ARCHIVES
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
172,
135-142 (1976)
and Properties of S-Adenosyl-L-Homocysteine from Leaves of Spinach Beet JONATHAN
Botany School, Oxford
E. POULTON
AND
VERNON
University, South Parks Road, Oxford Received June 18, 1975
Hydrolase
S. BUTT OX1 3RA Great Britain
S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) has been isolated from spinach-beet leaves and puritied 100-fold. The enzyme catalyzes both the hydrolysis of S-adenosyl-Lhomocysteine to adenosine and L-homocysteine and its synthesis from these compounds. The The equilibrium constant for the reaction is 1.8 x 10-O in relation to hydrolysis. enzyme shows optimum activity at pH 8.5. Enzyme preparations were stabilized by the addition of bovine serum albumin. The K, for S-adenosylhomocysteine was 41 WM in the hydrolysis reaction and for adenosine, nn-homocysteine, and L-homocysteine it was 13 pM, t2.2 mM, and 1.2 mhf, respectively. The enzyme was inhibited by S-adenosylmethionine, homocysteine, and adenine. These inhibitions and the K, values determined are discussed in relation to the regulation of the enzyme in vivo and especially its effect on methylation reactions using Sadenosylmethionine as methyl donor.
netic data, which might explain more exactly the relation between the hydrolase activity and the methylation reactions generating S-adenosylhomocysteine. The hydrolysis of S-adenosylhomocysteine is not easy to investigate in isolation because the reaction equilibrium favors the reverse direction, in which S-adenosylhomocysteine is synthesized from adenosine and L-homocysteine; hydrolysis is usually assisted by the destruction of adenosine by added adenosine deaminase (5, 7). In this paper, the purification of S-adenosyl-L-homocysteine hydrolase from spinach-beet leaves is described. Kinetic data for the reaction in each direction (“hydrolase” and “synthetase” activities, respectively) have been obtained, which have allowed conclusions to be drawn about the cellular conditions under which the hydrolysis of S-adenosylhomocysteine might proceed.
Euzymes likely to affect the intracellular levels of S-adenosyl-L-homocysteine are important in the study of methylation reactions, because it has been demonstrated that many methyltransferases are inhibited by S-adenosyl-L-homocysteine competitively with S-adenosyl-L-methionine (l-3). For example, an enzyme catalyzing the methylation of c&eic acid was purified from spinach-beet leaves and was found to be potently inhibited by S-adenosyl-L-homocysteine ‘(4). Crude extracts from these leaves were shown to contain Sadenosine-L-homocysteine hydrolase (EC 3.3.1.11, which catalyzed both the synthesis of S-adenosine-L-homocysteine from adenosine and homocysteine and its catabolism to these compounds (4). This enzyme has already been identified in extracts from animals, birds, and plants (5-101, and a more detailed study of its properties has been undertaken using partially purified preparations from rat liver (5, 8, 10) and yeast (7) cells. Although the existence of this enzyme in plant tissues is known (9, lo), no purification from plants has hitherto been reported. Further, there has been an absence of quantitative ki-
MATERIALS
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Materials Fresh leaves from spinach-beet (Beta vulgaris L. ssp. vulgaris) plants, which were grown outdoors, were harvested immediately before use. 135
Copyright All rights
AND
136
POULTON
All chemicals used were the purest available from British Drug Houses Ltd., Poole, Dorset, Great Britain. S-Adenosyl-L-homocysteine was purchased from Boehringer Corporation (London) Ltd., London, Great Britain, deoxyadenosine from KochLight Ltd., Colnbrook, Great Britain, and m-homocysteine from Fluka A.G., Buchs, Switzerland. LHomocysteine was prepared by hydrolysis of L-homocysteine thiolactone hydrochloride (Sigma Chemical Co.) with 5 N NaOH at room temperature (11). Adenosine deaminase was purchased from Sigma Chemical Co., London. S-[8J4C]adenosyl-L-homocysteine (47 mCi/mmol) was prepared enzymically using purified S-adenosyl-L-homocysteine hydrolase from spinach-beet leaves; 0.1 pmol of [8-i4C]adenosine (containing 5 &i) was incubated with 1 pmol of m-homocysteine, 0.3 pmol of P-mercaptoethanol, 5 pmol of Tris-HCl buffer (pH 8.4), and 15 m-units of purified S-adenosyl-L-homocysteine hydrolase in a total volume of 0.15 ml at 30°C for 30 min. The reaction was terminated by adding 10 ~1 of 80% (w/v) trichloroacetic acid and the mixture was centrifuged to remove precipitated protein. The supernatant liquid was applied in a band to silica G-25 sheets (0.25 mm layer; Macherey & Nagel). The chromatogram was developed with isopropyl alcohol-ethyl acetate-ammonia-water (23:27:4:4, by volume). The zone containing S-adenosyl+homocysteine was detected under uv light, scraped off the sheet and eluted with 1 ml of 1 mM dithioglycol at room temperature. The molar extinction coefficient of S-adenosyl-L-homocysteine in aqueous solution at 260 nm was taken to be 15,400 in determining the specific activity (12).
AND
Chromatographic
SEPARATION
Enzyme Assays (i) S-ao!enosyl-L-homocysteim synthetuse activity. This was assayed by measuring the rate of formation of S-[8J*C]adenosyl-L-homocysteine from 18*4C]adenosine and nL-homocysteine; 0.2 pmol of [8Yl]adenosine (2.2 x 105 dpm), 1.0 pmol of m-homocysteine (adjusted to pH 8 with KOH), 0.3 pmol of pmercaptoethanol, 5 pmol of Tris-HCl buffer (pH 8.5), and up to 30 ~1 of the enzyme preparation in a total volume of 0.09 ml were incubated at 30°C. Control vessels, in which either DL-homocysteine or enzyme preparation were omitted or in which a boiled enzyme preparation replaced the active extract were included. The reaction was terminated by I
OF S-ADENOSYL-L-HOMOCYSTEINE
Solvent
ADENOSINE
Adenosine
AND ADENINE~
ChroE;tonqaTphic
Rf S-adenosyl-Lhomocysteine
Methods
Thin-layer chromatography was performed on Polygram SIL N-HBUV254 sheets (Macherey-Nagel and Co., Duren, West Germany) with: (il isopropylalcohol-ethyl acetate-ammonia-water (23:27: 4:4, by volume) on Polygram CEL 300 UV254 sheets (Macherey-Nagel and Co.); (ii) water; and (iii) acetone-water (5:2, by volume). Solvent (iii) did not separate adenosine and inosine, so that its use was confined to the assay of hydrolytic activity of the hydrolase in the presence of adenosine deaminase, when no adenosine could be detected using solvent 1. Descending paper chromatography was performed on Whatman No. 1 paper with: (iv) n-butylalcohol-acetic acid-water (12:3:5, by volume). The R, values of the various reaction products are given in Table I.
