ANALYTICAL
BIOCHEMISTRY
207,68-72
(1992)
The Use of a Spectrophotometric Interaction of S-Adenosylmethionine with Methionine Analogues Ho Jin Kim, Department
Received
Thomas
of Chemistry,
May
J. Balcezak, Amherst
Steven
College, Amherst,
J. Nathin,
Heather
Massachusetts
F. McMullen,
and David
E. Hansen
01002
21, 1992
We have developed a continuous spectrophotometric assay for S-adenosylmethionine synthetase and, using this assay, have examined the interaction of five potential inhibitors with the E. coli enzyme. S-Vinylhomocysteine and S-allylhomocysteine were found to be substrates, while S-(methanethio)cysteine and S-(methanethio)homocysteine were found to be competitive inhibitors. S-Cyanohomocysteine is neither a substrate 0 1992 Academic Press, Inc. nor an inhibitor.
S-Adenosylmethionine synthetase (adenosine 5’triphosphate:L-methionine S-adenosyltransferase, EC 2.5.1.6) catalyzes the reaction MgATP
Assay to Study the Synthetase
+ L-methionine
+ H,O +
S-adenosyl-L-methionine
+ Pi + PPi.
The product S-adenosylmethionine is the primary methyl group donor in intermediary metabolism and is utilized by methyltransferases for the methylation of RNA, DNA, histones, proteins, polysaccharides, steroids, and numerous other metabolites. Methylation can occur on carbon, oxygen, sulfur, and nitrogen, and a number of metabolic functions including chemotaxis, restriction-modification of DNA, amino acid modification, and neurotransmitter biosynthesis depend upon S-adenosylmethionine (1). The concentration of Sadenosylmethionine, and hence the level of methylation activity, is controlled primarily by S-adenosylmethionine synthetase and by S-adenosylhomocysteine hydrolase, which degrades S-adenosylhomocysteine, a potent product inhibitor of most S-adenosylmethionine utilizing enzymes, to homocysteine and adenosine (2). Many analogues of methionine, S-adenosylhomocysteine, and S-adenosylmethionine have been studied as
possible substrates and inhibitors of the enzymes involved in S-adenosylmethionine metabolism. Ethionine and S-trifluoromethylhomocysteine are inhibitors of rat-liver S-adenosylmethionine synthetase (3), and the ethyl and propyl analogues of S-adenosylmethionine are substrates for some methyl transferases (4,5). Much effort has been expended on the synthesis of inhibitors of these enzymes, yet, with the important exception of a series of “multisubstrate analogues” synthesized by Anderson et al. (6) and by Vrudhula et al. (7), few rationally designed inhibitors have been made. Notably, in a study of the inhibition of S-adenosylmethionine synthetase derived from rat tumor, Lombardini and Sufrin (8) repoked that the tumor enzyme “displays kinetic and physical properties and responses to methionine analogue inhibitors or substrates that are different than those found for [S-adenosylmethionine synthetases] in normal tissues, thus implying altered active site regions” and suggested that these regions may “provide an exploitable target for cancer chemotherapy.” Given this interest in S-adenosylmethionine synthetase inhibitors, we initiated the study described herein. As a first step in our study of potential new inhibitors, we developed a continuous spectrophotometric assay of the enzyme. All of the studies discussed above utilized an assay for S-adenosylmethionine synthetase first described by Mudd et al. (9), in which the rate of incorporation of radiolabeled methionine into S-adenosylmethionine is measured. Our spectrophotometric assay monitors the production of the product pyrophosphate using a coupled enzyme system initiated by pyrophosphate-dependent fructose-6-phosphate kinase and culminating in the oxidation of 2 moles of NADH per mole of pyrophosphate. Argininosuccinate synthetase was the first pyrophosphate-producing enzyme assayed using this technique (10). For our purposes, the advantages of the spectrophotometric assay are twofold: one, the nonradioactive, continuous assay is far simpler to
68 All
Copyright 0 1992 rights of reproduction
0003-269’7192 $5.00 by Academic Press, Inc. in any form reserved.
SPECTROPHOTOMETRIC
ASSAY
OF
S-ADENOSYLMETHIONINE
‘:H3N+Y-s- ‘I:H3N+Y-sCO?
IV: H3N+TS,s,CH, Sushi, CO,’
COZ’ V: H,N+?S-CGN cop-
FIG. 1. The methionine analogues S-vinylhomocysteine (I), S-allylhomocysteine (II), S-(methanethiojcysteine (III), S-(methanethio)homocysteine (IV), and S-cyanohomocysteine (V).
run; and two, all of our analogues could be tested as substrates (as each will liberate pyrophosphate, but presumably a different S-adenosylmethionine analogue) with this one assay. We have examined the compounds I-V shown in Fig. 1 as potential inhibitors of Escherichia coli S-adenosylmethionine synthetase. (We chose the E. coli enzyme for our initial studies because of its ease of purification and kinetic simplicity (II).) Each is approximately isosteric with methionine, ethionine, or S-propylhomocysteine. Potentially, each of the analogues can act as a competitive inhibitor, as a substrate, or as a mechanism-based inhibitor (12,13). To date, no mechanism-based inhibitors of S-adenosylmethionine synthetase have been reported. Compounds I-V are each stable in the sulfide form, but will become reactive electrophiles if enzymatitally processed to the corresponding sulfonium salt. If instead the S-adenosyl sulfonium products are released from the enzyme-that is, if the methionine analogues act as substrates-their interaction with methyl transferases would be of great interest. For example, selective inhibition of catechol-0-methyltransferase would be of enormous therapeutic utility (14).
MATERIALS
Fox Chase Cancer Center
69 (Phila-
co*-
III: H,N+ 7
George D. Markham, delphia, PA).
