Binding energy in the one-electron reductive cleavage of S-adenosylmethionine in lysine 2,3-aminomutase, a radical SAM enzyme - PubMed (original) (raw)

. 2007 Nov 13;46(45):12889-95.

doi: 10.1021/bi701745h. Epub 2007 Oct 18.

Affiliations

Binding energy in the one-electron reductive cleavage of S-adenosylmethionine in lysine 2,3-aminomutase, a radical SAM enzyme

Susan C Wang et al. Biochemistry. 2007.

Abstract

The common step in the actions of members of the radical SAM superfamily of enzymes is the one-electron reductive cleavage of S-adenosyl-l-methionine (SAM) into methionine and the 5'-deoxyadenosyl radical. The source of the electron is the [4Fe-4S]1+ cluster characterizing the radical SAM superfamily, to which SAM is directly ligated through its methionyl carboxylate and amino groups. The energetics of the reductive cleavage of SAM is an outstanding question in the actions of radical SAM enzymes. The energetics is here reported for the action of lysine 2,3-aminomutase (LAM), which catalyzes the interconversion of l-lysine and l-beta-lysine. From earlier work, the reduction potential of the [4Fe-4S]2+/1+ cluster in LAM is -0.43 V with SAM bound to the cluster (Hinckley, G. T., and Frey, P. A. (2006) Biochemistry 45, 3219-3225), 1.4 V higher than the reported value for trialkylsulfonium ions in solution. The midpoint reduction potential upon binding l-lysine has been estimated to be -0.6 V from the values of midpoint potentials measured with SAM bound to the cluster and l-alanine in place of l-lysine, with S-adenosyl-l-homocysteine (SAH) bound to the cluster in the presence of l-lysine, and with SAH bound to the cluster in the presence of l-alanine or of l-alanine and ethylamine in place of l-lysine. The reduction potential for SAM has been estimated to be -0.99 V from the measured value for S-3',4'-anhydroadenosyl-l-methionine. The reduction potential for the [4Fe-4S] cluster is lowered 0.17 V by the binding of lysine to LAM, and the binding of SAM to the [4Fe-4S] cluster in LAM elevates its reduction potential by 0.81 V. Thus, the binding of l-lysine to LAM contributes 4 kcal mol-1, and the binding of SAM to the [4Fe-4S] cluster in LAM contributes 19 kcal mol-1 toward lowering the barrier for reductive cleavage of SAM from 32 kcal mol-1 in solution to 9 kcal mol-1 at the active site of LAM.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1. The currently accepted mechanism of action of LAM

Kinetic and EPR spectroscopic evidence for the radical complexes 1 and 3 and the radical Ado • as intermediates has been published (3,5,19). The present work relates to the energetics for the reductive cleavage of SAM ligated to the FeS-cluster.

Fig. 2

Fig. 2. Equilibrium in the reductive cleavage of _an_SAM at the active site of LAM

_an_SAM in place of SAM at the active site of LAM is reductively cleaved to the radical _an_Ado • upon reaction with lysine (19). The figure shows the increase with time of the EPR signal for _an_Ado • after addition of lysine to 34 µM LAM. The reaction proceeds to equilibrium at 2 µM _an_Ado •. Although lysine is a poor substrate and reacts forward very slowly, the cleavage of _an_SAM to the radical _an_Ado • approaches equilibrium. The detailed composition of the the reaction mixture is given in the Experimental section.

Fig. 3

Fig. 3. Energetics of one-electron reversible cleavage of SAM at the active site of LAM

The blue scale on the left illustrates the mid-point reduction potentials of the [4Fe–4S]2+/1+ cluster with cysteine as the ligand, with SAM as the ligand, and with lysine bound and SAM as the ligand. The red scale shows the potentials for irreversible one-electron reduction of trialkylsulfonium ions, such as SAM, in solution and for reversible one-electron reductive cleavage of SAM bound to the [4Fe–4S] cluster in the active site of LAM.

Scheme 1

Scheme 1

Similar articles

Cited by

References

    1. Sofia HJ, Chen G, Hetzler BG, Reyes-Spindola JF, Miller NE. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 2001;29:1097–1106. - PMC - PubMed
    1. Cheek J, Broderick JB. Adenosylmethionine-dependent iron-sulfur enzymes: versatile clusters in a radical new role. J. Biol. Inorg. Chem. 2001;6:209–226. - PubMed
    1. Frey PA, Booker SJ. Radical mechanisms of S-adenosylmethionine-dependent enzymes. Adv Protein Chem. 2001;58:1–45. - PubMed
    1. Fontecave M, Mulliez E, Ollagnier-de-Choudens S. Adenosylmethionine as a source of 5'-deoxyadenosyl radicals. Curr. Opin. Chem. Biol. 2001;5:506–511. - PubMed
    1. Frey PA, Magnusson OTh. S-Adenosylmethionine: A wolf in sheep’s clothing or a rich man’s adenosylcobalamin? Chem. Rev. 2003;103:2129–2148. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources