Journal
NUCLEIC ACIDS RESEARCH
Volume 51, Issue 9, Pages 4508-4518Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkad260
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By using simulations and calculations, we have elucidated the mechanism of methyl transfer catalyzed by a methyltransferase ribozyme. We have identified two transition states and a rate-controlling step, and predicted the activity-pH profile of the reaction.
A methyltransferase ribozyme (MTR1) was selected in vitro to catalyze alkyl transfer from exogenous O-6-methylguanine (O(6)mG) to a target adenine N1, and recently, high-resolution crystal structures have become available. We use a combination of classical molecular dynamics, ab initio quantum mechanical/molecular mechanical (QM/MM) and alchemical free energy (AFE) simulations to elucidate the atomic-level solution mechanism of MTR1. Simulations identify an active reactant state involving protonation of C10 that hydrogen bonds with O(6)mG:N1. The deduced mechanism involves a stepwise mechanism with two transition states corresponding to proton transfer from C10:N3 to O(6)mG:N1 and rate-controlling methyl transfer (19.4 kcal center dot mol(-1) barrier). AFE simulations predict the pK(a) for C10 to be 6.3, close to the experimental apparent pK(a) of 6.2, further implicating it as a critical general acid. The intrinsic rate derived from QM/MM simulations, together with pK(a) calculations, enables us to predict an activity-pH profile that agrees well with experiment. The insights gained provide further support for a putative RNA world and establish new design principles for RNA-based biochemical tools. [GRAPHICS] .
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