Biochem. J. (1964), 92, 497

Transfer of the Methyl Group from N5-Methyltetrahydrofolatesto Homocysteine in Escherichia coli

By J. R. GUEST, S. FRIEDMAN, M. A. FOSTER, GENOVEVA TEJERINA AND D. D. WOODSMicrobiology Unit, Department of Biochemistry, University of Oxford

(Received 23 January 1964)

Evidence was given in the two preceding papers

that strains of Escherichia coli possess alternativeenzymic mechanisms for the conversion of homo-cysteine into methionine, and that these mechan-isms differ at a point subsequent to the creation ofthe methyl group (Foster, Tejerina, Guest & Woods,1964b; Guest, Foster & Woods, 1964a). Onemechanism requires, specifically, N5-methyltetra-hydropteroyltriglutamate* as methyl donor and iscatalysed by an enzyme (B) which does not containcobalamin. In the other, either N5-methyl-H4PtGor N5-methyl-H4PtG3 serves as a methyl donor buta cobamide-containing enzyme (Foster, Jones &Woods, 1961) is an essential component of theenzyme system.The work now described is a detailed study of the

cofactor and other requirements associated with thetwo mechanisms. The term folate is used in generalreference to both mono- and tri-glutamate deriva-tives. A brief report of some of the observationshas been given by Guest, Friedman & Foster(1962).

MATERIALS AND METHODS

Sources of enzymes

Organis8M. The methods used for the maintenance andbulk growth of the three auxotrophic strains of E. coli usedwere as described by Guest, Helleiner, Cross & Woods(1960), as also was the preparation from them of ultrasonicextracts. E. coli 121/176 requires for growth eithermethionine or cobalamin, whereas strain 3/62 respondsonly to methionine; their content of relevant enzymes

under differing growth conditions is summarized in Table 1

of Guest et al. (1964a). E. coli PA15, which requires forgrowth either serine or glycine, is fully competent tosynthesize methionine from homocysteine and serine. Thebasal growth medium was normally supplemented withglycine or glycine plus cobalamin (strain PA 15), methionineor methionine plus cobalamin (strain 3/62), and methionineor cobalamin (strain 121/176); the concentrations usedwere: glycine, 10 mM; DL-methionine, 0 4 mM; cobalamin,18-5 femM.Enzyme preparations. An ultrasonic extract of E. coli

3/62 (grown with methionine) was used directly as source of

* Abbreviations for members ofthe folic acidgroup, basedon PtG for pteroylglutamate: H4PtG, tetrahydropteroyl-monoglutamate; H4PtG3, tetrahydropteroyltriglutamate;derivatives of these compounds are indicated as, e.g.,

N5-methyl-H4PtG.32

enzyme B in some experiments; it has the advantage ofbeing free from methylenetetrahydrofolate reductase(enzyme A) which could oxidize the N5-methyltetrahydro-folate used as methyl donor for the methylation of homo-cysteine. Preparations purified 7-12-fold were prepared bythe method of Foster et al. (1964b).An extract of E. coli 121/176 was sometimes used as

direct source of the cobamide-containing enzyme free fromenzyme B. Purified cobamide-containing enzyme was pre-pared from an aqueous extract of acetone-dried E. coliPA 15 (grown with cobalamin) by the modified procedure ofFoster et al. (1964b); it was finally precipitated by dialysisat 2° for 6 hr. against 20 vol. of (NH4)2SO4 solution (90%saturated, adjusted to pH 7-8 with NH3). After centrifug-ing for 5 min. at 25 000g, the bulk of the supernatant fluidwas removed and the sediment stored at 20 in the remainingfluid. This preparation had a half-life of 3 weeks and thespecific activity, determined according to the method ofFoster et al. (1961), indicated a purification of 140-180-foldwith an overall recovery of 5%. Before use samples of thesuspension were centrifuged; the pellet was dissolved in50 mM-potassium phosphate, pH 8-7, and the solutiondialysed for 2 hr. against the same buffer.

Extracts in which both the cobamide-containing enzymeand enzyme B were present were obtained from eitherE. coli 3/62 or PA15 after growth with cobalamin pluseither methionine or glycine respectively.The enzyme (or enzymes) designated protein fraction Y

were present in all three strains of E. coli irrespective ofwhether or not they had been grown with cobalamin.Material enriched 2-3-fold was obtained from E. coli PA 15during the purification of the cobamide-containing enzyme.The protein eluted from Celite 545 by 75 mM- and 120 mM-potassium phosphate, pH 7 8, was combined and precipita-ted by dialysis against (NH4)2SO4 solution (90 %saturated);it was stored and prepared for use as described for thepurified cobamide-containing enzyme.Removal of cofactors and metal ions. Ultrasonic extracts

used directly as sources of enzymes were dialysed against50 mM-phosphate buffer, pH 7-8, and treated with Dowex 1resin (X8; CF- form; 100-200 mesh) as described byKisliuk & Woods (1960) immediately before use. Bothcrude and purified preparations of enzyme B were alsotreated with charcval (acid-washed and ground Nuchar C;West Virginia Pulp and Paper Co., New York, U.S.A.): theamount of charcoal used/mg. of protein was 0-4 and 3-0 mg.respectively for the crude and purified material. Afterstirring for 20 min. at 00 the charcoal was removed by twicecentrifuging at 25 OOOg for 5 min.Four methods were used to remove metal ions from puri-

fied preparations of enzyme B; they were carried out in tri-ethanolamine buffer, pH 7-8. The preparation was treatedfor 2 hr. at 00 with either (1) potassium cyanide (10 mM) or