TABLE CHROMATOGRAPHIC
BUTT
Adenine
Inosine
i
0.05
0.40
0.55
0.16
ii
0.60
0.50
0.30
-
111
0.25
0.78
-
0.77
iv
0.18
0.42
-
-
Polygram SIL N-HB’UV254 sheets Polygram CEL 300 UV254 sheets Polygram CEL 300 UV254 sheets Whatman No. 1 paper
a S-adenosyl-L-homocysteine, adenosine, adenine and inosine were identified by thin-layer and paper chromatography using the following solvents: (i) isopropyl alcohol-ethyl acetate-ammonia-water (23:27:4:4, by vol.); (ii) water; (iii) acetone-water (5:2, by volume); (iv) n-butyl alcohol-acetic acid-water (12:3:5, by volume).
s-ADENOSYL-L-HOMOCYSTEINE adding 10 ~1 of ice-cold 50% (w/v) trichloroacetic acid. The mixture was allowed to stand at 0°C for 10 min and the mixture was centrifuged to remove precipitated protein. An aliquot (10 ~1) of the supernatant liquid was applied in a 1.5-cm band to a Polygram SIL N-HR/UV 254 sheet and the chromatogram was developed in solvent (i). The zone containing S-adenosyl-I,-homocysteine was detected under uv light, cut out, and counted in 2 ml of scintillation fluid, containing 0.5% (w/v) butyl phenyl biphenylyl-oxadiazole in toluene with a Tracerlab Corumatic/ scintillation counter. The total amount of radioactivity added to the assay system was determined by applying an aliquot (10 ~1) of a reaction mixture, to which trichloroacetic acid had been added, to a piece (1 x 1 cm) of the Polygram sheet and by counting directly as above. (ii) S-Adenosyl-L-homocysteine hydrolase activity. This was assayed by measuring the rate of formation of [8-14C]inosine from S-[8-*4C]adenosyl-L-homocysteine in the Ipresence of excess adenosine deaminase. 100 nmol of S-[8-14C]adenosyl-L-homocysteine (7.3 x 10“ dpm), 10 nmol of thiodiglycol, 0.2 units of commercial adenosine deaminase, 5 pmol of TrisHCl buffer (PI-I 8.51, and up to 20 ~1 of enzyme preparation in a total volume of 0.07 ml were incubated at 30°C. The reaction was terminated by boiling for 1.5 min and then cooling in an ice bath, after which the mixture was centrifuged to remove the precipitated protein. An aliquot (10 ~1) of the supernatant liquid was applied in a 1.5-cm band to a Polygram CEL 300 UV254 sheet and the chromatogram was developed with solvent (iii). The zone containing inosine was detected under uv light, cut out, and counted in scintillation fluid as above. The total amount of radioactivity added to the system was determined by applying an aliquot (10 ~1) of a heat-treated reaction mixture to a piece (1 x 1 cm) of the Polygram [sheet and by counting directly as above. Control vessels, in which a boiled enzyme preparation replaced the active extract, were routinely included. In some experiments, adenosine deaminase was omitted and [8J4Cladenosine was cut out from the developed chromatogram instead of [8-*4Clinosine. For comparison., the products were also separated on Polygram SIL N-HRJUV254 sheets using solvent (9.
Enzyme Purification Unless otherwise stated, all stages were carried out at 4°C. During the purification, the enzyme was assayed at each stage for synthetase activity. The washed laminae (200 g) of spinach-beet leaves were macerated in an Ato-mix with 250 ml of 0.1 M KH,PO,-K,HPO, buffer, pH 7.5. The macerate was squeezed through two layers of muslin and centrifuged at 30,OOOgfor 25 min. The supernatant liquid was brought to 45% saturation by the addition
HYDROLASE
FROM
SPINACH
BEET
137
of solid (NH&SO, over a period of 15 min, during which the pH of the solution was continuously adjusted to 7.4-7.6 by adding small volumes of dilute KOH solution. The mixture was allowed to stand for a further 25 min at 0°C and then centrifuged at 10,OOOg for 20 min. The supernatant liquid was brought to 80% saturation by the further addition of solid (NH&SO, over a period of 25 min, with adjustment of the pH as before. The mixture was allowed to stand for 20 min at 0°C and the precipitate was collected by centrifuging at 10,OOOgfor 25 min. This was redissolved in the minimum volume of 20 mM KH,PO, buffer, pH 7.5. The redissolved precipitate (7.5 ml) was applied to a Sephadex G-200 column (35 x 3 cm) that had been equilibrated with 20 mM KH,PO,-K,HPO, buffer, pH 7.5. The column was eluted with the same buffer and 7-ml fractions were collected. The fractions containing the enzyme were pooled, made 5 mM with respect to P-mercaptoethanol and applied to a DEAE-cellulose column (4 x 1.25 cm), which had been equilibrated with 20 mM KH,PO,-K,HPO, buffer, pH 7.6, containing 5 mM p-mercaptoethanol. After the column had been washed with this buffer, 40 mM KH,PO,-K,HPO, buffer, pH 7.6, containing 5 rnM P-mercaptoethanol, was passed through and 7ml fractions of eluate were collected. During elution, 5 ml of each fraction was removed and added quickly to 0.5 ml of an ice-cold solution of bovine serum albumin (22 mgiml), and the remainder of the fraction was used for protein determination. The purified enzyme was stored deep-frozen at -20°C and thawed when required.
Protein Estimation The protein content of cruder preparations was estimated by the Lowry method, as modified by Leggett Bailey (131, after precipitation from solution by 5% (w/v) trichloroacetic acid; crystalline bovine serum albumin, desiccated before use, was employed as a standard. The protein content of column eluates was determined by the method of Warburg and Christian (14). RESULTS
of PurifEation teine Hydrolase
S-Adenosyl-L-Homocys-
The specific activity of the enzyme was doubled when the crude leaf homogenate was centrifuged at 30,OOOg(Table II). The purification achieved by (NH&SO, precipitation over the range of 4580% saturation was limited, but this stage served to concentrate the preparation before the next step. When the resuspended precipitate was chromatographed on Sephadex G-200,
138
POULTON
AND
TABLE PURIFICATION
Stage
Crude Homogenate Supernatant after 30,000 g centrifugation precipitate (NH,),SO, (4580% saturated) Eluate from G-200 fractionation Fraction from DEAE-cellulose fractionation
OF SPINACH-BEET
Total protein
(mg)
BUTT
II
S-ADENOSYL-L-HOM~CYSTEINE
Total activity units)
(m-
HYDROLASE~
Yield (% initial)
Specific activity (m-units/mg of protein)
1150 580
32,534 29,120
100
89.5
28.3 50.2
206
15,756
48
76.5
60
13,187
40.5
1.8
5,129
218.3
16
2840
a One unit of S-adenosyl-L-homocysteine synthetase activity is defined as the amount of enzyme required to catalyze the formation of one pmol of S-adenosyl-L-homocysteine per minute under the assay conditions described.
the enzyme was found to be present asymmetrically in the first protein peak (Fig. 1); these fractions were stabilized by addition of p-mercaptoethanol (to a final concentration of 5 mM) before chromatography on DEAE-cellulose. It was necessary to add bovine serum albumin to the enzymically active fractions as they emerged from this column, since the enzyme was found to be unstable in solutions containing low concentrations of protein. The best preparation showed a loo-fold increase in specific activity relative to the homogenate. No loss in activity, measured by the synthetase reaction, was observed after the enzyme had been stored deep-frozen for several months at -20°C in the presence of 5 mM p-mercaptoethanol and bovine serum albumin (2 mg/ml). Properties of Synthetase Activity The pH optimum for the synthesis of Sadenosyl-L-homocysteine was about 8.5, but more than 50% of the maximum activity was observed between pH 6.5 and 10.0 (Fig. 2). The synthesis, in Tris-HCl buffer (pH 8.5), was proportional to protein concentration up to 30 ~1 of purified enzyme preparation (containing 7.0 pug protein) and the reaction was linear with time up to 30 min. The K, values for m-homocysmine, L-homocysteine, and adenosine determined from a Lineweaver-Burk plot, were 2.2 mM, 1.2 mM, and 12.8 PM, respec-
Froctlon
number
FIG. 1. Elution diagram of gel filtration on a column of Sephadex G-200. Enzyme units defined as for Table II.
