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Methionine metabolism in mammals: Regulatory effects of S-adenosylhomocysteine
ARCHIVES
OF BIOCHEMISTBY
AND
Methionine
165, 774-7i’9
BIOPHYSICS
Metabolism
(1974)
in Mammals:
Regulatory
of S- Adenosyl homocysteine JAMES
D. FINKELSTEIN,’
Veterans Administration
Hospital
WALTER
E. KYLE,
and Department of Medicine, Washington, D.C. 20422
Effects
l
AND
BARBARA
George Washington
J. HAQRIS University,
Received July 29, 1974 S-Adenosylhomocysteine inhibits betaine-homocysteine methyltransferase. The inhibition is nonlinear, competitive in relation to homocysteine, and noncompetitive in relation to betaine. S-Adenosylhomocysteine activates cystathionine synthase at all concentrations of the substrates, serine and homocysteine. By altering the distribution of homocysteine between these competing pathways, S-adenosylhomocysteine may be significant in the regulation of methionine metabolism in the intact animal.
In mammalian liver, homocysteine, derived from methionine, may be converted to cystathionine or may be remethylated to yield methionine. It is the distribution of homocysteine between these alternate pathways that may regulate and control methionine metabolism (l-3). At least four enzymes utilize homocysteine as a substrate. These are: cystathionine-P-synthase (EC 4.2.1.22); S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1); &methyltetrahydrofolate-homocysteine methyltransferase (5-methyltetrahydropteroyl-L-glutamate: L-homocysteine-S-methyltransferase, EC 2.1.1.13); and betaine-homocysteine methyltransferase (betaine: L-homocysteine S-methyltransferase, EC 2.1.1.5). Both the tissue content of these enzymes and the concentrations of substrates and products relative to the kinetic properties of the enzymes would serve to modulate the distribution of homocysteine among the competing reactions. In addition metabolites, other than substrates and products, may activity. S-Adenosylaffect enzyme homocysteine inhibits several classes of ‘Supported in part by Grant AM-13048 from the National Institutes of Health, U.S. Public Health Service. 2 Medical Investigator, Veterans Administration Hospital. 774 Copyright 0 1974 by Academic Press. Inc. All rights of reproduction in any form reserved.
transmethylation reactions that utilize Sadenosylmethionine as the methyl donor (4-6). S-Adenosylhomocysteine may facilitate the synthesis of &methyltetrahydrofolate (7) but inhibits 5-methyltetrahydrofolate-homocysteine methyltransferase prepared from porcine kidney (8) as well as betaine-homocysteine methyltransferase derived from rat liver (9). In the present paper we have studied, in greater detail, the inhibition of the betaine methyltransferase and present the additional observation that in vitro addition of Sadenosylhomocysteine stimulates the cystathionine synthase reaction. EXPERIMENTAL
PROCEDURE
Enzyme Preparation We have described our method for the partial purification of betaine-homocysteine methyltransferase from rat liver (9). Cystathionine synthase was partially purified by a modification of the method of Nakagawa and Kimura (10). We dissolved the first ammonium sulfate fraction in 10 mM potassium phosphate (pH 7.5) and treated the resulting solution by gel filtration with Sephadex G-25. The proteincontaining fractions were combined and pyridoxal phosphate was added to a final concentration of 1 DIM. We then heated the solution for 15 min at 60°C. After centrifugation and Sephadex G-25 filtration, we added 1 mg of calcium phosphate gel (water equilibrated) for each mg of protein. The suspension was
REGULATORY
EFFECTS
OF S-ADENOSYLHOMOCYSTEINE
stirred at 0°C for 60 min. Cystathionine synthase was not adsorbed and remained in the supernatant obtained by centrifugation. Using this method we obtained an over-all yield of approximately 50% and a specific activity of 12-24 times that of the initial extract.
Enzyme Assays The methods for assaying betaine-homocysteine methyltransferase and cystathionine synthase both depend on the synthesis of a radioactive product from a radioactive substrate with subsequent separation by ion-exchange chromatography. We have published the standard assay conditions (1, 9, 11). In addition to the previously-described components, the assay media for cystathionine synthase contained 5.0 mM dithiothreitol.