Bioch. 1964, 92

497

J. R. GUEST AND OTHERS(2) EDTA (disodium salt) (25 mM) and the reagent removedby dialysis against two changes of 20 vol. of 100 mM-triethanolamine buffer, pH 7-8. (3) The preparation (5 ml.;1 mg. of protein/ml.) was applied to a column (12 cm. x1-5 cm.2) of Sephadex G-25 (Pharmacia, Uppsala, Sweden)gel in 100 mM-triethanolamine buffer, pH 7-8. (4) The pre-paration was shaken gently for 2 hr. at 00 with Chelex-100(Dowex Al) chelating resin (K+ form; 50-100 mesh).

Test for methionine formationCobalamin-independent mechanism. The basal reaction

mixture, RI, contained (in final vol. 1 ml.): DL-homocysteine,8 /Lmoles; (± )-N'-methyl-H4PtG3, 1-5 iemoles; MgSO4,5 ,umoles; potassium phosphate buffer, pH 7-8, 100,umoles.Supplements used as stated in certain experiments were:ATP, 5fimoles; an enzymic reducing system (FAD, 0-1 IL-mole; NAD, 1 ,smole; ethanol, 40 temoles; crystallizedalcohol dehydrogenase, 150,tg.). In tests of the metal ionrequirements of the enzyme the phosphate buffer wasreplaced by 100lmoles of triethanolamine buffer, pH 7-8.The amount of enzyme B added (005-1-0 mg. of protein)depended on the purity of the preparation used and serumalbumin was included to bring the final protein concentra-tion to 2 mg./ml. Incubation was at 370 in air for 20-30 min.and the reaction was stopped by heating the mixture at1000 for 3 min. Precipitated protein was removed bycentrifuging and methionine estimated in the supernatantfluid by the microbiological assay of Gibson & Woods(1960). The value obtained (15-20,um-moles) in each testfor a control containing heated enzyme has been deductedfrom the experimental values recorded.

Cobalamin-dependent mechanism. The basal reactionmixture, R2, contained (in final vol. 1-6 ml.): DL-homo-cysteine, 10 ,umoles; (± )-N5-methyl-H4PtG, 2-5 ,moles, or( i)-N5-methyl-H4PtG3, 1-5 ,moles; ATP, 10,umoles;MgSO4, 10 ,umoles; FAD, 0-2 Hmole; NAD, 1 temole; ethanol,40 iemoles; alcohol dehydrogenase, 150,g. The source ofenzyme was either a mixture of purified cobamide-contain-ing enzyme (2040,jg. of protein) and protein fraction Y(04-1.0 mg. of protein) to which was added 1 mg. of bovineserum albumin, or an unpurified (with respect to protein)extract of organisms grown with cobalamin (5 mg. ofprotein). Incubation was for 1-5-4 0 hr. at 370 in an atmo-sphere of H2. The reaction was terminated and methionineassayed as described above; the value for a control withheated enzyme was about 10,um-moles.

ChemicalsFolic acid derivatives. (± )-N5-Methyltetrahydrofolates

were prepared and stored as described by Guest et al.(1964a) for the products of the chemical reduction of thecorresponding N5N'0-methylene derivatives. Solutions inpotassium phosphate buffer, pH 7-8, were standardizedspectrophotometrically at 290 m,u assuming an arbitrarymolar extinction coefficient of 25 000 cm.2/mole. With N5-methyl-H4PtG the product was used directly; the N5-methyl-H4PtG3, which had been prepared from the lesspure sample (II) of PtG3, was purified by chromatographyon triethylaminoethylcellulose (TEAE-cellulose).

Other materials. S-Adenosyl-L-homocysteine was a giftfrom Dr W. Sakami and S-adenosyl-L-methionine iodidewas obtained from the California Corp. for BiochemicalResearch, Los Angeles, U.S.A. The sources of other special

chemicals and enzymes were as given by Guest et al. (1964a).Potassium phosphate buffers were prepared from KH2PO4and K2HPO4. Triethanolamine buffer was made by adjust-ing a solution of recrystallized triethanolamine hydro-chloride to pH 7-8 with KOH.

