Methionine metabolism in mammals: Kinetic study of betaine-homocysteine methyltransferase

ARCHIVI:S

OF

HIOCHEMISTRY

.lND

Methionine

163,

BIOPHYSICS

Metabolism

320-321

in Mammals:

Betaine-Homocysteine JAMES Veterans

D. FINHELSTEIN,2 Administration

Hospital

(19i2)

Study

of

Methyltransferase’

BARBARA and

Kinetic

J. HARRIS

Department H.ashington, Received

AXD

of Medicine, DC 20422 June

George

WALTER H’ashington

E. KYLE I’niversityy,

23, 1972

We have studied the kinetic properties of betaine-homocysteine methgltrallsferase prepared from rat liver. The Michaelis constants for the substrates are Kkletaine = 48 PM and Iremethylated to yield mcthionine. The distribution of homocysteine betwcrn these two alternate pathw-ays may regulate and control mrthionine met,abolism in rat liver (l-3). At, least two enzyme systems can remethylatc homocysteine. It, is likely that, both reactions are significant in the regulation of the pathway. Hepatic levels of Nj-methyltctrahydrofolabe-homocysteinc methyltransfrrase increase with protein and methionine deprivat,ion (4). In contrast, the lrvels of betaine-homocysteinc methyltransferase (EC 2.1.1.5) mcreasc with protein and methioninc supplementation (4). Based on these observations, we suggested that the N”-metjhyltetrahydrofolate-homocystcinc methyltransferase reaction serves to conserve methionine while betaine-homocysteine methyltransferase funct’ions primarily t,o metabolize excess homocysteine and betainr. Studies of the tissue content of the methyl-

1 Supported in part the National Institutes Health Service. 2 Medical Investigator, tion.

by

grant AM 13048 from of Health, U.S. Public Veterans

Administra320

Copyright Ml rights

@ 1972 hy .lcademic Press, of reproduction in any form

Inc. reserved.

transfwascs wer(’ the basis for thcx above concept. Ho\vever, enzymatic activity in wo depends on the kin&c properties of the enzyme’ as well as on thr tissue content. For this reason, w have studied t)he properties of bctainc-homocystcine mcthgltransferase prcparcd from rat liver. MATERIALS

AND

METHODS

as&au. The method for assaying Enzyme betaine-homocysteine methyltransferase depends on the format,ion of [;lfe-l%]methionine from [.1fe-14C]betaine and t,he separation of product and substrate by ion-exchange chromatography (1). Unless specifically stated the reaction volume was 1.0 ml and contained 0.05 M Tricine (pH 8.0) and 1 mM dithiothreit,ol. I,-homocysteine was prepared from L-homocysteine thiolactone hydrochloride by the method of Duerre (5). The rolltine incubation period was 15 min. Atlalysis of data. We employed graphical representation, linear regression analysis and Wilkinson’s weighted and nonlinear regression methods (6) to evaluate individual kinetic experiments. To determine kinetic const,ants, as in the productinhibition studies, parameters derived by Wilkinson’s method were translated into the doublereciprocal equation. In turn these equations were used to define the Michaelis ronstsnts by the method of Florini and Vestling (7). However, mathematical solution of the equations, rather than graphical analysis, was used to determine

thr pt)int,s the i1xes.