Analyses of Data We employed graphical representation, linear regression analysis and Wilkinson’s weighted and nonlinear regression methods (12) to evaluate individual kinetic experiments. In most studies these several techniques of analysis yielded comparable results. Cleland’s nomenclature is used in the interpretation of the studies (13). RESULTS
Inhibition
of Betaine-Homocysteine Methyltransferase by S-Adenosylhomocysteine
The betaine-homocysteine methyltransferase reaction conforms to the ordered BiBi model. Homocysteine is the first substrate to add to the enzyme and methionine is the last product released. The reported Michaelis constants are K betalne = 48 PM and Khomocystelne= 12 PM (9). As indicated by the data in Table I, when homocysteine is the variable substrate, the addition of S-adenosylhomocysteine alters the slope but not the intercept of the double-reciprocal plot. We observed this effect in experiments with low (0.12 mM) or high concentrations (1.03 mM) of the fixed substrate-betaine. The graph of the slope against the concentration of inhibitor was hyperbolic and the plot of the reciprocal of the change in slope against the reciprocal of the inhibitor concentration was linear. Furthermore, we obtained nonlinear graphs when we plotted l/v versus the concentration of S-adenosyl-
775
homocysteine. Admittedly, we tested only a limited number of concentrations of Sadenosylhomocysteine. Nevertheless the results are compatible with nonlinear (hyperbolic) competitive inhibition. In Table II we present the results of a typical study in which we varied the concentration of betaine while we maintained the concentration of homocysteine at 25 pM. Under these conditions, the addition of S-adenosylhomocysteine altered both the slope and the intercept of the double-reciprocal graph. The change in intercept is a complex function of the concentration of inhibitor, and the relationship between slope and inhibitor concentration cannot be defined unequivocally from this experiment alone. The five points fit either a parabola or a straight line (correlation coefficient = .99). Additional studies indicated a linear relationship between the slope and the concentration of S-adenosylhomocysteine. This relationship was demonstrated most clearly in studies with low concentrations of homocysteine (10 PM). Thus, S-adenosylhomocysteine is a mixed noncompetitive inhibitor relative to betaine when the concentration of homocysteine is not saturating. Increasing concentrations of homocysteine (>5 mM) abolished the inhibition by S-adenosylhomocysteine (Table III). Activation
of Cystathionine Synthase S-Adenosylhomocysteine
by
S-Adenosylhomocysteine is not a substrate for cystathionine synthase. Nor does the addition of S-adenosylhomocysteine increase the recovery of [‘%]cystathionine incubated in a reaction mixture containing cystathionine synthase, L-homocysteine and L-serine as well as the other reagents in our routine assay system (11). Nonetheless, the formation of radioactive cystathionine from L-homocysteine and ~-(3-l%) serine was consistently increased in the presence of S-adenosylhomocysteine. In studies where serine was the variable substrate (0.25-5.0 mM) and the concentration of homocysteine was fixed (1.25. 2.50 or 6.25 mM), we observed that the addition of S-adenosylhomocysteine invariably in-
776
FINKELSTEIN,
KYLE
AND HARRIS
TABLE I S-ADENOSYLHOMOCYSTEINEINHIBITION OF BETAINE-HOMOCYSTEINE METHYLTRANSFERASEWITH HOMOCYSTEINE AS VARIABLE SUBSTRATES Betaine S-AdoHcy
1.03
0.12
(mM)
0
(mM)
0.38
Homocysteine (md 0.025 0.04 0.05 0.08 0.125 0.20 0.25 0.40 0.80
K,IV l/V(rm-‘)
Product formation 4.94 6.54 6.57 7.49 9.28 10.51 10.42 10.47 10.78
3.68
0
3.44
12.39
7.15
17.09
12.10
24.50
17.74
28.10
20.80
1.33 30.3
2.33 38.4
3.75
0.75
(nmoles/60*min)
3.32 4.37
6.28
5.10 5.95
7.88
7.62 8.14
9.75
9.05 9.41 10.59
2.96 86.2
4.18 87.7
5.33 89.3
6.16 89.3
DValues represent means of duplicate determinations of reaction rate at each concentration of homocysteine. The table presents the results of separate studies performed at two concentrations of betaine. Different enzyme preparations were employed in the two studies. TABLE
TABLE
II
S-ADENOSYLHOMOCYSTEINEINHIBITION OF BETAINE-HOMOCYSTEINE METHYLTRANSFERASEWITH BETAINE AS VARIABLE SUBSTRATE” S-AdoHcy 0 0.36 0.82 1.69 3.62
(mM)
K,IV 10.2 12.2 13.7 28.8 53.2
l/V(pm-l)
S-AdoHcy
creased the maximum velocity of the reaction. In most experiments the addition of S-adenosylhomocysteine also reduced the apparent Michaelis constant for serine. The net result was a consistent reduction in the slope of the double-reciprocal plot. Table IV illustrates some typical results. In a similar manner, we studied the effect of S-adenosylhomocysteine when the concentration of serine was 2.5 mM and the concentration of homocysteine was varied from 0.0 to 12.5 mM. When we added higher
(mM)
Betaine (mM)
97.0 120.5 139.7 91.0 89.3
“The concentration of homocysteine was fixed at 0.025 mM. For each concentration of S-adenosylhomocysteine we performed duplicate determinations of the reaction rate at five concentrations of betaine ranging from 0.034 mM to 0.245 mM.
III
FAILURE OF S-ADENOSYLHOMOCYSTEINETO INHIBIT BETAINE-HOMOCYSTEINE METHYLTRANSFERASEAT SATURATING CONCENTRATION OF HOMOCYSTEINE~
0.084 0.124 0.174 0.274 K,IV l/V(wrl)
0
2.56
Product formation (nmoles/60 min) 13.43 16.24 18.27 19.93 2.84 39.2
14.46 15.01 17.91 20.81 2.97 38.7
a The concentration of homocysteine was fixed at 5 Values represent means of duplicate determinations of reaction rate at each concentration of betaine. mM.
levels of S-adenosylhomocysteine (0.3-1.55 mM), we found an increase in maximum velocity together with a decrease in the apparent K, for homocysteine, causing a significant decrease in both the slope and the intercept of the double reciprocal graph. The effect of lower concentrations of S-adenosylhomocysteine on the maximum velocity was not significant statistically while the consistent decrease in K, was.
