TEE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 244, No. 16, Issue of August 25, PP. 4499-4509, 1969

Printed in U.S.A.

The Specificity of S-Adenosylmethionine Derivatives

in Methyl Transfer Reactions*

(Received for publication, March 10, 1969)

VINCEKZO ZAPPIA,$ CYNTHIA R. ZYDEK-CWICK, AND F. SCHLENK

From the Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60@9

SUMMARY

The preparation, purification, and analysis of a variety of new sulfonium derivatives of methionine is reported. De- amination of S-adenosyl-L-methionine with nitrous acid yielded, depending on experimental conditions, S-adenosyl L-(Z-hydroxy-4-methylthio)butyric acid or S-inosyl-L-(Z-hy- droxy - 4 - methylthio)butyric acid. S-Inosyl - L - methionine was obtained by enzymatic deamination of S-adenosyl-L- homocysteine to S-inosyl-L-homocysteine and methylation of the latter by methyl iodide. 4-Dimethylsulfonium-L-(Z- hydroxy)butyric acid has been prepared from S-methyl-L- methionine sulfonium salt. The sulfonium derivatives were purified by ion exchange chromatography and were charac- terized by ultraviolet spectrophotometry, paper and thin layer chromatography, electrophoresis, and hydrolysis to known fragments.

The new methionine sulfonium derivatives and S-adeno- syl-(5’)-3-methylthiopropylamine were investigated as methyl donors and as inhibitors with three purified methyltrans- ferases: histamine methyltransferase, acetylserotonin meth- yltransferase, and homocysteine methyltransferase. The replacement of the 2-amino group of the methionine moiety by the hydroxyl group resulted in complete loss of activity in all systems investigated; these deaminated derivatives were also inactive as inhibitors of S-adenosyl-L-methionine in the transmethylations. The methyl donor capacity of inosine derivatives and of decarboxylated S-adenosylme- thionine ranged between inactivity and an effect equal to that of the biological methyl donor. Homocysteine methyl- transferase was the least specific of the three enzymes. Ex- periments with S-inosylmethionine, racemic with respect to the sulfonium pole, established that both sulfonium dia- stereoisomers were utilized in the methylation of homo- cysteine.

S-Adenosyl-L-homocysteine was found to be a very effective inhibitor of the systems investigated; the enzymatic removal of its amino group in the adenine part led to S- inosylhomocysteine, which was without effect as an inhibi- tor. 5’-Methylthioadenosine, a catabolite of S-adenosyl-

* This research was performed under the auspices of the United States Atomic Energy Commission.

1 Visiting Scientist on leave from the Department of Biochem- istry, Second Chair, Medical School, University of Naples, Italy.

methionine, inhibited the methylation of histamine and acetylserotonin.

On the basis of these results, three binding sites for S-adenosylmethionine on the methyl transfer enzymes may be postulated.

Analogues and derivatives of metabolic intermediates have contributed much to the understanding of biochemical reaction mechanisms. The possibilities of modifying the biological methyl donor, S-adenosylmethionine, have been explored, so far, only with regard to the methyl sulfonium group. The sulfur atom has been replaced by selenium (1) and some propert,ies of the selenonium derivative have been described (2). S-Adenosyl- ethionine has become available by biosynthesis from ethionine with yeast (3); this derivat,ive has been investigated extensively by Stekol (2). Chemical synthesis of (+)S-adenosyl-L-methio- nine has permitted a study of the sulfonium stereoisomerism in relation to enzymatic activity in transmethylat,ions (4).

The present experiments deal with the modification of other regions of the S-adenosylmethionine molecule, specifically, the two amino groups and the carboxyl group. Several derivatives (Fig. 1) of S-adenosylmethionine (I) have been obtained. Re- placement of the 2-amino group of the methionine part by a hydroxyl group gave S-adenosyl-L-(2-hydroxy.&methylthio)- butyric acid (II); more rigorous deaminating conditions led to the totally deaminated derivative, S-inosyl-L-(2.hydroxy-4-

RI R2 -NH2 -CH2CH,CH(NH2) COOH I

--NH* -CH2CH&H(OH)COOH II IN’

CH3 -OH

,oq-i-“’ -oH

-CH2CH2CH(OH)CCOH DI

-CHHzCHpCH(NHP)COOH IY

w --NH2 -CH2CH2CH2NH2 P

OH OH

FIG. 1. S-Adenosylmethionine (1) and some of its derivatives (II to V).

4499

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meth!-lthio)l)ut\.ric acid (I/f). 111 a circuitous way, S-inosyl-L- mcthiolline (I l-) has been l)reparecl. For this, S-adeuosylhomo- c!-steilw 1~x8 roll\-crtctl 1)~ cnzymutic deamination to S-inosyl- homoq-atcille; the latter w:L.~ methylated to X-inos)-llliethiollille (IT-). 13~ cwz\-matic rcmoval of the carboql group, using the procctlure of ‘I’:lbor (5), S-atlc1los~-1-(5’)-3-1~leth~lthiopropyl- aniinc (l?) Iv:15 obtained. S-~Ieth!-I-L-lllcthiorlille sulfonium salt, which :I(+ as an enzymatic methyl donor toward L-homocysteine (6), has bccw inclutlctl in thi.s stud>-. Its deumination led to 4-

clir~~etll~lsulfo~li~~~~~llL-(2~h~~l~~o~~)b~~t~ric wid, (CH&&‘H~C:H~- CH(OII)cooII.

l’hwe t~l~zymcs, catalyzing nitrogen, oxygen, and sulfur methylatioll, n-ew wlcctctl for tcbting these corr~~~ou~~ls as nlethyl donors and as illhibitors; tile systenis were, histamine nlcthyl- tranxf’wwc, acctylserotonin illeth!-ltrullaferxse, and homocysteine meth\-ltlansfer~~sc, reslwctivc~ly.

S-~~~~lc~los)-lho~~~o~~~steillc is a product of all tmmlslneth~latiorls with S-:~tlciios~linctliioiiiilc as methyl donor (7, 8); similarly, S-iiloh~lllomoc)-stcine would result from tr:~rlsmeth~lutio~~s with S-irio~~l~nctllioi~i~lc. 5’.~Ieth-lthioadellosirle represents another enzynlatic dcri\-ativc formed from S-udcnos3rllncthiollille through separ:ttc pathways (9-l 1). The inhibitory effect of these reac- tion l)rotlucts 011 tr:~ll~tllcth!.latiolrs has been esamined.

I.:);~‘I.:Rlhll.:STAL PROCEDURE

Cowpounds-The quality of S-ndenos-1-L-methiolline wed as startiilg material for the preparation aiid isolation of derivatives iu of great im~wrtaiwc. The commercial rttdioactive preparations in acid solution wre satisfactory, but I~UIIIC~OUS iml)urities were found in SOI~L’ ~~onlabc~lctl samples b>- pa~w and thin layer chro- matography. I<)- clcc~:~rl~os~latio~~ or deamination of such mix- tures, the nunlber of i)rotlucth is increased beyond the l)ossibilit\ of rc-olutiotl 1)~ silqlc stcl) ion cschange chromatography. For the l)wswlt illvest igxtion, S-:tdcllosa-1Inethiollillc was l)rcpared by biobyllthcsis with Jxwst (12) :~ntl iholated by the procedures de- velo~wd in this lalwratory (13, 14). The material was concen- trated under ~~ctluc~~tl j~iwsurc: to a level of 15 to 20 pmoles l)cr ml, the pI-I \V:W atljustctl to a \alue between 2.5 and 3.0, and the solrltioll \ws kcl)t in the frozen btate. l\Iore rccentl~-, it satis- factory qualit\- of S-atlcilos?.lilietliioiiiiic sulfate has been made av:rilal)lc to th(t ;~uthors l)y tlw Rewnrch 1,:rboratories of lsoch- riiiger 1\I:uiiihrim.