tively. V was equal to 210 pm01 of homocysteineklmg protein. All plots were linear, showing no indication either of cooperative or of inhibition effects. Among the metabolites investigated for their effect on S-adenosyl+homocysteine synthesis (Table III), L-cysteine and L-methionine had no effect even at 10 mM concentration, but 2 mM adenine and 10 mM S-adenosyl-L-methionine inhibited the reaction considerably. Properties
of Hydrolase
Activity
When S-adenosyl-L-homocysteine was incubated with the enzyme in Tris-HCl buffer, pH 8.5, hydrolysis to adenosine and homocysteine did not proceed very far, since the equilibrium position was soon
s-ADENOSYL-L-HOMOCYSTEINE
l-y
* G
8
10
PH
FIG. 2. Effect of pH on the S-adenosyl-L-homocysteine “synthetase” activity of the purified enzyme. Dr.-Homocysteine and adenosine were incubated with 5 ~1 purified enzyme (1.3 pg protein) under conditions given under Methods using 56 mM concentrations of the following buffers: Glycine-NaOH (A-A), tricine-NaOH (O-O), Tris-HCl (O-O), and 56 InM K,HPO, with citric acid added to give the required pH (A-n). Enzyme activity is expressed as unite per milliliter.
reached (Fig. 3). The low specific activity of the substrate and its very limited conversion made determination of the equilibrium constant for the reaction difficult. In three separate determinations at pH 8.5 and 3O”C, the equilibrium constant was found to be 1.59, 1.80, and 1.95 PM, respectively. However, the hydrolysis of S-adenosylhomocysteine continued for over 90 min when adenosine deaminase was present in the reaction mixture, when the adenosine was rapidly converted to inosine (Fig. 3). To obtain measurable rates of reaction, this enzyme was included in all experiments. The hydrolase activity, in Tris-HCl buffer, showed a broad pH optimum with a maximum at around pH 8.5; over the range pH 7.6 to 8.8, the ratio of hydrolase and synthetase activities was almost constant. At pH 8.5, the hydrolase activity was proportional to protein concentration up to 15 ~1 of purified enzyme preparation (containing 3.5 ,ug protein) and the reaction was linear with time up to 15 min. The K, for S-adenosylhomocysteine was 41 PM, as determined from a Lineweaver-
HYDROLASE
FROM
SPINACH
139
BEET
Burk plot, which was found to be linear. V for the hydrolysis was equal to 22.4 pmol converted/h/mg protein. Among the metabolites investigated, neither deoxyadenosine nor L-methionine affected the hydrolysis of S-adenosylhomocysteine very much, but, like the synthetase activity, this reaction was severely affected by adenine and S-adenosyl-methionine at concentrations similar to those of the substrate (Table IV). The effect of DLhomocysteine was also appreciable and was analyzed further in relation to the time-course of the hydrolysis in the presence of adenosine deaminase. After incubation for 90 min with adenosine deaminase, the reaction rate declined virtually to zero (Fig. 3). Preincubation of either adenosine deaminase or S-adenosylhomocysteine hydrolase for 120 min at 30°C did not affect the subsequent tiecourse of the reaction, showing that the decline was not due to thermal inactivation of the enzymes. Inosine, at concentrations up to 2 mM, did not inhibit adenosine deaminase nor the hydrolase, nor was adenosine deaminase inhibited by m-homocysteine at concentrations up to 3.4 mM. However, m-homocysteine inhibited TABLE EFFECT
OF METABOLITES HOM~CYSTEINE
Addition
-
L-cysteine
10 1 10 1 2 0.2 10 1 3.3 0.33
Methionine Deoxyadenosine
ON S-ADENOSYL-LSYNTHESIS’
Final con- Specific activcentraity (mtion (mhr) units/mg of protein)
None
S-Adenosyl-nmethionine Adenine
III
Control activity (%)
;;8”; 12128
100
2102 2178 1178 2002 1453 2085 2156 2141 1778 2113
99 102 55 94 68 98 101 101 84 99
” The adenosine concentration in this assay only was reduced to 0.9 mM, in order to allow the easier detection of any inhibitory effects. Activity unite are as described for Table II.
140
POULTON 150 -
I-,--
T
I
/
80
LO Trne
120
(m!n)
FIG. 3. Time-course of S-adenosyl-L-homocysteine hydrolysis in the presence and absence of adenosine deaminase. In the presence of adenosine deaminase, the product was inosine (m-m, nanomoles); in the absence of adenosine deaminase, the product was adenosine (O-0, nanomoles).
the reaction increasingly up to 6 mM (Fig. 4).
at concentrations
DISCUSSION
S-Adenosyl-L-homocysteine hydrolase (EC 3.1.1.11, which catalyzed the synthesis of S-adenosyl-L-homocysteine from adenosine and nL-homocysteine, was purified loo-fold from crude extracts of spinachbeet leaves. The specific activity observed is the highest reported for a preparation of this enzyme from any source. The equilibrium for this reaction lies far in the direction of S-adenosyl-L-homocysteine synthesis. The equilibrium constant determined here is almost idc,ltical with that observed with the rat liver enzyme (51, but less than those reported using a preparation from rat brain (1) and calculated from the data for the yeast enzyme (7). The lowest figures are likely to be the most reliable, since the contamination of preparations with enzymes of even low activity which act upon either of the reaction products is likely to raise this value artificially; evidence for the absence of any other reaction products was not mentioned in connection with this data. The data derived from the LineweaverBurk plot for L- and DL-homocysteine sug-
AND
BUTT
gest that the enzyme is specific for the Lisomer only, and that the n-isomer does not inhibit the activity of the enzyme towards the L-isomer when a mixture is present. Although the tissue level of homocysteine in living organisms is unknown, it appears likely that the high K, value found here for this substrate is unfavorable for S-adenosyl-L-homocysteine synthesis, despite the low K, value for adenosine. Thus, the synthesis of S-adenosyl-Lhomocysteine might proceed in viva at a significant rate only when homocysteine accumulated. This suggestion is further strengthened by experiments with the rat liver enzyme (5, 8, lo), which showed not only an equally high K, value for adenosine, but also inhibition of the reaction by both substrates, by the product S-adenosyl-L-homocysteine, and by adenine. The spinach-beet enzyme was likewise inhibited by low concentrations of adenine and S-adenosyl-L-methionine. Determinations of the levels of S-adenosylmethionine and S-adenosylhomocysteine have shown that they are present in similar concentrations in rat liver (15) and at a ratio of about 2 to 1 in pea seedlings (16). The low Ki for S-adenosylhomocysteine relative to the K, for S-adenosylmeTABLE
IV
EFFECT OF METABOLITES ON S-ADENOSYL-LHOM~CYSTEINE HYDROLYSIS’ Addition
Ficn$;ntion (mM)
Spe;i$c(ztivunits/mg of protein)
Control activity (%)
None
-
162.3 169.6 1161.4
100
L-Methionine
10 1 10 1 2 0.5 2.86 0.71
165.4 163.3 14.6 79.2 49.8 95.2 141.5 159.6
102 101 9 49 31 59 88 99
S-Adenosyl-Lmethionine Adenine Deoxyadeonsine
(23ne unit of S-adenosyl-L-homocysteine hydrolase activity is defined as the amount of enzyme required to catalyse the formation of 1 kmol of inosine per minute from S-adenosyl-L-homocysteine under the assay conditions described.
