Extraordinary rates of transition metal ion-mediated ribozyme catalysis

Abstract

In pre-steady-state, fast-quench kinetic analysis, the tertiary-stabilized hammerhead ribozyme “RzB” cleaves its substrate RNA with maximal measured k _obs values of ∼3000 min⁻¹ in 1 mM Mn²⁺ and ∼780 min⁻¹ in 1 mM Mg²⁺ at 37°C (pH 7.4). Apparent pKa for the catalytic general base is ∼7.8–8.5, independent of the corresponding metal hydrate pKa, suggesting potential involvement of a nucleobase as general base as suggested previously from nucleobase substitution studies. The pH-rate profile is bell-shaped for Cd²⁺, for which the general catalytic acid has a pKa of 7.3 ± 0.1. Simulations of the pH-rate relation suggest a pKa for the general catalytic acid to be ∼9.5 in Mn²⁺ and >9.5 in Mg²⁺. The acid pKa's follow the trend in the pKa of the hydrated metal ions but are displaced by ∼1–2 pH units in the presence of Cd²⁺ and Mn²⁺. One possible explanation for this trend is direct metal ion coordination with a nucleobase, which then acts as general acid.

Keywords

• acid-base
• divalent ions
• hammerhead ribozyme
• ribozyme kinetics