S-.\tlcuo~;\l-r,-lioi~io(,~-~t~~iile was produwd by enzymatic s\-nthwi~ I’rom xl~wosine and r,-holllo(:T.::tcille (15) and isolated by ion escahalrgc c~h~~o~~l;~toglal)l-~\- (16).

S-l\Ictll~-l-r,-l~rc~tllio~lit~(~ suli’onium ‘aIt was obtained as iodide b\. tlrc, nwthod of l’ownies and Kolb (17). In the nitrous acid treatillcxirt usetl l’or the dcamillation of the co~npoulltl, iodide intcrfcrw lxc:~usc it is ositlizrd to iodine. The mat trial was conwrtcd, therefore, into the sulfate by adsorption on a wcakl~ acitlic wtion c~scllallgc rcsiil, followc,tl I)>- elution with sulfuric acitl. The l~rocc~lln~(~ also removes nlethioninc whirh occasion- all)- i. l)rcs;cl~t :I’*: a11 impurity in the sulfonium l)reparation. For the lws;cnt cqwimcnts, ‘-‘(‘TI,-labclrd material was used because 4-dinrt~th?-ls.ulfo!~irct~l-L-(2-h\-tlros~)l~~lt~ric acid (dcaminated S- meth\-l!llcthiollili(,) cannot be detwted by ninhydrin tests or sp~~~tl.o~,lloto!llctl’y. The lmrification stel)s arc exemplified I~>- the following lxoce~luw: 160 pmolcs of (14(‘H3)-S-mcthyl-L- methionillc su!fo~~ium ioditlr were applied to a column of .\mber- litc IN-50 (resin lw(1, 2 x 10 cm) in I-If form, previously washed

with water until the effluent sholr-ed a 1~1~ value above 5. The column TWS n-ashed with 200 ml of water, and fractions of 25 ml were collected and assayed for radioactivity. The contaniinat- ing ‘“C’H3-111Cthiollille wxs contained in the first 75 nil and the follo~x-ing fractions declined to an insignificant counting rate. The sulfonium conllx~und W:IS then eluted with 0.1 s I-12S01, ne:wl,x- all of it beiilg contained in the first 75 ml of cluatc as judgrl by tlicl radiowtivit\~. These fractions wcrc pooled and adjusted to 1)1-I 3 to 4 with 0.3 s l<a(OII)z, the prwil)itatc \vas re- mowd by c~,iltrifug:rtion, and the solution was conccntr:~tctl to a small volume. The wmpound was pure as judged by paper and thin la\-cr chrolli;ltogr:tl)li?- and by electrophoresis.

5’-~Icth\-lthio~\tlet~(~~i!l~ was Ixcpared by- hydrolysis ol S-aden- osylmcthionine (IS). Commercial L-homocysteinc thiolactone hydrochloride n-as conr-crted to L-honloq-steine by alkali (19). Histamine tlih~clrochlol~ide and S-acetylscrotonin were obtained froni l\Iaiiii.

Enzy~les and Alssny-Sonal)ecific adenosine dcamiilase (atho-

sine aminoh!-tlrolase, IX‘ 5.5.4.4) from Aspergillus oryzae (20) was prcparcd from Sanzyme (Sallkyo, Japan) which was obtained from C’albioc~hcm. The material ~-as purified according to the directions of Sharpless and Kolfenden (21) through the alcohol fractionation. This product was satisfactory for the deamina- tion of X-adellos~lhorrloc~steinc (22).

S--~~lenoa!-lmethiollilic decarbosylasc was purified l’rom I&x%- ericltia coli by the method of Tabor (5) including the first am- monium sulfate precipitation.

X-.~deno~~lmethiollillc: histamine S-lneth?-lt~lllsferase (EC 2.1.1.8) was purified by the lxocedure of I%rown, Tomchick, and .Iselrotl (23). Frozcl~ guinea pig brains Ivere obtained from Pel-Freez I<iolopicals, I tic., Rogers, .Irkansas. The labeled rnet2i\-lhi~t;~lrlillc i’ormcd in the reaction was mensurcd by tlie lxocedurc of Snyder, l~ultlessarini, and Xsclrotl (24), and the amount of “(‘-!neth!,lhihtarliilie produced was calculated on the basis of the hl)wific radioactivity of the methyl donor. The sulfoiiiuni wmI)ouiids tested as donors were retained quantitu- timely in thr aqueou> l)hasc, as .q h on-11 by the absence of radio- actirit>- in thr control espcrimcnts without xwptor. A\ slight correction x:15 new~s:~r~- in esperiments with S-;ldeilos!-l-(j’)-3- meth\-lthiol)rol)~l:~lllilrc bcc:u~s:c some tlecoml)osition occ~urred in the colitrol (qerim(wt‘: a11(1 traces of radioactirity were ex- tracted. _ . ‘~C-~IethT-lhistaliiiiie was measured also 1)~ radioactir- ity scanning after separation I,!- paper chro!~i:~togr~~l,h!-.

Acetylserotonin n~ethyltrallsfcrase (S-atlellos~l~nctlliollille:,~- acetylscrotonin O-lncth~ltl~an~fcr:rsc, EC 2 1 1 .4) was purified by the procedure of .Awlrod and n’eissbach (25) from pineal glands, obtained from I’cl-Frcez l~iologicnls. 14(:-Rlclatonin, separated from the methyl donor by estraction into CI-ICl:I at, neutrality (25), n-as rnca~wrcd by scintillation counting. The calculation of the yield of melatonin was based on the sl)ccific activit>- of the moth\~l tloiior.

S-.\tleno~~llrletllio~li~le:llo~~~oc~stei~~e S-meth?-ltl,aiisferase (EC 2. 1 1 lo), purified from Sncchnrowyces cerevisia.e (19), xv-as generously- furnished I)\- I jr. S. I<. Shapiro. Mcthionine forma- tion was mcwured by the tracer assay of Shapiro and Yphantis (26). All sulfonium tlcrirative.~ were retained by the l)o\~cs 50, Li+ form columns, except those without the 2-amino group of methioninc. For S-atle~lo,y+ and S-inos~l-r,-(2-hvdros~-4- methylthio)butyric acid, the llrocedure has been modified as follows. The chromato~lal)hic separation was performed with I)owvcs 50, H+-fornl columns;, instead of Lif form, as in the orig-

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inal method. The saml)les ~vcrc acitlified to pH 3 before chroma-

tography, and mcthionine ~~-aa elutcd with 8 ml of 1 s H,SO,, whereas the sulfonium c*ompou~lds wcrc retailled. The samples with 4-dimeth3-lsulfolliuln-L-(2-h~dros\-)but?-ric acid as Illethyl dollor ~vvrrc analyzed for labeled nlcthionillr and 2.hydras\--4- mcthylthiobutyric arid after l)apcr and thin laycxr chromstog- ral)liy of the incubation mixture.

In sonic experiments, trallslnethS-l:Ltioll \vah htudied I)y COW pling the methyl transfer reac+ion with tlcamination of the re- hulting :&nosine thioether. The dcan~i~~ation was followed spcctrol)lloto~~ic~tt~icall!- (22). Fol S-adt1ros~1-~-(2-h~tlros?--4- mc,rcal)to)hut!-i,ic acid the reacti\-it\. with tlcanrina~c ~~-as iml)lird in :u~ulog)- with other S-adcllos~lthioc,tllers (22).

I’rotcin concclltration was cstinialc~tl by the method of Lo\v~,J et al. (2i), with bovine dcrum albumin :I., ~;taiidartl.

‘1 nalyiical Procedures-For sl)ectrol)hotolnetric ahsay of l)urillc coni~~ounds, the, folio\\-ing molar absorbance3 were ued: for all adcnosyl compounds, 15,400 at 260 111~ at, or near llcutralit\-, and 14,700 at 257 111~ in strongly acid solution (12, 14); for all inosyl compounds, 12,200 at 249 nip in neutral holution, and 10,900 at 250 rnp ill acid solution (21, TJ).