of inter’sectiorl

and

the

interc,epts

011

Emyme preparnliou. Our routine preparation yielded large quantities of semipurified enzyme. WP did tlot attempt to obtaitl a homogeneorls p~,o~ein. The initial homogenate was prepared ill 3 vol of 0.01 M potassium phosphate buffer (pH 7.j). After ~~entrifrlgutiotl at 15,000~ for 20 mill :tt OY:, the s\Ipprnatant was heated at 80°C fol !)!I HP,‘. The supernatalit from this first heat treatrlretlt was treated by g;pl filtration (Sephades (i-25, s;rn,r h1f’Pl) and the protein fractions were c.,mrbilrrd. To these frartitrm we added watercyrlilihrat.ed c:tl(~i~un phosphate gel (Bio-Itad). The, qrlalltity of grl WV&S 20-30 mg for each milligram of protein and the mixture was eqrlilibrated for 3’) mill at 0°C. The enzyme is absorbed OII the gc.1 :uld is Ilot eluted by trratmellt with water, 0.01 M :tlrd 0.1 M potassium phosphate (pH 7.5). The r~raymr was cluted with 0.25 IV phosphate 1111ft’rr. Subsequent gel filtration (Sephadex (i-25) rcd~~c~xl the bufl’rr c,ollc,elrt~:ttiorl to 0.01 M. TOP c’rlzyrne :rc,tivity was Ilot absorbed b!* passage t Ilrorlgh :I c~1~1~1rml of TEAISkellulose equilibrated with the same hul’l‘er. This final product was ellric,hrd 1520 fold compared to the initial su~xrIr:itatlt The total yield was approximately 25’; III ot.her studies, we observed that the enzyme ww Ilot absorbed dllriug passage through C?\Im r~all~~losr eq~~ilibratcd with 0.01 M potassium phosl)hatr (pII 7.5). Either a sec,ond heat treatment at 81)“C X 93 sec. or ethanol fractionation (final roru.clltration = 30’~,) yielded an additional twoto five-fold purificatiotl. However, both methods gave \tnprrdirtablo and limited yields trf enzyme lvhicnh prec.luded their routine else. Equal purific,:ttioll, I)ut with higher yields, can be obtained bv (.haugillg the heat treatments to 60°C for 6 n;i~:. The addition of O.O(ii M I)L-honloc,SstriIlr dnring t,he heat treatment had no effect on rithel purifi(aatioll or yield.

Tlic~

rr%ction

rate

drpc&s

on

the

spc&c

huffw, thcl pH and the concentration of huffor ion (Tahlc I). With potassium phosphatcb buffer the rate of product formation incwased consistently as the pH KM incrcawd in incremclnts of 0.25 units from pH 7.0 IO pH 8.25. This upper valw is in a poor rangy for phosphatcl buffer. As shown in thcl table, product formation was grcbatwt in 0.05 M Tricinc (pH 8.0). Tris, TES and HEI’ES inhibited the reaction.

Buffer

Product formation* (‘“( control activity)

(pH)

Potassium phosphate Potassium phosphate Potassium phosphate Tris (S.1) (;lycilie (8.3) Tl<:S (8.35) Hlq:PES (8.15) Bicinc (8.3) Tric.ine (5.5) Tri(,illr (8.0) Tric,illc (8.4) 0.1 v Tricinr (i.5) 0.1 II Tric,iue (8.0) 0.1 M Tricine (8.1)

(7.0) (7.5)

47.X 72.(i

(8.X)

so. 1 5fi. 0 80 4 5u.:? 57.0 83.5 901 100.0 i!J .8 i(i .(i 82.6 41.5

” The assay system contained 0.05 rnM I,-homecystPine and 0.035 rnM hetaine. Buffer concentr:ttioll was 0.05 JI r~nlcss we specify another c.oncelitratiotl. b l’rodllct formation with 0.05 M Tricirre (pII 8.0) is arbitraril! assigned the value of 100’ ;.

Both r,-mcthionirw and dimclthylglycinc inhibit l)c~tainc.-homoc3,stt~ine marlwdl> mc~th\.ltrarlsfc~ra.sc. WV n-ill detail this inhibition by both products in a subscyucnt WCtion. The addition of wq~stath.ionine consistckntly incrcascd product formation to a limitcld chxtwt (Table IT). This may haw sorn(’significanw to the wgulation of homocystck mc~taholisminmammalian liwr and t lw mechanism of this cystathionino c#ect is thr subjwt of ongoing studies. In addition to tlw compounds listed in Tahlc II, VY studkd t auriw, cyst (tic acid, cvsteinclsulfina tc and sulfatcl in conwnt raiions to 0.01 M. Sonc significantly altcrcd the reaction rat(a; nor did glycincl, l~-sc~rinc~ or I,threoninc. Thiols could cwhanw product formation. Dithiothwitol consistc>ntl\- stimulated the reaction n-hen added to the waction mixture in concc~ntrations qua1 or greatw than 0.05 rnlt. Tlw magnitude of this &“ect varicld with the conwntration of substrate1 homocystrin(~. In kinetic stud& in lvhich