RESULTS

Cobalamin-independent methyl transferIt was shown in the two preceding papers

(Foster et al. 1964b; Guest et al. 1964a) thatextracts of E. coli containing enzyme B, but not thecobamide-containing enzyme, catalysed the trans-fer of the methyl group from N5-methyl-H4PtG3 tohomocysteine. Further exploratory experimentswith preparations of enzyme B treated to removecofactors, but not purified with respect to protein,established optimum conditions for the study ofthis reaction and also revealed that neither ATP nora reducing environment was apparently required.The rate of methionine formation (about 400ym-moles/mg. of protein/hr.) was almost four timesthat obtained under optimum conditions for theoverall enzymic conversion of serine plus homo-cysteine into methionine. Investigations werethereafter carried out with a preparation of enzymeB which had also been purified about tenfold withrespect to protein content; this is the highest degreeof purification so far achieved (Foster et al. 1964b).

Kinetics. The amount of methionine formedincreased linearly with time up to about 30 min.(Fig. 1) and was proportional to enzyme concentra-tion up to about 150,ug. of protein/ml. (Table 1).

m

0

g 300

o 200

r._ 010-) 100

Time (min.)

Fig. 1. Time-course of the methylation of homocysteine bypurified enzyme of the cobalamin-independent reaction.Reaction mixture R1 was incubated for the time stated with150 lg. of protein of a preparation of enzyme B that hadbeen purified tenfold.

1964498

METHYLATION OF HOMOCYSTEINEThe deviation from linearity beyond these pointswas probably due to the concentration of N5-methyl-H4PtG3 having fallen below the limitingvalue; it was not possible, with the limited supplies

Table 1. Effect ofenzyme concentration on methionineformation by the cobalamin-independent reaction

The preparation of enzyme B (purified tenfold) wasincubated for 20 min. in reaction mixture R1.

Enzyme B added(rIg. of protein)

75150225450

L-Methionineformed

(Im-moles)77140185245

Table 2. Substrate specificity and cofactor require-ments of the cobalamin-independent reaction

Reaction mixture R1 was modified as indicated andincubated for 30 min. with purified enzyme B (140,tg. ofprotein).

Substrate Modifications to(1.5 fimoles) reaction mixture

N5-Methyl-H4PtG3 NoneMgS04 omittedATP added'NADH2'* addedATP and 'NADH2'*addedATP and 'NADH2'*addedt

N5-Methyl-H4PtG NoneATP, 'NADH2'* andFAD addedt

L-Methionineformed

(um-moles)190132198202195

* 'NADH2' refers to the NADH2-generating systemcomprising NAD, ethanol and alcohol dehydrogenase (seethe Materials and Methods section).

t Incubated anaerobically.

Table 3. Effect of mgnesium ions on thecobalamin-independent reaction

A purified preparation of enzyme B was further treatedas indicated (for details see the Materials and Methodssection) and incubated (75 ilg. of protein) for 30 min. withand without MgSO4 (5 mM) in reaction mixture R1 modifiedby omitting MgSO4 and substituting triethanolaminebuffer for phosphate.

'I A

Buffer100 mM-Phos-phate buffer,pH 7-8100 mM-Tri-ethanolaminebuffer, pH 7-8

Treatment ofthe enzymepreparation

None

NoneCyanideEDTASephadex G-25Chelex-100

L-Methionine formed(jum-moles)

Mg2+ ions Mg2+ ionsabsent present135 135

available, to use this substrate at a higher initialconcentration.The optimum pH was not sharp; activity was

maximal at pH 8-1 but fell only to 70 and 88 % ofthe maximum at pH 6-0 and 8-7 respectively.

Substrate specificity. Enzyme B was active onlywith N5-methyl-H4PtG3; the monoglutamate ana-logue was not used (Table 2), even on the furtheraddition to the reaction mixture of ATP and areducing system, i.e. cofactors required for the useof this substrate by the cobamide-containingenzyme. Other potential methyl donors whichproved to be inactive were choline, betaine andcreatine, but just detectable amounts ofmethioninewere found chromatographically with S-methyl-cysteine.

Cysteine did not replace homocysteine as accep-tor of the methyl group of N5-methyl-H4PtG,, norwas the reaction with homocysteine as acceptorinhibited by cysteine.

Cofactor requirements. The use of the purifiedprotein fraction of the enzyme B preparation didnot reveal any accessory requirement other thanfor Mg2+ ions, the omission of which decreasedmethionine formation by about 30 % (Table 2). Theaddition of ATP, FAD and a NADH2-generatingsystem did not significantly increase methionineformation (Table 2). This was also the case with anaqueous extract of heated E. coli (Guest et al. 1960)which was tested as a general source of cofactorsnatural to this organism.

