glmS Riboswitch binding to the glucosamine-6-phosphate α-anomer shifts the pKa toward neutrality.

The glmS riboswitch regulates gene expression through a self-cleavage activity. The reaction is catalyzed with the assistance of the metabolite cofactor glucosamine-6-phosphate (GlcN6P), whose amino group is proposed to serve as the general acid during the reaction. This reaction is pH-dependent with a pK(a) that is lower than the observed pK(a) for the amine of GlcN6P in solution. GlcN6P, like other pyranose sugars, undergoes spontaneous and rapid interconversion between the α and β anomers at the C1 position. Here we demonstrate by NMR that the Bacillus anthracis glmS riboswitch selectively binds the α-anomer of GlcN6P with a maximum binding affinity of 0.36 mM and that binding is pH-dependent. We also report that the anomeric ratio between α and β is pH-dependent and the pK(a)s of the two amines differ by 0.5 pH units, α being the higher of the two (pK(a)=8.3). The pH dependence of binding reveals a pK(a) of 6.7, suggesting that the glmS RNA reduces the pK(a) of the GlcN6P amine by 1.6 units in the ground state. We reevaluated previously obtained kinetic data and found the reaction pK(a) is 6.9, within error of the binding data. The data support a model where the reaction pK(a) corresponds to that of the GlcN6P amine. This observation has broader relevance for considering how the microenvironment of an RNA, despite its anionic character, can reduce the pK(a)s of functional groups for use in catalysis.