s-ADENOSYL-L-HOMOCYSTEINE
2 Corm
L
G
8
of DL-hcmocysteme (mM)
4. Effect of m-homocysteine concentration on the initial rate of S-adenosyl+homocysteine hydrolysis by the purified enzyme preparation in the presence of excess adenosine deaminase. s is expressed as millimoles per liter. FIG.
thionine in many methylation reactions (1, 3, 4) emphasizes the importance attached to this balance in determining the rates of methylation. The characteristics and activities of the methyltransferases and hydrolase must be arranged so that the methylations proceed at a rate determined by the steady-state levels of the substrates Sadenosyl-L-methionine and S-adenosyl-Lhomocysteine. The catabolism of S-adenosyl-L-homocysteine was also studied here using the purified enzyme. Due to the equilibrium position, this reaction was especially governed by the disposal of the products, adenosine and homocysteine. Adenosine was routinely removed in the assay mixture by the action of adenosine deaminase, but the activity of this enzyme in spinach-beet extracts was very weak or absent (4). The hydrolysis of S-adenosyl-L-homocysteine was facilitated in crude preparations from these leaves by the action of adenosine nucleosidase (4). However, it seems more likely that adenosine is conserved in viuo by its conversion to ADP through the successive action of adenosine kinase and adenylate kinase, both of which have been demonstrated in spinach-beet leaf extracts (17). Both adenosine and L-homocysteine must be removed for the hydrolysis of Sadenosylhomocysteine to proceed; the accumulation of either of the products inhibits
HYDROLASE
FROM
SPINACH
BEET
141
the enzyme. This was shown here by the inhibition of the reaction by L-homocysteine when adenosine was deaminated by adenosine deaminase. It seems unlikely that the effect of homocysteine was due only to its reversal of the reaction, because attempts to detect labeled adenosine among the reaction products failed, even though the equilibrium constant for the reaction indicated that if homocysteine were reacting with adenosine, sufficient labeled adenosine should have been present for detection. Therefore, it is concluded that homocysteine exerts its effect by inhibiting the enzyme directly, and that its accumulation during the hydrolysis of Sadenosylhomocysteine in the presence of adenosine deaminase is sufficient after 90 min to stop the further progress of the reaction. Other evidence for inhibition of S-adenosyl-L-homocysteine hydrolysis by homocysteine was obtained with the rat liver enzyme (5). Furthermore, with the latter enzyme, adenosine was shown to become inhibitory when homocysteine was removed in vitro by methylation, using the dimethylthetin-homocysteine methyltransferase system in the reaction mixture. In uivo, L-homocysteine may be converted to cystathionine by condensation with serine (18), but it is more likely to be methylated by N5-methyltetrahydrofolate to methionine, already demonstrated in extracts of pea seedlings (19, 20) and spinach and barley leaves (21). Although the V for S-adenosyl-L-homocysteine hydrolysis was only one-tenth of that for synthesis, the lower K, value (41 PM) for S-adenosylL-homocysteine would tend to favor this reaction. It is of importance that the V for S-adenosyl-L-homocysteine hydrolysis is well in excess of that observed for the purified caffeic acid o-methyltransferase from spinach-beet leaves (4). However, S - adenosyl - L - homocysteine hydrolysis, like the synthesis of this compound, is also subject to a number of controls by the metabolites present. Adenine and S-adenosyl-L-methionine inhibited this reaction at concentrations similar to the substrate. A knowledge of the mode of inhibition by these compounds and of their intracellular concentration is required to
142
POULTON
estimate how strongly these metabolites might affect S-adenosyl-L-homocysteine hydrolysis in viva. The kinetic evidence provided shows that the enzyme is a sensitive regulator in the conversion of S-adenosyl-homocysmine, depending for its activity not only upon favorable concentrations of metabolites in relation to the equilibrium conditions but also upon the levels of S-adenosyl-methionine, adenosine, and L-homocysteine maintained within the system. These, in turn, will feed back to determine the rates of methylation reactions which are sensitive to the levels of S-adenosylhomocysteine. ACKNOWLEDGMENTS We thank Dr. M. A. Foster, Biochemistry Department, Oxford, for his suggestions in the initial stages of this work, and Prof. Dr. K. Hahlbrock for reading the manuscript. J. E. Poulton thanks the Science Research Council for a Research Studentship. REFERENCES 1. DEGUCHI, T., AND BARCHAS, J. (1971) J. Biol. Chem. 246,3175-3181. 2. COWARD, J. K., D’URSO-SCOTT, M., AND SWEET, W. D. (1971) Biochem. Pharmacol. 21, 12001203. 3. MANN, J. D., AND MUDD, S. H. (1963) J. Biol. Chem. 238,381-385. 4. POULTON, J. E. (1974) D. Phil. Thesis, University of Oxford, Great Britain.
AND
BUTT
5. DE LA HABA, G., AND CANTONI, G. L. (1959) J. Bid. Chem. 234, 603-608. 6. DUERRE, J. A. (1968) Arch. Biochem. Biophys. 124, 422-430. 7. KNUDSON, R. C., AND YALL, I. (1972) J. Bacteriol. 112, 569475. 8. FINKELSTEIN, J. D., AND HARRIS, B. (1973)Arch. Biochem. Biophys. 159, 160-165. 9. DODD, W. A., AND COSSINS, E. A. (1969) Arch. Biochem. Biophys. 133, 216-233. 10. WALKER, R. D., AND DUERRE, J. A. (1975) Can. J. Biochem. 53,312-319. 11. DUERRE, J. A., AND MILLER, C. H. (1966) Anal. Biochem. 17, 310-315. 12. SHAPIRO, S. K., AND EHNINGER, 0. J. (1966) Anal. B&hem. 15, 323-333. 13. LEGGETT BAILEY, J. (1962) In Techniques in Protein Chemistry, pp. 293-294. Elsevier Publishing Co., Amsterdam. 14. WARBURG, O., AND CHRISTIAN, W. (1941) Biothem. 2. 310, 384-421. 15. SALVATORE, F., ZAPPIA, W., AND SHAPIRO, S. K. (1968) Biochem. Biophys. Acta 158,461-464. 16. DODD, W. A., AND COSSINS, E. A. (1968) Phytochemistry 7, 2143-2145. 17. BARTLETT, D. J., AND BUTT, V. S. (1974), personal communication. 18. GIOVANELLI, J., AND MUDD, S. H. (1966) Biothem. Biophys. Res. Commun. 25,366-371. 19. WOODS, D. D., FOSTER, M. A., AND GUEST, J. R. (1965) in Transmethylation and Methionine Biosynthesis (Shapiro, S. K., and Schleuk, F., eds), pp. 138-154, University of Chicago Press, Chicago. 20. DODD, W. A., AND COSSINS, E. A. (1970) Biothem. Biophys. Acta 201, 461-470. 21. BURTON, E. G., AND SAKAMI, W. (1969) Biochem. Biophys. Res. Commun. 36, 228.
