Biosynthesis of adenosylmethionine from adenosylhomocysteine in Candida utilis

67

BIOCHIMICA ET BIOPtlYSICA ACTA

u~A 26075 BIOSYNTHESIS OF ADENOSYLMETHIONINE FROM ADENOSYLHOMOCYSTEINE IN CANDIDA UTILIS* STANLEY K. S H A P I R O AND DI1KIS J. E H N I N G E R

Division of Biological and 2VIedical Research, Argonne National Laboratory, Argonne, Ill. (U.S.A.) (Received August 28th, 1968)

SUMMARY

The mechanism of the biosynthesis of adenosylmethionine from adenosylhomocysteine in Ca~dida utilis involves decomposition of adenosylhomocysteine and reutilization of the products for the synthesis of adenosylmethionine. This conclusion is based on data which showed that the addition of adenine, adenosine, or homocysteine to a medium containing labeled adenosylhomocysteine results in a dilution of the specific radioactivity of the resultant adenosylmethionine only if the unlabeled supplement corresponds to the labeled moiety of the adenosylhomocysteine. The data do not support the hypothesis ot an exchange reaction between adenosylhomocysteine and its decomposition products because the specific radioactivity of adenosylmethionine increased as a function of time when labeled adenosylhomocysteine and its decomposition products were added to a culture of C. utilis. While methionine repressed 99-9 % of the endogenous incorporation of sulfate into adenosylmethionine, adenosylhomocysteine repressed only 2o % of this endogenous incorporation. Adenosylhomocysteine served as a sole source of sulfur, but sulfate repressed 8o% of the incorporation of label from adenosyl[carboxy-~C~homocysteine into adenosylmethionine.

INTRODUCTION

Adenosylhomocysteine is one of the products of all transmethylation reactions that involve adenosylmethionine as the methyl donor 1. Nevertheless, there have been very few studies concerned with the metabolic fate of adenosylhomocysteine in various cells. Dunm~E has described an enzyme that decomposes adenosylhomocysteine to adenine and ribosylhomocysteinez. SHAI~IRO has reported the enzymatic synthesis of methionine from adenosylhomocysteine and S-methylmethionine a. DUERRE AND SCHLENK have conducted experiments with whole yeast cells which suggested that adenosylhomocysteine may be regenerated directly to adenosylmethionine without decomposition *. In the studies reported below, we have reinvestigated ~he mechanism of conversion of adenosylhomocysteine to adenosylmethionine in yeast cells with the use ot a new quantitative method for the analysis of adenosylmethionine in the presence of adenosylhomocysteine~. * This work was presented in part at the 7th Intern. Congr. ]3iochem., Tokyo, August, 1967 ,{S. K. SHAPIRO, K. D. SPENeE A N n D. J. EBNINC-ER, Abstr. 4 (1967) 592).

Biochim. Biophys. Acts, 177 (1969) 67-77

65

So Ko SHAPIRO, Do ]o EHNINGER

MATERIALS AND METHODS

Culture conditions Candida utilis, ATCC 9950, was maintained on agar slants consisting of 2 % glucose, 2% tryptone (Difco), ~ O//oyeast extract (Difco), and x. 5 % agar {Difco). The formation of adenosylmethionine in C. ~tilis was studied in the following minimal medium, Medium M. It contained (per 1): z o g KH,,PO4, 5 g K~HPO,, ~ g (NH,)~SO4, i g trisodium citrate, o.o 3 g MgClz.6H~O, o.o~ g MnSO~'7H~O, o.ox g CaCI~, o.ox g ZnSO~. 7H~O; and ~5 g glucose, sterilized separately. In order to prepare a sulfur-free medium, Medium S, the sulfate salts were replaced by (per i): ~-35 g amrnonimn acetate, o.ox g MnCI~'4H.~O, and o.oz g zinc acetate, respectively. For every experiment, C. utiZfs was transferred to a fresh agar slant and incubated at 3°o for 24 h. The organism was then inoculated into 5 ml of Medium M or Medium S, and the culture was grown in a New Brunswick gyrotory shaker (270-3o0 rev./min) at 3 o° for ~8 h. This culture was used to inoculate (5 %) the final cuitures of Medium >f or Medimn S, and incubation was carried out for the desired 1ought of time in tt~e shaker at 3o%

Chemicals Adenosylhomocysteine (labeled and unlabeled was prepared according to the method of SHaPiRo _aNDEHNINGERSoAll other compounds were commercial products.