REGULATORY TABLE
EFFECTS
TABLE
IV
EFFECT OF S-ADENOSYLHOMOCYSTEINE ON CYSTATHIONINE SYNTHASE WITH SERINE AS THE VARIABLE SUBSTRATES
L-Homocysteine (mM) 1.25 1.25 2.50 2.50 6.25 6.25
S-AdoHcy (mM) 0.0 0.8 0.0 1.25 0.0 1.23
V (nmoles/ 30 min) 19.5 37.5 40.9 66.2 54.0 98.0
777
OF S-ADENOSYLHOMOCYSTEINE
K,IV 61.5 21.8 43.5 24.8 46.6 20.6
V
EFFECT OF S-ADENOSYLHOMOCYSTEINE ON CYSTATHIONINE SYNTHASE AT HIGHER CONCENTRATIONS OF SUBSTRATES~
Homocysteine (mM)
12.5
12.5
25.0
25.0
S-AdoHcy (m@
0
1.55
0
1.55
L-Serine (mid 2.5 15.0 27.5
Product formation 0.22 0.39 0.43
0.46 0.65 0.61
(rmolesl60 0.21
min) 0.41
“In each experiment we performed duplicate assays at each of five concentrations of L-serine (0.25-5.0 mM) in the presence or absence of added S-adenosylhomocysteine. K, and V were determined by Wilkinson’s method (12).
“Values are means of at least two determinations of the reaction rate with each combination of substrate concentrations.
With the serine concentration of 2.5 mM, maximum product formation occurs when the concentration of homocysteine is 12.5 mM. Even at these concentrations of the substrates, the addition of S-adenosylhomocysteine results in an increase in the reaction velocity (Table V). Conversely, with 12.5 mM homocysteine a concentration of serine of 15.0 mM is “saturating.” S-adenosylhomocysteine Nevertheless, causes a further increase in product formation.
adenosylhomocysteine inhibited 5-methyltetrahydrofolate-homocysteine methyltransferase derived from porcine kidney (8). The inhibition was competitive relative to homocysteine and noncompetitive relative to S-adenosylmethionine. The differences between our results and those of Burke et al. probably result from the relatively high, fixed concentrations of reactants in our system. Species or organ-specific differences are less-likely explanations.
Effect of S-Adenosylhomocysteine Other Enzymes
on
Our standard assay system for Sadenosylmethionine synthetase (methionine adenosyltransferase, EC 2.5.1.6) contains 0.1 mM L-methionine and 15 mM ATP (11). We found no significant change in the rate of product formation when we added 1.5 mM S-adenosylhomocysteine. Similarly, we observed no effect of 2.1 mM S-adenosylhomocysteine on the reaction velocity of 5-methyltetrahydrofolatehomocysteine methyltransferase. The concentrations of reactants in this assay were 0.25 mM L-homocysteine, 0.15 mM 5methyltetrahydrofolate, 0.15 mM Sadenosylmethionine and 0.5 mM L-methionine (14). In these experiments we employed an enzyme preparation partially purified from rat liver. In a detailed kinetic study, Burke et al. found that S-
DISCUSSION
In mammalian liver, methionine metabolism is a cycle with a single, major outlet at the cystathionine synthase reaction (Fig. 1, Reaction 4). As homocysteine is ultilized in this reaction, it is committed irreversibly to the transsulfuration sequence-the primary means for methionine catabolism (15). Alternatively, homocysteine may be methylated thereby maintaining the tissue content of methionine. There are two homocysteine methylases. 5-Methyltetrahydrofolate-homocysteine methyltransferase (Fig. 1, Reaction 8) is present in all rat tissues except the small intestinal mucosa (14). In contrast, only liver contains significant amounts of betaine-homocysteine methyltransferase (Fig. 1, Reaction 7) (14). Alterations in the dietary intake of protein (or methionine) have different effects on these two methyl-
778
FINKELSTEIN,
r-
KYLE
[L-METHIONINI ,
AND HARRIS
-‘@ Q-CH,SH+
UNKNOWN PRODVCT
I-l-S-ADENOSYL-L-METHIONINE KCEPTORS 0
METHYLATEOACCEPTORS
S-ADENOSYL-L-HOMOCYSTEINE
0
ADENOSINE _ mm~_---/ IL-~~OMOCYSTEINEI ! .a
1
L-SER,NE
-
0
L-HOMOCYSTINE
03 H>S + a-KETOBUTYRATE
IL-CYSTATHIONI~ 0
PROPIONATE
“-KETOSUTYRATE
IL-CYSTEINEI
so,
co2
0 i JSOfj
FIG. 1. Known pathways of mammalian metabolism of methionine and homocysteine. Abbreviations: ATP, adenosine 5’-triphosphate, PP,, inorganic pyrophosphate, P,, inorganic orthophosphate, FH,, tetrahydrofolic acid; mSFH,, 5-methyltetrahydrofolic acid.
