Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES
Communication
Sulfonium Ion Condensation: the Burden Borne by SAM Synthetase Charles A. Lewis, and Richard Wolfenden Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00477 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
Sulfonium Ion Condensation: the Burden Borne by SAM Synthetase Charles A. Lewis, Jr and Richard Wolfenden* From the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260, United States Supporting Information Placeholder +
but also to hydrolytic cleavage of the bonds that join substituents to the 2- and 6- positions of adenine.5,6 To avoid those complications, we investigated the hydrolysis of S-methylmethionine (SMM+) as a simple model for SAM+.
ABSTRACT: S-Adenosylmethionine (SAM ) serves as
the principal methylating agent in biological systems, but the thermodynamic basis of its reactivity does not seem to have been clearly established. Here, we show that methionine, methanol and H+ combine to form Smethylmethionine (SMM+) with a temperatureindependent equilibrium constant of 9.9 M-2. The corresponding group transfer potential of SMM+ (its free energy of hydrolysis at pH 7) is -8.2 kcal/mol. The “energy-rich” nature of sulfonium ions is related to the extreme acidity (pKa -5.4) of the S-protonated thioether produced by sulfonium hydrolysis, and the large negative free energy of deprotonation of that species in neutral solution (-16.7 kcal/mol). At pH 7, SAM synthetase requires the free energy released by cleavage of two bonds of ATP to reverse that process. __________________________
Scheme 1: S-methylmethionine (SMM+) and Sadenosylmethionine (SAM+).
+
S-Adenosylmethionine (SAM ), discovered by Cantoni in 1952,1 serves as the major donor of methyl groups in biosynthetic processes, and in the epigenetic modification of proteins and nucleic acids. SMethylmethionine (SMM+), a simpler analogue, was discovered in plants two years later (Figure 1).2 The effectiveness of sulfonium ions as alkylating agents has long been recognized by organic chemists. But the thermodynamic basis of sulfonium reactivity in aqueous surroundings does not appear to have been established. That reactivity is of special interest in view of the unusual strategy employed by SAM synthetase, which uses a single ATP molecule (a) to furnish the adenosyl group of SAM+ and (b) to drive the reaction by coupling product release to the cleavage of a pyrophosphate bond.3 In the present experiments, we set out to measure the group transfer potential of sulfonium ions, i. e. the free energy released by their hydrolytic demethylation in dilute solution at pH 7.4 In water, SAM+ is subject not only to demethylation
Experiments were conducted at elevated temperatures in quartz tubes sealed under vacuum, using 1H NMR to monitor the disappearance of SMM+ and the appearance of methanol (MeOH) and homocysteine (Hcy). In preliminary experiments, we found that SMM+ was stable indefinitely in potassium phosphate buffer (0.1 M, pH 7.0) at ordinary temperatures. But at temperatures above 100°, hydrolysis proceeded to more than 99% completion at pH 7, precluding direct determination of its equilibrium constant under those conditions. This reaction can be described as the hydrolysis of a sulfonium ion (SMM+) to form methanol and an S-protonated thioether (Scheme 2). But Sprotonated thioethers are known to be extremely strong acids: the pKa value of S-protonated dimethyl sulfide, for example, is -5.4.7 If experiments could be conducted under sufficiently acidic conditions, it might be possible, at least in principle, to reverse
1 ACS Paragon Plus Environment
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
SMM+ hydrolysis and observe the condensation of methanol with methionine. The concentration of SMM+, if that were formed to a measurable extent, would allow the position of equilibrium of sulfonium hydrolysis to be determined.
Page 2 of 4
the free energy of methyl transfer from MeFH4 to homocysteine and (b) De la Haba and Cantoni’s free energy of hydrolysis of SAH (+7.2 kcal/mol).
group transfer potentials G°’pH 7 (kcal/mol, 25°)
Scheme 2: Hydrolysis of SMM+ to produce Sprotonated methionine (MetSH+).