Analytical methods The uptake of label by the ceils was calculated by subtraction of the counts in the supernatant fluid and wash from the total counts added to each culture. Since the total recovery of radioactivity in all experiments was over 9o%, the estimanon of cellular uptake was subject ro an error of less than Io% due to volatile metabolites. The amount of adenosylmethionine in the yeast cells was determined on Dowex-5o(Na-i columns ~, For increased accuracy in the determination of the specific radioactivity of the isolated adenosylmethionine, the elution procedure was modified as follows The first xo-m! fraction of the 3 M H.~SO¢ e!uate was put aside. The second ~o-ml fraction was used for absorbance and radioactivity determinations for the calculation of the specific radioactivity; this fraction contained the highest concentration of adenosvlmethionine a. In order ~o determine the total yield of adenosylmethionine, another 7o-ml fraction was collected and combined with the first ~o-ml fraction. The incorporation of label into adenosylmethionine is expressed as ~oo > specific radioactivity of adenosybnethionine/specific radioactivity of the particular supplement used° tn ali experiments, the specific radioactivity of adenosylhomocysteine, methionine, adenine, and adenosine was adjusted to z-5"xo~ counts.rain per ~rnole The specific radioactivity of the ~a~S~sulfate was 6.7 • zo ~ counts.'min per ~mote. Under these conditions. the specific radioactivity of adenosvlmethionine m the second xo-ml fracnon was determined with an error of ~ 2%° A Beckman DU-z spectrophotometer was used for all absorbance readings. Radioactivity ~vas measured in a three-channel Beckman liquid scintillation spectrometer, Model LS-2oo, using a scintillation fluid of o.4% diphenyloxazole in equai volumes of abs. ethanol and toluene. For mixed isotope experiments, ~H and ~C were measured in the same samp!e by the foliowing procedure. The gain and the isosets ot Biochim. Biop]~i~s. dcta, x77 (~g69) 67-77

69

BIOSYNTHESIS OF ADENOSYLMETHIONINE

the scintillation counter were adjusted so that less than 1% of the 3H counts were measured in Channel C. At this setting, approx. 6o % of the ~C counts were measured in Channel C and approximately 20 % in Channel A. For each series of determinations, the percent distribution of ~4C and ~H in Channels A, 13, and C was determined with singly labeled samples. These samples were prepared in I.O ml of 3 M H2SO~ so as to correspond to the i.o-ml samples of 3 M HzSO~ eluates that contained the doubly labeled adenosylmethionine. The 1'C activity of such samples was calculated from the formula: C a l c u l a t e d x~C ~

Counts in C h a n n e l C % Counts of ~ C in C h a n n e l C

Since Channel B was adjusted to measure the total radioactivity of both 14C and ~H, the 3H activity was determined b y subtraction of the calculated 14C activity from the total counts in Channel B. This method of calculating 3H and 1~C activity in a mixed sample gave reproducible results with an error of 4- 2 %. RESULTS

Biosynthesis of adenosylmethionine from adenosylhomocysteine From the results presented in Table I, it was clear that the addition of adenosylhomocysteine to the medium resulted in the formation of adenosylmethJonine at a level above t h a t of the value obtained in an unsupplemented culture. It m a y be seen t h a t at the three lowest concentrations, adenosylhomocysteine was more effective than methionine in the stimulation of adenosylmethionine synthesis, both with respect to net yield and incorporation of label. At the two highest concentrations, methionine was more effective. Considering the amounts of each supplement taken up b y the TABLE I BIOSYNTHESIS OF ADENOSYLMETHIONINE FROM ADENOSYLHOMOCYSTEINE OR METHIONINE

C. util/is w a s g r o w n for 18 h a t 3 °o in 15 m l of M e d i u m M s u p p l e m e n t e d w i t h v a r i o u s c o n c e n t r a t i o n s of [Me-laC~meth ionine or adenosyl~carbo~y-14C~ h o m o c y s t e i n e . Supplement ([,moles/ml)

Cell yield (rag)

Label in cells (%)

Yield of adenosylmethionine (l~moles/g)

Incorporation of label into adenosylmethionine

56.8 57.9 53.6 5o.1 45-5

1.36 1.53 1.65 2.71 4.24

1.3 3.5 13.7 35.o 62.5

5oo 517 52o 515 522

23.3 16.1 8.5 1.6 1.5

1.6o 1.78 1.85 1.9 ° 2.24

12. 4 20.6 23.4 28.2 34.2

505

--

1.41

--

(%)