Radioactivity was mcasurcd in :I I?ccl~n~:~~~ Scintillation Spcc- trometer, niodcl LS II. i1 0.4% solution of 2.5.dil)henyl- osazole ill :I mixture of equal volumes of tolncne alltl ethanol was used. The quc,nching was corrected by ertcrnal ~;t:rildal~diz:~tioll. -1 l’ackard radiochrom;~togr:rm scanner, model 7200, \v:ls en- ploycd for scanning of the paper chrom:ltogr~tm2. In home in- stances, t.hin layer chromatograms wcrc esamincd for rntlionctiv- it!- by radioautography IT-ith Gevacrt ;Y-r:ly film::; an rrposure time of 5 days w:rs sufhicient for the ltvels of radioactivity used in thr csperilnents.

For ion cschullge chromatography, Do\~cx 50 rt>in, 8’ ; cross- linked, 100 to 200 mesh size, ~~1s nr~~~lo~-ctl ill moht experiments. The resill bed TV;IS 12 X 1 cm. For the clution of >ulfonium derivatives, hydrochloric acid was used and then remol-cd from the desired fractions by distillation under reduced 1)rea.s;lu.c. -in altcrnativc method is the elution with H$O, and isolation of the compounds by precipitation with l)hosl’hot~mg.~tic acid (12) or Reineckc acid (13). However, this method gnvc’ ~OW.Y yields because the precipitates of dcarnillatcd derivatives with the com- plesing agents are more solul~le than those of S-ntleno.~!-lmethio- nine. =\fter removal of HCl, tht oily residue-: lvere taken up twice in small quantities of water, re-evaporated, and krpt in the frozen state. These solutions still contained some KC1 which contributed to the stability of the sulfonium compoun(ls. All derivatives, like X-adriios)-lmethioninc (2S), have been found unstable when kept for prolonged prriods in neutral solution. If neutralization is required for rnzymatic experiments, the ])I% can be adjusted to the desired value with K2H1’04 or KHCOZ shortly before USC.

For paper chromatography, JYhatman So. 1 paper \v:I~ used with ascending technique. Silica gel plates (Eaatmnn (‘hromn- gram Sheets 6060, rrith fluorescent indicator) were cmplo\-cd fol thin layer chromatography. Before use, the C’hromagram Sheets were activated by heating for 20 min at 80”. The solvent sys- terns had the following coml)ositioll, b>- volun~c: Solvent -1, I-butanol-acetic acid-water (12:3:5); Solvrllt B, 1 liter of 4.55 M

(NH&S04-0.1 XI sodium phosl)hatc, pH 6.8, + 20 ml of 2- propanol; Solvent C, 1-but~Liiol-ethanol-l)rol~iollic a&-water (10:5:2:5); Solvent, D, 2-I)ropanol-\~ater (16:9); Sol\& E, 2-l)ropallol-HCl-~~-ater (170:41 :39); Solvent E’, 2! 4-lutidine-

TAHLE I Paper and thin layer chromatography of suljoniunz compounds and

their structural units

Compounds

S-Adenosyl-L-methioniile (Z)

S-Adenosyl-L-(2.hydrox~- 4-methylthio)butyric acid (II)

S-Illosyl-L-(2.hydroxy-4- methyIthio)butyric arid (117)

S-Irlosyl-L-methiorline (ZP S-Adenosy-(5’).a-methyl-

t hiopropylamine (1;) S-Methyl-L-methionine. 4-Dimethl-Isulfonillm-L-(2-

hydroxy)but)-ric acid.

S-Adenosyl-L-homoc)-s- teine.

S-Illosyl-r,-homoc:rsteillc S-lLibosyl-r,-honlocysteille. 5’.Methylthioaderrosine . 5’.Methylthioinosine.,

RF values (X 100) and solvent systems

Paper chromatog

Thin layer chromatography

raphy (silica plates)

5

32

17 15 20 ti(j 56 -!

37

27

GO 85

39 9G

89

15 38 SO

7 24

-

A

19

21

18 10

19 14

19

41 31 23 30 71

B

50

30

(is 71

49 73

32

30

G 1s

23

19

1s 17

21 17

20

50 38 52 8G 80

7

14

20 14

8 3

19

56 55

93 92

55

45 4!)

47 52

G4

GO

/’ -

18

27 20

4

8

87 58 33

90 -

2,4,6-collidine-water (6:5:5). Ul compounds wcrc neutralized to pH 7 before the chromxtographic separation. For dctcction, ultraviolet quenching (254 mF), ninh!-drin spray-, platinum iodide spray (29), and radioactivity scanning were used. Table I gives the RF values of the lnincipal compounds alld of some of their hydrolytic products.

For paper ionophoresis, a %vant high voltage clectrophoresis apparatus, model LT-489, n-:1.< used with :I distance of 90 cm between the electrodes. The separation- (Thblc II) were performed for 1 or 2 hours at 3000 volts on stril)s of Wh:Ltman n-0. 3MM paper. ;U’ter migration, the com~~ounds were identified as described above.

Sulfoniunr Conzpounds-Dcniiiillatioii of S-3denosi?-lmethiolline by enzymes would be the method of choice, but experiments with various dcaminascs including the nonsprrific adcnosine deaminase from A. oryxne, which dcaminates S-adcnosylhomo- cysteine, were unsuccrssful (22). Like\vise, IJ-amillo acid oxidase from snake venom, while active toward L-methionine, is without effect on the amino group of rnethionine when the latter is bound in the sulfonium structure (30). It was necessary, therefore, to employ nitrous acid as the dcnminating agent.

Cantoni (30) has shown that the amino group of the adenine part of S-adenosylmethioninc can be removed by nitrous acid, but, the product was not isolated. The pK ralurs of ihe two amino groups in S-adello~;3lmethiolline arc himilar to those in adenosinc and mcthionine, rcspectivcly. Klce and Mudd (31) have reported pK 3.5 for the amino group of the adeninc, and pK 7.8 for the 2-amino groul) of the methioninc part of the sulfonium compound. We have taken advantage or this difference, and by reducing the acitlity, nitrite concelltration,

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TABLE II

Electrophorelic mobility of S-adenosylmethionine and related suljonium compounds

The separations were carried out at 25” on Whatman No. 3MM paper in a Savant high voltage electrophoresis apparatus. Nega- tive numbers indicate migration toward the anode. Each spot contained 20 mpmoles.

S-Adenosyl-L-methionine (I) S-Adenosyl-L-(2.hydroxy-4-

methylthio)butyric acid (II) S-Inosyl-L-(2.hydroxy-4-

methylthio)butyric acid (III S-Inosyl-L-methionine (IV). . S-Adenosyl-(5’).3-methylthio-

propylamine (V) S-RlethSl-L-methionine 4-Dimethylsulfonium-L-(2.hy-

droxy)butyric acid

1

-

I 1 _

Buffer and pH values

0.2 M

I I

phos- 0.1 M

PFF? borate,

8.5

1.9 1.1 4.0 3.6

6.8 5.6 6.4 6.2

2.6 2.2

I

cm volt-'hr-' x 103

1.8

0.9

1.0 2.2

3.4 4.3

2.0 i

-3.4

-2.2 -1.4

2.4 1.7

1.2

I I 0 I COOH PURlNEtRlBOSE + S + CH$H$H(

I 1 CH3 1 NH2 I I I

(Cl) (5) Cc)

FIG. 2. Hvdrolyses of X-adenosylmethionine and its deriva- tives. Details are given in the text.

and reaction time, it5 compared with the conditions leading to complete dcnmination, we have succeeded in making the removal of the amino group of the methionine the predominant reaction.