Requirement for Mg2+ ions. This was investigatedfurther by so treating the enzyme preparation as toremove or form complexes with any metal ionspresent. Treatment with Sephadex G-25 gel,Chelex-100 resin, EDTA and cyanide all resulted ina 70-80 % loss of activity that was completelyrestored by the addition of Mg2+ ions (Table 3). Ithad been found previously that cobalamin-indepen-dent methyl transfer was more sensitive to cyanidethan the mechanism dependent on cobalamin(Foster et al. 1964b); this may therefore have beendue to the formation of complexes of the metal ionrequired by enzyme B.At low concentrations Mn2+ ions were more

effective than Mg2+ ions, but concentrations ofMn2+ ions greater than 1 mm were inhibitory andthe highest levels of methionine formation wereobtained with Mg2+ ions (5 mm) (Fig. 2). Otherbivalent metal ions tested at 5 mm were onlyweakly active (Fe2+, Ca2+ and Zn2+ ions) or inactive(Co2+ ions); no steps were taken to purify the saltsused.Adenosine triphosphate. There was no require-

135 135 ment for ATP under the present test conditions30 150 with defined substrates and purified enzyme40 140 (Table 2). In earlier experiments a 2-3-fold stimu-25 140 latio-n by ATP had been found when cobalamin-

32-2

Vol. 92 499

J. R. GUEST AND OTHERSindependent methylation of homocysteine wasstudied in more complex systems with serine as Cdonor and H4PtG as cofactor or with N5N'0-methylene-H4PtG3 as direct C, donor (Guest, 1960;Foster et al. 1964b). Further investigation of thefirst of these systems, with a mixture of extracts ofstrains 121/176 and 3/62 as enzyme source, hasshown that the ATP requirement depends on thenature of the reducing system supplied and that is

GO(

o0

:._

o

0

0

Conen. of Mg2+ or Mn2+ ions (mM)

Fig. 2. Effect of Mg2+ and Mn2+ ions on the activity of thepurified enzyme of the cobalamin-independent reaction.Enzyme B (purified tenfold) was treated consecutively withSephadex G-25 and Chelex-100; 75,ug. (protein) was incu-bated in reaction mixture R1 containing 100 mM-tri-ethanolamine buffer, pH 7-8, instead of phosphate andbivalent metal ion at the concentrations indicated. 0,Mg2+ ions; 0, Mn2+ ions.

Table 4. Assay of the enzyme of the cobalamin-independent reaction by independent methods

The fractions containing enzyme B were obtained fromE. coli 3/62 as described by Foster et al. (1964b) and wereassayed (a) as described in that paper with extracts ofE. coli 121/176 and serine as C. donor and (b) by the methoddescribed in the present paper with N5direct methyl donor in reaction mixture

Crude e:ProtamiDEAE-4Dialyse(Alumina

L-Methionine(,emoles/mj

enzyme B prot

Fraction Method (a) Mextract 0-64ine supernatant 1-56cellulose eluent 2-75d eluent 3-45%Cy-gel super- 3-85r

natant (firt treatment)Alumina Cy-gel eluent(after second treatment)

DEAE-celluloseconcentrate

750

790

necessary for the reduction of the C, donor systemfrom hydroxymethyl to methyl level. ATP was notrequired if FAD were present together with aNADH2-generating system comprising NAD pluseither ethanol and alcohol dehydrogenase orhexose diphosphate plus endogenous enzymes. It isconcluded that ATP was required only for the phos-phorylation of glucose or the conversion of FMNinto FAD or both; in the earlier experiments eitherglucose had been used as source of reducing poweror FMN as source of added flavin.

Phosphate. Replacement of phosphate buffer bytriethanolamine buffer of the same pH did notdiminish the yield of methionine (Table 3); theenzyme preparation and other reagents had alsobeen prepared in the latter buffer. There is thereforeno major requirement for phosphate ion in thereaction.

Alternative assay. Evidence was sought bearingon the question of whether enzyme B has only oneenzymic component by measuring its apparentactivity, by two different methods, at all stages ofits isolation. One method was that employedthroughout this section, i.e. the specific catalysis ofthe transfer of the methyl group from N5-methyl-H4PtG3 to homocysteine. The other was an assess-ment of its ability to complete the enzymic constitu-tion of extracts of E. coli 121/176 (which lackenzyme B) and thus bring about the overall forma-tion of methionine from homocysteine and serineby such extracts (Foster et al. 1964b). The ratio ofthe specific activities in each assay was virtuallyconstant throughout the purification (Table 4).

Distribution. The relative activities of the strainsof E. coli used in this work in catalysing the forma-tion of methionine from homocysteine and N5-methyl-H4PtG3 were compared with extracts un-purified with respect to protein. The valuesobtained were (,um-moles of L-methionine/mg. ofprotein/hr.): strain 3/62, 330; strain PA15, 180;strain 121/176, 0.