[Me-14C] M e t h i o n i n e o.i 0.25 0.5 i.o 2.0

493 5Io 486 469 448

A d e n o s y l [carboxy-14C]h o m o c y s t e i n e o.1 0.25 0.5 1.o 2.0 No s u p p l e m e n t

t?iochim. Biophys. Aeta, 177 (1969) 67-77

S, K. SHAPIR0~ D, j,, EHN:NG, ER

7°

cells, adenosylhomocysteine was far more effective than methionine in the stimulation of adenosylmethionine synthesis. In Fig. ~ the amount of adenosyh~ethionine formed b y C. u~iZis is shown as a function of time. When z.o ~mole/ml of adenosylhomocysteine was added to ~Iediuu~ M, the production of adenosylmethionine reached a m a x i m u m between 3o and 44 b ~ ~o [ - 4 - r

,

,

T

1

~g ~i -

-

E

,

g 2o

I

~ _z

~

,

~ 05 ~n w ~

~L

___~ ............ ~

1.5 10

~

,

~

s

~"

~

,

-,

: Z222

I~ Adenosyih°m°ws~elne Adenosylhomocysteine +Adenosine--

2~o ~@ HOURS OF INCUBATION

I

40

5b

~'ig. L Biosynthesis of adenosylmethionine as a f u n c t i o n of time. C. utilis was g r o w n in zo ml of Medium M suppIemen~ed w i t h L o # m o l e / m l of a d e n o s y l h o m o c y s t e i n e ([]), or Lo #mole/m1 of adenosine ( ~ ) , or ~.o #raole/ml of a d e n o s y l h o m o c y s t e i n e + i.o ~ m o l e / m l of adenosine (l!I), or no s u p p l e m e n t (O).

The presence of eqnimo!ar concentrations of adenosine m the medimn did not affect the yield of adenosylmethionine at any time. In order to test the effect of methionine and adenosylhomocysteine on the endogenous formation of adenosylmethionine, the cells were grown in Medium S supplemented xvith [a~Sisulfate (o.8 ~mole/ml) as the sole source of sulfur. As shown in Table H, the addition of methionine to the medium resulted in virtually compiete repression of the incorporation of a~S into adenosylmethionine. This effect has beep. TABLE II INCONPORATION OF [aS$]SULF.~TE INTO ADENOSYLM~THiONIN1~ IN THE PRESENCE OF M E T t t ! O N i N E OR ADENOSYL~OMOCYSTEINE

C. utilis was grow~ for ~8 h at 3o ~ in x5 ml of Medium S snpplemen~ed w i t h o~8 ~ m o t e / m t of ~a~S]sulfate plus methionine or adenosylhomocysleine, The specific radioactivity of ~aaS]suifaze was 4" IO* counts/rain per ~mote.

Suppleme~¢$ (2.0 ~moles/m~)

Cell yield (rag)

Label in cells (%)

Y,;eld of ~de~osylmethio~i~e (~moles/g)

Zncofpoeatio~,z of a~S into ~de~osylmet~do*d~e

(%)

No s u p p l e m e n t

532 527

5o.3 55.2

i.oo x.o2

93.o 92.8

?¢[ethionine

482 488

x, 2 2,3

5.85

5,82

o. z 8 o.~9

Adenosy!homocysteine 537 542

50.7 49.5

0.97 o,94

75.I 72.9

Biochim, Biophys. Acts, z77 (x969) 67-77

BIOSYNTHESIS OF ADENOSYLMETHIONINE

71

reported previously by SCHLENI¢AN~ ZYI)EK". This repression is reflected by a 25-fold decrease in the uptake of [~S~sulfate by the cells.The addition of adenosylhomocysteine to the medium resulted in only 19 % represssion of incorporation of ~aS into adenosylmethionine and only a slight decrease in the uptake of [a~S~sulfate by the cells. However, it should be noted that there was no net increase in synthesis of adenosylmethionine in the presence of adenosylhomocysteine as there was in Medium M (Table I, Fig. I).

Mechanism orformation of adenosylmethioninefrom adenosylhomocysteine Experiments were performed in order to determine whether adenosylmethionine is synthesized from adenosylhomocysteine directly or after the decomposition of adenosylhomocysteine to intermediary compounds. Cells were grown in Medium M supplemented with various levels of adenosine and with adenosylhomocysteine labeled in the adenosine or the homocysteine moiety. When the cells were grown on [8-~4C~adenosylhomocysteine plus adenosine, the specific radioactivity of the isolated adenosylmethionine was diluted in relation to the concentration of adenosine in the ~nedium (Fig. 2). On the other hand, when the cells were grown on adenosyl[carboxy~C~homocysteine plus adenosine, there was no dilution of the specific radioactivity of the isolated adenosylmethionine. I00 -