For identification of the new sulfonium compounds, the procedures elaborated for S-adenosylmethionine were used (28, 31). Like the parent substance, the derivatives were not obtained in the cry&lline state, and the analytical tests were restricted by the small quantities available. Spectrophotom- etry permitted us to distinguish between adenosine and inosine dcrivatirex; failure to respond to the ninhydrin reagent indicated deamination of the 2-amino group of the methionine moiety. Hydrolyses nnalogouq to those of X-ndenosylmethionine were carried out as indicn,ted in Fig. 2. For hydrolysis Split a, 0.1 IV NaOH at 25” for 10 min is sufficient. With the same hydrolytic conditions at loo”, Split b occurs in addition to Split a; the pentove is destroyed under these conditions. Hydrolysis at pH 4 at 100“ for 25 min leads almost exclusively to Split c. The products, adenine, hyposanthine, 5’.melhylthioadenosine, 5’- methylthioinosinc, methionine, 2-hydroxy-4-methylthiobutyric acid, and homoserine were nvnilablc as reference substances for the chromatographic ljrocedures given in Tables I and II. The results of these tests were taken as evidence for the structure of the sulfonium compounds.

Sdderrosyl-r.-(d-Hydrozy-4-~nethyltkio)butyric d&&The par- tial dcamination of S-adenosylmcthionine was carried out in citrate-phosphate buffer solution at 111-I 2.5 to 3.0 with a IO-

70 20 30 40 50

FRACTION NUMBER

FIG. 3. Chromatography of deaminated derivatives of S-adeno- sylmethionine. For the quantity of 70 hmoles of mixed sulfonium compounds, a Dowex 50-H+ column, as described under “Experi- mental Procedure,” was used. For elution, an acid gradient was provided by 200 ml of 0.5 N HCl in the mixing flask and 400 ml of 2 N HCl in the feeding reservoir. Fractions, 13 ml each, were collected and assayed by spectrophotometry at 250 and 257 rnp. The elution was finished by a gradient obtained with 100 ml of 1.8 N IICl and 100 ml of 3.5 N HCl. The material in the elution peaks was identified by spectrophotometry and paper chroma- tography as: A, S-inosyl-L-(2-hydroxy-4-methylthio)butyric acid (Amax = 250 mp); B, S-adenosyl-L-@hydroxy-4-methylthio)- butyric acid; and C, residual, nondeaminated S-adenosylmethio- nine.

fold excess of NaK02. The buffer solution was similar to that specified by >IcIlvaine (32); it contained 4.4 ml of 1.0 M citric acid and 1.1 ml of 1.0 M Na2H1’04, diluted with water to 10 ml; the pI-I was 2.5. A quantity of 48 mg of KaKOs was dissolved with minimal agitation in 3.0 ml of this buffer in an ice bath. To this, 4.0 ml of cold S-adenosylrnethionine solution (17.5 pmoles per ml; pH 2.3) were added and the mixture was kept without agitation at 25” for 45 min. The ,final pH was 2.9. Residual nitrous acid was removed from the reaction mixture by evaporation under reduced pressure to one-half of its original volume. The material was transferred to a cation exchange column (H+ form), and the elution was carried out with HCl as shown in Fig. 3. The fractions of clution Peak B were concen- trated under reduced pressure to the dry state; the material was taken up in a few milliliters of water and stored in the frozen state. The yields in several prcparalions ranged from 60 to 80%. ~4C-Methyl-labeled material was prepared from the labeled sulfonium precursor without modifications.

The compound was identified by the following tests: spectro- photometry gave EZGO ,,,LL = 15,400 in 0.02 M phosphate at pH 7.4, and tzsl ,np = 14,700 in 1 JS HzSO+ These values are identical with those of X-adenosylmethionine (14, 33) which can be considered as evidence that the adenosine rnoiety was not altered. Paper and thin layer chromatography (Table I), as well as paper electrophoresis (Table II), gave only one spot which distinguished the material from the parent substance, and its failure to respond to the ninhydrin reagent indicated the absence of the 2-amino group. The presence of the sulfonium structure was indicated by the hydrolytic behavior, which

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resembled closely t’hat of other adenosine sulfonium compounds in the extreme lability of t’he glycosidic bond in the alkaline range and unusual stability in the acid range (28, 33).

Hydrolysis according to Fig. 2, under the conditions specified above, yielded adenine (Split a), 2-hydroxy&mcthylthiobutyric acid (Split b), and 5’-methylthioadenosine (Split c). The release of adenine by alkaline hydrolysis was shown by spectro- photometry (~269 ,np = 12,300) and paper chromatography. X- Pentos)-l-(2-hydroxy-4-methylthio)butyric acid was an addi- tional product of alkaline hydrolysis at low temperature; it was located on the paper chromatograms by anisidine spray (RF 0.12 in Solvent System A, and RF 0.95 in Solvent System l3). 2- Hydrox&methylthiobutyric acid was formed in 0.1 N NaOH at 100” aft.er 10 min; it was identified by paper chromatography and comparison with a commercial sample, giving RF 0.84 and 0.57 in Solvent Systems A and B. No other spots were found. These data characterized the new compound as S-adenosyl-L- (2.hydrosy-4-methylthio)butyric acid (Formula II in Fig. 1).

S-Inosyl-L-(d-hydrozy-J-methylthio)butyric &id-For the prep- aration of doubly deaminated S-adenosylmethionine (34), a quantity of 50 Kmoles of S-adenosylmethionine in 3.4 ml of H20 was mixed wit,h 0.6 ml of glacial acetic acid and cooled in ice. One milliliter of 3 M XaNOz was added, drop by drop, over a period of 10 min. The solution was kept in ice for 24 hours. Paper chromatography of 5.~1 samples wit,h Solvent System B showed that all S-adenosylmethionine had disappeared after 30 min, while X-adcnosyl-(2.hydroxy+methylthio)butyric acid had become the principal product. The latter gradually was converted to S-inosy-L-(2-hydrox&methylthio)butyric acid. After 24 hours, the excess of nitrous acid was removed by con- centrating the solution under reduced pressure to one-half of its original Tolume. It was transferred to a Dowex 50-H+ column as specified under “Analytical Procedures.” The development was carried out with HCl in a gradient provided by 100 ml of H20 in the mixing chamber and 300 ml of 2 N I-ICI in the feeding reservoir. Initially, a small peak of 5’-methylthioinosine was observed by monitoring the eluates at 250 rnp; S-inosyl-L-(2- hydrosy+methylthio)butyric acid was elutcd with 1.0 to 1.3 s HCl. The requisite fractions were pooled, concentrated, and stored in the frozen state.

The yields ranged between 70 and 80yL:, and the same pro- cedure n-as used to obtain the labeled derivative from 14CH3-S- adenosylmethionine. The fully deaminated derivative of X- adenosSlmcthioniIle n-as identified by paper and thin layer chromatogr:rl)hy (Table I), paper electrophoresis (Table II), and by its abrorbance spectrum (~2~~ n,lr = 10,900 in 1.0 x H,SO,; 6249 II,,, = 12,200 in 0.02 ;\I phosphate buffer). Alkaline hy- drolysis at 100” for 10 min (Split b, Fig. 2) gave hypoxanthine and 2-hydroxy-4-methylthiobutyric acid. Hydrolysis at pH 4 yielded 5’.methylthioinosine (35, 36). No ot,her spots were detected with the reagents employed.

X-Inosl/l-L-~~~etlzioni~ze-X-8denosyl-L-homocysteille was con- verted to S-inosyl-L-homocysteine; the methyl sulfonium deriva- tive of the latter was obtained by treatment with methyl iodide.

Deaminntion of S-adenosylhomocysteine to X-inosylhomo- cysteine cannot bc accomplished in a satisfactory way with nitrous acid, because experimental conditions that lead to replacement of the B-amino group of adenine remove the amino group of the homocysteine part as well. The thioether group is also affected by nitrous acid, leading to the sulfoxide. It was

possible, however, to convert S-adenosylhomocysteine to X- inosylhomocysteine by enzymatic deamination with the non- specific adenosine deaminase from 8. oryzae. This enzyme did not remove the amino group of the homocysteine part, and S-inosylhomocysteine \yas obtained (22). This compound was characterized by the absorption spectrum, which was typical of an inosine compound, and by a positive response to nin- hydrin which showed the presence of the 2-amino group of the homocysteine part. For further identification, the product was hydrolyzed with 0.1 N HCl for 30 min at 100”. Paper chroma- tography (see Table I) showed the formation of hypoxanthine and S-ribosylhomocysteine; a reference sample of the latter was obtained from X-adenosylhomocysteine (16, 37).