Cobalamin-dependent methyl transfer

-\ethyl-H4PtG3 as Further investigation of this mechanism, inR1. which N5-methyl-H4PtG as well as N5-methyl-

formed H4PtG3 is an effective substrate, was carried out

gf of with a preparation of cobamide-containing enzymetein/hr.) (derived from E. coli PA 15 grown with cobalamin)

(a): (b) purified about 160-fold with respect to protein. Infthod (b) ratio view of its greater availability N5-methyl-H4PtG0 38 1 70 was used for most of the work. During purification,0190 1-75 the assay of the cobamide-containing enzyme was

1-98 1-75 in terms of its ability to promote methionine forma-2-19 1-75 tion from homocysteine and serine in the presence

4-86 1-55of unpurified preparations of E. coli PA 15 specifi-cally devoid of cobamide-containing enzyme

4-48 1-75 (Foster et al. 1961). Previous studies with N5-methyl-H4PtG as direct methyl donor were with an

500 1964

METHYLATION OF HOMOCYSTEINEunpurified preparation of the cobamide-containingenzyme (Guest et al. 1964a). It was not knowntherefore whether other enzymes, possibly presentin the bacterial preparations used, were required inaddition to the cobamide-containing enzyme.Need for accessory enzyme(s). A batch of purified

cobamide-containing enzyme was prepared by themodified method of Foster et al. (1964 b) and materialat successive steps in the fractionation tested for itsability to catalyse transmethylation from N5-methyl-H4PtG to homocysteine (Table 5). Thesupernatant fluid after treatment with protaminesulphate (fivefold purification) was fully active,but the final material obtained after chromato-graphy on Celite 545 (150-fold purification) wasalmost inactive. Activity was restored by theaddition of other fractions from the Celite column,those eluted by 75 mM- and 120 mM-potassiumphosphate buffer, pH 7-8, being responsible for thegreater part of the effect. These fractions werepooled and designated protein fraction Y, whichhad a specific activity 1-5-3 times that of theoriginal extract; its essential requirement, in addi-tion to cobamide-containing enzyme, for methionineformation is shown in Table 6.

Kinetics. The rate of formation of methioninefrom homocysteine and N6-methyl-H4PtG wasdirectly proportional to the amount of cobamide-containing enzyme with a constant amount of pro-tein fraction Y, and directly proportional to theamount ofthe latter when the amount ofcobamide-containing enzyme was held constant. However,even with both enzyme sources present at optimumconcentrations there was always a lag, varyingerratically from 30 to 90 min., before methionineformation commenced; thereafter it was directlyproportional to time for about 4 hr. It was notpossible in these circumstances to make a quantita-tive study of other aspects of the kinetics of thereaction.

Cofactors. Use of the purified sources of bothcobamide-containing enzyme and protein fractionY permitted a clear-cut demonstration of the co-factor requirements of the reaction. The singleomission from the reaction mixture of ATP, Mg2+ions, FAD or the NADH2-generating systemdecreased methionine formation to about 10 % orless (Table 6), allowing for the controls withoutsubstrate. FMN had only about one-fifth of theactivity of the same concentration ofFAD and wasinhibitory at higher concentrations. ATP wasreplaced byADP to only a limited extent, and AMPand adenosine were inactive (Table 7).

Substrate specificity. Similar results to those ofTable 6 were obtained when N5-methyl-H4PtG wasreplaced as substrate by the corresponding tri-glutamate derivative, although considerably lessmethionine was formed, probably owing to the use

Table 5. Requirement of the purified cobamide-containing-enzyme system for accessory enzyme(s)

The fractions possessing the cobamide-containingenzyme were obtained from E. coli PA 15 as described byFoster et al. (1961) and were incubated (6 units of enzymein each case) in reaction mixture R2. The extract (ultrasonic)of E. coli 3/62 after growth with methionine was dialysed,but not otherwise purified.

Source of cobamide-containing enzyme

Crude extractpH 5-0 supernatantProtamine sulphatesupernatant

Celite 545 eluentExtract of E. coli 3/62Celite 545 eluent +extract of E. coli 3/62

Specific activity L-Methionine(units/mg. of formed

protein) (gm-moles)1-2 4103-6 3906-0 350

180-0 355

400

Table 6. Components of the cobalamin-dependentsystem for the methylation of homocysteine

Purified cobamide-containing enzyme (5,ug. of protein)plus protein fraction Y (0-6 mg. of protein) were incubatedfor 4 hr. in reaction mixture R2. Enzymes, substrates andcofactors were omitted as indicated.

L-Methionineformed

Component omitted (,m-moles)None 615FAD 85'NADH2'* 25ATP 75Mg2+ ions 80Homocysteine 25N5-Methyl-H4PtG 15Cobamide-containing enzyme 25Protein fraction Y 30

* 'NADH2' refers to the NADH2-generating systemcomprising NAD, ethanol and alcohol dehydrogenase.

Table 7. Effect of adenosine triphosphate andhomocysteine on the cobamide-containing-enzymesystem

The general test conditions were as for Table 6 with themodifications to reaction mixture R2 indicated. Supple-ments used were ADP (5 ,umoles), AMP (5 ,&moles), adeno-sine (5 umoles), and S-adenosylhomocysteine (4 i&moles)plus 2-mercaptoethanol (20 ,umoles).