-10
Upon heating methionine hydrochloride (0.1 M) with methanol (MeOH) (1 M) in aqueous HCl (1 M) at 120 °C for 18 h in quartz tubes sealed under vacuum (SI), we observed substantial conversion to SMM+. The approach to equilibrium was slow at lower temperatures (t1/2 = 60 h at 100 °C), but equilibrium was achieved overnight at 120° (Figure S1). When the initial concentrations of methionine (Met) and methanol (MeOH) were varied independently at 120 °C, the ratio of SMM+ to Met in the product mixture varied in direct proportion to the concentration of MeOH remaining in the product mixture, and also in proportion to the activity of H+ in HCl solutions with effective pH values between -3.0 and +2.0.8
(SMM+) (Met)(MeOH)(H+)
sulfate monoester
Me-S +Me2 (-8.2)
sulfonium ion (SMM +, SAM+)
acetyl-CoA (-7.5) ATP (-7.3) -5
0
Me-O-CO-CH 3 (-4.7) acetate ester Me-O-PO 3 (-3.3)
phosphate monoester
Me-OH (0) Me-FH 4 (+0.1)
methanol 5-methyltetrahydrofolate
Me-SMet (+7.2)
thioether (Met, SAH)
5
Conducting reactions in triplicate in the range between 120° and 150 °C, and taking the activity of water as 1.0 by convention,9 we obtained values of the equilibrium constant (Kformation) (equation 1) that did not vary significantly for conversion of Met + MeOH + H+ to SMM+: 9.74, 10.05, 10.17, and 9.56 M-2 at 120, 130, 140 and 150 °C, respectively (Figure S2). The constancy of these values with changing temperature indicates that ∆H for this reaction is essentially zero, consistent with a Kformation value of approximately 9.9 M-2 at 25 °C. Kformation =
Me-OSO3- (-8.9)
Figure 1: Group transfer potentials (in brackets) of methyl-bearing molecules. The broken vertical line indicates the bond undergoing hydrolysis. Table 1 shows that the value of ∆G°'pH 7 for SMM+ (-8.2 kcal/mol) resembles those of several other highenergy molecules including ATP, acetyl-CoA, and methyl sulfate, which range from -7.5 to -8.9 kcal/mol. These molecules vary, however, in the strengths of the acids released by their hydrolysis, and the contributions of ionization effects (∆Gionization) to their respective ∆G°'pH 7 values (Table 1).13 ATP, acetyl-CoA, and methyl sulfate release products with pKa values in the conventional pH range. But sulfonium ion hydrolysis generates a very strong acid (pKa -5.4), and the negative free energy of sulfonium ion hydrolysis includes that released by the loss of a proton from MetH+ at pH 7 (∆Gionization = -16.9 kcal/mol) as diagrammed in Figure 2. Without that driving force, sulfonium ion hydrolysis would be quite unfavorable (∆G’ = +8.6 kcal/mol).
(equation 1)
The reciprocal of that value, divided by 10-7 M, yields an equilibrium constant of 1.1 x 106 M for SMM+ hydrolysis at pH 7, equivalent to a group transfer potential (∆G°'pH 7) of -8.2 kcal/mol. Figure 1 compares the present ∆G°'pH 7 value with those of several other methyl-bearing molecules in biological systems.9 On this scale, the value for MeOH is zero by definition. The value for thioethers is based on the free energy of hydrolysis of Sadenosylhomocysteine (SAH) reported by De la Haba and Cantoni.11 The near-zero value for 5methyltetrahydrofolate (MTHF) represents the sum of (a) Rüdiger and Jaenicke’s value (-7.0 kcal/mol)12 for
ACS Paragon Plus Environment
2
Page 3 of 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biochemistry
fore SAM can dissociate.3 This obligatory order of product release, imposed by structural constraints within the enzyme-substrate complex, permits the free energy captured from two ATP cleavage events to be coupled to the costly formation of a sulfonium ion, tending to drive the methionine cycle in a productive direction.
∆G°'
pKa
∆Gionization
∆G’
pH 7
ref.
SMM
-5.4
-16.9
8.6
-8.2
Present work
CH3-SO4-
2.0
-6.8
-2.1
-8.9
10
acetyl-CoA
4.7
-3.1
-4.4
-7.5
9
ATP
7.2
(0)
-7.3
-7.3
9
+
ASSOCIATED CONTENT Supporting Information Experimental procedures; 1H NMR spectra of methionine and S-methylmethionine; effects of temperature on Kformation; free energies of interconversion of members of the methionine cycle. The Supporting Information is available free of charge on the ACS Publications website.