SYNTHETASE
AND
METHODS
General Melting points are uncorrected. IR spectra were measured with a Bruker IFS 66 instrument, and ‘H and 13C NMR spectra were measured with a Bruker AC250 or JEOL FX-100 instrument. Enzymatic rates were determined with a Perkin-Elmer Lambda 3B spectrophotometer with a thermostatted cell holder. Gel electrophoresis was run using a Pharmacia PhastSystern. Elemental analyses were performed by the Microanalysis Laboratory at the University of Massachusetts (Amherst, MA). All chemicals were obtained from Aldrich Chemical CO. or Sigma Chemical Co. and were used without further purification. The E. coli K-12 strain EWH205/pCC27-37 was a gift from Professor
Enzyme
Purification
and Assays
S-Adenosylmethionine synthetase was purified from an overproducing E. coli K-12 strain described by Markham et al. (11) except that the final DEAE-A50 Sephadex column was omitted and that the enzyme was assayed spectrophotometrically as described below. Purified enzyme migrated as one band on denaturing polyacrylamide gel electrophoresis and was assumed, as previously reported (ll), to have an OD,,, of 1.3 for a 1 mg/ml solution, path length 1 cm. S-Adenosylmethionine synthetase was assayed by spectrophotometrically measuring pyrophosphate production as coupled to fructose-6-phosphate kinase (pyrophosphate dependent from P. shermanii), aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase with concomitant oxidation of 2 moles of NADH per mole of pyrophosphate. These enzymes and requisite substrates are available as a prepackaged kit from Sigma Chemical Co. Thus, a typical assay contained 240 mM imidazole-HCl buffer, pH 8.4, pyrophosphate assay mix (300 ~1 of mix reconstituted in 4 ml water), adenosine triphosphate (10 mM), andmethionine (1 mM), containing 20 mM MgCl, and 100 mM KCI, in a final volume of 1 ml, 30°C. (Reagents in the pyrophosphate assay mix, upon dilution into our assay, gave imidazole (13.5 mM), citrate (0.15 mM), EDTA (0.03 mM), MgCl, (0.6 mM), MnCl, (60 FM), CoCl, (6 PM), NADH (0.24 mM), D-fructose 6-phosphate (3.6 mM), bovine serum albumin (1.5 mg), Ficoll (1.5 mg), D-fructose-6-phosphate kinase, pyrophosphate-dependent (0.15 units), aldolase (2.25 units), a-glycerophosphate dehydrogenase (1.5 units), and triosephosphate isomerase (15 units).) A background rate, measured in the absence of methionine, was subtracted from all enzymatic rates. One unit of S-adenosylmethionine synthetase gives an OD,,, change of 12.44/min (1 cm path length), and the assay is linear for enzyme concentrations of O-0.002 units and 0.003-0.01 units (with a slight break in linearity at approximately 0.0025 units). With respect to time, the assay is linear, after an initial lag phase, for between 5 and 20 min, depending on enzyme and substrate concentration. All kinetic constants were determined from Lineweaver-Burk plots. The data were statistically analyzed using the programs HYPER and COMP and the standard errors determined as described by Cleland (15). The K,,, and relative V,,, for the substrates L-methionine, L-ethionine, S-vinyl-DL-homocysteine, and Sallyl-DL-homocysteine were determined using the assay described above; in addition each was determined with a buffer concentration of 150 mM, pH 8.4. The Ki for the inhibitors DL-homocysteine (at concentrations of 10
70
KIM
ET
and 15 mM), S-(methanethio)-L-cysteine (0.6 and 1 mM), and S-(methanethio)-DL-homocysteine (1 and 2 mM) were determined using the assay described above, as were tests of S-cyano-L-homocysteine as a substrate and competitive inhibitor (at concentrations of 2 and 4 mM). Where racemic compounds were used, it was assumed that the D-isomer had no effect on the kinetics. The Ki for the inhibitor cycloleucine (at concentrations of 5 and 10 mM) was determined using the assay described above, except MgCl, concentration was 6 mM, the ATP concentration was 4.3 mM, and the buffer concentration was 13.5 mM. Irreversible mechanism-based inhibition was assayed by two methods. In the first, enzyme was incubated at 25°C with ATP (10 mM) and inhibitor (20 mM) in 166 mM imidazole buffer, pH 8.4, containing 20 mM MgCl, and 100 mM KCl, total volume 200 ~1. Every 20 min for 2 h, a 20-/*l aliquot was removed and assayed for enzymatic activity as described above. As a control, the enzyme was incubated with ethionine (20 mM). In the second, the above reaction mixture was incubated for 60 min at 25°C dialyzed against 50 mM Tris-HCl, pH 8.0, containing 50 mM KCl, 1 mM EDTA, and 0.1% 2-mercaptoethanol, and assayed for enzymatic activity. As controls, the enzyme was incubated with ethionine (20 mM) and in the absence of a sulfur-containing substrate. Substrates and Inhibitors L-Methionine, L-ethionine, DL-homocysteine, and cycloleucine were from Sigma; S-(methanethio)-L-cysteine (3-(methyldithio)-L-alanine) was synthesized as described by Smith et al. (16); S-vinyl-DL-homocysteine (S-ethenyl-DL-homocysteine)
was
synthesized
as de-
scribed by Leopold et al. (17); S-cyano-L-homocysteine ((S)-2-amino-4-thiocyanatobutanoic acid) was a gift from Professor Charlotte Ressler, University of Connecticut Health Center. The ‘H and 13CNMR spectra of these compounds are consistent with their structures. S-Allyl-DL-homocysteine (S-(2-propenyl)-DL-homocysteine) was synthesized by a modification of the procedure of Kolenbrander (18). DL-Homocysteine thiolactone.