Modifications to the reaction mixture

Omissions AdditionsNone NoneATP NoneATP ADPATP AMPATP AdenosineHomocysteine NoneHomocysteine S-Adenosylhomocysteineand ATPHomocysteine S-AdenosylhomocysteinE

L-Methionineformed

(,m-moles)6157512570502527

88

Vo]. 92 501

e

,e

J. R. GUEST AND OTHERS

of sub-optimum concentrations of the triglutamate,which was in short supply. The requirement forATP and the reducing system made it clear, how-ever, that the reaction then studied could not havebeen due to the presence of any enzyme B which, asshown above, does not require these cofactors.S-Adenosylhomocysteine replaced homocysteine

as methyl acceptor only to a very limited extentthat was still dependent on the presence of ATP(Table 7). Further, S-adenosylmethionine, the pre-sumptive product of methylation, did not yieldmethionine when incubated, either alone or in thepresence of homocysteine, with unpurified bacterialextracts with or without the cobamide-containingenzyme; these extracts were derived from E. coli3/62 grown with or without cobalamin respectively.

Substitution of cysteine for homocysteine in thenormal reaction mixture did not yield any S-methylcysteine detectable either by chromato-graphy on paper or by radioautography withN5-['4C]methyl-H4PtG as methyl donor.

Properties of protein fraction Y. The activity ofthis material is presumed to be enzymic in naturesince it is non-diffusible and destroyed by heatingat 700 for 6 min.; it is, however, relatively thermo-stable since it is not affected by a temperature of60° for 6 min. It was not replaced by an aqueousextract of heated whole organisms.An unpurified extract of E. coli 3/62, grown with

niethionine and devoid of the cobamide-containingenzyme, was also able to activate the purified pre-paration of cobamide-containing enzyme (Table 5).It is clear therefore that the production during

growth of the enzyme(s) of protein fraction Y isnot linked to the capacity to form the cobamide-containing enzyme from the apoenzyme. The apo-enzyme is present in methionine-grown E. coli3/62, as it is in extracts of strains W, PA 15and 121/176 which also have fraction Y activitywhen derived from organisms grown without addedcobalamin.

It was found by visual observation that proteinfraction Y caused the reduction of FAD to FADH2in the presence of NADH2; FADH2 did not, how-ever, alone support methionine formation. BothATP and reduced flavin are known to be requiredfor the biosynthesis from cobalamin of dimethyl-benzimidazolylcobamide coenzyme (Brady & Bar-ker, 1961; Peterkofsky, Redfield & Weissbach,1961); the coenzyme was therefore tested for abilityto replace protein fraction Y plus ATP and thereducing system in the methylation of homo-cysteine, but it had no significant activity.

DISCUSSION

The results as a whole further strengthen theview that there are two enzymic mechanisms for themethylation ofhomocysteine in E. coli; they are setout in Scheme 1, which also takes into account theresults of the two preceding papers (Foster et at.1964b; Guest et al. 1964a).The transfer of methyl groups specifically from

N5-methyl-H4PtG3 to homocysteine catalysed bypreparations of enzyme B (cobalamin-independentpathway) showed no requirement for any addi-

CobalaminMethionine Homocysteine

Cobamide enzyme IATP, FAD, NADH2,

Formaldehyde Mg2+ ions and fraction Y

H4PtG(3) -N N-5NNh-MethyIene-H4PtG(3) 15-Methyl-H4PtG(3)

Serinhydroxymethyl- N5N"-Methylene-

transferase tetrahydrofolateSerine ang2y ions Glycine FADH2 reductase FAD

and pyridoxalGyinphosphate

/ / ~~~~~~~~~~~~~~~~~~~IIH PtG FormaldehydeNANIo-Methylene-HPtG N5-Methyl-H PtG3

Enzyme BMg2' ions

Methionine Homocysteine

Scheme 1. The two pathways for the methylation of homocysteine with serine as the ultimate donor of the C1unit. I, Cobalamin-dependent pathway; H4PtG(3) refers to either H4PtG or H4PtG3. II, Cobalamin-independentpathway.