ases. High protein feeding or supplementation with methionine results in an increase in the hepatic content of the bemine enzyme. Simultaneously, the level of 5methyltetrahydrofolate-homocysteine methyltransferase decreases. On the basis of these observations, we suggested that the folate enzyme was more significant in the maintenance of the tissue concentration of methionine while the betaine enzyme might be of primary importance in the catabolism of choline (14). This hypothesis is supported by the finding that a decreased level of tissue methionine characterizes patients with decreased activity of 5-methyltetrahydrofolate-homocysteine methyltransferase (3). The protein (and methionine) content of the diet also regulates the hepatic level of cystathionine synthase (1, 16). The activity of this enzyme is increased by either increased protein intake or the administration of supplemental methionine. In summary, rats fed a low-methionine diet may conserve this amino acid by reducing the flow of homocysteine into the transsulfuration sequence (decreased cystathionine
synthase) and by increasing the resynthesis of methionine (increased &methyltetrahydrofolate-homocysteine methyltransferase). In rats fed excessive methionine we observed, on the other hand, an increase in cystathionine synthase and a decrease in the folate methyltransferase. Whether the latter changes in enzyme activity would allow increased transsulfuration and reduced homocysteine methylation is less clear since excess dietary methionine also increases the activity of betaine-homocysteine methyltransferase. Conceivably the decrease in methylation from 5-methyltetrahydrofolate would be balanced by an increase in methylation from betaine. As illustrated by the present paper as well as by earlier studies, the distribution of homocysteine between cystathionine synthase and betaine-homocysteine methyltransferase may be regulated by the kinetic properties of these two competing enzymes. The Km for homocysteine in the betaine reaction is 12 pM (9) while the K, for this substrate in the cystathionine synthase reaction has been reported to approximate 10 mM (10, 17-19). As the concentra-
REGULATORY
EFFECTS
OF S-ADENOSYLHOMOCYSTEINE
tion of homocysteine increases we would anticipate that a greater fraction would be utilized for the synthesis of cystathionine. Distribution in favor of cystathionine synthase would be facilitated by other properties of the two enzymes. Since homocysteine does not occur in dietary protein, homocysteine accumulation is likely to occur in the normal animal only as the result of the ingestion of excessive methionine. Furthermore, the hydrolase reaction (Fig. 1, Reaction 3) which generates homocysteine is reversible and the equilibrium favors the synthesis of S-adenosylhomocysteine rather than its hydrolysis (20). For these reasons, the accumulation of homocysteine in tissues should be associated with a parallel increase in methionine and S-adenosylhomocysteine. Methionine is a competitive inhibitor of homocysteine in the betaine-homocysteine methyltransferase reaction (9) and in the present study we have demonstrated that S-adenosylhomocysteine has similar properties. The accumulation of these two metabolites might inhibit the betaine reaction until the tissue concentration of homocysteine beSimultaneously. incame “saturating.” creased concentrations of S-adenosylhomocysteine might activate cystathionine synthase independent of the level of homocysteine. ACKNOWLEDGMENT We are indebted to Ann-Marie Pick for her assistance in many aspects of this study. REFERENCES 1. FINKELSTEIN, J. D., AND MUDD, S. H. (1967) J. Biol. Chem. 242, 873-880.
779
2. FINKELSTEIN, J. D. (1971) in Inherited
3. 4. 5. 6.
7. 8. 9. 10. 11.
12. 13. 14. 15.
16.
Disorders of Sulphur Metabolism (Carson, N. A. J., and Raine, D. N., eds.), pp 1-13, Churchill Livingstone, London. FINKELSTEIN, J. D. (1974) Metabolism 23, 387-398. ZAPPIA, V., ZYDEK-CWICK, C. R., AND SCHLENK, F. (1969). J. Biol. Chem. 244, 4499-4509. DEGUCHI, T., AND BARCHAS, J. (19711 J. Viol. Chen. 246,3175-3181. COWARD, J. K., D’URSO-SCOTT, M., AND SWEET, W. D. (1972) Biochem. Pharm. 21, 1200-1203. KUTZBACH, C., AND STOKSTAD, E. L. R. (1967) Biochem. Biophys. Acta 139, 217-220. BURKE, G. T., MANGUM, J. H., AND BRODIE, J. D. (1971) Biochemistp 10, 3079-3085. FINKELSTEIN, J. D., HARRIS, B. J., AND KYLE, W. E. (1972) Arch. Biochem. Biophys. 153, 320-324. NAKAGAWA, H., AND KIMURA, H. (1968) Biochem. Biophyy. Res. Commun. 32, 208-214. MUDD, S. H., FINKELSTEIN, J. D., IRREVERRE, F., AND LASTER, L. (1965) J. Biol. Chem. 240, 4382-4392. WILKINSON, G. N. (1961) Biochem. J. 80, 324-332. CLEI,AND, W. W. (1971) Ann. Rec. Biochem. 36, 77-112. FINKELSTEIN, J. D., KYLE, W E., AND HARRIS, B. J. (1971) Arch. Biochem. Biophys. 146, 84-92. LASTER L., MUDD, S. H., FINKELSTEIN, J. D., AND IRREVERRE, F. (1965) J. Clin. Invest. 44, 1708-1719. FINKELSTEIN, J. D. (1967) A&. Biochem. Biophys. 122,583-590.
17 KASHIWAMATA, S., AND GREENBERC, D. M. (1970) Biochem. Biophys. Acta. 212, 48%500. 18 BROWN, F. C., ANDGORDON, P. H. (1971) Canad. J. Biochem. 49, 484-491. 19. BRAUNSTEIN, A. E., GORYACHENKO~A, E. V., TOLOSA, E. Z., WILLHARDT, I. H., AND YEFREMOVA, L. L. (1971) Biochim. Biophys. Acta. 242, 247-260. 20. DE LA HABA, G., AND CANTONI, G. L. (1959) J. Viol. ’ Chem. 234, 603-608.
