Chiral instability at sulfur of S-adenosylmethionine

Wu, Su Er; Huskey, W. Phillip; Borchardt, Ronald T.; Schowen, Richard L.

doi:10.1021/bi00281a009

Cited by 84 publications

(51 citation statements)

References 29 publications

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“…This compound is derived from AdoMet metabolism in the polyamine biosynthetic pathway, and can also arise from nonenzymatic hydrolysis of AdoMet under physiologic conditions. 24 Hence, we evaluated the effect of MTA on OA-induced apoptosis. Pretreatment of hepatocytes with MTA abolished the apoptotic effect of OA (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1 Exogenous AdoMet can also undergo nonenzymatic hydrolysis in vivo into MTA and homoserine. 9,24 It has been suggested that the beneficial effects of AdoMet in liver damage could be attributed in part to its conversion to MTA. 9,10 Additionally, we have recently shown that MTA mimics AdoMet's effect on gene expression in cultured rat hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

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S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells

et al. 2002

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S-adenosylmethionine (AdoMet) is an essential compound in cellular transmethylation reactions and a precursor of polyamine and glutathione synthesis in the liver. In liver injury, the synthesis of AdoMet is impaired and its availability limited. AdoMet administration attenuates experimental liver damage, improves survival of alcoholic patients with cirrhosis, and prevents experimental hepatocarcinogenesis. Apoptosis contributes to different liver injuries, many of which are protected by AdoMet. The mechanism of AdoMet's hepatoprotective and chemopreventive effects are largely unknown. The effect of AdoMet on okadaic acid (OA)-induced apoptosis was evaluated using primary cultures of rat hepatocytes and human hepatoma cell lines. AdoMet protected rat hepatocytes from OA-induced apoptosis dose dependently. It attenuated mitochondrial cytochrome c release, caspase 3 activation, and poly(ADP-ribose) polymerase cleavage. These effects were independent from AdoMet-dependent glutathione synthesis, and mimicked by 5-methylthioadenosine (MTA), which is derived from AdoMet. Interestingly, AdoMet and MTA did not protect HuH7 cells from OA-induced apoptosis; conversely both compounds behaved as proapoptotic agents. AdoMet's proapoptotic effect was dose dependent and observed also in HepG2 cells. In conclusion, AdoMet exerts opposing effects on apoptosis in normal versus transformed hepatocytes that could be mediated through its conversion to MTA. These effects may participate in the hepatoprotective and chemopreventive properties of this safe and welltolerated drug. (HEPATOLOGY 2002;35:274-280.)

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells

et al. 2002

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“…In the absence of DTB, AdoMet is bound to biotin synthase with low affinity [ Figure A major concern in assessing AdoMet binding by equilibrium dialysis is the instability of AdoMet toward both hydrolytic degradation and racemization at the chiral sulfonium (37). To minimize the effect of these processes on our binding results, we performed dialysis at 20 °C to slow racemization and used HPLC analysis to separate AdoMet from the various degradation products.…”

Section: Substratementioning

confidence: 99%

Control of Adenosylmethionine-Dependent Radical Generation in Biotin Synthase: A Kinetic and Thermodynamic Analysis of Substrate Binding to Active and Inactive Forms of BioB

