Lincoln Scott - Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme.

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  Nat Chem Biol. 2009   May;5(5):351-7. doi: 10.1038/nchembio.156. Epub 2009 Mar 29.


  Active site guanines are critical for self-cleavage reactions of   several ribozymes, but their precise functions in catalysis are   unclear. To learn whether protonated or deprotonated forms of   guanine predominate in the active site, microscopic pKa values   were determined for ionization of 8-azaguanosine substituted for   G8 in the active site of a fully functional hairpin ribozyme in   order to determine microscopic pKa values for 8-azaguanine   deprotonation from the pH dependence of fluorescence. Microscopic   pKa values above 9 for deprotonation of 8-azaguanine in the   active site were about 3 units higher than apparent pKa values   determined from the pH dependence of self-cleavage kinetics.   Thus, the increase in activity with increasing pH does not   correlate with deprotonation of G8, and most of G8 is protonated   at neutral pH. These results do not exclude a role in proton   transfer, but a simple interpretation is that G8 functions in the   protonated form, perhaps by donating hydrogen bonds.

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