HCl(O.765
g, 5.0 mmol)
was added
to a solution
of
sodium methoxide (0.27 g, 11.7 mmol sodium metal in 21 ml methanol) at 0°C and the solution was stirred for 30 min. Ally1 chloride (407 ~1,5.0 mmol) was then added over a period of 5 min, and the resulting mixture was stirred for 1.25 h. The reaction mixture was filtered, and the filtrate was concentrated to a volume of 2 ml by evaporation under reduced pressure. Then, 1 N NaOH (30 ml) was added, and the resulting solution refluxed for
1.5 h, concentrated
to 5 ml by evaporation
under
reduced pressure, and cooled to 0°C. The pH was lowered to 5 by addition of concentrated HCl, and the resulting suspension was filtered. The precipitate was dis-
AL. TABLE
Comparison
1
of Kinetic Constants for Methionine with Old and New Assay K,,, (methionine) (mM)
Assay [YSlMethionine (11) [35S]Methionine (20) Spectrophotometric
0.08
Specific activityb (pmol/min/mg) 2.2 0.75 1.2
0.03-0.08’
’ During the course of this work, K,,, values measured for methionine varied in the range shown with standard errors of approximately 10% for each determination. * All specific activities determined at 25°C.
solved in hot water (4 ml), filtered, and absolute ethanol (16 ml) was added to the filtrate. Cooling at -20°C for 2 days yielded crystalline S-allyl-DL-homocysteine, which was obtained by vacuum filtration (0.150 g, 0.86 mmol, 17% yield, mp 200-205’C dec.); IR (CsI) v 3500-2700, 2105, 1698, 1582, 1435, 1415 cm-‘; ‘H NMR (250 MHz, D,O/DCl) 6 5.89 (m, lH), 5.27 (m, 2H), 4.32 (t, J = 6.4 Hz, lH), 3.31 (d, J = 7.2 Hz, 2H), 2.76 (t, J = 7.9 Hz, 2H), 2.33 (m, 2H) ppm; 13CNMR (62.9 MHz) 6 174.3, 136.5, 120.5, 54.5, 36.1, 32.0, 27.7 ppm. Anal. Calcd for C,H,,NO,S: C, 47.97; H, 7.49; N, 7.99; S, 18.29%. Found: C, 45.96; H, 7.19; N, 7.88; S, 18.13%. S-(Methanethio)-DL-homocysteine ((R,S)-2-amino4-(methyldithio)butanoic acid) was synthesized by a modification of the procedure of Smith et al. (16). DLHomocysteine (0.340 g, 2.5 mmol) was dissolved in water (13 ml), and the resulting solution was cooled to 0°C. To this solution was added methyl methanethiosulfonate (0.370 g, 2.9 mmol) in ethanol (5 ml) over a period of 10 min with stirring; a white precipitate formed immediately. After stirring for an additional 60 min, the precipitate was collected by vacuum filtration and washed with cold ethanol (2 X 5 ml) and then cold water (2 X 5 ml) to yield S-(methanethio)-DL-homocysteine (0.210 g, 1.4 mmol, 56% yield, mp 225-230°C dec.); IR (CsI) u 3500-2700,2105,1653,1581,1412,1342 cm-‘; ‘H NMR (250 MHz, D,O/DCl) S3.33 (t, J = 6.6 Hz, lH), 1.97 (t, J = 6.3 Hz, 2H), 1.50 (s, 3H), 1.48 (m, 2H) ppm; 13C NMR
(25.1 MHz)
6 174.3, 54.7, 34.9, 32.2, 25.1 ppm.
Anal. Calcd for C,H,,NO,S,: C, 33.12; H, 6.13; N, 7.73; S, 35.37%. Found: C, 32.78; H, 6.10; N, 7.44; S, 33.88%. RESULTS We have developed a continuous spectrophotometric assay for S-adenosylmethionine synthetase and have studied the E. coli enzyme. We have determined the specific activity for the enzyme and the K,,, for methionine, and our values are in good agreement with those determined using radiolabeled methionine and reported previously
(Table
1). In addition,
Km and relative V,, ble 2).
we have
determined
the
for the substrate ethionine (Ta-
SPECTROPHOTOMETRIC TABLE
ASSAY
OF
S-ADENOSYLMETHIONINE
71
SYNTHETASE
2
Kinetic Constants for Substrates of E. coli S-Adenosylmethionine Synthetase Substrate Methionine Ethionine S-Vinylhomocysteine S-Allylhomocysteine
K,,, (mM)’ 0.07
V,,,,, (rel)”
k 0.004 4 s 0.5
6+1 10 * 2
100 k 2 62 + 2 16 f 2
10 * 1
V,,,,, (rel)*
K,,, (mM)b 0.04
* 0.003 6&l 8f2 8-+1
100 f 2 44 * 4 14 * 2 5 + 0.3
a240
mM imidazolebuffer. * 150 mM imidazole buffer.