OF
BIOCHEMISTRY
Purification
AND
BIOPHYSICS
172,
135-142 (1976)
and Properties of S-Adenosyl-L-Homocysteine from Leaves of Spinach Beet JONATHAN
Botany School, Oxford
E. POULTON
AND
VERNON
University, South Parks Road, Oxford Received June 18, 1975
Hydrolase
S. BUTT OX1 3RA Great Britain
S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) has been isolated from spinach-beet leaves and puritied 100-fold. The enzyme catalyzes both the hydrolysis of S-adenosyl-Lhomocysteine to adenosine and L-homocysteine and its synthesis from these compounds. The The equilibrium constant for the reaction is 1.8 x 10-O in relation to hydrolysis. enzyme shows optimum activity at pH 8.5. Enzyme preparations were stabilized by the addition of bovine serum albumin. The K, for S-adenosylhomocysteine was 41 WM in the hydrolysis reaction and for adenosine, nn-homocysteine, and L-homocysteine it was 13 pM, t2.2 mM, and 1.2 mhf, respectively. The enzyme was inhibited by S-adenosylmethionine, homocysteine, and adenine. These inhibitions and the K, values determined are discussed in relation to the regulation of the enzyme in vivo and especially its effect on methylation reactions using Sadenosylmethionine as methyl donor.
netic data, which might explain more exactly the relation between the hydrolase activity and the methylation reactions generating S-adenosylhomocysteine. The hydrolysis of S-adenosylhomocysteine is not easy to investigate in isolation because the reaction equilibrium favors the reverse direction, in which S-adenosylhomocysteine is synthesized from adenosine and L-homocysteine; hydrolysis is usually assisted by the destruction of adenosine by added adenosine deaminase (5, 7). In this paper, the purification of S-adenosyl-L-homocysteine hydrolase from spinach-beet leaves is described. Kinetic data for the reaction in each direction (“hydrolase” and “synthetase” activities, respectively) have been obtained, which have allowed conclusions to be drawn about the cellular conditions under which the hydrolysis of S-adenosylhomocysteine might proceed.
Euzymes likely to affect the intracellular levels of S-adenosyl-L-homocysteine are important in the study of methylation reactions, because it has been demonstrated that many methyltransferases are inhibited by S-adenosyl-L-homocysteine competitively with S-adenosyl-L-methionine (l-3). For example, an enzyme catalyzing the methylation of c&eic acid was purified from spinach-beet leaves and was found to be potently inhibited by S-adenosyl-L-homocysteine ‘(4). Crude extracts from these leaves were shown to contain Sadenosine-L-homocysteine hydrolase (EC 3.3.1.11, which catalyzed both the synthesis of S-adenosine-L-homocysteine from adenosine and homocysteine and its catabolism to these compounds (4). This enzyme has already been identified in extracts from animals, birds, and plants (5-101, and a more detailed study of its properties has been undertaken using partially purified preparations from rat liver (5, 8, 10) and yeast (7) cells. Although the existence of this enzyme in plant tissues is known (9, lo), no purification from plants has hitherto been reported. Further, there has been an absence of quantitative ki-
MATERIALS
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Materials Fresh leaves from spinach-beet (Beta vulgaris L. ssp. vulgaris) plants, which were grown outdoors, were harvested immediately before use. 135
Copyright All rights
AND
136
POULTON
All chemicals used were the purest available from British Drug Houses Ltd., Poole, Dorset, Great Britain. S-Adenosyl-L-homocysteine was purchased from Boehringer Corporation (London) Ltd., London, Great Britain, deoxyadenosine from KochLight Ltd., Colnbrook, Great Britain, and m-homocysteine from Fluka A.G., Buchs, Switzerland. LHomocysteine was prepared by hydrolysis of L-homocysteine thiolactone hydrochloride (Sigma Chemical Co.) with 5 N NaOH at room temperature (11). Adenosine deaminase was purchased from Sigma Chemical Co., London. S-[8J4C]adenosyl-L-homocysteine (47 mCi/mmol) was prepared enzymically using purified S-adenosyl-L-homocysteine hydrolase from spinach-beet leaves; 0.1 pmol of [8-i4C]adenosine (containing 5 &i) was incubated with 1 pmol of m-homocysteine, 0.3 pmol of P-mercaptoethanol, 5 pmol of Tris-HCl buffer (pH 8.4), and 15 m-units of purified S-adenosyl-L-homocysteine hydrolase in a total volume of 0.15 ml at 30°C for 30 min. The reaction was terminated by adding 10 ~1 of 80% (w/v) trichloroacetic acid and the mixture was centrifuged to remove precipitated protein. The supernatant liquid was applied in a band to silica G-25 sheets (0.25 mm layer; Macherey & Nagel). The chromatogram was developed with isopropyl alcohol-ethyl acetate-ammonia-water (23:27:4:4, by volume). The zone containing S-adenosyl+homocysteine was detected under uv light, scraped off the sheet and eluted with 1 ml of 1 mM dithioglycol at room temperature. The molar extinction coefficient of S-adenosyl-L-homocysteine in aqueous solution at 260 nm was taken to be 15,400 in determining the specific activity (12).
AND
Chromatographic
SEPARATION
Enzyme Assays (i) S-ao!enosyl-L-homocysteim synthetuse activity. This was assayed by measuring the rate of formation of S-[8J*C]adenosyl-L-homocysteine from 18*4C]adenosine and nL-homocysteine; 0.2 pmol of [8Yl]adenosine (2.2 x 105 dpm), 1.0 pmol of m-homocysteine (adjusted to pH 8 with KOH), 0.3 pmol of pmercaptoethanol, 5 pmol of Tris-HCl buffer (pH 8.5), and up to 30 ~1 of the enzyme preparation in a total volume of 0.09 ml were incubated at 30°C. Control vessels, in which either DL-homocysteine or enzyme preparation were omitted or in which a boiled enzyme preparation replaced the active extract were included. The reaction was terminated by I
OF S-ADENOSYL-L-HOMOCYSTEINE
Solvent
ADENOSINE
Adenosine
AND ADENINE~
ChroE;tonqaTphic
Rf S-adenosyl-Lhomocysteine
Methods
Thin-layer chromatography was performed on Polygram SIL N-HBUV254 sheets (Macherey-Nagel and Co., Duren, West Germany) with: (il isopropylalcohol-ethyl acetate-ammonia-water (23:27: 4:4, by volume) on Polygram CEL 300 UV254 sheets (Macherey-Nagel and Co.); (ii) water; and (iii) acetone-water (5:2, by volume). Solvent (iii) did not separate adenosine and inosine, so that its use was confined to the assay of hydrolytic activity of the hydrolase in the presence of adenosine deaminase, when no adenosine could be detected using solvent 1. Descending paper chromatography was performed on Whatman No. 1 paper with: (iv) n-butylalcohol-acetic acid-water (12:3:5, by volume). The R, values of the various reaction products are given in Table I.
