Structure of the divalent metal ion activator binding site of S-adenosylmethionine synthetase studied by vanadyl(IV) electron paramagnetic resonance.
The structure of the divalent metal ion binding site of S-adenosylmethionine synthetase from Escherichia coli has been studied by using the vanadyl(IV) ion (VO2+) as probe. VO2+ binds at a single site per subunit in the presence or absence of substrates. Single turnover experiments measuring S-adenosylmethionine (AdoMet) formation from methionine and the ATP analogue 5'-adenylyl imidodiphosphate show that complexes containing VO2+ and either Mg2+ or Ca2+ as a second metal ion are catalytically active, while a complex containing VO2+ alone is inactive. Electron paramagnetic resonance spectra of the enzyme-VO2+ complex, as well as complexes also containing AdoMet or methionine, indicate the coordination of two water molecules and at least two protein ligands to the VO2+. In complexes with polyphosphate substrates or products (e.g., enzyme-VO2+-ATP-methionine, enzyme-VO2+-PPi-Mg2+), EPR spectral changes reveal ligand substitutions on the VO2+, and 8.5-G isotropic superhyperfine coupling to two 31P nuclei can be resolved. 17O superhyperfine coupling from [17O]pyrophosphate indicates coordination of two oxygen atoms of PPi to the VO2+ ion. Thus the polyphosphate compounds are bidentate ligands to the VO2+, demonstrating that the VO2+ binds at the active site and suggesting a catalytic role for the protein-bound metal ion.
D001665:Binding Sites; D002413:Cations, Divalent; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008716:Methionine Adenosyltransferase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D014166:Transferases / Q000378:metabolism*; D014639:Vanadium / Q000494:pharmacology*
D002413:Cations, Divalent; D014639:Vanadium; D014166:Transferases; D008716:Methionine Adenosyltransferase