Since enzymatic processing of the five analogues I-V to yield a reactive sulfonium salt is a requisite for mechanism-based inhibition, each was tested as a substrate for S-adenosylmethionine synthetase. S-Vinylhomocysteine and S-allylhomocysteine were found to be substrates, while S-(methanethio)cysteine, S-(methanethio)homocysteine, and S-cyanohomocysteine were not. The values for K,,, and relative V,,,,, for S-vinylhomocysteine and S-allylhomocysteine are listed in Table 2. Since these two compounds are substrates and thus presumably form the reactive sulfonium salts discussed above, they were tested as mechanism-based inhibitors. The S-vinyl or the S-ally1 analogue was incubated with the enzyme in the presence of ATP and the essential metal ions, and enzymatic activity was assayed every 20 min for 2 h by diluting a small volume of incubation solution into a large excess of assay mix. No loss of enzymatic activity, as compared with incubation with ethionine as a control, was observed. (Note that in all three incubations a slow loss of activity-approximately 10% per hour-was observed.) In addition, incubation for 1 h followed by dialysis and subsequent assay showed no loss of activity as compared with incubation with ethionine and with incubation in the absence of a sulfurcontaining substrate as controls. (Again, a small loss of activity was observed in all of the incubations including the controls.) Thus, the vinyl and ally1 analogues are not mechanism-based inhibitors. The three nonsubstrates S-(methanethio)cysteine, S-(methanethio)homocysteine, and S-cyanohomocysteine were tested as competitive inhibitors with respect
TABLE
3
Inhibition Constants for Inhibitors of E. coli S-Adenosylmethionine Synthetase Inhibitor S-(Methanethio)cysteine S-(Methanethio)homocysteine S-Cyanohomocysteine Cycloleucine Homocysteine
K; (mM) 0.6 k 0.2 0.6 + 0.1 2 + 0.3 5 2 0.4
-20
-10
0
10
l/fmethlonine]
(1imM)
20
30
FIG.
2. Inhibition of S-adenosylmethionine synthetase by S-(methanethio)homocysteine. Methionine concentrations were 0.05, 0.10, 0.25, 0.50, and 1.0 mM with fixed S-DL-(methanethio)homocysteine concentrations of zero (squares), 1.0 (triangles), and 2.0 was calcu(circles) mM. The K, for S- L -( me th anethio)homocysteine lated to be 0.6 + 0.1 mM. (The units of l/u are arbitrary.)
to methionine, and the Ki values listed in Table 3 were determined (the S-cyano compound was not an inhibitor). Each value is based upon data obtained at two different inhibitor concentrations. A Lineweaver-Burk plot demonstrating of the S-(methanethio)homocysteine inhibition data is shown in Fig. 2. In addition, Ki values for homocysteine and the known inhibitor cycloleucine were determined (Table 3). The Ki value for cycloleucine (2 mM) agrees well with that measured with rat liver isozymes (0.5, 0.3, and 1.6 mM) (8). DISCUSSION
We have developed a spectrophotometric assay for the enzyme S-adenosylmethionine synthetase and have used it to study a number of substrates and inhibitors for this enzyme. Specifically, we have examined the interaction of the methionine analogues I-V shown in Fig. 1 with E. coli S-adenosylmethionine synthetase. Each is roughly isosteric with methionine, ethionine, or S-propylhomocysteine, and each could act as a competitive inhibitor, as a substrate, or as a mechanism-based inhibitor. The data reported here indicate that the Svinyl and S-ally1 derivatives I and II are substrates (Table 2), and the disulfide derivatives III and IV are competitive inhibitors with respect to methionine (Table 3). The S-cyano derivative V is neither a substrate nor an inhibitor. The failure of the vinyl and ally1 analogues I and II to irreversibly inactivate the enzyme, despite their being substrates, is intriguing. Most likely, a nucleophilic amino-acid side chain is not in proximity with the enzyme bound sulfonium salt, a reasonable expectation
72
KIM
given the electrophilic nature of S-adenosylmethionine itself. Of course, we had hoped that the additional electrophilic sites on our analogues would allow for mechanism-based inhibition. Currently, we are attempting to elucidate the structure of the products produced from analogues I and II, and in addition, to test if they are catechol-0-methyltransferase inhibitors. The Ki, 0.6 mM, of each of the disulfide derivatives III and IV is comparable to that of a number of competitive inhibitors previously reported with rat liver isozymes (8). That the cyano derivative V shows no interaction with the enzyme may be due to the linear structure of the thiocyanate group (19), whereas the thio functionality in each of the other inhibitors is bent. Future work will include the attempted synthesis of additional analogues, such as S-ethynylhomocysteine and S-cyclopropylhomocysteine, that may function as mechanism-based inhibitors, and the study of the interaction of the analogues I-V with a mammalian synthetase. ACKNOWLEDGMENTS The authors are grateful to the Amherst College Research Award Program for support of this research. D.E.H. is a National Science Foundation Presidential Young Investigator. We also are indebted to Professor George D. Markham for numerous helpful conversations and for his gift of the engineered E. coli strain. Finally, we are pleased to thank Professor Charlotte Ressler for a sample of S-cyanohomocysteine.
REFERENCES 1. Usdin, E., Borchardt, R. T., and Creveling, Biochemistry of S-Adenosylmethionine pounds, Macmillan & Co., London. 2. Salvatore,
F., Borek,
E., Zappia,
C. R. (Eds.) and Related
V., Williams-Ashman,
(1982) Com-
ET
AL. Schlenk, F. (Eds.) (1977) The Biochemistry ine, Columbia Univ. Press, New York.
3. Cox, R., and Smith, R. C. (1969) Arch. Biochem. Biophys. 129, 615-619. 4. Parks, L. W. (1958) J. Biol. Chem. 232,169-176. 5. Schlenk, F., and Dainko, J. L. (1975) Biochim. Biophys. Acta 385, 312-323. 6. Anderson, G. L., Bussolotti, Med. Chem. 24, 1271-1277. I. Vrudhula, V. M., Chem. 30,88&894. 8. Lombardini, 489-495.
9. Mudd, J. Biol. 10. O’Brien,
D. L., and Coward,
Kappler,
F., and
J. B., and Sufrin,
Hampton,
R. R. (1983)
S. H., Finkelstein, J. D., Irreverre, Chem. 240,4382-4392. W. E. (1976)
Anal.
Biochem.
J.
J. Med.
Phurm.
32,
L. (1965)
76,423-430.
R. R., Brock, 3229-3231.
13. Walsh,
Reu. Biochem.
Annu.
A. (1987)
F., and Laster,
12. Helmkamp, G. M., Rando, (1968) J. Biol. Chem. 243, C. T. (1984)
J. K. (1981)
Biochem.
11. Markham, G. D., Hafner, E. W., Tabor, (1980) J. Biol. Chem. 255,9082-9092.
C. W., and
Tabor,
H.
D. J. H., and Bloch,
K.
53,493-535.
14. Backstrom, R., Honkanen, E., Pippuri, A., Kairisalo, P., Pystynen, J., Heinola, K., Nissinen, E., Linden, I.-B., Mannisto, P. T., Kaakkola, S., and Pohto, P. (1989) J. Med. Chem. 32,841846. 15. Cleland, W. W. (1979) Methods Enzymol. 63, 103-138. 16. Smith, D. J., Maggio, try 14,766-771.
E. T., and Kenyon,
G. L. (1975)
Biochemis-
17. Leopold, W. R., Miller, J. A., and Miller, E. C. (1982) Cancer 42,4364-4374. 18. Kolenbrander, H. M. (1969) Can. J. Chem. 47,3271-3273. 19. Beard, 936. 20.
H., and
of Adenosylmethion-
C. I., and Dailey,
Markham, G. D., Hafner, (1983) Methods Enzymol.
B. P. (1949)
J. Am.
E. W., Tabor, 94,219-222.
Chem.
Res.
Sot. 71,929-
C. W., and
Tabor,
H.
BIOCHEMISTRY
207,68-72
(1992)
The Use of a Spectrophotometric Interaction of S-Adenosylmethionine with Methionine Analogues Ho Jin Kim, Department
Received
Thomas
of Chemistry,
May
J. Balcezak, Amherst
Steven
College, Amherst,
J. Nathin,
Heather
Massachusetts
F. McMullen,
and David
E. Hansen
01002
21, 1992
We have developed a continuous spectrophotometric assay for S-adenosylmethionine synthetase and, using this assay, have examined the interaction of five potential inhibitors with the E. coli enzyme. S-Vinylhomocysteine and S-allylhomocysteine were found to be substrates, while S-(methanethio)cysteine and S-(methanethio)homocysteine were found to be competitive inhibitors. S-Cyanohomocysteine is neither a substrate 0 1992 Academic Press, Inc. nor an inhibitor.
S-Adenosylmethionine synthetase (adenosine 5’triphosphate:L-methionine S-adenosyltransferase, EC 2.5.1.6) catalyzes the reaction MgATP
Assay to Study the Synthetase
+ L-methionine
+ H,O +
S-adenosyl-L-methionine
+ Pi + PPi.
The product S-adenosylmethionine is the primary methyl group donor in intermediary metabolism and is utilized by methyltransferases for the methylation of RNA, DNA, histones, proteins, polysaccharides, steroids, and numerous other metabolites. Methylation can occur on carbon, oxygen, sulfur, and nitrogen, and a number of metabolic functions including chemotaxis, restriction-modification of DNA, amino acid modification, and neurotransmitter biosynthesis depend upon S-adenosylmethionine (1). The concentration of Sadenosylmethionine, and hence the level of methylation activity, is controlled primarily by S-adenosylmethionine synthetase and by S-adenosylhomocysteine hydrolase, which degrades S-adenosylhomocysteine, a potent product inhibitor of most S-adenosylmethionine utilizing enzymes, to homocysteine and adenosine (2). Many analogues of methionine, S-adenosylhomocysteine, and S-adenosylmethionine have been studied as
possible substrates and inhibitors of the enzymes involved in S-adenosylmethionine metabolism. Ethionine and S-trifluoromethylhomocysteine are inhibitors of rat-liver S-adenosylmethionine synthetase (3), and the ethyl and propyl analogues of S-adenosylmethionine are substrates for some methyl transferases (4,5). Much effort has been expended on the synthesis of inhibitors of these enzymes, yet, with the important exception of a series of “multisubstrate analogues” synthesized by Anderson et al. (6) and by Vrudhula et al. (7), few rationally designed inhibitors have been made. Notably, in a study of the inhibition of S-adenosylmethionine synthetase derived from rat tumor, Lombardini and Sufrin (8) repoked that the tumor enzyme “displays kinetic and physical properties and responses to methionine analogue inhibitors or substrates that are different than those found for [S-adenosylmethionine synthetases] in normal tissues, thus implying altered active site regions” and suggested that these regions may “provide an exploitable target for cancer chemotherapy.” Given this interest in S-adenosylmethionine synthetase inhibitors, we initiated the study described herein. As a first step in our study of potential new inhibitors, we developed a continuous spectrophotometric assay of the enzyme. All of the studies discussed above utilized an assay for S-adenosylmethionine synthetase first described by Mudd et al. (9), in which the rate of incorporation of radiolabeled methionine into S-adenosylmethionine is measured. Our spectrophotometric assay monitors the production of the product pyrophosphate using a coupled enzyme system initiated by pyrophosphate-dependent fructose-6-phosphate kinase and culminating in the oxidation of 2 moles of NADH per mole of pyrophosphate. Argininosuccinate synthetase was the first pyrophosphate-producing enzyme assayed using this technique (10). For our purposes, the advantages of the spectrophotometric assay are twofold: one, the nonradioactive, continuous assay is far simpler to
68 All
Copyright 0 1992 rights of reproduction
0003-269’7192 $5.00 by Academic Press, Inc. in any form reserved.
SPECTROPHOTOMETRIC
ASSAY
OF
S-ADENOSYLMETHIONINE
‘:H3N+Y-s- ‘I:H3N+Y-sCO?