I

I

/

~]~

Uncorrected Values

I

I

40

I

rected V~lues

~2 z

SO

~_ #

8o

~ _J

m

I10

60

I00

s o

-150

~0

~ ~0 _~ ~1

30

- - ,~

<

~H ~

~o,o~ 0~l 0

~"~ 0.5

I 10

I L5

Z0 0

I 0.5

I [0

~

I~ ~ A5 0 1.5 2 0 °

~MOLKS ADKNOSINK/ML CULTURE

Fig. 2. Incorporation of label into adenosylmethionine from adenosylhomocysteine. C. utilis was grown for 18 h at 3°0 in io ml of Medium M plus various levels of adenosine and either I.O/~mole/ml of [8-14C]adenosylhomocysteine (O) or i.o/*mole/ml of adenosyl[carboxy-14C]homocysteine (&). Corrected values: values obtained after subtraction of endogenous adenosylmethionine formation in the absence of supplements.

*

]Data supporting the hypothesis that adenosylhomocysteineis decomposed prior to the formation of adenosylmethionine are shown in Table III. At the level used for adenine and homocysteine, there was an increase of 9% and 6%, respectively, in the amounts of adenosylmethionine formed compared to the unsupplemented culture. When adenosylhomocysteine was added to the medium, the production of adenosylmethionine was similar in all cases. The percent incorporation of label from [8-14C]adenosylhomocysteine and from adenosyl[carboxy-~aC]homocysteinewas also similar. This suggested that both parts of the molecule are incorporatedinto adenosylmethionine t3iochim. Biophys. Acta, i77 (1969) 67-77

72

s . K . SHAP~RO~ D. J~ EHN!NGER

at i d e n t i c a l rates. Yet, when adenine was a d d e d to t h e m e d i u m s u p p l e m e n t e d w i t h a d e n o s y l h o m o c y s t e i n e , o n l y t h e p e r c e n t i n c o r p o r a t i o n of label from. [8-~¢C]adenosyi h o m o c y s t e i n e into a d e n o s y l m e t h i o n i m e was g r e a t l y r e d u c e d (5o°/;). T h e pereer_,~c i n c o r p o r a t i o n of label from adenosyl[cafboxF-~C]homocysteine into adeno~@~ m e t h i o n i n e was also r e d u c e d (~5 %) when adenine was present~ This was p r o b a b l y due to t h e increased u n l a b e l e d a d e n o s y l m e t h i o n i n e p r o d u c e d as a result of t h e presence of ~.o ~ m o l e / m l of adenine. A t lower tevels of adenine, t h e r e was ~o significant reductior~ in i n c o r p o r a t i o n of label from [8-~C] a d e n o s y t h o m o c y s t e i n e or a d e n o s y t [ c a z b o ~ - ~ C ] ~ h o m o c y s t e i n e into a d e n o s y l m e t h i o n i n e . W h e n h o m o c y s t e i n e was presenL o n l y the p e r c e n t i n c o r p o r a t i o n of label from adenosyl[carboxy-~C]homocysteine into adenosyim e t h i o n i n e was reduced. These ~esuits suggest t h a t a d e n o s y l h o m o c y s t e i n e is decomp o s e d a n d t h a t t h e r e u t i l i z a t i o n of t h e adenosine a n d h o m o c y s t e i n e moieties occurs at a similar r a t e d u r i n g t h e p e r i o d of i n c u b a t i o n . TABLE III BIOSYNTHESIS

OF ADEIWOSYLI~I]ETIZfIONINE

FROM

ADENOSYLI-IOMOCYSTEINE

(A#~

O R A ~ e)

C. ~tilis was grown for 44 h a~ 3o ° in ~o ml of M e d i u m IV[ pl~s ~he supplements indicated. The concentration of lhe supplements was: A * ~ or AH*, ~.o ~nole/mi; ~I~gdenine, ~.o ~mole/ml; homocysteine, o.4/~mo]e/ml. Abbreviations: A*ll, [8-~C~adenosylhomocysteine; AI-I*, adenosyL [cafbo~yA~C]homocysteine.

Supplements

Cell yield (rag)

Label i~ cells (%) ~C

None ~aH]Adenine I-Iomocysteine A*H A*I~ + ~aH]adenine A*H + homocysteine All* All* + [all,adenine All* + homocysteine

387 39o 39 x 384 4oi 394 367 397 374

aH

6.6 8.6 4.2 7-9 7.I 7-4 4 .1

Yield of adenosylmethionine ([~moles/g)