TABLE CHROMATOGRAPHIC
BUTT
Adenine
Inosine
i
0.05
0.40
0.55
0.16
ii
0.60
0.50
0.30
-
111
0.25
0.78
-
0.77
iv
0.18
0.42
-
-
Polygram SIL N-HB’UV254 sheets Polygram CEL 300 UV254 sheets Polygram CEL 300 UV254 sheets Whatman No. 1 paper
a S-adenosyl-L-homocysteine, adenosine, adenine and inosine were identified by thin-layer and paper chromatography using the following solvents: (i) isopropyl alcohol-ethyl acetate-ammonia-water (23:27:4:4, by vol.); (ii) water; (iii) acetone-water (5:2, by volume); (iv) n-butyl alcohol-acetic acid-water (12:3:5, by volume).
s-ADENOSYL-L-HOMOCYSTEINE adding 10 ~1 of ice-cold 50% (w/v) trichloroacetic acid. The mixture was allowed to stand at 0°C for 10 min and the mixture was centrifuged to remove precipitated protein. An aliquot (10 ~1) of the supernatant liquid was applied in a 1.5-cm band to a Polygram SIL N-HR/UV 254 sheet and the chromatogram was developed in solvent (i). The zone containing S-adenosyl-I,-homocysteine was detected under uv light, cut out, and counted in 2 ml of scintillation fluid, containing 0.5% (w/v) butyl phenyl biphenylyl-oxadiazole in toluene with a Tracerlab Corumatic/ scintillation counter. The total amount of radioactivity added to the assay system was determined by applying an aliquot (10 ~1) of a reaction mixture, to which trichloroacetic acid had been added, to a piece (1 x 1 cm) of the Polygram sheet and by counting directly as above. (ii) S-Adenosyl-L-homocysteine hydrolase activity. This was assayed by measuring the rate of formation of [8-14C]inosine from S-[8-*4C]adenosyl-L-homocysteine in the Ipresence of excess adenosine deaminase. 100 nmol of S-[8-14C]adenosyl-L-homocysteine (7.3 x 10“ dpm), 10 nmol of thiodiglycol, 0.2 units of commercial adenosine deaminase, 5 pmol of TrisHCl buffer (PI-I 8.51, and up to 20 ~1 of enzyme preparation in a total volume of 0.07 ml were incubated at 30°C. The reaction was terminated by boiling for 1.5 min and then cooling in an ice bath, after which the mixture was centrifuged to remove the precipitated protein. An aliquot (10 ~1) of the supernatant liquid was applied in a 1.5-cm band to a Polygram CEL 300 UV254 sheet and the chromatogram was developed with solvent (iii). The zone containing inosine was detected under uv light, cut out, and counted in scintillation fluid as above. The total amount of radioactivity added to the system was determined by applying an aliquot (10 ~1) of a heat-treated reaction mixture to a piece (1 x 1 cm) of the Polygram [sheet and by counting directly as above. Control vessels, in which a boiled enzyme preparation replaced the active extract, were routinely included. In some experiments, adenosine deaminase was omitted and [8J4Cladenosine was cut out from the developed chromatogram instead of [8-*4Clinosine. For comparison., the products were also separated on Polygram SIL N-HRJUV254 sheets using solvent (9.
Enzyme Purification Unless otherwise stated, all stages were carried out at 4°C. During the purification, the enzyme was assayed at each stage for synthetase activity. The washed laminae (200 g) of spinach-beet leaves were macerated in an Ato-mix with 250 ml of 0.1 M KH,PO,-K,HPO, buffer, pH 7.5. The macerate was squeezed through two layers of muslin and centrifuged at 30,OOOgfor 25 min. The supernatant liquid was brought to 45% saturation by the addition
HYDROLASE
FROM
SPINACH
BEET
137
of solid (NH&SO, over a period of 15 min, during which the pH of the solution was continuously adjusted to 7.4-7.6 by adding small volumes of dilute KOH solution. The mixture was allowed to stand for a further 25 min at 0°C and then centrifuged at 10,OOOg for 20 min. The supernatant liquid was brought to 80% saturation by the further addition of solid (NH&SO, over a period of 25 min, with adjustment of the pH as before. The mixture was allowed to stand for 20 min at 0°C and the precipitate was collected by centrifuging at 10,OOOgfor 25 min. This was redissolved in the minimum volume of 20 mM KH,PO, buffer, pH 7.5. The redissolved precipitate (7.5 ml) was applied to a Sephadex G-200 column (35 x 3 cm) that had been equilibrated with 20 mM KH,PO,-K,HPO, buffer, pH 7.5. The column was eluted with the same buffer and 7-ml fractions were collected. The fractions containing the enzyme were pooled, made 5 mM with respect to P-mercaptoethanol and applied to a DEAE-cellulose column (4 x 1.25 cm), which had been equilibrated with 20 mM KH,PO,-K,HPO, buffer, pH 7.6, containing 5 mM p-mercaptoethanol. After the column had been washed with this buffer, 40 mM KH,PO,-K,HPO, buffer, pH 7.6, containing 5 rnM P-mercaptoethanol, was passed through and 7ml fractions of eluate were collected. During elution, 5 ml of each fraction was removed and added quickly to 0.5 ml of an ice-cold solution of bovine serum albumin (22 mgiml), and the remainder of the fraction was used for protein determination. The purified enzyme was stored deep-frozen at -20°C and thawed when required.
Protein Estimation The protein content of cruder preparations was estimated by the Lowry method, as modified by Leggett Bailey (131, after precipitation from solution by 5% (w/v) trichloroacetic acid; crystalline bovine serum albumin, desiccated before use, was employed as a standard. The protein content of column eluates was determined by the method of Warburg and Christian (14). RESULTS
of PurifEation teine Hydrolase
S-Adenosyl-L-Homocys-
The specific activity of the enzyme was doubled when the crude leaf homogenate was centrifuged at 30,OOOg(Table II). The purification achieved by (NH&SO, precipitation over the range of 4580% saturation was limited, but this stage served to concentrate the preparation before the next step. When the resuspended precipitate was chromatographed on Sephadex G-200,
138
POULTON
AND
TABLE PURIFICATION
Stage
Crude Homogenate Supernatant after 30,000 g centrifugation precipitate (NH,),SO, (4580% saturated) Eluate from G-200 fractionation Fraction from DEAE-cellulose fractionation
OF SPINACH-BEET
Total protein
(mg)
BUTT
II
S-ADENOSYL-L-HOM~CYSTEINE
Total activity units)
(m-
HYDROLASE~
Yield (% initial)
Specific activity (m-units/mg of protein)
1150 580
32,534 29,120
100
89.5
28.3 50.2
206
15,756
48
76.5
60
13,187
40.5
1.8
5,129
218.3
16
2840
a One unit of S-adenosyl-L-homocysteine synthetase activity is defined as the amount of enzyme required to catalyze the formation of one pmol of S-adenosyl-L-homocysteine per minute under the assay conditions described.
the enzyme was found to be present asymmetrically in the first protein peak (Fig. 1); these fractions were stabilized by addition of p-mercaptoethanol (to a final concentration of 5 mM) before chromatography on DEAE-cellulose. It was necessary to add bovine serum albumin to the enzymically active fractions as they emerged from this column, since the enzyme was found to be unstable in solutions containing low concentrations of protein. The best preparation showed a loo-fold increase in specific activity relative to the homogenate. No loss in activity, measured by the synthetase reaction, was observed after the enzyme had been stored deep-frozen for several months at -20°C in the presence of 5 mM p-mercaptoethanol and bovine serum albumin (2 mg/ml). Properties of Synthetase Activity The pH optimum for the synthesis of Sadenosyl-L-homocysteine was about 8.5, but more than 50% of the maximum activity was observed between pH 6.5 and 10.0 (Fig. 2). The synthesis, in Tris-HCl buffer (pH 8.5), was proportional to protein concentration up to 30 ~1 of purified enzyme preparation (containing 7.0 pug protein) and the reaction was linear with time up to 30 min. The K, values for m-homocysmine, L-homocysteine, and adenosine determined from a Lineweaver-Burk plot, were 2.2 mM, 1.2 mM, and 12.8 PM, respec-
Froctlon
number
FIG. 1. Elution diagram of gel filtration on a column of Sephadex G-200. Enzyme units defined as for Table II.