IV: H3N+TS,s,CH, Sushi, CO,’
COZ’ V: H,N+?S-CGN cop-
FIG. 1. The methionine analogues S-vinylhomocysteine (I), S-allylhomocysteine (II), S-(methanethiojcysteine (III), S-(methanethio)homocysteine (IV), and S-cyanohomocysteine (V).
run; and two, all of our analogues could be tested as substrates (as each will liberate pyrophosphate, but presumably a different S-adenosylmethionine analogue) with this one assay. We have examined the compounds I-V shown in Fig. 1 as potential inhibitors of Escherichia coli S-adenosylmethionine synthetase. (We chose the E. coli enzyme for our initial studies because of its ease of purification and kinetic simplicity (II).) Each is approximately isosteric with methionine, ethionine, or S-propylhomocysteine. Potentially, each of the analogues can act as a competitive inhibitor, as a substrate, or as a mechanism-based inhibitor (12,13). To date, no mechanism-based inhibitors of S-adenosylmethionine synthetase have been reported. Compounds I-V are each stable in the sulfide form, but will become reactive electrophiles if enzymatitally processed to the corresponding sulfonium salt. If instead the S-adenosyl sulfonium products are released from the enzyme-that is, if the methionine analogues act as substrates-their interaction with methyl transferases would be of great interest. For example, selective inhibition of catechol-0-methyltransferase would be of enormous therapeutic utility (14).
MATERIALS
Fox Chase Cancer Center
69 (Phila-
co*-
III: H,N+ 7
George D. Markham, delphia, PA).
SYNTHETASE
AND
METHODS
General Melting points are uncorrected. IR spectra were measured with a Bruker IFS 66 instrument, and ‘H and 13C NMR spectra were measured with a Bruker AC250 or JEOL FX-100 instrument. Enzymatic rates were determined with a Perkin-Elmer Lambda 3B spectrophotometer with a thermostatted cell holder. Gel electrophoresis was run using a Pharmacia PhastSystern. Elemental analyses were performed by the Microanalysis Laboratory at the University of Massachusetts (Amherst, MA). All chemicals were obtained from Aldrich Chemical CO. or Sigma Chemical Co. and were used without further purification. The E. coli K-12 strain EWH205/pCC27-37 was a gift from Professor
Enzyme
Purification
and Assays
S-Adenosylmethionine synthetase was purified from an overproducing E. coli K-12 strain described by Markham et al. (11) except that the final DEAE-A50 Sephadex column was omitted and that the enzyme was assayed spectrophotometrically as described below. Purified enzyme migrated as one band on denaturing polyacrylamide gel electrophoresis and was assumed, as previously reported (ll), to have an OD,,, of 1.3 for a 1 mg/ml solution, path length 1 cm. S-Adenosylmethionine synthetase was assayed by spectrophotometrically measuring pyrophosphate production as coupled to fructose-6-phosphate kinase (pyrophosphate dependent from P. shermanii), aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase with concomitant oxidation of 2 moles of NADH per mole of pyrophosphate. These enzymes and requisite substrates are available as a prepackaged kit from Sigma Chemical Co. Thus, a typical assay contained 240 mM imidazole-HCl buffer, pH 8.4, pyrophosphate assay mix (300 ~1 of mix reconstituted in 4 ml water), adenosine triphosphate (10 mM), andmethionine (1 mM), containing 20 mM MgCl, and 100 mM KCI, in a final volume of 1 ml, 30°C. (Reagents in the pyrophosphate assay mix, upon dilution into our assay, gave imidazole (13.5 mM), citrate (0.15 mM), EDTA (0.03 mM), MgCl, (0.6 mM), MnCl, (60 FM), CoCl, (6 PM), NADH (0.24 mM), D-fructose 6-phosphate (3.6 mM), bovine serum albumin (1.5 mg), Ficoll (1.5 mg), D-fructose-6-phosphate kinase, pyrophosphate-dependent (0.15 units), aldolase (2.25 units), a-glycerophosphate dehydrogenase (1.5 units), and triosephosphate isomerase (15 units).) A background rate, measured in the absence of methionine, was subtracted from all enzymatic rates. One unit of S-adenosylmethionine synthetase gives an OD,,, change of 12.44/min (1 cm path length), and the assay is linear for enzyme concentrations of O-0.002 units and 0.003-0.01 units (with a slight break in linearity at approximately 0.0025 units). With respect to time, the assay is linear, after an initial lag phase, for between 5 and 20 min, depending on enzyme and substrate concentration. All kinetic constants were determined from Lineweaver-Burk plots. The data were statistically analyzed using the programs HYPER and COMP and the standard errors determined as described by Cleland (15). The K,,, and relative V,,, for the substrates L-methionine, L-ethionine, S-vinyl-DL-homocysteine, and Sallyl-DL-homocysteine were determined using the assay described above; in addition each was determined with a buffer concentration of 150 mM, pH 8.4. The Ki for the inhibitors DL-homocysteine (at concentrations of 10
70
KIM
ET
and 15 mM), S-(methanethio)-L-cysteine (0.6 and 1 mM), and S-(methanethio)-DL-homocysteine (1 and 2 mM) were determined using the assay described above, as were tests of S-cyano-L-homocysteine as a substrate and competitive inhibitor (at concentrations of 2 and 4 mM). Where racemic compounds were used, it was assumed that the D-isomer had no effect on the kinetics. The Ki for the inhibitor cycloleucine (at concentrations of 5 and 10 mM) was determined using the assay described above, except MgCl, concentration was 6 mM, the ATP concentration was 4.3 mM, and the buffer concentration was 13.5 mM. Irreversible mechanism-based inhibition was assayed by two methods. In the first, enzyme was incubated at 25°C with ATP (10 mM) and inhibitor (20 mM) in 166 mM imidazole buffer, pH 8.4, containing 20 mM MgCl, and 100 mM KCl, total volume 200 ~1. Every 20 min for 2 h, a 20-/*l aliquot was removed and assayed for enzymatic activity as described above. As a control, the enzyme was incubated with ethionine (20 mM). In the second, the above reaction mixture was incubated for 60 min at 25°C dialyzed against 50 mM Tris-HCl, pH 8.0, containing 50 mM KCl, 1 mM EDTA, and 0.1% 2-mercaptoethanol, and assayed for enzymatic activity. As controls, the enzyme was incubated with ethionine (20 mM) and in the absence of a sulfur-containing substrate. Substrates and Inhibitors L-Methionine, L-ethionine, DL-homocysteine, and cycloleucine were from Sigma; S-(methanethio)-L-cysteine (3-(methyldithio)-L-alanine) was synthesized as described by Smith et al. (16); S-vinyl-DL-homocysteine (S-ethenyl-DL-homocysteine)
was
synthesized
as de-
scribed by Leopold et al. (17); S-cyano-L-homocysteine ((S)-2-amino-4-thiocyanatobutanoic acid) was a gift from Professor Charlotte Ressler, University of Connecticut Health Center. The ‘H and 13CNMR spectra of these compounds are consistent with their structures. S-Allyl-DL-homocysteine (S-(2-propenyl)-DL-homocysteine) was synthesized by a modification of the procedure of Kolenbrander (18). DL-Homocysteine thiolactone.
