The glmS Ribozyme Tunes the Catalytically Critical pKa of Its Coenzyme Glucosamine-6-phosphate

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• 作者：Bo Gong ; Daniel J. Klein ; Adrian R. Ferr茅-D鈥橝mar茅 ; Paul R. Carey
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：September 14, 2011
• 年：2011
• 卷：133
• 期：36
• 页码：14188-14191
• 全文大小：728K
• 年卷期：v.133,no.36(September 14, 2011)
• ISSN：1520-5126

文摘

The glmS ribozyme riboswitch is the first known natural catalytic RNA that employs a small-molecule cofactor. Binding of glucosamine-6-phosphate (GlcN6P) uncovers the latent self-cleavage activity of the RNA, which adopts a catalytically competent conformation that is nonetheless inactive in the absence of GlcN6P. Structural and analogue studies suggest that the amine of GlcN6P functions as a general acid鈥揵ase catalyst, while its phosphate is important for binding affinity. However, the solution pK_a of the amine is 8.06 卤 0.05, which is not optimal for proton transfer. Here we used Raman crystallography directly to determine the pK_a鈥檚 of GlcN6P bound to the glmS ribozyme. Binding to the RNA lowers the pK_a of the amine of GlcN6P to 7.26 卤 0.09 and raises the pK_a of its phosphate to 6.35 卤 0.09. Remarkably, the pK_a鈥檚 of these two functional groups are unchanged from their values for free GlcN6P (8.06 卤 0.05 and 5.98 卤 0.05, respectively) when GlcN6P binds to the catalytically inactive but structurally unperturbed G40A mutant of the ribozyme, thus implicating the ribozyme active site guanine in pK_a tuning. This is the first demonstration that a ribozyme can tune the pK_a of a small-molecule ligand. Moreover, the anionic glmS ribozyme in effect stabilizes the neutral amine of GlcN6P by lowering its pK_a. This is unprecedented and illustrates the chemical sophistication of ribozyme active sites.