Table 1: Effects of product ionization on group transfer potentials (kcal/mol) of “high-energy” biomolecules.
AUTHOR INFORMATION Corresponding Author Richard Wolfenden, phone: 919-966-1203. Fax: (919)-966-2852.
ORCID Richard Wolfenden: 0000-00002-3745-9099
Notes The authors declare no competing financial interests. Author Contributions Figure 2: Influence of pH on ∆G for hydrolysis of SMM+, acetyl-CoA and ATP.
C.A.L. and R.W. performed the experiments and wrote this paper.
It is of interest to consider the present findings in the context of the methionine cycle (diagrammed in Figure S3). We have seen that the cost in free energy of forming a sulfonium ion (SAM+) by direct condensation of a thioether (Met) with an alcohol (the 5’-OH group of adenosine) would be substantial (+8.2 kcal/mol). SAM synthetase overcomes this barrier by harnessing that condensation to the cleavage of ATP at two positions. Remarkably, that coupling is believed to be indirect, i.e. at no point do the elements of ATP seem to become linked covalently to the constituents of SAM+. After the substrates ATP and Met have been bound, the adenosyl moiety of ATP is transferred to SAH to form bound SAM+ and bound inorganic triphosphate (PPPi). Bound PPPi is then cleaved to inorganic phosphate (Pi) and pyrophosphate (PPi). Both Pi and PPi must then be released be-
ABBREVIATIONS
SAM+, S-adenosylmethionine; SMM+, Smethylmethionine; Hcy, homocysteine; SAH, Sadenosylhomocysteine.
References: (1) Cantoni, G.L. (1952) The nature of the active methyl donor formed enzymatically from L-methionine and adenosine triphosphate. J. Am. Chem. Soc 74, 2942-1943. (2) McRorie, R. A., Sutherland, G. L., Lewis, M. S., Barto, A. D., Glazener, M. R., Shive, W. (1954) Isolation and identification of a naturally occurring analog of methionine. J. Amer. Chem. Soc. 76, 115-118.
ACS Paragon Plus Environment
3
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(3) McQueeney, M. S., Anderson, K. S., Markham, G. D. (2000), Energetics of S-Adenosylmethionine Synthetase Catalysis, Biochemistry 39, 4443-4454. (4) Lipmann, F. (1941) Metabolic generation and utilization of phosphate bond energy, Adv. Enzymol. Related Areas Mol. Biol., 1, 99-162. (5) Frick, L., MacNeela, J. P., Wolfenden, R. (1987) Transition state stabilization by deaminases: rates of non-enzymatic hydrolysis of adenosine and cytidine, Bioorg. Chem. 15, 100-108. (6) Stockbridge, R. B., Schroeder, G. K., Wolfenden, R. (2010). The rate of spontaneous cleavage of the glycosidic bond of adenosine. Bioorg. Chem. 38, 224228. (7) Arnett, E. M. (1963) Quantitative comparison of weak organic bases, Progress in Physical Organic Chemistry 1, 223-403. (8) Paul, M. A., Long, F. A. (1957) H0 and related indicator acidity functions, Chem. Rev. 1190. (9) Jencks, W. P. (1968) in “Handbook of Biochemistry and Biophysics” (ed. Sober, H. A.) pp. 579-583, Chemical Rubber Co. (10) Wolfenden, R., Yang, Y. (2007) Monoalkyl sulfates as alkylating agents in water, arylsulfatase rate enhancements, and the “energy-rich” nature of sulfate half-esters. Proc. Natl. Acad. Sci. U. S. A 104, 83-86. (11) De la Haba, G., Cantoni, G. L. (1959) The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine, J. Biol. Chem. 234, 603-608. (12) Rüdiger, H., Jaenicke, (1969) L. Methionine synthesis: demonstration of the reversibility of the reaction, FEBS Letters 4, 316-318. (13) Carpenter, F. H. (1960) The free energy change in hydrolytic reactions: the non-ionized compound convention, J. Am. Chem. Soc. 82, 1111-1121.
Insert Table of Contents artwork here
ACS Paragon Plus Environment
4
Page 4 of 4