The synthesis of S-inosylmethionine was accomplished as follows. S-Adenosyl-L-homocysteine (45 mg) was dissolved in 45 ml of 0.01 M potassium phosphate buffer, pH 6.8, and 0.3 ml of adenosine deaminase purified from A. oryzae (see “Enzymes and Assay”), 2.0 mg of protein per ml, was added. The progress of deamination was followed by spectrophotomctry at 265 rnl.r (22). -1fter 20 min the readings were constant and the absorbance at 265 rnp was 407, of the initial value. The solution was passed through an amberlite IX-50 column, resin bed 8 X 1 cm, previously treated with 100 ml of 1 N HCl, followed by water until the effluent was at or above pH 5. X-Inosylhomo- cysteine was not adsorbed to the resin; its elution was completed by water, and spcctrophotometry at 250 rnp showed the material to be present in the first 100 ml of eluate; the enzyme was re- tained by the column. After the addition of 0.15 ml of 88% formic acid to the eluate, S-inosylhomocysteine was isolated by evaporation to the dry state. Its methylation was patterned after the procedure outlined by Tocnnies and Kolb (17) for the synthesis of S-methylmethionine. The material was taken up in 0.65 ml of 88% formic acid and 0.2 ml of glacial acetic acid. After cooling in ice, 0.1 ml of CH,I was added, the container was sealed tightly and kept at room temperature for 4 days with exclusion of light. After dilut,ion with a few milliliters of HzO, the residual CHPI was removed by distillation under reduced pressure. The solution was cooled and neutralized to pH 6.2 by slow addition of about 24 ml of 1.0 M K2HP04. The separation of S-inosyl-L-methionine from S-inosyl-L-hort;ocystcine is illustrated in Fig. 4. The yield of S-inosylmcthionine was 48.4 pmolcs (41 y0 of the theoretical value).

The product had the typical absorbance sl)ectrum of an inosine compound (~2~~ lnll = 10,900 in 1.0 x H,SO,; e249 ,np = 12,200 in 0.02 M phosphate buffer, pH 7.4). 13y the hydrolytic procedures outlined in Fig. 2, hyposanthinc, methionine, 5’- mcthylthioinosine, and homoscrinc n-as obtained. The presence of the sulfonium group in t,he molecule rendered the glycosidic bond labile. After 10 min in 0.1 r\~ NaOH at 25”, the absorbance shifted to that of hypoxanthine (~263 mp = 11,500).

Baddiley and Jam&on (38) have shown that the mcthylation of S-adenosylhomocysteine with methyl iodide leads to S- adenosylmethionine which is racemic at the sulfonium center. In analogy, the compound obtained in the present experiments should be designated as (&)X-inosy1-L-mcthionine.

S-Adenosyl-(5’) -S-methylthiopropylamine-S -Adenosylmethio - nine was decarboxylated with the specific dccarboxylase obtained from E. coli (5) which converted 60 to 85% of the starting material. For detection of the residual S-adenosylmethionine, paper electrophoresis was used; especially at low pH values,

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r

< 0.1 M ,“?, 0.5-40N ./’ // : ,

> I NaCl HCI

/

/’ j I

/’ /

A2 ;

/ / : I 0 -I

A

0 Ll I -L-j0 0 I 5 1 20 30

FRACTION NUMBER

FIG. 4. Chromntographic separation of S-inos~lhomocvs~eille from S-inosylrnethionine. A Dowex 50 (Na+) colrimn. r&n bed 1 X 12 cm, was prepared as described by Shapiro ani Ehninger (14). S-Illos?:lhomocysteine (Peak A ) \vas elnted by 0.1 SC SaCl in 25.ml fractions. After rinsing the colrmm with 25 ml of HzO, the elution was continried with HCl, ranging in a gradieiit from 0.5 to 3.0 N. Fractions of 11.5 ml u-ere collected; the fractions of Peak B contained S-inosylmethionine.

I I I 7

0 20 40 60 80

FRACTION NUMBER

FIG. 5. Chromatogrsphic separation of S-adenosyl-r-methio- nine (A) and S-adenosyl-(5’).3-methylthiopropylamine (B). For the experimental procedlire see the text.

decarbosylatcd S-adellos~lmcthio~line migrates toward the cathode faster than its parent material (Table II). RESULTS

-1 solution of 5.0 pmoles of S-adenos\-I-L-lnethiollillc-l’~I~I~, Various sulfonium con~l~ounds lvere tested for their ability to 0.3 PLci per fimole, 1 mmole of Tris-I-ICY buffer, 1~1-1 i.4, 40 mg of act as methyl douors in the formatiori of methylhistaniiiie decarboxylnting enzyme, and 0.1 mmolc of MgSO.,, in a final (Table III). In addition to the natural mrthyl donor, S- volume of f.0 ml, was incubated for 2 hours at 37”. The mixture adenos~l-L-rllethiolline, only S-illosyl-L-~lletlliollille showed sig- n-as deproteinized with 2.0 ml of 1.5 s perchloric acid. -1fter nificant activity. The other compounds were found to be centrifugation, the supernatant fluid was applied to a 110~ex inactive within the limits of accuracy of the analytical tests. 50-H+ column as described under “Analytical Procedures.” The same compounds were checked for their inhibitory action After initial treatment with 300 ml of 2 x HCl, an acid gradient in the preseuce of X-adcllosylmethionin(: (Table IV). Inhibition

ranging from 3 x to 3.5 s I-ICI was applied which separutctl S- adenosylmethionine and its decarbosylatcd product (Fig. 5). The fractions of clution Peuk B were pooled and cvwporated under reduced pressure; the material was pure as judged by ionophoretic aiialysis. The specific radioactivity of the lrarcnt material, X-adel~os~l-L-lncthioniIle-~~~I~l~, \v:rs rccovcrcd iii the dccarboxylated compound and the yield n-as 65$/L of the .+t:irting material.

The glycosidic bond of S-adenosSl-(5’).3-mcthylthioprol))’la- mint showed the characteristic stability of X-adenos~lsulfollium compounds toward acid, and unusual lability toward alkali. Chromatography with strong acid ~vas tolerated for prolonged periods. Hydrolysis in 0.1 s SaOH for 10 min at 25” hevcred adeniric, which was obscrvcd by the shift of the mazinium of absorbance from 260 to 268 mp, and by the drop of the niolar absorbance \-aluc. The spectrophotomc~tric data of the de- carbosylated compound resembled those shown by- S~atlc~~~+)-l- methionine (14) and by S-adenos~~l-L-(2-h~dros?--4-meth-lthio)- butyric acid. H~drolyeis for 25 min at 100” and l)H 4 yielded 5’.methylthioadcnosine and 3-aminol~~~o~~:tnol. The :rl)sence of homoserine iii the hydrolysate further l~ovcd the removal of X-adenosylmcthioninc from the decarboxylatcd lnoduct. Sol- vent System I (Table I) permitted the separation of homo~crine (RF 0.31) and 3-arnillol)ropanol (RF 0.2 1).

Denminafion oj S-dIethyl-r,-,tletkionine Sdjonium SW-The conditions of dcamination of S-adenos?lnlethiollirle by nitrous acid have been found adequate also for the removal of the amino group of S-n~eth~lmethio~lil~e. To a solution of 52 pmolcs of the sulfate of purified 14C’11~-~S-r~ietliyl-L-rnetlii~~r~iiie sulfonium (see ‘LConipounds”), 9 mmoles of acetic acitl were added to a final volume of 6.5 ml. After cooling iii a11 ice bath, 1.0 ml of 3 M Nan-02 was added, drop by thol), without agitation’. The mixture was removed from the ice bath and left at room temperature for 30 min. Residual nitrous arid was removed by concentration of the solution under reduced pressure to about one-half of the original volume. The material was tran~fcrred to a column of Uowes 50-H+ resin. Gradient elution with HCl, ranging from 0 to 1.0 K, was started, and fractions of 14 ml were collected and assayed for radioactivity. Most of the latter appeared in the Fractions 8 to 10, with an acidity near 0.5 N; they were pooled and evaporated under reduced Ilressure. The material was taken up twice in a few milliliters of water and re- evaporated. It was dissolved in 2.0 ml of water and stored in the frozen state. The yield of the draminated product, as judged by radioactivity, was 40.3 ~molcs. Its structure as 4- dimcthylsulfonium-L-(2-hydroxy)-butyri(* acid was indicated by the absence of ninhydrin response and by the recovery in a single fraction by ion exchange chromatography. The material proved to be uniform, as judged by radioactivity scanning of paI:er chromatograms and electrophoretogtsms (Tables I and II).