0.6

3.o

Incorporation of label into adenosylmethio~dne( ~ ) 1'~C

~.78 ~.95 L 89 2.6o 2.62 2.74 2.3I 2.37 2-33

aH

~3.9 8o.~ § 4o.5 § 76.~ § 82.5 § 7o-'~ § 5z.~ §

x~.9 -12.2 -

§Values calculated after subtraction of endogenous synthesis of adenosytmethionine. I n o r d e r to test t h e p o s s i b i l i t y of an exchange r e a c t i o n b e t w e e n adenosy!h o m o c y s t e i n e a n d adenosine a n d h o m o c y s t e i n e , t i m e studies were c o n d u c t e d on the f o r m a t i o n of a d e n o s y l m e t h i o n i n e from ~8-1~C[adenosylhomocysteine. It m a y be seen in Fig. 3 t h a t t h e specific r a d i o a c t i v i t y of t h e a d e n o s y h n e t h i o n i n e f o r m e d from [8-1~C]adenosylhomocysteine increased with time in t h e presence or absence of [~H]adenosine. T h e i n c o r p o r a t i o n of [ S H ] a d e n o s i n e - - i n t h e absence of adenosv!h o m o c y s t e i n e into a d e n o s y l m e t h i o n i n e also increased w i t h time. I n the e x p e r i m e n t w i t h [8-1~C]adenosylhomocysteine a n d Jail]adenosine, t h e t o t a l percent i n c o r p o r a t i o n of labels a p p r o x i m a t e d the i n c o r p o r a t i o n o b t a i n e d i~ the e x p e r i m e n t w i t h [8-a~C~a d e n o s y l h o m o c y s t e i n e alone. A similar e x p e r i m e n t i n v o l v i n g t h e h o m o c y s t e i n e m o i e t y of a d e n o s y I h o m o c y s t e i n e is shown in Fig. 4. T h e yield of a d e n o s v l m e t h i o n i n e was

Biochim. Biophys. Acta, ~77 (I969) 67-77

BIOSYNTHESIS OF ADENOSYLMETIqlONINE

73

a l w a y s t h e s a m e w h e t h e r or n o t hornocysteine was p r e s e n t in a d d i t i o n to a d e n o s y l [carboxyJ~C~hornocysteine. The specific r a d i o a c t i v i t y of t h e a d e n o s y l r n e t h i o n i n e f o r m e d increased w i t h t i m e w h e t h e r or n o t h o r n o c y s t e i n e was present. Thus, t h e r e was no significant exchange b e t w e e n a d e n o s y l h o m o c y s t e i n e a n d hornocysteine or adenosine because this w o u l d h a v e r e s u l t e d in d i l u t i o n of t h e a d e n o s y l h o r n o c y s t e i n e a n d t h e r e s u l t a n t a d e n o s y l m e t h i o n i n e b y t h e u n l a b e l e d hornocysteine or adenosine as a f u n c t i o n of time. ~ Q 60

~

~

-

......

~

T

~ ~o ~,l-AH*÷HomocysteJne

( i~-C

_

3t4c+ ~H ~(~ + ~)

fi

z/z~D

~d

~,

>~ u~

zo 6e

g" z

oo , ~ O,E3-AH~

i i - - i ] (8-14C) Adenosyl homocysteine • .,A (8-14C) Aaenosylhomocysfeine + (SB) Adenosine

z/

/ _o

~

~

~

5

z_

~ 14C H

.J ~0

~ 2C

<

u

~_

l 50

HOURS OF INCUBATION

iI

-. ~,~

~,~ HOURS OF INCUBATION

Fig. 3- Incorporation of label into adenosylmethionine from [~H]adenosine and/or [8-~aC]adenosyl homocysteine as a function of time. C. utilis was grown in IO ml of Medium M supplemented with either i.o #mole/ml of [8-I~C]adenosylhomocysteine alone or with I.O/~mole/ml [8-1aC]adenosyl t~omocysteine + i.o/~mole/ml of [~H]adenosine. ~ , percent incorporation of I~C into adenosylmethionine in cultures supplemented with [8-~C]adenosylhomocysteine alone; ~,, & and ~], percent incorporation of label into adenosylmethionine in cultures supplemented with [8-1~C]adenosylhomocysteine + /all/adenosine; ~ , percent incorporation of 1~C; /~, percent incorporation of ~H ; ~ , sum of percent incorporation of ~C and ~tt. Percent incorporation of label into adenosylmethionine was calculated after subtraction of endogenous synthesis of adenosylmethionine. Fig. 4. Incorporation of label into adenosylmethionine from adenosyl[carbozy-~C~homocysteine as a function of time. C. utilis was grown in io ml of Medium 3£ supplemented with I.O/~mole/ml of adenosyl[cafbozy-l~C]homocysteine alone (©, [~ ) or with I.O/,mole/ml adenosyl[cafboxy-l~C]homocysteine + 0. 5/,mole/rill homocysteine (O, ~1). The circles represent percent incorporation of I~C, and the squares represent the/,moles of adenosylmethionine formed/g of ceils (wet weight). Percent incorporation of label into adenosylmethionine was calculated after subtraction of endogenous synthesis of adenosylmethionine.