OF BIOCHEMISTBY
AND
Methionine
165, 774-7i’9
BIOPHYSICS
Metabolism
(1974)
in Mammals:
Regulatory
of S- Adenosyl homocysteine JAMES
D. FINKELSTEIN,’
Veterans Administration
Hospital
WALTER
E. KYLE,
and Department of Medicine, Washington, D.C. 20422
Effects
l
AND
BARBARA
George Washington
J. HAQRIS University,
Received July 29, 1974 S-Adenosylhomocysteine inhibits betaine-homocysteine methyltransferase. The inhibition is nonlinear, competitive in relation to homocysteine, and noncompetitive in relation to betaine. S-Adenosylhomocysteine activates cystathionine synthase at all concentrations of the substrates, serine and homocysteine. By altering the distribution of homocysteine between these competing pathways, S-adenosylhomocysteine may be significant in the regulation of methionine metabolism in the intact animal.
In mammalian liver, homocysteine, derived from methionine, may be converted to cystathionine or may be remethylated to yield methionine. It is the distribution of homocysteine between these alternate pathways that may regulate and control methionine metabolism (l-3). At least four enzymes utilize homocysteine as a substrate. These are: cystathionine-P-synthase (EC 4.2.1.22); S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1); &methyltetrahydrofolate-homocysteine methyltransferase (5-methyltetrahydropteroyl-L-glutamate: L-homocysteine-S-methyltransferase, EC 2.1.1.13); and betaine-homocysteine methyltransferase (betaine: L-homocysteine S-methyltransferase, EC 2.1.1.5). Both the tissue content of these enzymes and the concentrations of substrates and products relative to the kinetic properties of the enzymes would serve to modulate the distribution of homocysteine among the competing reactions. In addition metabolites, other than substrates and products, may activity. S-Adenosylaffect enzyme homocysteine inhibits several classes of ‘Supported in part by Grant AM-13048 from the National Institutes of Health, U.S. Public Health Service. 2 Medical Investigator, Veterans Administration Hospital. 774 Copyright 0 1974 by Academic Press. Inc. All rights of reproduction in any form reserved.
transmethylation reactions that utilize Sadenosylmethionine as the methyl donor (4-6). S-Adenosylhomocysteine may facilitate the synthesis of &methyltetrahydrofolate (7) but inhibits 5-methyltetrahydrofolate-homocysteine methyltransferase prepared from porcine kidney (8) as well as betaine-homocysteine methyltransferase derived from rat liver (9). In the present paper we have studied, in greater detail, the inhibition of the betaine methyltransferase and present the additional observation that in vitro addition of Sadenosylhomocysteine stimulates the cystathionine synthase reaction. EXPERIMENTAL
PROCEDURE
Enzyme Preparation We have described our method for the partial purification of betaine-homocysteine methyltransferase from rat liver (9). Cystathionine synthase was partially purified by a modification of the method of Nakagawa and Kimura (10). We dissolved the first ammonium sulfate fraction in 10 mM potassium phosphate (pH 7.5) and treated the resulting solution by gel filtration with Sephadex G-25. The proteincontaining fractions were combined and pyridoxal phosphate was added to a final concentration of 1 DIM. We then heated the solution for 15 min at 60°C. After centrifugation and Sephadex G-25 filtration, we added 1 mg of calcium phosphate gel (water equilibrated) for each mg of protein. The suspension was
REGULATORY
EFFECTS
OF S-ADENOSYLHOMOCYSTEINE
stirred at 0°C for 60 min. Cystathionine synthase was not adsorbed and remained in the supernatant obtained by centrifugation. Using this method we obtained an over-all yield of approximately 50% and a specific activity of 12-24 times that of the initial extract.
Enzyme Assays The methods for assaying betaine-homocysteine methyltransferase and cystathionine synthase both depend on the synthesis of a radioactive product from a radioactive substrate with subsequent separation by ion-exchange chromatography. We have published the standard assay conditions (1, 9, 11). In addition to the previously-described components, the assay media for cystathionine synthase contained 5.0 mM dithiothreitol.
