Direct Measurement of the Ionization State of an Essential Guanine in the Hairpin Ribozyme

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise function(s) in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pK(a) values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme to determine microscopic pK(a) values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pK(a) values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pK(a) 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.