322

FINKELSTEIN, TABLE

EFFECT

HARRIS,

II

OF METHIONINE METABOLITES BETliINE-HOnaOCYSTEINE

ON

METHYLTRANSFERASE~

Compound added bmoles)

None L-methionine (0.05) S-adenosyhnethionine S-adenosylhomosysteine L-cystathionine (0.84) n-homoserine (0.05) Choline (0.075) N, N-dimethylglycine L-cysteine (0.05) L-cysteine (0.4) L-cystine (0.05) Glutathione (reduced)

Product formation (% control)

(0.05) (0.05)

(0.075)

(1.0)

100 7 85 84 125 94 94 4 120 83 41 102

0 Concentration of substrates was: L-homocysteine = 0.02 mM, betaine = 0.035 mM. The total volume in each reaction was 1.0 ml.

the concentration of bet,aine was 0.035 mM and homocysteine varied from 2.5 /IM to 10 mM, the addit’ion of 1 mM dithiothreitol decreased the apparent K, (from 103.2 to 51.4 PM) without effecting v (2.18 and 2.24 nmoles/l5 min). Dithiothreitol could activate the enzyme directly or it could funct,ion by maintaining an optimal concentration of homocysteine. Either effect might be minimized by increased concentrations of homocysteine. In an attempt to further delineate the mechanism, we preincubat’ed t,he enzyme in 5 mM dithiothreitol for 15 min at 37°C. Subsequent’ly t,he free thiol was removed by gel filtration and the resultant enzyme preparation was assayed with or wibhout, added dithiothreitol. Preincubation wit,h dithiothreitol caused a three-fold increase in enzyme specific activity when compared to preincubation in water. In addition, dithiothreitol added to the assay system caused no further increase in product formation by enzyme preincubated with this compound. Thus, it is likely that dithiothreitol directly effects the enzyme but we cannot exclude an additional effect on substrate concentration. The effects of n-cysteine and of reduced glutathione were inconstant and varied

AND

KYLE

inexplicably in experiments using diff errnt enzyme preparations. The moat consistent pattern was a stimulatjion of product formation at low concentrations of Lcysteine. With increasing cystcinc conccntration, the degree of stimulation decreased and progressed to inhibition when the cysteine concentration was 20 t,imes greater than the homocvsteine concentration. LCysteine ncvcr increased product formation when the assay system contained optimal concentrations of dithiothreitol. Indeed, under these conditions, L-cystcine caused only inhibition. Addition of Dcysteinc enhanced product formation and X-methylcysteine did not alter the reaction rate. E’rom these studies, we concluded that both stereoisomers of cystcine possess the general ability of the thiols to augment enzyme activit,y. However, L-cysteine specifically inhibits bet’aine-homocysteine methyltransferase. Since this inhibition requires the L configuration it cannot be due to the chemical formation of the cy-steine-homocysteine mixed disulfide. The free thiol group is required for inhibition since Smethylcysteine, L-serine and L-alanine do not share this property. In contrast to the specific effect’s of Lcysteine, both D- and L-cystinc markedly inhibit’ed betaine-homocysteinc methyltransferase. In studies with low concentrations of substrate (betaine, 0.035 mM and n-homocysteine, 0.05 mM) there was 25% inhibition at 0.05 m&I D- or L-cystine and 90% inhibition at concentrations of 0.3 rnlf. Although t,he formation of homocysteine-cysteine mixed disulfide was not demonstrated, the lack of stereospecificity for the cystine inhibition suggests t)he possibi1it.y of a direct chemical effect on either enzyme or substrate. Kinetic