2003

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Biotin synthase (BS) is an AdoMet-dependent radical enzyme that catalyzes the insertion of sulfur into saturated C6 and C9 atoms in the substrate dethiobiotin. To facilitate sulfur insertion, BS catalyzes the reductive cleavage of AdoMet to methionine and 5′-deoxyadenosyl radicals, which then abstract hydrogen atoms from the C6 and C9 positions of dethiobiotin. The enzyme from Escherichia coli is purified as a dimer that contains one [2Fe-2S] 2+ cluster per monomer and can be reconstituted in vitro to contain an additional [4Fe-4S] 2+ cluster per monomer. Since each monomer contains each type of cluster, the dimeric enzyme could contain one active site per monomer, or could contain a single active site at the dimer interface. To address these possibilities, and to better understand the manner in which biotin synthase controls radical generation and reactivity, we have examined the binding of AdoMet and DTB to reconstituted biotin synthase. We find that both the [2Fe-2S] 2+ cluster and the [4Fe-4S] 2+ cluster must be present for tight substrate binding. Further, substrate binding is highly cooperative, with the affinity for AdoMet increasing >20-fold in the presence of DTB, while DTB binds only in the presence of AdoMet. The stoichiometry of binding is ca. 2:1:1 AdoMet:DTB:BS dimer, suggesting that biotin synthase has a single functional active site per dimer. AdoMet binding, either in the presence or in the absence of DTB, leads to a decrease in the magnitude of the UV-visible absorption band at ∼400 nm that we attribute to changes in the coordination environment of the [4Fe-4S] 2+ cluster. Using these spectral changes as a probe, we have examined the kinetics of AdoMet and DTB binding, and propose an ordered binding mechanism that is followed by a conformational change in the enzyme-substrate complex. This kinetic analysis suggests that biotin synthase is evolved to bind AdoMet both weakly and slowly in the absence of DTB, while both the rate of binding and the affinity for AdoMet are increased in the presence of DTB. Cooperative binding of AdoMet and DTB may be an important mechanism for limiting the production of 5′-deoxyadenosyl radicals in the absence of the correct substrate.Biotin synthase (BS) 1 catalyzes formation of the thioether ring in the essential vitamin biotin (for reviews, see refs 1 and 2). Thioether ring formation requires substitution of sulfur for hydrogen atoms at the saturated C6 and C9 positions of the substrate dethiobiotin. Functionalization of these relatively unreactive carbon atoms in dethiobiotin is likely initiated by hydrogen atom abstraction (3), generating carbon radicals at one or both positions. Biotin synthase is purified from Escherichia coli as a dimeric iron-sulfur protein (4) that requires S- † This research has been supported by NIH Research Grant GM59175 (J.T.J.).* To whom correspondence should be addressed: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, 905B Stellar-Chance Laboratories, Philadelphia, PA 19104-6059...

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“…In this respect, it can be hypothesized that D-aspartyl-containing peptides could be methylated in vitro by PCMT using AdoMet, with opposite chirality at sulfur center (R), as methyl donor. Such an AdoMet enantiomer has been shown to be present in living cells [35].…”

Section: Discussionmentioning

confidence: 99%

Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate

Galletti

Ingrosso

Manna

et al. 1988

European Journal of Biochemistry

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Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595-6021, while very little is know concerning the structural requirements of the protein substrate.In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J . Biol. Chem. 261, 11 503 -11 51 11; (2) the isoaspartyl residue is not recognized unless its cc-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methylesterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.Protein carboxyl methyl esterification, catalyzed by the enzyme S-adenosylmethionine : protein carboxy-0-methyl transferase (PCMT) has been recently proposed to be a crucial step in the repair or disposal of proteins containing altered aspartyl residues [l, 21. The above suggestion is supported by the experimental evidence that in eukaryotic proteins only L-isoaspartyl [3,6] residues are selectively recognized and modified by this enzyme.An enzyme playing such physiological roles should be endowed with a broad substrate specificity. A wide range of proteins can indeed be enzymatically methyl-esterified in vitro, although only to a substoichiometric extent [7], in that only a subpopulation of the protein substrate contains methylatable sites. Furthermore L-isoaspartyl-containing natural and synthetic peptides are stoichiometrically methylated in vitro by PCMT, regardless of the sequences surrounding the modification site [8 -111.Correspondence fo P. Galletti, Istituto di Biochimica delle Macromolecole, I Facolta di Medicina e Chirurgia, Via Costantinopoli 16, 1-80138 Napoli Abbreviations. PCMT, protein carboxyl-0-methyl transferase; AdoMet, S-adenosyl-L-methionine;...

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Chiral instability at sulfur of S-adenosylmethionine

Cited by 84 publications

References 29 publications

S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells

S-adenosylmethionine and methylthioadenosine are antiapoptotic in cultured rat hepatocytes but proapoptotic in human hepatoma cells

Control of Adenosylmethionine-Dependent Radical Generation in Biotin Synthase: A Kinetic and Thermodynamic Analysis of Substrate Binding to Active and Inactive Forms of BioB

Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate

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