Molecular simulations of the complex between M5 and its rRNA substrate have thus demonstrated the plausibility of the experimentally suggested complex and inferred active site structure, which are-provided a small displacement of the scissile phosphate and the use of specialized force fields-stable and with a reasonable reactive pathway. Combining advanced enhanced sampling techniques and dynamic QM/MM-MD reaction path exploration, we have uncovered a rich conformational landscape in the active site and characterized different mechanistic routes associated with various orientations of key active site residues. The most favorable mechanism features deprotonation of the nucleophilic water molecule by a Mg 2+ -bound glutamate (Glu96). The formed hydroxide ion, stabilized by the Mg 2+ , then attacks the scissile phosphate in an associative mechanism with a pentavalent phosphate at the transition state. The leaving group is later reprotonated by a water molecule, whose acidity is exacerbated by coordination to both Mg 2+ ions. Such a mechanism is not possible from the main conformational basin, so that the system must wait for a conformational fluctuation to bring it in a reactive conformation.