title: Structural studies of inhibition of S-adenosylmethionine synthetase by slow, tight-binding intermediate and product analogues.
abstract: S-Adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a two-step reaction in which tripolyphosphate (PPPi) is a tightly bound intermediate. Diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3); PNPNP), a non-hydrolyzable analogue of PPPi, is the most potent known inhibitor of AdoMet synthetase with a K(i) of 2 nM. The structural basis for the slow, tight-binding inhibition by PNPNP has been investigated by spectroscopic methods. UV difference spectra reveal environmental alterations of aromatic protein residues upon PNPNP binding to form the enzyme.2Mg(2+).PNPNP complex, and more extensive changes upon formation of the enzyme.2Mg(2+).PNPNP.AdoMet complex. Stopped-flow kinetic studies of complex formation revealed that two slow isomerizations follow PNPNP binding in the presence of AdoMet, in contrast to the lower affinity, rapid-equilibrium binding in the absence of AdoMet. (31)P NMR spectra of enzyme complexes with PNPNP revealed electronic perturbations of each phosphorus atom by distinct upfield chemical shifts for each of the three phosphoryl groups in the enzyme.2Mg(2+).PNPNP complex, and further upfield shifts of at least 2 resonances in the complex with AdoMet. Comparison of the chemical shifts for the enzyme-bound PNPNP with the enzyme complexes containing either the product analogue O(3)P-NH-PO(3) or O(3)P-O-PO(2)-NH-PO(3) indicates that the shifts on binding are largest at the binding sites corresponding to those for the alpha and gamma phosphoryl groups of the nucleotide (-3.1 to -4.1 ppm), while the resonance at the beta phosphoryl group position shifts by -2.1 ppm. EPR spectra of Mn(2+) complexes demonstrate spin coupling between the two Mn(2+) in both enzyme.2Mn(2+).PNPNP and enzyme.2Mn(2+).PNPNP.AdoMet, indicating that the metal ions have comparable distances in both cases. The combined results indicate that formation of the highest affinity complex is associated with protein side chain rearrangements and increased electron density at the ligand phosphorus atoms, likely due to ionization of an -NH- group of the inhibitor. The energetic feasibility of ionization of a -NH- group when two Mg(2+) ions are bound to O(3)P-NH-PO(3) is supported by density functional theoretical calculations on model chelates. This mode of interaction is uniquely available to compounds with P-NH-P linkages and may be possible with other proteins in which multiple cations coordinate a polyphosphate chain.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111-2497, USA. GD_Markham@fccc.edu
authors.forename: George D
authors.initials: GD
authors.lastname: Markham
authors.forename: Robert S
authors.initials: RS
authors.lastname: Reczkowski
mesh_terms: D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D001667:Binding, Competitive; D004164:Diphosphonates / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008716:Methionine Adenosyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010759:Phosphorus Isotopes; D011122:Polyphosphates / Q000737:chemistry; D011485:Protein Binding; D011522:Protons; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D004164:Diphosphonates; D004791:Enzyme Inhibitors; D009587:Nitrogen Isotopes; D010759:Phosphorus Isotopes; D011122:Polyphosphates; D011522:Protons; D000266:Adenylyl Imidodiphosphate; C016514:imidodiphosphonic acid; D008345:Manganese; D012436:S-Adenosylmethionine; D008716:Methionine Adenosyltransferase; D008274:Magnesium