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TABLE III

SulJoonirrm compounds as methyl donors for histamine A-methyl- transjerase

Pruified histamine methyltransferase, 0.4 mg of prokill, was incubated in the prcscncc of 0.2 Hmole of histamine, 50 pmoles of sodirml phosphate bluffer, pH 7.5, and the labeled methyl donor as indicated in the table; the final volume was 0.2 ml. lteaction mixtures without histamine were prepared as controls. The mixtuws were incubated for 30 min at 37” in glass-stoppered t,ubes. The samples were assayed for methylhist amine as olltlillcd under “Experimental Procedlkre.”

3lethyl donor, ‘4CH3-labeled

S-Adellosvl-L-methionine

S-Adclros~l-~-(2-hydrox?l-4- met hylt hio)but.yric arid

S-Il,os\~l-r,-methionilrc

S-Irlos~l-L-(2.hytlroxy-4- methylthio)butyric acid

S-Adellosyl-(5’).3-methyl- thiopropylamine

S-&let hyl-L-methionitle 4.l)imeth~lsulfollium-L-(2-

hytlroxy)butyric acid

c

-

Specific radio- activity

Pn$w$yl

1.4

1.5

1.8

1.1

1.4

1.G 1.G

C

e

Zoncentration used

mw

0.25 0.50 1.00 1 .ooa

0.25 0.5 0.50 0.s 1.00 1.8 l.OOU <0.05

1.ooa

1.ooa 0.50

Methyl- hisstaae2

L~?ldtX/SLWtpl~

4.5 8.1

14.2 <0.05

<0.2

(0.05 <0.05

a The same result was obtained with concentrations of 0.25 and 0.5 mM.

TABLE IV Inhibition of hislamine methylation by S-adenosy-L-methionine

analoyues and derivalives

The conditions of the reaction are reported in Table III. The cor~et~tration of S-adelrosSl-L-methionille was 0.25 mM in each experiment.

Additions

None S-Adct1os~l-1,-(2-hydros~--L-

rnetllylthio)butyric acid S-Irlos~l-L-nielhiollille

S-Iuos~l-I,-(2.hydroxy-l- nlethylthio)~)l~tyric acid

S-Adcnosyl-(5’).3-mcthylthio- propylamille

S-Met hyl-I,-mcthionine

S-Adenos?l-L-hornoc~s~eille S-Illosyl-r,-hornocysteille 5’.Mel hylt hioadellosilre

i

Concentra- tion

t&cd

0.25 1.00 0.45 1.00 0.25 1.00 0.50 1.00 0.50

w.moles/ Salltplt?

4.58 4.58 4.55 4.12 3.66 4.57 4.5G 3.75 3.34 4.57

0.25 1.28 0.50 4.21 0.45 3.(.X 1.00 3.65

Inhibition

%

0 0.7

10” 20”

0.3 0.4

180 27a

0.3

7% Iv

2w 2oa

4 For these values the inhibition is statistically significant

o-L 75 100

THIOETHER x 1O-5 M

FIG. 6. Effect of S-adenosyl-L-homocysteine (0) and S-inosyl- L-homocysteine (A) on histamine methylation. The reaction conditions and methylhistamine assay are reported in Tables III and IV alld lmder “Experimental Procedllre.”

was observed with S-adenosyl-(5’).3-mctllylthiol~ro1~~larnine (decarbosylated S-adenosylmethionine) and with X-inosyl-L- methionine. In the latter case, a correction has been made for the low methJ-1 donor activity of this compound (Table III). Several thioethers, derived from the sulfonium compounds, were included in the ,survcy. S-Adcllos\-1-L-homocysteine was found to be an effective inhibitor. The corresponding dcaminated compound, S-illoR3.1.L-hoiiloc)-steine, gave only negligible inhibi-

tion which illustrates the importance of the 6-amino group of the purine 1):rrt of the molecule. 5’.Methylthiondcnoxinc also inhibited the methylatiou of histamine significantly (Table IV).

The difference bctwrcn S-inosyl-L-homocystcinc and S- aclcno~~l-L-holnoc~-stciilc as inhibitors was esamincd in more detail (Fig. 6). At a concentration of lo-” nr the iwcttion was strongly inhibited by S-adenosyl-z-homocystcinc whcrtas the

inosyl deril-utivc escrted only 1O0/C inhibition. -\t :, lcwl of 5 x lo-” 11, S-:~tlenos!l-L-lioIIIoc~steille still c:~uscd :I significant inhibitioll.

Espcrimcnts on the methylation of :Lcct,)-lscrolollirl by the requisite meth?-1trallsfcr;Ise arc summarized in ‘rablc V. Of the enz>-mes tested in this investigation, this was the most specific. X11 derivatives of S-iLderlos~lmethiorlillc mere found

to be inactive as methyl donors. When S-atlerlos~lrrlethiolline was used as methyl donor, high concentrations of S-inosyl- methiollinc, S-atle~lo~~l-(5’)-3-methylthiol~rol~~l:~mi~~c, or 5’.

meth!-lthioudello~itl(~ were required for inhibition of the cnz)-me. S-Illo~\-lholnoc!-steillc ditl llot cause significant inhibition while S-atlellox!-lhonic,c!-steiiic was found to be a very cffwtivc in-

hibitor (Fig. i). This again emphasizes the significuucc of the G-amino groul, of the 1)lwine moiet?- for this cl:~ss of rcnctione.

The ~neth?-lsulfonilul1li~~~ll donor specificity ill the cnzynatic methylation of L-homo(Lysteine is much wider (Table VI). In fact, all wlfonium coml)ounds examined acted as methyl donors IT-ith the csccption of those that lacked the 2-amino groul) of the

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TABLE V

S-Adenosylmethionine derivatives as substrates and as inhibitors for acetylserotonin methyltransferase

Purified acetylserotonin methyltransferase (50 rg of protein) was incubated with 30 pmoles of sodium phosphate buffer, p1-I 7.9, 0.2 pmole of N-acetylserotonin, and the labeled methyl donors (specific activity 2.8 X 103 cpm per mpmole) in the concentrations listed in the table; the final volume was 0.2 ml. The concentra- tion of the additions is indicated in the table. Reaction mix- tures without N-acetylserotonin were prepared as controls. After 1 hour of incubation at 37”, the reaction mixtures were as- sayed for labeled melatonin as described under “Experimental Procedure”’

Substrate, ‘4CHj-labeled

S-Adenosyl-n- methionine

S-Adenosyl-r-(2-hy- droxy-4-methyl- thio)butyric acid

S-Adenosyl-n-me- thionine

S-Inosyl-L-methio- nine

S-Adenosyl-n-me- thionine

S-Adenosyl- (5’) -3- methylthiopropyl- amine

S-Adenosyl-r-me- thionine

S-Inosyl-L+hy- droxy-4-methyl- thio)butyric acid

X-Methyl-L-methio- nine

S-Adenosyl-n-me- thionine

oncen ration

?tLM

0.05 0.10 0.10’

0.05

O.l@

0.05 0.05 0.05

0.10’

0.05 0.05 0.05

0.19

0.19

0.05 0.05 0.05 0.05

Additions

S-Adenosyl-r- (2-hydroxy- 4-methyl- thio)butyril acid

S-Inosyl-r- methionine

S-Adenosyl- (5’)-3-meth- ylthiopro- pylamine

S-Adenosyl- n-homo- cysteine

5’-Methyl- thioadeno- sine

T

oncen ration

Mela- tonin

formed Inhi- bition

THIOETHER X lO-5 M

YDrlesl 2.10 4.32

:0.05

% FIG. 7. Effect of S-adenosyl-n-homocysteine (0) and S-inosyl- L-homocysteine (0) on the methylation of N-acetylserotonin. The experimental conditions are reported in Table V and under “Experimental Procedure.” The concentration of S-adenosyl-L- methionine was 0.05 mM in all experiments.