Analyses of Data We employed graphical representation, linear regression analysis and Wilkinson’s weighted and nonlinear regression methods (12) to evaluate individual kinetic experiments. In most studies these several techniques of analysis yielded comparable results. Cleland’s nomenclature is used in the interpretation of the studies (13). RESULTS
Inhibition
of Betaine-Homocysteine Methyltransferase by S-Adenosylhomocysteine
The betaine-homocysteine methyltransferase reaction conforms to the ordered BiBi model. Homocysteine is the first substrate to add to the enzyme and methionine is the last product released. The reported Michaelis constants are K betalne = 48 PM and Khomocystelne= 12 PM (9). As indicated by the data in Table I, when homocysteine is the variable substrate, the addition of S-adenosylhomocysteine alters the slope but not the intercept of the double-reciprocal plot. We observed this effect in experiments with low (0.12 mM) or high concentrations (1.03 mM) of the fixed substrate-betaine. The graph of the slope against the concentration of inhibitor was hyperbolic and the plot of the reciprocal of the change in slope against the reciprocal of the inhibitor concentration was linear. Furthermore, we obtained nonlinear graphs when we plotted l/v versus the concentration of S-adenosyl-
775
homocysteine. Admittedly, we tested only a limited number of concentrations of Sadenosylhomocysteine. Nevertheless the results are compatible with nonlinear (hyperbolic) competitive inhibition. In Table II we present the results of a typical study in which we varied the concentration of betaine while we maintained the concentration of homocysteine at 25 pM. Under these conditions, the addition of S-adenosylhomocysteine altered both the slope and the intercept of the double-reciprocal graph. The change in intercept is a complex function of the concentration of inhibitor, and the relationship between slope and inhibitor concentration cannot be defined unequivocally from this experiment alone. The five points fit either a parabola or a straight line (correlation coefficient = .99). Additional studies indicated a linear relationship between the slope and the concentration of S-adenosylhomocysteine. This relationship was demonstrated most clearly in studies with low concentrations of homocysteine (10 PM). Thus, S-adenosylhomocysteine is a mixed noncompetitive inhibitor relative to betaine when the concentration of homocysteine is not saturating. Increasing concentrations of homocysteine (>5 mM) abolished the inhibition by S-adenosylhomocysteine (Table III). Activation
of Cystathionine Synthase S-Adenosylhomocysteine
by
S-Adenosylhomocysteine is not a substrate for cystathionine synthase. Nor does the addition of S-adenosylhomocysteine increase the recovery of [‘%]cystathionine incubated in a reaction mixture containing cystathionine synthase, L-homocysteine and L-serine as well as the other reagents in our routine assay system (11). Nonetheless, the formation of radioactive cystathionine from L-homocysteine and ~-(3-l%) serine was consistently increased in the presence of S-adenosylhomocysteine. In studies where serine was the variable substrate (0.25-5.0 mM) and the concentration of homocysteine was fixed (1.25. 2.50 or 6.25 mM), we observed that the addition of S-adenosylhomocysteine invariably in-
776
FINKELSTEIN,
KYLE
AND HARRIS
TABLE I S-ADENOSYLHOMOCYSTEINEINHIBITION OF BETAINE-HOMOCYSTEINE METHYLTRANSFERASEWITH HOMOCYSTEINE AS VARIABLE SUBSTRATES Betaine S-AdoHcy
1.03
0.12
(mM)
0
(mM)
0.38
Homocysteine (md 0.025 0.04 0.05 0.08 0.125 0.20 0.25 0.40 0.80
K,IV l/V(rm-‘)
Product formation 4.94 6.54 6.57 7.49 9.28 10.51 10.42 10.47 10.78
3.68
0
3.44
12.39
7.15
17.09
12.10
24.50
17.74
28.10
20.80
1.33 30.3
2.33 38.4
3.75
0.75
(nmoles/60*min)
3.32 4.37
6.28
5.10 5.95
7.88
7.62 8.14
9.75
9.05 9.41 10.59
2.96 86.2
4.18 87.7
5.33 89.3
6.16 89.3
DValues represent means of duplicate determinations of reaction rate at each concentration of homocysteine. The table presents the results of separate studies performed at two concentrations of betaine. Different enzyme preparations were employed in the two studies. TABLE
TABLE
II
S-ADENOSYLHOMOCYSTEINEINHIBITION OF BETAINE-HOMOCYSTEINE METHYLTRANSFERASEWITH BETAINE AS VARIABLE SUBSTRATE” S-AdoHcy 0 0.36 0.82 1.69 3.62
(mM)
K,IV 10.2 12.2 13.7 28.8 53.2
l/V(pm-l)
S-AdoHcy
creased the maximum velocity of the reaction. In most experiments the addition of S-adenosylhomocysteine also reduced the apparent Michaelis constant for serine. The net result was a consistent reduction in the slope of the double-reciprocal plot. Table IV illustrates some typical results. In a similar manner, we studied the effect of S-adenosylhomocysteine when the concentration of serine was 2.5 mM and the concentration of homocysteine was varied from 0.0 to 12.5 mM. When we added higher
(mM)
Betaine (mM)
97.0 120.5 139.7 91.0 89.3
“The concentration of homocysteine was fixed at 0.025 mM. For each concentration of S-adenosylhomocysteine we performed duplicate determinations of the reaction rate at five concentrations of betaine ranging from 0.034 mM to 0.245 mM.
III
FAILURE OF S-ADENOSYLHOMOCYSTEINETO INHIBIT BETAINE-HOMOCYSTEINE METHYLTRANSFERASEAT SATURATING CONCENTRATION OF HOMOCYSTEINE~
0.084 0.124 0.174 0.274 K,IV l/V(wrl)
0
2.56
Product formation (nmoles/60 min) 13.43 16.24 18.27 19.93 2.84 39.2
14.46 15.01 17.91 20.81 2.97 38.7
a The concentration of homocysteine was fixed at 5 Values represent means of duplicate determinations of reaction rate at each concentration of betaine. mM.
levels of S-adenosylhomocysteine (0.3-1.55 mM), we found an increase in maximum velocity together with a decrease in the apparent K, for homocysteine, causing a significant decrease in both the slope and the intercept of the double reciprocal graph. The effect of lower concentrations of S-adenosylhomocysteine on the maximum velocity was not significant statistically while the consistent decrease in K, was.