tively. V was equal to 210 pm01 of homocysteineklmg protein. All plots were linear, showing no indication either of cooperative or of inhibition effects. Among the metabolites investigated for their effect on S-adenosyl+homocysteine synthesis (Table III), L-cysteine and L-methionine had no effect even at 10 mM concentration, but 2 mM adenine and 10 mM S-adenosyl-L-methionine inhibited the reaction considerably. Properties
of Hydrolase
Activity
When S-adenosyl-L-homocysteine was incubated with the enzyme in Tris-HCl buffer, pH 8.5, hydrolysis to adenosine and homocysteine did not proceed very far, since the equilibrium position was soon
s-ADENOSYL-L-HOMOCYSTEINE
l-y
* G
8
10
PH
FIG. 2. Effect of pH on the S-adenosyl-L-homocysteine “synthetase” activity of the purified enzyme. Dr.-Homocysteine and adenosine were incubated with 5 ~1 purified enzyme (1.3 pg protein) under conditions given under Methods using 56 mM concentrations of the following buffers: Glycine-NaOH (A-A), tricine-NaOH (O-O), Tris-HCl (O-O), and 56 InM K,HPO, with citric acid added to give the required pH (A-n). Enzyme activity is expressed as unite per milliliter.
reached (Fig. 3). The low specific activity of the substrate and its very limited conversion made determination of the equilibrium constant for the reaction difficult. In three separate determinations at pH 8.5 and 3O”C, the equilibrium constant was found to be 1.59, 1.80, and 1.95 PM, respectively. However, the hydrolysis of S-adenosylhomocysteine continued for over 90 min when adenosine deaminase was present in the reaction mixture, when the adenosine was rapidly converted to inosine (Fig. 3). To obtain measurable rates of reaction, this enzyme was included in all experiments. The hydrolase activity, in Tris-HCl buffer, showed a broad pH optimum with a maximum at around pH 8.5; over the range pH 7.6 to 8.8, the ratio of hydrolase and synthetase activities was almost constant. At pH 8.5, the hydrolase activity was proportional to protein concentration up to 15 ~1 of purified enzyme preparation (containing 3.5 ,ug protein) and the reaction was linear with time up to 15 min. The K, for S-adenosylhomocysteine was 41 PM, as determined from a Lineweaver-
HYDROLASE
FROM
SPINACH
139
BEET
Burk plot, which was found to be linear. V for the hydrolysis was equal to 22.4 pmol converted/h/mg protein. Among the metabolites investigated, neither deoxyadenosine nor L-methionine affected the hydrolysis of S-adenosylhomocysteine very much, but, like the synthetase activity, this reaction was severely affected by adenine and S-adenosyl-methionine at concentrations similar to those of the substrate (Table IV). The effect of DLhomocysteine was also appreciable and was analyzed further in relation to the time-course of the hydrolysis in the presence of adenosine deaminase. After incubation for 90 min with adenosine deaminase, the reaction rate declined virtually to zero (Fig. 3). Preincubation of either adenosine deaminase or S-adenosylhomocysteine hydrolase for 120 min at 30°C did not affect the subsequent tiecourse of the reaction, showing that the decline was not due to thermal inactivation of the enzymes. Inosine, at concentrations up to 2 mM, did not inhibit adenosine deaminase nor the hydrolase, nor was adenosine deaminase inhibited by m-homocysteine at concentrations up to 3.4 mM. However, m-homocysteine inhibited TABLE EFFECT
OF METABOLITES HOM~CYSTEINE
Addition
-
L-cysteine
10 1 10 1 2 0.2 10 1 3.3 0.33
Methionine Deoxyadenosine
ON S-ADENOSYL-LSYNTHESIS’
Final con- Specific activcentraity (mtion (mhr) units/mg of protein)
None
S-Adenosyl-nmethionine Adenine
III
Control activity (%)
;;8”; 12128
100
2102 2178 1178 2002 1453 2085 2156 2141 1778 2113
99 102 55 94 68 98 101 101 84 99
” The adenosine concentration in this assay only was reduced to 0.9 mM, in order to allow the easier detection of any inhibitory effects. Activity unite are as described for Table II.
140
POULTON 150 -
I-,--
T
I
/
80
LO Trne
120
(m!n)
FIG. 3. Time-course of S-adenosyl-L-homocysteine hydrolysis in the presence and absence of adenosine deaminase. In the presence of adenosine deaminase, the product was inosine (m-m, nanomoles); in the absence of adenosine deaminase, the product was adenosine (O-0, nanomoles).
the reaction increasingly up to 6 mM (Fig. 4).
at concentrations
DISCUSSION
S-Adenosyl-L-homocysteine hydrolase (EC 3.1.1.11, which catalyzed the synthesis of S-adenosyl-L-homocysteine from adenosine and nL-homocysteine, was purified loo-fold from crude extracts of spinachbeet leaves. The specific activity observed is the highest reported for a preparation of this enzyme from any source. The equilibrium for this reaction lies far in the direction of S-adenosyl-L-homocysteine synthesis. The equilibrium constant determined here is almost idc,ltical with that observed with the rat liver enzyme (51, but less than those reported using a preparation from rat brain (1) and calculated from the data for the yeast enzyme (7). The lowest figures are likely to be the most reliable, since the contamination of preparations with enzymes of even low activity which act upon either of the reaction products is likely to raise this value artificially; evidence for the absence of any other reaction products was not mentioned in connection with this data. The data derived from the LineweaverBurk plot for L- and DL-homocysteine sug-
AND
BUTT
gest that the enzyme is specific for the Lisomer only, and that the n-isomer does not inhibit the activity of the enzyme towards the L-isomer when a mixture is present. Although the tissue level of homocysteine in living organisms is unknown, it appears likely that the high K, value found here for this substrate is unfavorable for S-adenosyl-L-homocysteine synthesis, despite the low K, value for adenosine. Thus, the synthesis of S-adenosyl-Lhomocysteine might proceed in viva at a significant rate only when homocysteine accumulated. This suggestion is further strengthened by experiments with the rat liver enzyme (5, 8, lo), which showed not only an equally high K, value for adenosine, but also inhibition of the reaction by both substrates, by the product S-adenosyl-L-homocysteine, and by adenine. The spinach-beet enzyme was likewise inhibited by low concentrations of adenine and S-adenosyl-L-methionine. Determinations of the levels of S-adenosylmethionine and S-adenosylhomocysteine have shown that they are present in similar concentrations in rat liver (15) and at a ratio of about 2 to 1 in pea seedlings (16). The low Ki for S-adenosylhomocysteine relative to the K, for S-adenosylmeTABLE
IV
EFFECT OF METABOLITES ON S-ADENOSYL-LHOM~CYSTEINE HYDROLYSIS’ Addition
Ficn$;ntion (mM)
Spe;i$c(ztivunits/mg of protein)
Control activity (%)
None
-
162.3 169.6 1161.4
100
L-Methionine
10 1 10 1 2 0.5 2.86 0.71
165.4 163.3 14.6 79.2 49.8 95.2 141.5 159.6
102 101 9 49 31 59 88 99
S-Adenosyl-Lmethionine Adenine Deoxyadeonsine
(23ne unit of S-adenosyl-L-homocysteine hydrolase activity is defined as the amount of enzyme required to catalyse the formation of 1 kmol of inosine per minute from S-adenosyl-L-homocysteine under the assay conditions described.
