Extraordinary rates of transition metal ion-mediated ribozyme catalysis.
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 approximately 3000 min(-1) in 1 mM Mn(2+) and approximately 780 min(-1) in 1 mM Mg(2+) at 37 degrees C (pH 7.4). Apparent pKa for the catalytic general base is approximately 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 Cd(2+), 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 approximately 9.5 in Mn(2+) and >9.5 in Mg(2+). The acid pKa's follow the trend in the pKa of the hydrated metal ions but are displaced by approximately 1-2 pH units in the presence of Cd(2+) and Mn(2+). One possible explanation for this trend is direct metal ion coordination with a nucleobase, which then acts as general acid.
Roychowdhury-Saha
Manami
M
Department of Chemistry, Indiana University, Bloomington, Indiana 47405, USA.
Burke
Donald H
DH
D001483:Base Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008673:Metals, Alkaline Earth / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D028561:Transition Elements / Q000378:metabolism*
D008673:Metals, Alkaline Earth; D016337:RNA, Catalytic; D028561:Transition Elements; C427204:hammerhead ribozyme; D012313:RNA