Constants

of the Reaction

Table III illustrates one of several kinetic studies. In all studies graphic solutions, linear regression analyses and solutions by Wilkinson’s method (6) were in close accord. The data in Table III yield Kbetaine = 43 IAM, Khomocyskine = 12 I.LM and KAB = 0.0036 PM. In four additional experiments covering wider concentration ranges for both substrates we found t,hat Kbetaine

BETAINE-HOMOCYSTEINE TABLE KINETIC

STUDY

323

METHYLTRANSFERASE III

OF BET.\IN~-HOMOCYST~INR

~!~THYLTRANsFERASE’

-

Homocysteine Betaine

(mM)

0.025

(mM)

Product

0.027 0.067 0.117 0.217 ParametersC

Formation

0.87 1.80 2.53 3.44 Km/ V l/V

a All reaction tubes contained * Parameters for double-reciprocal for l/V are pmoles-*. c Parameters for double-reciprocal

26.56

166

(nmoles/lS

2.14 3.63 4.87 5.96

13.1

9.26 129

0.5 mM dithiothreitol. equation at varying equation

constant

at varying

by Products

Table IV rcpresent,s a summary of t’he st’udies of the inhibition of bet.aine-homocysteine methyltransferase by L-methionine and N, N-dimethylglycine. Wit,h the concentration of homocysteine (fixed substrate) equal or less than 0.05 mM the kinetics of inhibition by L-methionine wera noncompetitive. As we increased the homocysteine concent,ration, the effect of methioninc on t,he intercept became less apparent and the pattern approached t,hat of competitive inhibition. Methionine did not inhibit the reaction if the homocystcine concentration was greater than 0.2 rnw In contrast, methionine was a competitive inhibitor in studies at all concentrations of betainc as t,he fixed substrate. Dimethylglycine inhibition varied in type depending on the conccntrat,ion of fixed substrate. With the beta& concent,ration fixed at less t,han 0.1 mM, the inhibition was noncompetitive. At higher concentrations of betaine the inhibition became uncompetitive. When the concentration of homocysteine (as the fixed substrate) was greater than 0.2 mM, dimethylglycine was a noncompetitive inhibitor. At lower concentjrations of homocysteine the inhibition pattern was competitive for the dimethylglycine-bctaine pair. We also performed inhibition st,udies at

Parameter@

min)

1.60 2.90 3.91 4.94 145

ranged from 39 to 56 PM. Similarly, we found Khomoeysteinovalues from 15 to 21 PM. There was no suggestion that 4ther suhstrate activated or inhibited t’htx enzyme. Inhibition

0.125

0.065

constant

-

bet,aine homocysteine

substrate

Methionine Dimethylglycine

300 205 158 137

The

units

concentrations. IV

INHIBITION

~~OMOCYSTICINE

Fixed

20.8 8.74 5.97 3.92

concentrations.

TABLE

(concentration)

l/V

.-

PRODUCT

Variable substrate

K,lV

OF

BET.IINE-

&‘IETHYLTRANSFERASEa Homocysteine

Betaine

Betaine 0.025 nm

C NC

Ilomocysteine 1.0 InM

0.02 m&f

5.0 InM

C UC

NC C

NC

a 0 = no inhibition; C = competitive tion; NC = noncompetitive inhibition; uncompetitive inhibition.

0

inhibiUC =

high concentSrations of both homocysteine (5 mM) and betaine (1 mM). Marked rcduction in product radioactivity made it impossible to study higher concentrations of betaine. Rlethionine, in the concentration range of 0.2-10 mM, did not inhibit product formation. With 20 mM met,hionine there was 35 % inhibition. In cont,rast, inhibition by dimethylglycinc was marked. At 0.02 mM, inhibition was 19 %, at 0.1 mM -76 %, and at 1 mM -90%. These patterns of inhibition of betainehomocysteine methyltransferase by N, Ndimethylglycine and L-methionine are compatible wit,h an Ordered Bi Bi mechanism (8). L-Homocysteine is the first substrate to add to the enzyme and methionine is released aft,er the dimethylglycine. The ability of dimethylglycine to inhibit product formation at high concentrations of both substrates confirms the order of product release (9).