HCl(O.765
g, 5.0 mmol)
was added
to a solution
of
sodium methoxide (0.27 g, 11.7 mmol sodium metal in 21 ml methanol) at 0°C and the solution was stirred for 30 min. Ally1 chloride (407 ~1,5.0 mmol) was then added over a period of 5 min, and the resulting mixture was stirred for 1.25 h. The reaction mixture was filtered, and the filtrate was concentrated to a volume of 2 ml by evaporation under reduced pressure. Then, 1 N NaOH (30 ml) was added, and the resulting solution refluxed for
1.5 h, concentrated
to 5 ml by evaporation
under
reduced pressure, and cooled to 0°C. The pH was lowered to 5 by addition of concentrated HCl, and the resulting suspension was filtered. The precipitate was dis-
AL. TABLE
Comparison
1
of Kinetic Constants for Methionine with Old and New Assay K,,, (methionine) (mM)
Assay [YSlMethionine (11) [35S]Methionine (20) Spectrophotometric
0.08
Specific activityb (pmol/min/mg) 2.2 0.75 1.2
0.03-0.08’
’ During the course of this work, K,,, values measured for methionine varied in the range shown with standard errors of approximately 10% for each determination. * All specific activities determined at 25°C.
solved in hot water (4 ml), filtered, and absolute ethanol (16 ml) was added to the filtrate. Cooling at -20°C for 2 days yielded crystalline S-allyl-DL-homocysteine, which was obtained by vacuum filtration (0.150 g, 0.86 mmol, 17% yield, mp 200-205’C dec.); IR (CsI) v 3500-2700, 2105, 1698, 1582, 1435, 1415 cm-‘; ‘H NMR (250 MHz, D,O/DCl) 6 5.89 (m, lH), 5.27 (m, 2H), 4.32 (t, J = 6.4 Hz, lH), 3.31 (d, J = 7.2 Hz, 2H), 2.76 (t, J = 7.9 Hz, 2H), 2.33 (m, 2H) ppm; 13CNMR (62.9 MHz) 6 174.3, 136.5, 120.5, 54.5, 36.1, 32.0, 27.7 ppm. Anal. Calcd for C,H,,NO,S: C, 47.97; H, 7.49; N, 7.99; S, 18.29%. Found: C, 45.96; H, 7.19; N, 7.88; S, 18.13%. S-(Methanethio)-DL-homocysteine ((R,S)-2-amino4-(methyldithio)butanoic acid) was synthesized by a modification of the procedure of Smith et al. (16). DLHomocysteine (0.340 g, 2.5 mmol) was dissolved in water (13 ml), and the resulting solution was cooled to 0°C. To this solution was added methyl methanethiosulfonate (0.370 g, 2.9 mmol) in ethanol (5 ml) over a period of 10 min with stirring; a white precipitate formed immediately. After stirring for an additional 60 min, the precipitate was collected by vacuum filtration and washed with cold ethanol (2 X 5 ml) and then cold water (2 X 5 ml) to yield S-(methanethio)-DL-homocysteine (0.210 g, 1.4 mmol, 56% yield, mp 225-230°C dec.); IR (CsI) u 3500-2700,2105,1653,1581,1412,1342 cm-‘; ‘H NMR (250 MHz, D,O/DCl) S3.33 (t, J = 6.6 Hz, lH), 1.97 (t, J = 6.3 Hz, 2H), 1.50 (s, 3H), 1.48 (m, 2H) ppm; 13C NMR
(25.1 MHz)
6 174.3, 54.7, 34.9, 32.2, 25.1 ppm.
Anal. Calcd for C,H,,NO,S,: C, 33.12; H, 6.13; N, 7.73; S, 35.37%. Found: C, 32.78; H, 6.10; N, 7.44; S, 33.88%. RESULTS We have developed a continuous spectrophotometric assay for S-adenosylmethionine synthetase and have studied the E. coli enzyme. We have determined the specific activity for the enzyme and the K,,, for methionine, and our values are in good agreement with those determined using radiolabeled methionine and reported previously
(Table
1). In addition,
Km and relative V,, ble 2).
we have
determined
the
for the substrate ethionine (Ta-
SPECTROPHOTOMETRIC TABLE
ASSAY
OF
S-ADENOSYLMETHIONINE
71
SYNTHETASE
2
Kinetic Constants for Substrates of E. coli S-Adenosylmethionine Synthetase Substrate Methionine Ethionine S-Vinylhomocysteine S-Allylhomocysteine
K,,, (mM)’ 0.07
V,,,,, (rel)”
k 0.004 4 s 0.5
6+1 10 * 2
100 k 2 62 + 2 16 f 2
10 * 1
V,,,,, (rel)*
K,,, (mM)b 0.04
* 0.003 6&l 8f2 8-+1
100 f 2 44 * 4 14 * 2 5 + 0.3
a240
mM imidazolebuffer. * 150 mM imidazole buffer.