REGULATORY TABLE
EFFECTS
TABLE
IV
EFFECT OF S-ADENOSYLHOMOCYSTEINE ON CYSTATHIONINE SYNTHASE WITH SERINE AS THE VARIABLE SUBSTRATES
L-Homocysteine (mM) 1.25 1.25 2.50 2.50 6.25 6.25
S-AdoHcy (mM) 0.0 0.8 0.0 1.25 0.0 1.23
V (nmoles/ 30 min) 19.5 37.5 40.9 66.2 54.0 98.0
777
OF S-ADENOSYLHOMOCYSTEINE
K,IV 61.5 21.8 43.5 24.8 46.6 20.6
V
EFFECT OF S-ADENOSYLHOMOCYSTEINE ON CYSTATHIONINE SYNTHASE AT HIGHER CONCENTRATIONS OF SUBSTRATES~
Homocysteine (mM)
12.5
12.5
25.0
25.0
S-AdoHcy (m@
0
1.55
0
1.55
L-Serine (mid 2.5 15.0 27.5
Product formation 0.22 0.39 0.43
0.46 0.65 0.61
(rmolesl60 0.21
min) 0.41
“In each experiment we performed duplicate assays at each of five concentrations of L-serine (0.25-5.0 mM) in the presence or absence of added S-adenosylhomocysteine. K, and V were determined by Wilkinson’s method (12).
“Values are means of at least two determinations of the reaction rate with each combination of substrate concentrations.
With the serine concentration of 2.5 mM, maximum product formation occurs when the concentration of homocysteine is 12.5 mM. Even at these concentrations of the substrates, the addition of S-adenosylhomocysteine results in an increase in the reaction velocity (Table V). Conversely, with 12.5 mM homocysteine a concentration of serine of 15.0 mM is “saturating.” S-adenosylhomocysteine Nevertheless, causes a further increase in product formation.
adenosylhomocysteine inhibited 5-methyltetrahydrofolate-homocysteine methyltransferase derived from porcine kidney (8). The inhibition was competitive relative to homocysteine and noncompetitive relative to S-adenosylmethionine. The differences between our results and those of Burke et al. probably result from the relatively high, fixed concentrations of reactants in our system. Species or organ-specific differences are less-likely explanations.
Effect of S-Adenosylhomocysteine Other Enzymes
on
Our standard assay system for Sadenosylmethionine synthetase (methionine adenosyltransferase, EC 2.5.1.6) contains 0.1 mM L-methionine and 15 mM ATP (11). We found no significant change in the rate of product formation when we added 1.5 mM S-adenosylhomocysteine. Similarly, we observed no effect of 2.1 mM S-adenosylhomocysteine on the reaction velocity of 5-methyltetrahydrofolatehomocysteine methyltransferase. The concentrations of reactants in this assay were 0.25 mM L-homocysteine, 0.15 mM 5methyltetrahydrofolate, 0.15 mM Sadenosylmethionine and 0.5 mM L-methionine (14). In these experiments we employed an enzyme preparation partially purified from rat liver. In a detailed kinetic study, Burke et al. found that S-
DISCUSSION
In mammalian liver, methionine metabolism is a cycle with a single, major outlet at the cystathionine synthase reaction (Fig. 1, Reaction 4). As homocysteine is ultilized in this reaction, it is committed irreversibly to the transsulfuration sequence-the primary means for methionine catabolism (15). Alternatively, homocysteine may be methylated thereby maintaining the tissue content of methionine. There are two homocysteine methylases. 5-Methyltetrahydrofolate-homocysteine methyltransferase (Fig. 1, Reaction 8) is present in all rat tissues except the small intestinal mucosa (14). In contrast, only liver contains significant amounts of betaine-homocysteine methyltransferase (Fig. 1, Reaction 7) (14). Alterations in the dietary intake of protein (or methionine) have different effects on these two methyl-
778
FINKELSTEIN,
r-
KYLE
[L-METHIONINI ,
AND HARRIS
-‘@ Q-CH,SH+
UNKNOWN PRODVCT
I-l-S-ADENOSYL-L-METHIONINE KCEPTORS 0
METHYLATEOACCEPTORS
S-ADENOSYL-L-HOMOCYSTEINE
0
ADENOSINE _ mm~_---/ IL-~~OMOCYSTEINEI ! .a
1
L-SER,NE
-
0
L-HOMOCYSTINE
03 H>S + a-KETOBUTYRATE
IL-CYSTATHIONI~ 0
PROPIONATE
“-KETOSUTYRATE
IL-CYSTEINEI
so,
co2
0 i JSOfj
FIG. 1. Known pathways of mammalian metabolism of methionine and homocysteine. Abbreviations: ATP, adenosine 5’-triphosphate, PP,, inorganic pyrophosphate, P,, inorganic orthophosphate, FH,, tetrahydrofolic acid; mSFH,, 5-methyltetrahydrofolic acid.