Utilization of adenosylhomocysteine as the sole source of sulfur I n o r d e r to s t u d y t h e conversion of a d e n o s y l h o r n o c y s t e i n e to a d e n o s y l m e t h i o n i n e in C. utilis, it seemed of i n t e r e s t to increase t h e low i n c o r p o r a t i o n of a d e n o s y l h o r n o cysteine into t h e cells (Tables I a n d III). W h e n a d e n o s y l h o m o c y s t e i n e served as t h e sole source of sulfur (Mediurn S), t h e r a t e of cellular u p t a k e of a d e n o s y l h o r n o c y s t e i n e was 39-7 %, a n d t h e i n c o r p o r a t i o n of adenosylEcarboxyJ~C~hornocysteine into a d e n o s y l methJonine was 61.2% (Table IV). I n c o n t r a s t , t h e cellular u p t a k e of a d e n o s y l ~carboxy-l~CJhornocysteine in Mediurn M was 7.I % (Table I I I ) . Adenosylhornocysteined i d n o t a p p r e c i a b l y affect t h e cellular u p t a k e of [a~S]sulfate, a l t h o u g h t h e r e was a. decrease in t h e % i n c o r p o r a t i o n of label from [~sS]sulfate into a d e n o s y l m e t h i o n i n e (Table IV). On t h e o t h e r h a n d , sulfate d r a s t i c a l l y r e d u c e d t h e cellular u p t a k e o f adenosyl[carbox'.y-~C]hornocysteine a n d t h e i n c o r p o r a t i o n of label from a d e n o s y l h o r n o c y s t e i n e i n t o adenosylrnethion~ne (Table IV).

Biochim. Biophys. Acta, 177 (I969) 67-77

74

S.. K. SHAPIRO, D. J o EHN~NGER

T A B L E IV BIOS~ZNTHESIS OF ADENOSYLMETHiONIN~ FROM ADENOS~(LHOMOCYSTEINE (2~}~ OR ~}~:~)*

C. utilis was g r o w n for 24 h a t 3 °0 in ~o n~l of M e d i u m S pI~s t h e s u p p i e m e n ~ s i n d i c a t e d , e a c h a t a c o n c e n t r a t i o n of ~.o ~ m o l e / m l .

Su~aplements

Cell yield (rag)

Label in cells (%)

Yield of adenosylme~h.ionine (~moles/g)

Incorporation of label into adeq¢osytmeXhionine (~)

lass ] S u l f a t e

344

46. i

z. I o

84 .6

[a~S]Sulfate + A H

348

44.0

~.24

7L2

AH*

365

39.7

z.86

6~.2

AH* + sulfate

351

7,7

~.23

~4-5

* A b b r e v i a t i o n s : AH, a d e n o s y l h o m o c y s t e i n e ; AH*, adenosyl~ca~boxy-iaC~homocysteine.

In order to determine the optimum yield of adenosylmethionine in Medium S~ C. utilis was grown on various levets of adenosylhomocysteine as ~he sole so~rce o2

sulfur. It m a y be seen (Table V) that at the three concentrations used, ce!lular uptake of adenosylhomocysteine decreased rapidly while the yield of adenosytmethionine and the incorporation of label into adenosylmethionine remained fairly cons~an~ after incubation for ~8 and 44 ~. The addition of ~.aHiadenosine did no~ affect the latter results because adenosine was not taken up b y the ceils. However, under the same conditions, [~H~adenine was taken up b y the cells in Medium S and increased the yield of adenosylmethionine above that oI the unsupplemented culture [Table VB. Similari?,, when adenine was added to ~8-~aQadenosythomocysteine or adenosyi[carboxy-~'~C!. TABLE V ~31OSYNTHESIS

OF ADIgNOSYLMETHIONLNE

SYLHOMOCYSTEINE

FROM

[8~I4C~ADENOSYLHOMOCYSTEINE

C. ~tiliS w a s g r o w n for ~ 8 h a n d 4 4 h a t 3 o ~ i n ~ o m l of NIedium S ~ s at various concentrations.

[8-x*C]Adenosyl homocystei~e (ixmoles/ml)

o. 5 i.o 2.o i. o @ [aK]Adenosine* 0.5 ~.o 2.o ~.o + [aH] Adenosine•

\XZITH[~-I4C]ADENO ~

AS THE SOLE SOURCE OF SL'LFL'R

Time of incubation (h)

Cell yield (rag)

Label in cells (%)

~8-~C]adenosyl~omocysteine

Yield of adenosylmethionine (~moXes!g)

I~,corporatios~of label i~to adenosylmelhiom~e

(%)

x8 i8 ~8

342 340 345

6o.3 30.7 ~4.5

1.6I x.82 L83

71.2 74.5 76.4

~8 44 44 44

349 359 354 367

29.~ 7 ~.6 35.2 ~5.6

~.8~ 1.67 L92 L93

75~8 74.8 76.3 79.9

44

366

3o.8

~.97

7 x~8

* I.O ~ m o l e / m l ; t h e r e w a s < adenosy!methionine.

