Examining the role of structural features in reactivity requires to ensure proper sampling of active site conformations in simulations, as recent works increasingly suggest the interplay between conformational dynamics and reactivity to be central to understand enzyme catalysis with, for instance, conformational selection now being used as a guiding principle to enzyme design. In this work, we thus investigated using various molecular simulation approaches the structural stability of the M5:rRNA complex and characterized the conformational dynamics of this interface. We show that fluctuations of the interface impact key structural features of the active site and we identify distinct conformations, whose structures appear pre-organized to favor distinct mechanisms. Finally, we characterize using dynamic QM/MM-MD simulations the reaction mechanism and free energy profile in each of the identified conformational basins, identify the most favorable route and provide a comprehensive picture of the interplay between conformational fluctuations and reactivity of this RNase.