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 kobs 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 Mn 2+ 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. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 RNA Society.