Since enzymatic processing of the five analogues I-V to yield a reactive sulfonium salt is a requisite for mechanism-based inhibition, each was tested as a substrate for S-adenosylmethionine synthetase. S-Vinylhomocysteine and S-allylhomocysteine were found to be substrates, while S-(methanethio)cysteine, S-(methanethio)homocysteine, and S-cyanohomocysteine were not. The values for K,,, and relative V,,,,, for S-vinylhomocysteine and S-allylhomocysteine are listed in Table 2. Since these two compounds are substrates and thus presumably form the reactive sulfonium salts discussed above, they were tested as mechanism-based inhibitors. The S-vinyl or the S-ally1 analogue was incubated with the enzyme in the presence of ATP and the essential metal ions, and enzymatic activity was assayed every 20 min for 2 h by diluting a small volume of incubation solution into a large excess of assay mix. No loss of enzymatic activity, as compared with incubation with ethionine as a control, was observed. (Note that in all three incubations a slow loss of activity-approximately 10% per hour-was observed.) In addition, incubation for 1 h followed by dialysis and subsequent assay showed no loss of activity as compared with incubation with ethionine and with incubation in the absence of a sulfurcontaining substrate as controls. (Again, a small loss of activity was observed in all of the incubations including the controls.) Thus, the vinyl and ally1 analogues are not mechanism-based inhibitors. The three nonsubstrates S-(methanethio)cysteine, S-(methanethio)homocysteine, and S-cyanohomocysteine were tested as competitive inhibitors with respect
TABLE
3
Inhibition Constants for Inhibitors of E. coli S-Adenosylmethionine Synthetase Inhibitor S-(Methanethio)cysteine S-(Methanethio)homocysteine S-Cyanohomocysteine Cycloleucine Homocysteine
K; (mM) 0.6 k 0.2 0.6 + 0.1 2 + 0.3 5 2 0.4
-20
-10
0
10
l/fmethlonine]
(1imM)
20
30
FIG.
2. Inhibition of S-adenosylmethionine synthetase by S-(methanethio)homocysteine. Methionine concentrations were 0.05, 0.10, 0.25, 0.50, and 1.0 mM with fixed S-DL-(methanethio)homocysteine concentrations of zero (squares), 1.0 (triangles), and 2.0 was calcu(circles) mM. The K, for S- L -( me th anethio)homocysteine lated to be 0.6 + 0.1 mM. (The units of l/u are arbitrary.)
to methionine, and the Ki values listed in Table 3 were determined (the S-cyano compound was not an inhibitor). Each value is based upon data obtained at two different inhibitor concentrations. A Lineweaver-Burk plot demonstrating of the S-(methanethio)homocysteine inhibition data is shown in Fig. 2. In addition, Ki values for homocysteine and the known inhibitor cycloleucine were determined (Table 3). The Ki value for cycloleucine (2 mM) agrees well with that measured with rat liver isozymes (0.5, 0.3, and 1.6 mM) (8). DISCUSSION
We have developed a spectrophotometric assay for the enzyme S-adenosylmethionine synthetase and have used it to study a number of substrates and inhibitors for this enzyme. Specifically, we have examined the interaction of the methionine analogues I-V shown in Fig. 1 with E. coli S-adenosylmethionine synthetase. Each is roughly isosteric with methionine, ethionine, or S-propylhomocysteine, and each could act as a competitive inhibitor, as a substrate, or as a mechanism-based inhibitor. The data reported here indicate that the Svinyl and S-ally1 derivatives I and II are substrates (Table 2), and the disulfide derivatives III and IV are competitive inhibitors with respect to methionine (Table 3). The S-cyano derivative V is neither a substrate nor an inhibitor. The failure of the vinyl and ally1 analogues I and II to irreversibly inactivate the enzyme, despite their being substrates, is intriguing. Most likely, a nucleophilic amino-acid side chain is not in proximity with the enzyme bound sulfonium salt, a reasonable expectation
72
KIM
given the electrophilic nature of S-adenosylmethionine itself. Of course, we had hoped that the additional electrophilic sites on our analogues would allow for mechanism-based inhibition. Currently, we are attempting to elucidate the structure of the products produced from analogues I and II, and in addition, to test if they are catechol-0-methyltransferase inhibitors. The Ki, 0.6 mM, of each of the disulfide derivatives III and IV is comparable to that of a number of competitive inhibitors previously reported with rat liver isozymes (8). That the cyano derivative V shows no interaction with the enzyme may be due to the linear structure of the thiocyanate group (19), whereas the thio functionality in each of the other inhibitors is bent. Future work will include the attempted synthesis of additional analogues, such as S-ethynylhomocysteine and S-cyclopropylhomocysteine, that may function as mechanism-based inhibitors, and the study of the interaction of the analogues I-V with a mammalian synthetase. ACKNOWLEDGMENTS The authors are grateful to the Amherst College Research Award Program for support of this research. D.E.H. is a National Science Foundation Presidential Young Investigator. We also are indebted to Professor George D. Markham for numerous helpful conversations and for his gift of the engineered E. coli strain. Finally, we are pleased to thank Professor Charlotte Ressler for a sample of S-cyanohomocysteine.
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