0.20 2.11 0 TABLE VI Sulfonium compounds as methyl donors for S-adenosylmethionine-

homocysteine methyltransferase

:0.05

2.10 2.09 1.83

:0.05

0 0.5

13b

Purified S-adenosylmethionine-homocysteine methyltrans- ferase (1.4 mg of protein) was incubated with the i*CH3-labeled sulfonium compounds in the concentrations indicated above, 10 pmoles of n-homocysteine, 0.06 pmole of Zn++, 60 pmoles of sodium phosphate buffer, pH 7.2, in a final volume of 0.6 ml. The incu- bation time was 1 hour at 37”. The specific radioactivity of the sulfonium compounds ranged between 1.4 and 1.8 X lo6 cpm per fitmole.

Methyl donor, ‘“CHs-labeled ClXKeIl- tration

TKUIS- methyla-

tion

0.10 2.06 0.2 0.20 1.85 126 0.50 1.70 19b

pmole/hr % 0.28 31

<O.Ol 0 b.23 26

:0.05

:0.05 7

S-Adenosyl-n-methionine. S-Adenosyl-n-(2-hydroxy-4-

methylthio)butyric acid. S-Inosyl-L-methionine.. S-Inosyl-n-(2-hydroxy-4-

methylthio)butyric acid. S-Adenosyl-(5’)-3-methylthio-

propylamine . S-Methyl-n-methionine. 4-Dimethylsulfonium-L-(2.hy-

droxy) butyric acid. .

<O.Ol

0.19 0.306

<O.Ol

0

21 33

0 0.05 0.53 75b 0.05 1.95 7 0.25 1.60 24” 0.50 1.49 29”

n2.w

1.5

3.00 1.5

3.0s

1.5 1.5

3.4” -

a The same result was obtained at a concentration of 1.5 mM. b The value represents the total formation of methionine ac-

cording to the equation, S-methyl-L-methionine + L-homocys- teine -+ 2 n-methionine + H+. Thus, one-half of the methionine is formed by transmethylation, the other by demethylation of the donor.

a The same result was obtained with 0.05 mM concentration. 5 For these values the inhibition is significant on a statistical

basis.

methionine moiety, namely, X-adenosyl- and S-inosyl-L+hy-

droxy-4-methylthio)butyric acid, and 4-dimethylsulfonium-(2- hydroxy)butyric acid. In addition to ion exchange chromatog- raphy, the formation of methionine was tested by radioactivity scanning of paper chromatograms and autoradiography of thin

layer plates. A spectrophotometric test, based on the deamina- tion of S-adenosyl-n-homocysteine and related thioethers (22), was also used (Fig. 8). As transmethylation progresses and the S-adenosylthioether is formed, deamination of the latter by added deaminase can be observed by spectrophotometry at 265 mp. In accord with the other analytical technique, S- adenosyl-n-(2-hydroxy-4-methylthio)butyric acid was found to

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1 i 0

I I ’ --i-- 2

INCUBATION TIME (he)

FIG. 8. Spectrophotometric assay of S-adenosylmethionine- homocysteine methyltransferase with S-adenosyl-L-methionine (Cl) and S-adenosyl-L-(2.hydroxy-4-methylthio)butyric acid (0) as methyl donors. The progress of transmethylation was followed by deamination of the resulting adenosine thioether and measure- ment of the inosine derivative at 265 rnF (22). The samples con- tained, 0.25 pmole of potassium phosphate buffer, pH 6.8, 0.05 pmole of Zn++, 3 pmoles of mercaptoethanol, 3.6 pmoles of L-homo- cysteine, 0.35 &mole of S-adenosyl-L-methionine or 0.29 pmole of S-adenosyl-L-(2-hydroxy-4-methylthio)butyric acid, 0.9 mg of purified methyl transferase, and 35 pg of purified deaminase in a volume of 3.0 ml. In the control experiment with S-adenosyl-L- methionine (A), L-homocysteine was omitted. The reaction was observed in a spectrophotometer cell at 25”.

0.2

---I>

0.1

0

I I I

- S-ADENOSYL-(5’)-3-METHYL A MERCAPTOPROPYLAMINE Km 64.9x 1O-5 M

/

MERCAPTOPROPYLAMINE Km 64.9x 1O-5 M

0.5 0.5 1.0 1.0 1.5 1.5 2.0 2.0 I I

[C] x lO-5 M [C] x lO-5 M

FIG. 9. Methyl donor concentration and reaction velocity in the S-adenosylmethionine-homocysteine methyltransferase system. Reciprocal plots of initial reaction velocity and concentration of S-adenosyl-L-methionine or its decarboxylated derivative are shown. The enzyme (0.4 mg) was incubated with the labeled methyl donor, 0.5 rmole of L-homocysteine, 0.03 pmole of Zn++, 30 pmoles of phosphate buffer, pH 7.2, in a volume of 0.3 ml. After IO-min incubation at 37”, the reaction was stopped by immersion

INCUBATION TIME (mid

FIG. 10. Utilization of (+)-S-inosyl-L-methionine by S-adeno- sylmethionine-homocysteine methyltransferase. Purified en- zyme, 10 mg (0) and 5 mg (A) of protein, was incubated at 37” with 0.4 pmole of WHJabeled (&)-S-inosyl-L-methionine of a specific activity 5 X lo6 cpm per pmole, 4 Mmoles of r,-homocys- teine, 0.08 rmole of Zn++, 80 pmoles of sodium phosphate buffer, pH 7.2, in a volume of 0.8 ml. In a control experiment L-homo- cysteine was omitted. Aliquots of 0.1 ml were taken at different times and assayed for 14CII~-methionine according to the method of Shapiro and Yphantis (2G).

be inactive as a methyl donor toward L-homocysteine whereas

70% transmethylation was attained under the sa.me conditions with X-adcnosyl-L-methionine (Fig. 8).

The met,hionine sulfonium compounds active as methyl donors in this system were studied in some detail with respect to their

interaction with the enzyme. The results of the kinetic experi-

ments are illustrated in Fig. 9. The K, value of decarboxylated S-adenosylmethionine as compared with that of S-adenosyl-

methionine is relatively high. A K, value of 21.3 X lo-” M

was ca.lculated for S-inosyl-L-methionine in the same manner.

The methyl donor activity of X-inosyl-L-methioniile in the

biosynthesis of methionine was studied in more detail to clarify

the relation between the steric configuration of the sulfonium pole and the enzymatic activity (Fig. 10). The synthesis of

this compound by methylation of X-inosyl-L-homocysteine, in analogy to the earlier synthesis of S-adenosylmethionine from

S-adenosylhomocysteine (4, 38), results in a compound racemic

with respect to the sulfonium pole. With an excess of enzyme, (=t)S-inosyl-L-methionine gave virtually complete transmethyla-

tion (Fig. lo), which indicates that both diastereoisomers are utilized by this methyltransferase. This is in agreement with the results obtained with (=t)S-adenosyl-L-methionine in this

reaction (39); only the (-)-sulfonium component was found

active in several other transmethylations (4).

in Dry Ice. The quantity of WHa-methionine formed was as- sayed with 0.1 ml of the incubation mixture by the method speci- fied in Table VI and under “Experimental Procedure.” The reac- tion velocity has been expressed as millimicromoles of methionine formed per mg of protein per hour. The lines were fitted by the methods of the least squares.