~o~o u p t a k e of aH i n t o t h e cells a n d no i n c o r p o r a t i o n i n t o

Biochim. Biophys. Acta, z77 (~969) 67-77

BIOSYNTHESIS OF ADENOSYLMETHIONINE

75

TABLE VI BIOSYNTHESIS

OF

ADENOSYLMETtIIONINE

ADENOSYLHOMOCYSTEINE

AS THE

SOLE

FROM

(A*H OR AH*)

ADENOSYLHOMOCYSTEINE

SOURCE

WITH

OF SULFUR

C. uXilis w a s g r o w n for 18 h a t 3 °o in Io m l of M e d i u m S plus t h e s u p p l e m e n t s i n d i c a t e d , e a c h a t a c o n c e n t r a t i o n of 1.o/~mole/ml. A b b r e v i a t i o n s : A ' H , a d e n o s y l [carboxy-l~C]homocysteine.

Supplements

Cell yield (mg)

Label in cells (%)

[ S A ~ C ] a d e n o s y l h o m o c y s t e i n e ; AH*,

Yield of adenosylmethionine

(l~moles/g)

~C

3H

Incorporation of label into adenosylmethionine (%) ~aC

~H

A*H A'It + [~H]adenine AH* AH* + E~H]a d e n i n e

361

35.5

--

1-97

77 .0

--

355 365

26.3 41.o

12.5 --

2.62 1.75

58.9 49.2

7.9 --

374

43 .1

12.4

2.49

56.7

Sulfate

353

- -

- -

o.82

- -

- -

331 339

---

15.6 --

1.5I 3.33

---

17.8 --

Snfate + [3H]adenine ttomocysteine A*H + homocysteine AH* + homocysteine

9.7

349

3.3

--

3.o7

11.5

--

355

o.8

--

3.2I

5.6

--

homocysteine, the yield of adenosylmethionine was increased b y the same amount as above. When ~8-1~Cjadenosylhomocysteine was used, the incorporation of label into adenosylmethionine was decreased when adenine was present. On the other hand, with adenosyl[carboxyA~C~homocysteine in the medium, the addition of adenine resulted in an increase in the incorporation of 1~C into adenosylmethionine. When homocysteine was added to the medium, the yield of adenosylmethionine in the presence or absence of adenosylhomocysteine was virtually the same ( + 5 %), but the incorporation of label from adenosylhomocysteine into the cells was lowered to lO% of that observed when adenosylhomocysteine was the sole source of sulfur. Nevertheless, the incorporation of 1~C from adenosylhomocysteine into adenosylmethionine was twice as high with [SAaC~adenosylhomocysteine as compared to adenosyl[carboxy~C~homocysteine. These results are consistent with the hypothesis that adenosylhomocysteine is decomposed and the resultant fragments used for the synthesis of adenosylmethionine. DISCUSSION

I t is clear from the results described in this paper that the addition of adenosylhomocysteine to a minilnalmedium stimulated the biosynthesis of adenosylmethioninein C. utilis. This stimulation is more effective than that caused b y methionine at concentrations below I.O/~mole/ml (Table I). In terms of the internal cell concentration of the two supplements, adenosylhomocysteine is 6 - I 2 times more effective than methionine in the stimulation of adenosylmethionine synthesis. This effect m a y be the Biochim. Biophys. Acta, 177 (1969) 6 7 - 7 7

