Molecular mechanism of the site-specific self-cleavage of the RNA phosphodiester backbone by a twister ribozyme

Katarzyna Świderek,Iñaki Tuñón,Juan Bertran,Sergio Marti,Vicent Moliner

doi:10.1007/s00214-017-2060-8

Katarzyna Świderek, Iñaki Tuñón + Show 3 more

Open Access

https://doi.org/10.1007/s00214-017-2060-8

Copy DOI

Abstract

The catalytic activity of some classes of natural RNA, named as ribozymes, has been discovered just in the past decades. In this paper, the cleavage of the RNA phosphodiester backbone has been studied in aqueous solution and in a twister ribozyme from Oryza sativa. The free energy profiles associated with a baseline substrate-assisted mechanism for the reaction in the enzyme and in solution were computed by means of free energy perturbation methods within hybrid QM/MM potentials, describing the chemical system by the M06-2× functional and the environment by means of the AMBER and TIP3P force fields. The results confirm that this is a stepwise mechanism kinetically controlled by the second step that involves the P–O5′ breaking bond concomitant with the proton transfer from the OP1 atom to the leaving O5′ atom. 18O kinetic isotope effects on the nucleophile and leaving oxygen atoms, in very good agreement with experiments, also support this description. Nevertheless, the free energy profiles in the enzyme and in solution are almost coincident which, despite that the rate-limiting activation free energy is in very good agreement with experimental data of counterpart reactions in solution, rule out this substrate-assisted catalysis mechanism for the twister ribozyme from O. sativa. Catalysis must come from the role of alternative acid–base species not available in aqueous solution, but the rate-limiting transition state must be associated with the P–O5′ bond cleavage.

Highlights

Chemical reactions are traditionally known to be catalysed in nature by protein enzymes, but in the early 80s it was discovered that natural RNA molecules, named as ribozymes, present catalytic activity [1, 2]
The molecular mechanism for the cleavage of the RNA phosphodiester backbone has been studied in aqueous solution and catalysed by a twister ribozyme from Oryza sativa
The present paper shows the results of exploring the baseline or substrate-assisted mechanism by means of quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations where the QM subset of atoms is described by a DFT functional (M06-2X)

Summary

Introduction

Chemical reactions are traditionally known to be catalysed in nature by protein enzymes, but in the early 80s it was discovered that natural RNA molecules, named as ribozymes, present catalytic activity [1, 2]. According to their size, these natural ribozymes can be divided into three families: small self-cleaving RNAs (less than 200 nucleotides), medium-sized self-splicing introns and larger catalytic ribonuclear protein (RNP) complexes [3]. The activation of the nucleophile and leaving groups, if occurred, has been proposed to take place through nucleosides located in the active site as general acid or base catalysts (“AH” and “B” in Scheme 1)

Methods

Results

Conclusion