title: NMR-based reappraisal of the coordination of a metal ion at the pro-Rp oxygen of the A9/G10.1 site in a hammerhead ribozyme.
abstract: In the identification of a metal-binding site within enzymes, kinetic analyses based on thio-effects and Cd(2+)-rescues are widely used. In those analyses, kinetic studies using a phosphorothioate have been discussed on the premise that the substitution by a sulfur atom does not change the conformation of a ribozyme. However, our present NMR structural analysis demonstrates the change of the conformation at the metal-binding site by Rp-sulfur but not by Sp-sulfur substitution and warns against incautious interpretations of thio-effects and rescue phenomena in kinetic studies using a phosphorothioate. Our analysis further demonstrates that, in solution, a Cd(2+) ion can interact with an Rp-phosphorothioate (in support of the controversial McKay's structure, Nature 1994, 372, 68-74) and with an Sp-phosphorothioate (in support of the controversial Scott's structure, Cell 1995, 81, 991-1002) at the metal-binding A9/G10.1 site and that, in the former case, the bound Cd(2+) ion can return the ribozyme to an active conformation and rescue its enzymatic activity.
authors.affiliation: Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba Science City, 305-8585, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Suzumura
authors.forename: Yasuomi
authors.initials: Y
authors.lastname: Takagi
authors.forename: Masaya
authors.initials: M
authors.lastname: Orita
authors.forename: Kazunari
authors.initials: K
authors.lastname: Taira
mesh_terms: D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009690:Nucleic Acid Conformation; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism
chemical_list: D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D002104:Cadmium; D013455:Sulfur; D010100:Oxygen