502 1964

METHYLATION OF HOMOCYSTEINE

tional enzyme or cofactor other than Mg2+ ions.Although the degree of purification achieved forthis enzyme was low, it is probable that only a singleenzyme takes part in the reaction since the abilityto catalyse methyl transfer parallels, duringfractionation, the ability to complete the enzymicconstitution of the cobalamin auxotroph (strain121/176), which is almost certainly an organismwith a single metabolic lesion; enzyme B is then aN5 - methyltetrahydropteroyltriglutamate - homo -cysteine methyltransferase. It is, however, con-ceivable that after purification there remainspresent in excess in the active fraction a boundcofactor or associated enzyme other than oneof those implicated in the cobalamin-dependentpathway.The apparent simplicity of the cobalamin-

independent pathway contrasts sharply with thatdependent on cobalamin, where strict requirementswere found for ATP, a reducing system containingFAD and a protein fraction additional to the pre-viously recognized cobamide-containing enzyme.Two further major differences are the inability ofenzyme B to use N5-methyl-H4PtG as substrate,and the absence of cobalamin from this enzyme(Foster et al. 1964b). The mechanisms of methyltransfer in the two systems appear therefore todiffer radically; with enzyme B it is possible thatthe terminal glutamate residues of the triglutamateparticipate in labilizing the methyl group, a roletaken over in the other pathway by a complexcobalamin system.The cobalamin-dependent pathway studied here

is apparently the same as that described briefly byBuchanan, Larrabee, Cathou & Rosenthal (1961)and Rosenthal & Buchanan (1962), who investigat-ed E. coli 113/3, an auxotroph similar to strain121/176 in responding to cobalamin or methionine.The enzyme fraction D of these authors is similar toprotein fraction Y in its catalysis of the reduction ofFAD. Exploratory experiments on the furtherpurification of fraction Y suggested that this frac-tion has a necessary activity other than in FADH2formation. This has since been found to be the case,as reported briefly by Foster, Dilworth & Woods(1964a), who found that fraction Y also convertsATP plus methionine into S-adenosylmethionine;the latter is required in catalytic amounts formethyl transfer from N5-methyl-H4PtG to homo-cysteine. Evidence was also obtained that methylgroups from the folate are transferred to homo-cysteine via protein-bound S-adenosylmethionineand 'methylcobalamin' (the methyl analogue ofthe cobamide coenzymes); the latter is known toact as a methyl donor to homocysteine, a reactioncatalysed by purified cobamide-containing enzymeand with no detectable cofactor requirements(Guest, Friedman, Woods & Smith, 1962; Guest,

Friedman, Dilworth & Woods, 1964b). A possiblemechanism of the cobalamin-dependent trans-methylation is fully discussed by Foster et al.(1964a) and Guest et al. (1964b); it takes into ac-count also the present findings that free S-adenosyl-homocysteine is a poor substrate and that there isno net formation of methionine from S-adenosyl-methionine.While the present work was in progress, several

short reports have described the methylation ofhomocysteine by N5-methyl-H4PtG with enzymesfrom mammalian liver. The cofactors required(ATP, a reducing agent and Mg2+ ions) are similarto those noted above for the cobalamin-dependentpathway (Sakami & Ukstins, 1961; Mangum &Scrimgeour, 1962; Rooze, Sakami & Blair, 1962;Rohrbaugh, 1962).

SUMMARY

1. The two reactions by which the methyl groupof N5-methyltetrahydrofolates is transferred tohomocysteine by Eacherichia coli have been furtherstudied with purified preparations of the relevantenzymes.

2. Enzyme B, now characterized specifically asa N5-methyltetrahydropteroyltriglutamate-homo-cysteine methyltransferase, requires only Mg2+ ionsfor full activity; Mn2+ ions are also active, but donot support optimum methionine formation.Enzyme B, at several stages of purification, com-pleted the enzymic constitution of a methioninemutant with a single metabolic lesion, thus sup-porting the view that it is a single protein.

3. The cobamide-containing enzyme, which canuse as substrate both N5-methyltetrahydropteroyl-monoglutamate and -triglutamate, requires foractivity the presence of another protein fractio(which may be complex) as well as ATP, Mg2+ ions,FAD and a NADH2-generating system. S-Adeno-sylhomocysteine replaces homocysteine as methylacceptor to only a limited extent, and S-adenosyl-methionine does not yield methionine even withunpurified extracts of E. colti. FADH2, whoseformation from FAD is catalysed by the accessoryprotein fraction, does not replace that fraction, thefunction of which is not yet clear.

4. The results further emphasize the radical dif-ference in mechanism of the two transmethylationreactions.

We are grateful to the Nuffield Foundation for a granttowards the expenses of this work, which was also aided bygrants to the Department from the Rockefeller Foundationand the U.S. Department of Health, Education andWelfare. J. R. G. and M. A. F. were Guinness ResearchFellows in Microbiological Biochemistry; S.F. is indebtedto the National Foundatior (U.S.) for a Fellowship, andG. T. to the Juan March Foundation (Spain) for aResearch Fellowship.

Vol. 92 503

504 J. R. GUEST AND OTHERS 1964

REFERENCES

Brady, R. 0. & Barker, H. A. (1961). Biochem. biophys. Res.Commun. 4, 464.

Buchanan, M. J., Larrabee, A. R., Cathou, R. E. &Rosenthal, S. (1961). Ab8tr. Pap. Amer. chem. Soc.,140th Meet., p. 8c.

Foster, M. A., Dilworth, M. J. & Woods, D. D. (1964a).Nature, Lond., 201, 39.

