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
Author(s) : Atsushi Inoue, Yasuomi Takagi, Kazunari Taira , Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1‐1‐1 Higashi, Tsukuba Science City, Ibaraki 305‐8562, Japan. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113‐8656, Japan. IGENE Therapeutics, Inc., C\O National Institute of Advanced Industrial Science and Technology (AIST), 1‐1‐1 Higashi, Tsukuba Science City, Ibaraki 305‐8562, Japan. .
Summary : 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 NH
4+ 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, K
d > 800 mM) exists and\\or the predominant inactive complex converts to a minor active complex before the cleavage reaction.
Fig. 1. (A) The sequences and secondary
structures of a hammerhead ribozyme (R32) and its substrate (S11)
used in this study. (B) The proposed two-stage folding scheme for
hammerhead ribozyme – substrate complex. The higher
affinity Mg2+ drives the formation of domain II which
contains non-Watson-Crick pairings and the lower affinity
Mg2+ rotates around the helix I, forming the catalytic
core.
Fig. 2. (A) The equation for conversion
of the apparent isotope effect to the intrinsic one, and a table of
the apparent isotope effects, pKa values used in the
equation and the intrinsic isotope effects in the indicated
cations. The Mg2+-mediated, NH4+-mediated and
Li+-mediated reactions yielded in values for intrinsic
isotope effect of approximately 1, 2 and 1, respectively. (B)
Schematic representation of a proposed hammerhead ribozyme
reaction. The 2'-hydoxyl activated by catalyst attacks the adjacent
phosphate nucleophilically, and then the
5'-oxygen – phosphorus bond is cleaved. And the
developing negative charge on the leaving 5'-oxygen is stabilized
by another catalyst.
Fig. 3. Dependency of the
R32 – catalyzed reactions on Mg2+ ions. Even
at very high concentrations of Mg2+ ions, the
experimental data can be fitted to the linear line with a slope of
0.8. The activity increases with the concentration of
Mg2+, and it did not reach saturating points even when
the concentration reached 800 mM.
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