem., 230 (1958) 295. 11 G. H. SPRAY, L. A. STOCKEN AND R. H. S. THOMPSON, Biochem. J., 41 (1947) 362. IS R. A. PETERS, G. H. SPRAY, L. A. STOCKEN, C. H. COLLIE, M. A. GRACE AND G. A. WHEATLEY, Biochem. dr., 41 (1947) 37 o. 13 S. D. SIMPSON AND L. YOUNG, Biochem. J., 46 (195o) 634. it A. I. GUNINA, Bull. Exptl. Biol. Med. (Moskva), 43 (1957) 176. 15 F. CHALLANGER AND J. M. WALSHE, Biochem. J., 59 (1955) 372. 18 F. BINKLEY, J . Biol. Chem., 186 (1957) 287. 1~ C. J. WATSON, Ann. Internal Med., 31 (1949) 405 • 18 G. LJUNGGREN AND n . NORBERG, Acta Physiol. Scand., 5 (1943) 248. 13 j . F. DANIELLI, M. DANIELLI, J. ]3. FRASER, P. D. MITCHELL, L. N. OWEN AND G. SHAW, Biochem. J., 41 (1947) 325 .

Biochim. Biophys. Acta, 46 (1961) 217-224