The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous Gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, the detailed reaction mechanism as well as the protonation state of the glmS reactive form still remains elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the Thermoanaerobacter tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40(-) seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase in K-m with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and Gl purines in structural stabilization of the A-1(2'-01-I) in-line attack conformation. On the basis of the identified dominant protonation state of the reaction precursor, we propose a hypothesis of the self-cleavage mechanism in which A-1(2'-OH) is activated as a nucleophile by the Gl (pro-R-p) nonbridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the Gl(O5') leaving group.