324

FINKELSTEIN,

HARKIS,

I)ISCUHSION

earlier reports, we ctmphasizctd the significance of homocgstcinc: rcmcthylation in the regulation of rntlt hioninct metabolism (1-3). The conwrsion of homocystc%w to cystath.ioninc> is irwwrsiblc. In contrast, remethylation provides mclthioniw for ut ilization in the synthesis of protein or S-ad(w)sylmethionint:. Th(b distribution of homocysteine bctwcrn the transsulfuration and. remethylation pathways should bc governed by the tissue content of the enzymes involved as w.41 as by their kirwtic propclrties. The tissue levels of two homocysteine mcthylt,ransferases change ind.epcndrntly in response to age, diet and tjrc~aimc~nt with hormones (4). Thus, dietary supplc~mcnt s of methioninc depress hrpatic lcvcls of N”methylt~etrahydrofolatePhomocystcGnc methyltransfcrasc and simultaneously lead to a marked. increase in hctaino-homocysttinc: mrtjhyltransfwaw. E’rom this observation, wc suggested that thr mcth.yltetrahydrofolatc enzyme functions to conscrw met hionine by returning h.omocysteinc to the mcthionino pool. In cant rast), t hc bctaine enzymcb serves to prcwnt the accumulation of homocysteine (and bet ainc) during pwiods of mctjhionine excess. While t,hr original studios supported the above hypoth.esis the kinetic propcrtiw of betainc-homocystcinc methyltransfcrasc~, which wc present in this report, are not obviously in accord. The Alichaclis constant for L-homocysteine is 15 pM. This value is of t,hc same order of magnitude as the Rlichaclis constant for homocystcinc with N5 - mcthylt~etrahydrofolate - homocystcine m&hyltransferase prepared from rat liver or porcine kidney (3, 10). If betaine--homocysteinr metjhyltransferasc functions to prcvent, the accumulation of homocystcinc, we might expect) a higher K, for homocystoine in this reaction. Similarly one would not predict, that methionine would bc an inhibitor. These observations can be rccontiled wit’h the initial concept ‘by recognizing t,hat the betaine-homocysteine met,hylt,ransferase react,ion is an essential step in t.hc catabolism of bctainc (and the biosynthesis of sarcosine). Then, the relatively low K, for homocystrinc may be rationalized by the requirement for continued betaine metabolism-won at, low t,issuc: lovcls of homoIn

ANI)

KYLE

cysteinc. When the level of dietary mct,hionine incwaws, the low K, would effcct,ively exclude a significant role for betainchomocyst cGw mc~thyltransfwasc in thr prcvention of homocystc$nc~ accumulation. This could tw achieved only by an incwasc: in chnzymo contc,nt and this is t hc phcnomrnon which was observc:tl. WC, may intctrpwt our ohscrvat ion of mclthionirw inhibition in a similar manner. Duo to it,s position as the swond product released in this Ordwcd Bi Bi reacf.ion, methioninc is compc~titivt: with homocysteino at all 1r:wls of bctaino. 3lcthioninc does not inhibit, t 110 waction if the concentration of h(Jm(JCyStPin(! is saturating. Thus, the c,ffcctivwwss of mcthioninc~ in preventing its own synthesis is limited and homocgsteine accumulation will reverse this effect. Conversely, mcthionino inhibition may prcwntJ excws consumption of homocystrinc h) this reaction in a situation where brt aine conct~ntration is increased and homocystcine conwntration is limiting. The “conscrvat ion” of homocystcino may be necessary since t)his compound is an essential reactant in folatc metabolism as well as in the t ranssulfuration pathway.

1. FINI