ases. High protein feeding or supplementation with methionine results in an increase in the hepatic content of the bemine enzyme. Simultaneously, the level of 5methyltetrahydrofolate-homocysteine methyltransferase decreases. On the basis of these observations, we suggested that the folate enzyme was more significant in the maintenance of the tissue concentration of methionine while the betaine enzyme might be of primary importance in the catabolism of choline (14). This hypothesis is supported by the finding that a decreased level of tissue methionine characterizes patients with decreased activity of 5-methyltetrahydrofolate-homocysteine methyltransferase (3). The protein (and methionine) content of the diet also regulates the hepatic level of cystathionine synthase (1, 16). The activity of this enzyme is increased by either increased protein intake or the administration of supplemental methionine. In summary, rats fed a low-methionine diet may conserve this amino acid by reducing the flow of homocysteine into the transsulfuration sequence (decreased cystathionine
synthase) and by increasing the resynthesis of methionine (increased &methyltetrahydrofolate-homocysteine methyltransferase). In rats fed excessive methionine we observed, on the other hand, an increase in cystathionine synthase and a decrease in the folate methyltransferase. Whether the latter changes in enzyme activity would allow increased transsulfuration and reduced homocysteine methylation is less clear since excess dietary methionine also increases the activity of betaine-homocysteine methyltransferase. Conceivably the decrease in methylation from 5-methyltetrahydrofolate would be balanced by an increase in methylation from betaine. As illustrated by the present paper as well as by earlier studies, the distribution of homocysteine between cystathionine synthase and betaine-homocysteine methyltransferase may be regulated by the kinetic properties of these two competing enzymes. The Km for homocysteine in the betaine reaction is 12 pM (9) while the K, for this substrate in the cystathionine synthase reaction has been reported to approximate 10 mM (10, 17-19). As the concentra-
REGULATORY
EFFECTS
OF S-ADENOSYLHOMOCYSTEINE
tion of homocysteine increases we would anticipate that a greater fraction would be utilized for the synthesis of cystathionine. Distribution in favor of cystathionine synthase would be facilitated by other properties of the two enzymes. Since homocysteine does not occur in dietary protein, homocysteine accumulation is likely to occur in the normal animal only as the result of the ingestion of excessive methionine. Furthermore, the hydrolase reaction (Fig. 1, Reaction 3) which generates homocysteine is reversible and the equilibrium favors the synthesis of S-adenosylhomocysteine rather than its hydrolysis (20). For these reasons, the accumulation of homocysteine in tissues should be associated with a parallel increase in methionine and S-adenosylhomocysteine. Methionine is a competitive inhibitor of homocysteine in the betaine-homocysteine methyltransferase reaction (9) and in the present study we have demonstrated that S-adenosylhomocysteine has similar properties. The accumulation of these two metabolites might inhibit the betaine reaction until the tissue concentration of homocysteine beSimultaneously. incame “saturating.” creased concentrations of S-adenosylhomocysteine might activate cystathionine synthase independent of the level of homocysteine. ACKNOWLEDGMENT We are indebted to Ann-Marie Pick for her assistance in many aspects of this study. REFERENCES 1. FINKELSTEIN, J. D., AND MUDD, S. H. (1967) J. Biol. Chem. 242, 873-880.
779
2. FINKELSTEIN, J. D. (1971) in Inherited
3. 4. 5. 6.
7. 8. 9. 10. 11.
12. 13. 14. 15.
16.
Disorders of Sulphur Metabolism (Carson, N. A. J., and Raine, D. N., eds.), pp 1-13, Churchill Livingstone, London. FINKELSTEIN, J. D. (1974) Metabolism 23, 387-398. ZAPPIA, V., ZYDEK-CWICK, C. R., AND SCHLENK, F. (1969). J. Biol. Chem. 244, 4499-4509. DEGUCHI, T., AND BARCHAS, J. (19711 J. Viol. Chen. 246,3175-3181. COWARD, J. K., D’URSO-SCOTT, M., AND SWEET, W. D. (1972) Biochem. Pharm. 21, 1200-1203. KUTZBACH, C., AND STOKSTAD, E. L. R. (1967) Biochem. Biophys. Acta 139, 217-220. BURKE, G. T., MANGUM, J. H., AND BRODIE, J. D. (1971) Biochemistp 10, 3079-3085. FINKELSTEIN, J. D., HARRIS, B. J., AND KYLE, W. E. (1972) Arch. Biochem. Biophys. 153, 320-324. NAKAGAWA, H., AND KIMURA, H. (1968) Biochem. Biophyy. Res. Commun. 32, 208-214. MUDD, S. H., FINKELSTEIN, J. D., IRREVERRE, F., AND LASTER, L. (1965) J. Biol. Chem. 240, 4382-4392. WILKINSON, G. N. (1961) Biochem. J. 80, 324-332. CLEI,AND, W. W. (1971) Ann. Rec. Biochem. 36, 77-112. FINKELSTEIN, J. D., KYLE, W E., AND HARRIS, B. J. (1971) Arch. Biochem. Biophys. 146, 84-92. LASTER L., MUDD, S. H., FINKELSTEIN, J. D., AND IRREVERRE, F. (1965) J. Clin. Invest. 44, 1708-1719. FINKELSTEIN, J. D. (1967) A&. Biochem. Biophys. 122,583-590.
17 KASHIWAMATA, S., AND GREENBERC, D. M. (1970) Biochem. Biophys. Acta. 212, 48%500. 18 BROWN, F. C., ANDGORDON, P. H. (1971) Canad. J. Biochem. 49, 484-491. 19. BRAUNSTEIN, A. E., GORYACHENKO~A, E. V., TOLOSA, E. Z., WILLHARDT, I. H., AND YEFREMOVA, L. L. (1971) Biochim. Biophys. Acta. 242, 247-260. 20. DE LA HABA, G., AND CANTONI, G. L. (1959) J. Viol. ’ Chem. 234, 603-608.