The glmS ribozyme is a small, self-cleaving ribozyme found in many gram-positive bacteria. This class of ribozymes cleaves through a general acid-general base mechanism wherein the 2’-hydroxyl at the cleavage site attacks the adjacent phosphate, resulting in a 2’,3’-cyclic phosphate and a 5’-hydroxyl product. The small molecule, glucosamine-6-phosphate (GlcN6P), is required for glmS ribozyme cleavage, making it a riboswitch-ribozyme. In glmS ribozyme cleavage, GlcN6P is thought to serve as the general acid that protonates the leaving group [1], and G33, a conserved guanine positioned in the active site of the ribozyme, is well-positioned to act as the general base. Evidence from previous studies, however, suggests that G33(N1) exhibits a pKa shifted away from neutrality (≥10 compared to the pKa of 9.2 for free guanine in solution) [2], a shift in the direction opposite from that expected for a mechanistically-participating functional group. However, as demonstrated in recent work from our labs [3], this basic-shifted pKa may aid the function of the general base, wherein a pKa that more closely matches the high pKa of the 2’-hydroxyl nucleophile is more apt to abstract its proton. Theoretical studies and pKa calculations suggest that a deprotonated G33(N1) serves as the hydrogen bond acceptor of the 2’-hydroxyl nucleophile until attack of the scissile phosphate begins, whereupon the pKa of the 2’-hydroxyl is lowered enough for complete transfer of the proton to G33(N1) to occur, an event facilitated by the elevated pKa of G33. Recent work that explores new roles of exogenous species, including the GlcN6P cofactor and metal ions, in the glmS ribozyme mechanism will be presented. Preliminary data suggest that GlcN6P serves multiple catalytic roles and that metal ions participate directly within the active site of the ribozyme. [1] Viladoms, J. & Fedor, M. J. J. Am. Chem. Soc. 2012, 134, 19043-9. [2] Viladoms, J.; Scott, L. G.; Fedor, M. J., J. Am. Chem. Soc. 2011, 133, 18388-96. [3] Zhang, S., Ganguly, A., Goyal, P., Bingaman, J. L., Bevilacqua, P. C., and Hammes, Schiffer, S. J. Am. Chem. Soc. 2015, 137, 784-98.

Investigation of the glmS ribozyme: New catalytic roles for the GlcN6P cofactor and metal ions

251st National Meeting (2016)

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