s-ADENOSYL-L-HOMOCYSTEINE
2 Corm
L
G
8
of DL-hcmocysteme (mM)
4. Effect of m-homocysteine concentration on the initial rate of S-adenosyl+homocysteine hydrolysis by the purified enzyme preparation in the presence of excess adenosine deaminase. s is expressed as millimoles per liter. FIG.
thionine in many methylation reactions (1, 3, 4) emphasizes the importance attached to this balance in determining the rates of methylation. The characteristics and activities of the methyltransferases and hydrolase must be arranged so that the methylations proceed at a rate determined by the steady-state levels of the substrates Sadenosyl-L-methionine and S-adenosyl-Lhomocysteine. The catabolism of S-adenosyl-L-homocysteine was also studied here using the purified enzyme. Due to the equilibrium position, this reaction was especially governed by the disposal of the products, adenosine and homocysteine. Adenosine was routinely removed in the assay mixture by the action of adenosine deaminase, but the activity of this enzyme in spinach-beet extracts was very weak or absent (4). The hydrolysis of S-adenosyl-L-homocysteine was facilitated in crude preparations from these leaves by the action of adenosine nucleosidase (4). However, it seems more likely that adenosine is conserved in viuo by its conversion to ADP through the successive action of adenosine kinase and adenylate kinase, both of which have been demonstrated in spinach-beet leaf extracts (17). Both adenosine and L-homocysteine must be removed for the hydrolysis of Sadenosylhomocysteine to proceed; the accumulation of either of the products inhibits
HYDROLASE
FROM
SPINACH
BEET
141
the enzyme. This was shown here by the inhibition of the reaction by L-homocysteine when adenosine was deaminated by adenosine deaminase. It seems unlikely that the effect of homocysteine was due only to its reversal of the reaction, because attempts to detect labeled adenosine among the reaction products failed, even though the equilibrium constant for the reaction indicated that if homocysteine were reacting with adenosine, sufficient labeled adenosine should have been present for detection. Therefore, it is concluded that homocysteine exerts its effect by inhibiting the enzyme directly, and that its accumulation during the hydrolysis of Sadenosylhomocysteine in the presence of adenosine deaminase is sufficient after 90 min to stop the further progress of the reaction. Other evidence for inhibition of S-adenosyl-L-homocysteine hydrolysis by homocysteine was obtained with the rat liver enzyme (5). Furthermore, with the latter enzyme, adenosine was shown to become inhibitory when homocysteine was removed in vitro by methylation, using the dimethylthetin-homocysteine methyltransferase system in the reaction mixture. In uivo, L-homocysteine may be converted to cystathionine by condensation with serine (18), but it is more likely to be methylated by N5-methyltetrahydrofolate to methionine, already demonstrated in extracts of pea seedlings (19, 20) and spinach and barley leaves (21). Although the V for S-adenosyl-L-homocysteine hydrolysis was only one-tenth of that for synthesis, the lower K, value (41 PM) for S-adenosylL-homocysteine would tend to favor this reaction. It is of importance that the V for S-adenosyl-L-homocysteine hydrolysis is well in excess of that observed for the purified caffeic acid o-methyltransferase from spinach-beet leaves (4). However, S - adenosyl - L - homocysteine hydrolysis, like the synthesis of this compound, is also subject to a number of controls by the metabolites present. Adenine and S-adenosyl-L-methionine inhibited this reaction at concentrations similar to the substrate. A knowledge of the mode of inhibition by these compounds and of their intracellular concentration is required to
142
POULTON
estimate how strongly these metabolites might affect S-adenosyl-L-homocysteine hydrolysis in viva. The kinetic evidence provided shows that the enzyme is a sensitive regulator in the conversion of S-adenosyl-homocysmine, depending for its activity not only upon favorable concentrations of metabolites in relation to the equilibrium conditions but also upon the levels of S-adenosyl-methionine, adenosine, and L-homocysteine maintained within the system. These, in turn, will feed back to determine the rates of methylation reactions which are sensitive to the levels of S-adenosylhomocysteine. ACKNOWLEDGMENTS We thank Dr. M. A. Foster, Biochemistry Department, Oxford, for his suggestions in the initial stages of this work, and Prof. Dr. K. Hahlbrock for reading the manuscript. J. E. Poulton thanks the Science Research Council for a Research Studentship. REFERENCES 1. DEGUCHI, T., AND BARCHAS, J. (1971) J. Biol. Chem. 246,3175-3181. 2. COWARD, J. K., D’URSO-SCOTT, M., AND SWEET, W. D. (1971) Biochem. Pharmacol. 21, 12001203. 3. MANN, J. D., AND MUDD, S. H. (1963) J. Biol. Chem. 238,381-385. 4. POULTON, J. E. (1974) D. Phil. Thesis, University of Oxford, Great Britain.
AND
BUTT
5. DE LA HABA, G., AND CANTONI, G. L. (1959) J. Bid. Chem. 234, 603-608. 6. DUERRE, J. A. (1968) Arch. Biochem. Biophys. 124, 422-430. 7. KNUDSON, R. C., AND YALL, I. (1972) J. Bacteriol. 112, 569475. 8. FINKELSTEIN, J. D., AND HARRIS, B. (1973)Arch. Biochem. Biophys. 159, 160-165. 9. DODD, W. A., AND COSSINS, E. A. (1969) Arch. Biochem. Biophys. 133, 216-233. 10. WALKER, R. D., AND DUERRE, J. A. (1975) Can. J. Biochem. 53,312-319. 11. DUERRE, J. A., AND MILLER, C. H. (1966) Anal. Biochem. 17, 310-315. 12. SHAPIRO, S. K., AND EHNINGER, 0. J. (1966) Anal. B&hem. 15, 323-333. 13. LEGGETT BAILEY, J. (1962) In Techniques in Protein Chemistry, pp. 293-294. Elsevier Publishing Co., Amsterdam. 14. WARBURG, O., AND CHRISTIAN, W. (1941) Biothem. 2. 310, 384-421. 15. SALVATORE, F., ZAPPIA, W., AND SHAPIRO, S. K. (1968) Biochem. Biophys. Acta 158,461-464. 16. DODD, W. A., AND COSSINS, E. A. (1968) Phytochemistry 7, 2143-2145. 17. BARTLETT, D. J., AND BUTT, V. S. (1974), personal communication. 18. GIOVANELLI, J., AND MUDD, S. H. (1966) Biothem. Biophys. Res. Commun. 25,366-371. 19. WOODS, D. D., FOSTER, M. A., AND GUEST, J. R. (1965) in Transmethylation and Methionine Biosynthesis (Shapiro, S. K., and Schleuk, F., eds), pp. 138-154, University of Chicago Press, Chicago. 20. DODD, W. A., AND COSSINS, E. A. (1970) Biothem. Biophys. Acta 201, 461-470. 21. BURTON, E. G., AND SAKAMI, W. (1969) Biochem. Biophys. Res. Commun. 36, 228.
