Importance of magnesium ions in the mechanism of catalysis by a hammerhead ribozyme: strictly linear relationship between the ribozyme activity and the concentration of magnesium ions.
Analysis, based on kinetic solvent isotope effects, demonstrated that no proton transfer occurs in reactions catalyzed by a 32-mer hammerhead ribozyme (R32) in the presence of magnesium ions, whereas proton transfer occurs in reactions catalyzed by R32 in the presence of high concentrations of monovalent NH4+ ions without metal ions, demonstrating that the detailed mechanism of action of the hammerhead ribozyme might change depending on the environment. Importantly, when the concentration of magnesium ions was gradually increased from 1 mM to up to 800 mM, the R32 ribozyme activity increased linearly without reaching a plateau value. This phenomenon can be explained by a model in which a catalytic magnesium ion with a very low affinity (dissociation constant, Kd > 800 mM) exists and/or the predominant inactive complex converts to a minor active complex before the cleavage reaction.
Gene Function Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba Science City, Ibaraki 305-8562, Japan.
Atsushi
A
Inoue
Yasuomi
Y
Takagi
Kazunari
K
Taira
D001483:Base Sequence; D002384:Catalysis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D016337:RNA, Catalytic / Q000378:metabolism*
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D008274:Magnesium