Foster, M. A., Jones, K. M. & Woods, D. D. (1961).Biochem. J. 80, 519.

Foster, M. A., Tejerina, G., Guest, J. R. & Woods, D. D.(1964b). Biochem. J. 92, 476.

Gibson, F. & Woods, D. D. (1960). Biochem. J. 74, 160.Guest, J. R. (1960). D.Phil. Thesis: University of Oxford.Guest, J. R., Foster, M. A. & Woods, D. D. (1964a).

Biochem. J. 92, 488.

Guest, J. R., Friedman, S., Dilworth, M. J. & Woods, D. D.(1964b). Ann. N.Y. Acad. Sci. 112, 774.

Guest, J. R., Friedman, S. & Foster, M. A. (1962). Biochem.J. 84, 93P.

Guest, J. R., Friedman, S., Woods, D. D. & Smith, E. L.(1962). Nature, Lond., 195, 340.

Guest, J. R., Helleiner, C. W., Cross, M. J. & Woods, D. D.(1960). Biochem. J. 76, 396.

Kisliuk, R. L. & Woods, D. D. (1960). Biochem. J. 75, 467.Mangum, J. H. & Scrimgeour, K. G. (1962). Fed. Proc. 21,

242.Peterkofsky, A., Redfield, B. & Weissbach, H. (1961).

Biochem. biophys. Res. Commun. 5, 213.Rohrbaugh, P. (1962). Fed. Proc. 21, 4.Rooze, V., Sakami, W. & Blair, D. (1962). Fed. Proc. 21, 4.Rosenthal, S. & Buchanan, M. J. (1962). Fed. Proc. 21, 470.Sakami, W. & Ukstins, I. (1961). J. biol. Chem. 236, Pc5O.

Biochem. J. (1964), 92, 504

Effect of Cysteine on Respiration and Catalase Synthesisby Saccharomyces cerevisiae

BY C. BHUVANESWARAN* AND A. SREENIVASAN*Central Food Technological Research Institute, Mysore

AND D. V. REGEUniversity Department of Chemical Technology, Bombay, India

(Received 16 September 1963)

Catalase synthesis can be induced in anaerobi-cally grown cells of Saccharomyce8 cerevi8tae byaeration (Chantrenne, 1954). In a study of theeffect of various nitrogenous substances on theability of anaerobically grown yeast cells to syn-thesize catalase, it was found that cysteine inhibit-ed the catalase induction severely (Bhuvaneswaran,Rege & Sreenivasan, 1961). In the present paper,the effect of cysteine on catalase synthesis and onrespiration is examined in more detail.

MATERIALS AND METHODS

Yeast culture8 and preparation of 8u8pension8. Theorganism used was a brewing strain of Saccharomycescerevi8iae which was maintained by bi-weekly transfer onagar slants consisting of 2% (w/v) glucose, 1% (w/v)Bactopeptone and 0 3% yeast extract with 2% (w/v) agar.For experimental purposes the organism was grown in a

broth of the same composition but with the omission ofagar for 20 hr. at 300 in stationary flasks. Practicallyanaerobic conditions were maintained by using 500 ml.conical flasks filled to the neck. Most of the yeast cells wereat the bottom of the flask at the end of the incubation

period. The cells were harvested and washed twice with ice-cold distilled water by suspension and centrifuging at 100and used at once.The organism also grew well on a synthetic medium con-

sisting (w/v) of NH4Cl (1%), glucose (1%), KH2PO4 (1%),MgSO4,7H20 (0.01 %) and CaCl2 (0-01 %). This mediumafter the omission of NH4Cl served as the non-growthmedium in aeration experiments; the pH was adjusted to6-8.

Yeast cells (about 60-80 mg. dry wt.) were suspended in10 ml. of this medium and were aerated in 50 ml. conicalflasks by shaking in a reciprocating shaker at 60 oscillations/min. for 4 hr. A colorimeter calibrated in terms of dryweight facilitated the adjustment of yeast concentration.The cells were then separated by centrifugation, washedwith distilled water and resuspended in 10 ml. of water.Catalase activity of the cells was determined after ruptureby repeated freezing and thawing.A8say of catala8e. Catalase was assayed according to a

procedure based on the titanium colour reaction forhydrogen peroxide (Patti & Bonnet Maury, 1953) anddeveloped in this Laboratory by S. P. Manjrekar & A.Sreenivasan (unpublished work). The titanium reagent wasprepared as follows: 1 g. of TiO2 and 10 g. of K2S04 weremixed and digested with 150 ml. of conc. H2S04 for 5-6 hr.The digest was then cooled and diluted to 1-5 1. with distil-led water. This served as the stock solution and could bestored at room temperature. The working solution wasobtained by diluting the stock solution fivefold with10% (v/v) H2S04.

* Present address: Biochemistry and Food TechnologyDivision, Atomic Energy Establishment (Trombay),Byculla, Bombay 8, India.