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X-Adenosylmethionine Derivatives Vol. 244, No. 16

DISCUSSION

The preparation of three dcaminated derivatives of S-adenosy- methionine and the availability of its decarboxylation product has made possible an examination of the function of these groups in the activity of S-adenosylmethionine as methyl donor. The structure of the deaminated compounds was established by hydrolysis to known fragments. However, the possibility of racemization or Walden inversion at carbon atom 2 of the amino acid moiety of X-adenosylmethionine in the process of deamina- tion has to be considered. Conclusive proof for the retention of the L-configuration has not yet been provided, and the formula- tion of the 2-hydroxy derivatives as belonging to t,he L-series is tentative. However, for t,hc deamination of amino acids Brewster et al. (40) have furnished proof for the retention of the configuration; the presence of the adjoining carboxyl group was found to be important. The problem has been reviewed ex- tensively by Greenstein and Winitz (41). The different be- havior of P-protonated amino acids and amino acid esters has been reported recently by Yamada, Taniguchi, and Koga (42).

Deaminated derivatives, particularly of cocnzyme.~, have played an important role in investigations on the interaction of the amino group with enzymes. Extensive studies with I)PN, TPN, ATP, and FAD (43-46) have shown that the B-amino group of the adenine moiety is essential for biological activity in many instances.

The occurrence of decarboxylated X-adcnosi3-lmethiollille in nature and its function in enzymatic transfer of the prop>-lamino group has been established by Tabor, Rosenthal, and Tabor (47), but there are, as yet, no indications that the deaminated deriva- tivcs of S-adellosylmethioninc are natural compounds.

The enzymes used for testing the methionine sulfonium dcrivat,ives as methyl donors and inhibitors provide examples of methyl transfer to oxygen, nitrogen, aud sulfur atoms of the acceptors (6, 25, 48, 49).

In the present experiments, the 2-amino group of the mcthio-

FIG. 11. Proposed binding sites for S-adenosylmethionine in methyl transfer enzymes.

nine moiety has been found indispensable in all three methyl transfer reactions, and the 2-deamino derivatives are inactive as methyl donors as well as inhibitors. Similarly, deaminated S- methylmethionine is inactive in the methylation of homocysteine. There are exceptions, however, to the requirement of an amino group for methyl donor activity in transmethylation, since

dimethylacetothetin, (CH&CHnCOOH, and dimethylpro- +

piothetin, (CH&SCH&H2COOH, methylatc homocysteine in the presence of the specific enzyme (50). It appears, therefore, that the amino group of methionine is required for binding to the protein rather than for an interaction with the sulfonium group to facilitate methyl transfer.

The B-amino group of the adenine part of S-adenosylmcthio- nine was found indispensable in acetylserotonin methyltrans- ferase. In histamine methyltransferasc, S-inosylmethionine showed about one-tenth of thr methyl donor activity of S- adenosylmethionine; the doubly dearninated derivative, how- ever, was inactive. In the S-adenosylmethionine-homocysteine methyltransferase system, the ljrcsence of the adenosine moiety is not a necessity; S-inosylmethionine was found actirc al- though the h’, value is higher t,han that of S-adenosylmcthio- nine. The lack of specificity is not surprising because S- methylmethionine is also an cfhcient rncthyl donor in this system. The sulfonium pole and the 2-arnino group al)l:car to be the essential requirements in this case.

Decarboxylation of S-adenosylmethioniue occurs in micro- organisms (11) as well as in mammals (51). The ability of this compound to act as a methyl donor apparently has not been previously examined. Here again, the inactivity in the hista- mine methyltransferase and the acetylserotonin mcthyltrans- ferase systems is contrasted with its activity as methyl donor in the enzymatic formation of methionine from homocystcine; however, in the latter system the K, value is relatively high coml)arcd with that of X-adenosylmethionine. On the other hand, in the O-methyl- and N-meth~ltrailsferase~, the de- carboxylated derivative exhibits a significant iilhibition OII the reaction with S-adenosylmethionine as methyl donor. This suggests that in cellular metabolism adenosylmethionille de- carboxylase not only competes with the methyl transfer rcac- tions for the utilization of the same substrate, but also intlircctly inhibits this class of reactions.

The inhibitory effect of S-adenosylhomocysteine in the X- adenosylmethionine-homocysteine methyltransferase system had been observed earlier (52). In the present study on &V- and 0-methyltransferase, the effect is particularly evident. This product inhibition seems to bc a general feature of mcthyl- t,ransferase systems, and a regulation of this class of reactions by S-adcnosylhomocysteine is indicated. The recent observa- tion by Salvatore, Zappia, and Shapiro (53) that the concentra- tions of this thioether in liver are of the same order of m::gnitude as those of S-adenosylmcthionine further suggests the biological significance of this inhibition.

The inhibitory effect of 5’-mcthylthioadenosine on the methyla- tion of histamine and AT-acetylserotonin is of particular interest considering the origin of this compound from X-adenosyl- methionine (g-11).

The role of the amino groups and of the carboxyl group of S-adenosylmethionine in relation to the enzyme systems studied is represented in a schematized form in Fig. 11. Binding site S appears to be particularly important in all instances. In the

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Issue of August 25, 1969 V. Zappia, C. R. Zydelc-Cwick, and F. Schlenlc 4509

homocvsteirle-S-rneth?-ltr~~llsfelase, binding sites 1 and /t appear .\SD N. 0. K~PI,.~N (Editors), Methods in enzymology, Vol.

to be of minor importance. Attachment of the sulfonium group 12A, Academic Press, New York, 1967, p. 126.

at site 2 is not readily amenable to tests. However, if the 22. SCHLENK, F., .\ND ZYDEIC-CWICK, C. lL., Biochem. Biophys.

Res. Commun.. 31. 427 (1968). sulfonium group by itself would be a major factor in the attach- 23. BROXN, 1). l).,’ T~IXCXI&<, li., .\ND AxELI~OD, J., J. uio/,,

ment of the methyl dollor to the enzyme proteins, one would Chem., 234, 2948 (1959).

expect strong inhibition of S-adenosylmethionine-dependent 24. SXYDER, S. H., B.U,DESS.~RINI, R. J., AND AxELI~~D, J., J.

transmethylations by the dcaminated derivatives. The scheme Pharmacol. Ezp. Therap., 163, 544 (1966).

25. AXELROD, J., AND WEISSH~CH, H., J. Biol. Chem., 236, 211 does not take into consideration additional binding sites which (1961).

determine the acceptor specificity. 26. SH.SPIRO, S. K., ll~~ YPIUNTIS, U. A., Biochim. Biophys.

Acta, 36, 241 (1959). 27. LO~-RY, 0. H., ROSEI~ROUGH,N. J., FRIAR, A. L., AND I<.\SDILL,

R. J., J. Biol. Chem., 193, 2G5 (1951). PERKS, L. W., ‘IND SCIILENK, F., J. Biol. Chem., 230, 295 (1958). TOENNIES, G., .~ND KOLB, J. J., Anal. Chem., 23, 823 (1951). C.~NTON, G. L., J. Biol. Chem., 204, 403 (1953). KLEE, W. A., )~ND M~DD, S. H., Biochemistry, 6, 988 (1967). MCILV.UNE, T. C., J. Biol. Chem., 49, 183 (1921). SCHLENK, F., in L. ZECI~MEISTER (Editor), Progress in the

chemistry of organic natural products, Vol. 23, Springer Verlag, New York, 1965, p. 61.

SCHLENIC, F., AND ZYDEIC~VICIC, C. IL., Arch. Biochem. Bio- phys., 123, 438 (1968).

Bch-nowledgments-The advice of Dr. John F. Thomson in the kin&c experiments is greatly appreciated. The help of Mr. M. H. nipert in writing the computer program for the statistical

elaboration of the results is gratefully acknowledged.

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Vincenzo Zappia, Cynthia R. Zydek-Cwick and F. SchlenkReactions

-Adenosylmethionine Derivatives in Methyl TransferSThe Specificity of

1969, 244:4499-4509.J. Biol. Chem. 

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