70

So K. SHAPIRO~ Do ]~ EHNINGER

result of direct conversion of adenosylhomocysteine to adenosyhne~hionine or ibmresult of the slow release of homocysteine from adenosylhomocysteine, tn ti~e latter case, methionine would be synthesized slowly and would not be available for protein synthesis. On the other hand, when methionine is availab!e at low !eve!s in she medimn, most of it is probably used, preferentially, for protein synthesis. Since the percent incorporation of labe! from [8-~Ciadenosylhomocysteine and adenosyl[carboxiy-~¢C]homocysteine into adenosylmethionine was similar (Tabie 121):~ the hypothesis of direct regeneration of adenosylmethionine from adenosylhomoocysteine seemed to fit the data. Ho~vever, further experiments showed that the addition of adenine, adenosine, or homocysteine to the culture medimu resulted in dilution of the specific radioactivity of the corresponding moiety of the isolated adenosylmethionine. The latter results, however, could be consistent with the hypothesis tlmt adenosyhnethionine is directly regenerated from adenosy!homocysteine if there were an exchange reaction between adenosylhomocysteine and adenosine and homocysteineo If there is an exchange reaction of X%, then as [8-~lC]adenosylhomocysteine (Fig. 3) ,or adenosyl [carbox:y-a~C]homocysteine (Fig. 4) is directly converted to [8-~C] adenosylo methionine or adenosyl[carboxy-i4C]methionine, more of the added unlabeled adenosine or homocysteine would be exchanged with the [8-UCiadenosylhomocysteine or adenosyl[carbo~F-liC]homocysteine in order to maintain the exchange rate. This would result in a gradual dilution of the specific radioactivity of the [8-~.~!adenosyi homocysteine or adenosyi[carbo~v-~Cjhomocysteine and the resultant [8-~aCadenosylmethionine or adenosyl[cafboxy-~C]methionine. However, the data of Figs. 3 and 4 showed the opposite effect which is consistent with the hypothesis that as [8-1~Ciadenosylhomocysteine or adenosyl[cm'~ox2-~Clhomocysteine is decomposed, the resultant radioactive adenosine or homocysteine gradually increases the specific radioactivity of the unlabe!ed adenosine or homocysteine and the resultant [g_r~¢~.. adenosylmethionine or adenosyl~carbox3~-~'SC~methionine. One of the problems in studies on the metabolism of adenosylhomocysteine is the very low incorporation of the compound by the cells. This is not due simpty to limited cel!ular permeability since there was a ~o-fold increase in the incorporation of adenosylhomocysteine when_ the thioether was the sole source of sulfur (Table Vi). Under these conditions, there was approximately a z-fold increase in adenosylmethio~ nine synthesis compared to cultures with equimolar sulfate, and the percent incorporation of label from both moieties of adenosylhomocysteine into adenosylmethionine was over double that observed in lV[edium N. The addition of adenine or homocysteine ~o Medium S supplemented with [8-~C!adenosylhomocysteine or adenosyl[car~ox2~£1homocysteine resulted in dilution of the specific radioactivity of the corresponding m o i e t y of the isolated adenosylmethionine. When adenine was added to Medinrn S with adenosy![carboxiy~C~homocysteine, the incorporation of 1abel from_ adenosyt[carboxy-t~C~homocysteine into adenosylmethionine was increased. This is probably ~due to increased adenosylmethionine production and increased incorporation of the [carboxiy-~C]homocysteine moiety of adenosylhomocysteine into adenosylmethionineo Thus, it is conc!uded that under the conditions of the experiments reported with C. uHZis (Medium ~ or Medium S), adenosylhomocysteine is decomposed into intermediate products which may the~_ be incorporated into the adenosylmethionine molecule. A similar conclusion has been reached, recently, from different experimen*s with Saccharomyces cerevisiad. Whether adenosylmethionine may be directly regene~ ~iochi~, Bi@l~ys. Ac¢a, ~77 (z969) 67-77

BIOSYNTHESIS OF ADENOSYLMETHIONINE

77

rated from adenosylhomocysteine under different conditions or in other cells remains to be determined. ACKNOWLEDGEMENT

This work was supported by the U.S. Atomic Energy Commission. ~EFE~ENCES i S. ~-{. SHAPIRO AND F. SCHLENK, Transrne~hylation and Methio*dne Biosynthesis, The U n i v e r s i t y of Chicago Press, Chicago, I965. 2 J. A. DUERRE, Arch. Biochem. Biophys., 96 (1962) 7 o. 3 S. IK. SHAPIRO,in S. K. SHAPIRO AND F. SCHLENK,Transmethyl~tion and Methionine Biosynthesis, The U n i v e r s i t y of Chicago Press, Chicago, 1965. p. 200. 4 J. A. DI~ERRE AND F. SCHLEI~K, Arch. Biochem. Biophys., 96 (I962) 575. 5 S. K. SH~.PIRO AND D. J. ]EHNINGER, Anal. Biochem., 15 (1966) 323. 6 F. SCHLENK AND C. R. ZYD]ZK, f . Labelled Comds., 3 (1967) 1377 J- A. DUERRE, Arch. Biochem. Biophys., 124 (1968) 422.

Biochim. Biophys. Acta, 177 (1969) 67-77