glmS Riboswitch Binding to the Glucosamine-6-phosphate α-Anomer Shifts the p K a toward Neutrality

Department of Molecular Biophysics and Biochemistry, Yale University, 260 Whitney Avenue, New Haven, Connecticut 06520-8114, USA.
Biochemistry (Impact Factor: 3.02). 08/2011; 50(33):7236-42. DOI: 10.1021/bi200471c
Source: PubMed


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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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-base catalyst, while its phosphate is important for binding affinity. However, the solution pK(a) of the amine is 8.06 ± 0.05, which is not optimal for proton transfer. Here we used Raman crystallography directly to determine the pK(a)'s of GlcN6P bound to the glmS ribozyme. Binding to the RNA lowers the pK(a) of the amine of GlcN6P to 7.26 ± 0.09 and raises the pK(a) of its phosphate to 6.35 ± 0.09. Remarkably, the pK(a)'s of these two functional groups are unchanged from their values for free GlcN6P (8.06 ± 0.05 and 5.98 ± 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.
    Preview · Article · Aug 2011 · Journal of the American Chemical Society
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2'-OH nucleophile. Apparent pK(a) values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK(a) values obtained from functional assays and microscopic pK(a) values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state.
    Full-text · Article · Sep 2011 · Journal of the American Chemical Society
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The hepatitis delta virus (HDV) ribozyme and related RNAs are widely dispersed in nature. This RNA is a small nucleolytic ribozyme that self-cleaves to generate products with a 2',3'-cyclic phosphate and a free 5'-hydroxyl. Although small ribozymes are dependent on divalent metal ions under biologically relevant buffer conditions, they function in the absence of divalent metal ions at high ionic strengths. This characteristic suggests that a functional group within the covalent structure of small ribozymes is facilitating catalysis. Structural and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pK(a) that serves as a general acid to protonate the leaving group. The reaction of the HDV ribozyme in monovalent cations alone never approaches the velocity of the Mg(2+)-dependent reaction, and there is significant biochemical evidence that a Mg(2+) ion participates directly in catalysis. A recent crystal structure of the HDV ribozyme revealed that there is a metal binding pocket in the HDV ribozyme active site. Modeling of the cleavage site into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg(2+) ion can serve as a Lewis acid, facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies in its cleavage reaction.
    Preview · Article · Nov 2011 · Biochemistry
Show more